WO2003008625A2 - Structural and cytoskeleton-associated proteins - Google Patents

Structural and cytoskeleton-associated proteins Download PDF

Info

Publication number
WO2003008625A2
WO2003008625A2 PCT/US2002/022866 US0222866W WO03008625A2 WO 2003008625 A2 WO2003008625 A2 WO 2003008625A2 US 0222866 W US0222866 W US 0222866W WO 03008625 A2 WO03008625 A2 WO 03008625A2
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
seq
polypeptide
sequence
amino acid
Prior art date
Application number
PCT/US2002/022866
Other languages
French (fr)
Other versions
WO2003008625A3 (en
Inventor
Karen Anne Jones
Anita Swarnakar
Ann E. Gorvad
April J.A. Hafalia
Bridget A. Warren
Craig H. Ison
Cynthia D. Honchell
Danniel B. Nguyen
Ines Barroso
Debopriya Das
Erika A. Lindquist
Ernestine A. Lee
Henry Yue
Ian J. Forsythe
Jayalaxmi Ramkumar
Jennifer A. Griffin
Joana X. Li
Junming Yang
Mariah R. Baughn
Mark L. Borowsky
Michael Thornton
Monique G. Yao
Narinder K. Chawla
Neil Burford
Preeti G. Lal
Rajagopal Gururajan
Sally Lee
Sean A. Bulloch
Shanya D. Becha
Thomas W. Richardson
Vicki S. Elliott
William W. Sprague
Y. Tom Tang
Yalda Azimzai
Yan Lu
Yeganeh Zebarjadian
Original Assignee
Incyte Genomics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Incyte Genomics, Inc. filed Critical Incyte Genomics, Inc.
Priority to AU2002320621A priority Critical patent/AU2002320621A1/en
Publication of WO2003008625A2 publication Critical patent/WO2003008625A2/en
Publication of WO2003008625A3 publication Critical patent/WO2003008625A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to novel nucleic acids, structural and cytoskeleton-associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of cell motility disorders, developmental disorders, cell proliferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and structural and cytoskeleton-associated proteins.
  • the cytoskeleton is a cytoplas ic network of protein fibers that mediate 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.
  • Major cytoskeletal fibers include the microtubules, the microfilaments, and the intermediate filaments.
  • Motor proteins including myosin, dynein, and kinesin, drive movement of or along the fibers.
  • the motor protein dynamin drives the formation of membrane vesicles. Accessory or associated proteins modify the structure or activity of the fibers while cytoskeletal membrane anchors connect the fibers to the cell membrane.
  • Microtubules cytoskeletal fibers with a diameter of about 24 nm, have multiple roles in the cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing 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 are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
  • Microtubules are polymers of GTP-binding tubulin protein subunits. Each subunit is a heterodimer of ⁇ - and ⁇ - tubulin, multiple isoforms of which exist.
  • 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 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.
  • Gamma tubulin present in the MTOC 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 ⁇ .
  • Type I MAPs which include MAP1A and MAP1B, are large, filamentous molecules that co-purify with microtubules and are abundantly expressed in brain and testes.
  • Type I MAPs 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 MAP1B 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 MAP1A, MAP1B, and microtubules.
  • LC3 is synthesized from a source other than the MAPIA or MAP1B transcripts, and that the expression of LC3 maybe important in regulating the microtubule binding activity of MAPIA and MAP1B during cell proliferation (Mann, S.S. et al. (1994) J. Biol. Chem. 269:11492-11497).
  • Type ⁇ 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
  • STOP stable tubule only polypeptide
  • STOP proteins are associated with axonal microtubules, and are also abundant in neurons (Guillaud, L. et al. (1998) J. Cell Biol. 142:167-179). STOP proteins are necessary for normal neurite formation, and have been observed to stabilize microtubules, in vitro, against cold-, calcium-, or drug-induced disassembly (Margolis, R.L. et al. (1990) EMBO 9:4095-502).
  • the protein caldendrin is a calcium-binding protein selectively localized to the somatodendritic compartment of neurons and is highly enriched at postsynaptic structures. At least one isoform of caldendrin is found to be associated with neuronal cytoskeleton. Sequence alignment studies showed that caldendrin displays significant similarity with calmodulin and contains two EF-hand motifs (Seidenbecher, C. I. et al. (1998) J. Biol. Chem. 273:21324-21331).
  • Microfilaments cytoskeletal filaments with a diameter of about 7-9 nm, 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
  • 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
  • Actin-associated proteins have roles in cross-linking, 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.
  • Actin-interacting proteins (AJPs) participate in the regulation of actin filament organization.
  • Other actin-associated proteins such as TARA, a novel F-actin binding protein, function in a similar capacity by regulating actin cytoskeletal organization.
  • Calmodulin-like calcium-binding domains in actin cross-linking proteins allow calcium regulation of cross-linking.
  • Group I cross-linking proteins have unique actin-binding domains and include the 30 kD protein, EF-la, fascin, and scruin.
  • Group II cross-linking proteins have a 7,000-MW actin-binding domain and include villin and dematin.
  • Group HI cross-linking proteins have pairs of a 26,000-MW actin-binding domain and include fimbrin, spectrin, dystrophin, ABP 120, and filamin.
  • 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 and tropomodulin.
  • the proteins thymosin and profilin sequester actin monomers in the cytosol, allowing a pool of unpolymerized actin to exist.
  • Microtubule and actin filament networks cooperate in processes such as vesicle and organelle transport, cleavage furrow placement, directed cell migration, spindle rotation, and nuclear migration.
  • Microtubules and actin may coordinate to transport vesicles, organelles, and cell fate determinants, or transport may involve targeting and capture of microtubule ends at cortical actin sites.
  • These cytoskeletal systems maybe bridged by myosin-kinesin complexes, myosin-CLIP170 complexes, formin-homology (FH) proteins, dynein, the dynactin complex, Kar9 ⁇ , coronin, ERM proteins, and kelch repeat-containing proteins (for a review, see Goode, B.L. et al.
  • the kelch repeat is a motif originally observed in the kelch protein, which is involved in formation of cytoplasmic bridges called ring canals. A variety of mammalian and other kelch family proteins have been identified. The kelch repeat domain is believed to mediate interaction with actin (Robinson, D.N. and L. Cooley (1997) J. CeU Biol. 138:799-810).
  • the formin homology (FH) proteins are nuclear phosphoproteins which contain proline-rich sequences similar to Src homology 3 (SH3)-binding domains.
  • SH3 region is a small protein domain present in a large group of proteins, including cytoskeletal elements and signaling proteins.
  • FH proteins are linked to cell signaling pathways by interactions with members of the Rho family of GTP- binding proteins.
  • the FH proteins appear to play a crucial role in coodinating cytokinesis with nuclear division, as well as participating in actin-mediated processes affecting cell polarity and shape (Wasserman, S. (1998) Trends CellBiol. 8:111-115).
  • the formins are encoded by alternatively spliced transcripts arising from the limb deformity (Id) locus of the mouse. Mutations in this locus disrupt embryonic pattern formation, leading to reduction and fusion of the distal bones and digits of all limbs and to variable incidence of renal aplasia (Woychik, R.P. et al. (1990) Nature 346:850-853; Jackson-Grusby, L. et al. (1992) Genes Dev. 6:29-37).
  • a mutation in the human FH protein hDIAl was recently found to be the cause of an autosomal dominant hearing disorder (Wasserman, supra).
  • ADF/cofilins are a family of conserved 15-18 kDa actin-binding proteins that play a role in cytokinesis, endocytosis, and in development of embryonic tissues, as well as in tissue regeneration and in pathologies such as ischemia, oxidative or osmotic stress.
  • LBVI kinase 1 downregulates ADF (Carlier, M.F. et al. (1999) J. Biol. Chem. 274:33827-33830).
  • LIM is an acronym of three transcription factors, Lin-11, Isl-1, and Mec-3, in which the motif was first identified.
  • the LIM domain is a double zinc-finger motif that mediates the protein- protein interactions of transcription factors, signaling, and cytoskeleton-associated proteins (Roof, D.J. et al. (1997) J. Cell Biol. 138:575-588). These proteins are distributed in the nucleus, cytoplasm, or both (Brown, S. et al. (1999) J. Biol. Chem. 274:27083-27091). Recently, ALP (actinin-associated LIM protein) has been shown to bind alpha-actinin-2 (Bouju, S. et al. (1999) Neuromuscul. Disord. 9:3-10).
  • Frabin protein is another example of an actin-filament binding protein (Obaishi, H. et al. (1998) J. Biol. Chem. 273:18697-18700).
  • Frabin FGDl-related F-actin-binding protein
  • FAB actin-filament binding
  • DH Dbl homology
  • PH pleckstrin homology
  • Fablp, YOTB, Vaclp, and EEA1 early endosomal antigen 1
  • Frabin has shown GDP/GTP exchange activity for Cdc42 small G protein (Cdc42), and indirectly induces activation of Rac small G protein (Rac) in intact cells. Through the activation of Cdc42 and Rac, Frabin is able to induce formation of both filopodia- and lamellipodia-like processes (Ono, Y. et al. (2000) Oncogene 19:3050-3058).
  • Rho family of small GTP-binding proteins are important regulators of actin-dependent cell functions including cell shape change, adhesion, and motility.
  • the Rho family consists of three major subfamilies: Cdc42, Rac, and Rho.
  • Rho family members cycle between GDP-bound inactive and GTP-bound active forms by means of a GDP/GTP exchange factor (GEF) (Umikawa, M. et al. (1999) J. Biol. Chem. 274:25197-25200).
  • GEF GDP/GTP exchange factor
  • Interaptin is an actin-binding protein of the ⁇ -actinin superfamily (Rivero, F. et al. (1998) J. Cell Biol. 142:735-750).
  • the protein from Dictyostelium discoideum is 1737 amino acids in length and contains three domains, an N-terminal F-actin-binding domain of about 250 amino acids, a central rod domain of 1180 amino acids, which is predicted to have a coiled-coil oc-helical structure, and a C- terminal membrane-association domain of about 120 amino acids.
  • Interaptin localizes to the nuclear envelope and the endoplasmic reticulum. The expression level of interaptin in Dictyostelium is developmentally regulated.
  • Cyclic AMP which is known to regulate the expression of many developmentally controlled genes in Dictyostelium discoideum, stimulates the expression of interaptin.
  • actin binding and membrane association domains indicates that interaptin may provide a link between the intracellular membrane and actin cytoskeleton and play a role in membrane trafficking.
  • Titin may serve as a scaffold protein for the assembly of sarcomeric proteins during the development of striated muscle and also has a role in muscle elasticity (Trinick, J. and Tskhovrebova, L. (1999) Trends Cell Biol. 9:377-380). It is found exclusively in skeletal and cardiac muscle, and not in smooth muscle. It binds to actin and myosin and other sarcomeric proteins, including actinin, T-cap, M-protein and myomesin.
  • Drosophila melangaster D-titin contains fifty-three immunoglobulin C2 domains, six fibronectin HI domains, two PEVK (rich in proline, glutamic acid, valine, and lysine residues) domains, and a kinase domain (Zhang, Y. et al. (2000) J. Cell Sci. 3101-3115).
  • titin is required for the formation of multi-nucleate syncitia and the organization of actin-myosin filaments into striated arrays.
  • Nebulin is a large sarcomeric protein that interacts with actin filaments in skeletal muscle (Wang, K. et al. (1996) J. Biol. Chem. 271:4304-4314). Nebulin contains 185 or more copies of a 35- residue module that has a consensus sequence and a predicted oc-helical structure. The 35-residue module comprises an actin-binding domain. In the central region of nebulin, the 35-residue modules exhibit a seven module super-repeat pattern. This super-repeat pattern is not present in the C-terminal 100 kDa region of nebulin.
  • the N-teiminal region of nebulin contains 8 linker modules and an 8 kDa acidic domain.
  • the C-terminal region is distinct and contains an SH3 domain.
  • nebulin is oriented with its C-terminus located at the Z-line, and its N-terminus at the pointed slow- growing end of thin filaments in the acto-myosin overlap region.
  • the size of nebulin is tissue- and species-specific and is developmentally regulated. Based on the observation that isoform size correlates with the length of Ihin filaments in skeletal muscle, nebulin is proposed to play a role as a molecular ruler that regulates the length of thin filaments.
  • Each nebulin 35-residue module may associate with one actin monomer; thus, isoforms with different numbers of modules could determine the length of thin filaments.
  • Tropomodulin caps actin at the pointed end of thin filaments and maintains filament length by preventing actin monomer dissociation or addition.
  • Nebulin is absent from cardiac muscle, but related proteins with nebulin-like modules may provide similar functions. Nebulette, for example, is specifically expressed in heart and has a C- terminal region containing twenty-three 35-residue nebulin-like modules (Moncman, C.L. and K. Wang (2000) J. Muscle Res. Cell Motil. 21:153-169; Millevoi, S. et al. (1998) J. Mol. Biol. 282:111- 123). The domain structure of nebulette is similar to nebulin, though it is a smaller protein of only 107 kDa.
  • nebulette has an acidic N-terminal domain, a repeat domain containing nebulin-like modules, a linker domain, and an SH3 domain.
  • the repeat domain of nebulette is about one-tenth the size of that of nebulin.
  • the 35-residue modules of nebulette have a consensus motif, and a subfamily of modules 15- 22 share a conserved motif. Unlike nebulin, nebulette modules do not display a super-repeat pattern. Nebulette binds to actin as well as other sarcomeric proteins including myosin, calmodulin, tropomyosin, troponin, and ⁇ -actinin (Moncman, C.L. and K.
  • N-RAP Nebulin-related anchoring protein
  • cardiac and skeletal muscle (Luo, G. et al. (1997) Cell Motil. Cytoskeleton 38:75-90). It is a 133 kDa protein found at the ends of myofibrils at muscle myotendon junctions and intercalated disks.
  • the C-terminal region of N-RAP has 27 copies of the 35-residue nebulin-like modules. Seventeen of the modules are organized in a super- repeat pattern.
  • the N-terminal region contains a cysteine-rich LIM domain. LIM domains bind two zinc ions in two adjacent zinc finger-like structures and are known to mediate protein-protein interactions.
  • N-RAP may mediate interactions between actin filaments of myofibrils and other sarcomeric proteins.
  • N-RAP binds to actin, talin, and vinculin (Luo, G. et al. (1999) Biochemistry 38:6135-6143). It interacts with actin and vinculin through its super-repeat region and with talin through its LIM domain.
  • Talin and vinculin are also located at myotendon junctions and together with N-RAP may provide a link between actin filaments of the myofibril and the sarcolemma and transmit tension from the myofibril to the extracellular matrix.
  • Intermediate filaments are cytoskeletal fibers with a diameter of about 10 nm, intermediate between that of microfilaments and microtubules. IPs serve structural roles in the cell, reinforcing cells and organizing cells into tissues. IFs are particularly abundant in epidermal cells and in neurons. IFs are extremely stable, and, in contrast to microfilaments and microtubules, do not function in cell motility.
  • Type I and Type II proteins are the acidic and basic keratins, respectively. Heterodimers of the acidic and basic keratins are the building blocks of keratin IFs. Keratins are abundant in soft epithelia such as skin and cornea, hard epifhelia such as nails and hair, and in epithelia that line internal body cavities.
  • keratin genes lead to epithelial diseases including epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white sponge nevus.
  • Some of these diseases result in severe skin blistering.
  • Type HI JF proteins include desmin, glial fibrillary acidic protein, vimentin, and peripherin.
  • M-Protein is a structural constituent of myofibrillar M-band in striated muscle, and is a member of the cytoskeletal protein superfamily along with immunoglobulin/fibronectin repeats (Noguchi, J. et al. (1992) J. Biol. Chem. 267:20302-20310).
  • Glial fibrillary acidic protein filaments are found in the glial cells that surround neurons and astrocytes. Vimentin filaments are found in blood vessel endothelial cells, some epithelial cells, and mesenchymal cells such as fibroblasts, and are commonly associated with microtubules.
  • Vimentin filaments may have roles in keeping the nucleus and other organelles in place in the cell.
  • Type IN IFs include the neurofilaments and nestin. Neurofilaments, composed of three polypeptides, NF-L, NF-M, and NF-H, are frequently associated with microtubules in axons. Neurofilaments are responsible for the radial growth and diameter of an axon, and ultimately for the speed of nerve impulse transmission. Changes in phosphorylation and metabolism of neurofilaments are observed in neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease (Julien, J.P. and W.E. Mushynski
  • Type V IFs the lamins, are found in the nucleus where they support the nuclear membrane.
  • the nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane.
  • Lamins are type V intermediate filament (IF) proteins.
  • IF intermediate filament
  • Prelamin A is a prenylated and methylated nuclear protein (Barton, R.M. and Worman, H.J. (1999) J. Biol. Chem.
  • Prenylation is a post-translational modification that is necessary for membrane attachment.
  • 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 are particularly closely associated.
  • IFAPs include BPAG1, plakoglobin, desmoplakin I, desmoplakin II, plectin, ankyrin, filaggrin, and lamin B receptor.
  • Trichohyalin is a major structural protein in the inner root sheath cells and medulla layer of hair follicles (Tarcsa, E. et al. (1997) J. Biol. Chem. 272:27893-27901). It is also present in other specialized epithelia such as human foreskin epidermis, hard palate, and nail bed. In hair follicle cells, THH is a large insoluble protein that forms droplets mixed with keratin intermediate filaments.
  • THH undergoes post-translational modification by transglutaminases and peptidyl-arginine deirninases that convert THH to a more soluble form.
  • THH may serve as a keratin intermediate filament matrix protein and/or as a constituent of the cell envelope and may serve barrier functions in hardened epithelia.
  • Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These attachments are important for mamtaining cell shape and for muscle contraction.
  • the spectrin-actin cytoskeleton is attached to the 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.
  • the protein dystrophin links actin filaments to the plasma membrane; mutations in the dystrophin gene lead to Duchenne muscular dystrophy.
  • Focal adhesions are specialized structures in the plasma membrane involved in the adhesion of a cell to a substrate, such as the extracellular matrix (ECM). Focal adhesions form the connection between an extracellular substrate and the cytoskeleton, and affect such functions as cell shape, cell motility and cell proliferation.
  • Transmembrane integrin molecules form the basis of focal adhesions. Upon ligand binding, integrins cluster in the plane of the plasma membrane. Cytoskeletal linker proteins such as the actin binding proteins ⁇ -actinin, talin, tensin, vinculin, paxillin, and filamin are recruited to the clustering site.
  • integrins mediate aggregation of protein complexes on both the cytosolic and extracellular faces of the plasma membrane, leading to the assembly of the focal adhesion.
  • Many signal transduction responses are mediated via various adhesion complex proteins, including Src, FAK, paxillin, and tensin.
  • IFs are also attached to membranes by cytoskeletal-membrane anchors.
  • the nuclear lamina is attached to the inner surface of the nuclear membrane by the lamin B receptor.
  • Vimentin IFs are attached to the plasma membrane by ankyrin and plectin.
  • Desmosome and hemidesmosome membrane junctions hold together epithelial ceUs of organs and skin. These membrane junctions aUow shear forces to be distributed across the entire epithelial ceU layer, thus providing strength and rigidity to the epitlielium.
  • IFs in epithelial ceUs are attached to the desmosome by plakoglobin and desmoplakins. The proteins that link IFs to hemidesmosomes are not known.
  • Desmin IFs surround the sarcomere in muscle and are linked to the plasma membrane by paranemin, synemin, and ankyrin.
  • Smoothelin proteins are smooth muscle-specific cytoskeleton-associated proteins that have been shown to be co-distributed with filamin and desmin (Wehrens, X.H. et al. (1997) FEBS Lett. 405:315-320).
  • Two types of smoothelin have been identified: smoothelin-A, expressed in visceral tissues, and smoothelin-B, expressed in vascular tissues (Rensen, S. et al. (2000) Cytogenet. CeU Genet. 89:225-229).
  • Pinin is a ceU adhesion-associated and nuclear protein that localizes near intermediate filament /desmosomal junctures. These proteins are associated with mature desmosomes of the epithelia (Ouyang, P. and Sugrue, S.P. (1992) J. CeU Biol. 118:1477-1488), and may function to reinforce the interaction between intermediate filaments with desmosomal membranes (Ouyang, P. and Sugrue, S.P. (1996) J. CeUBiol. 135:1027-1042). Motor Proteins Mvosin-related Motor Proteins
  • Myosins are actin-activated ATPases, found in eukaryotic ceUs, that couple hydrolysis of ATP with motion. Myosin provides the motor function for muscle contraction and intraceUular movements such as phagocytosis and rearrangement of ceU contents during mitotic ceU division (cytokinesis).
  • the contractile unit of skeletal muscle termed the sarcomere, consists of highly ordered arrays of thin actm-containing filaments and thick myos -containing filaments. Crossbridges form between the thick and thin filaments, and the ATP-dependent movement of myosin heads within the thick filaments puUs the thin filaments, shortening the sarcomere and thus the muscle fiber.
  • Myosins are composed of one or two heavy chains and associated light chains.
  • Myosin heavy chains contain an ammo-terminal motor or head domain, a neck that is the site of light-chain binding, and a carboxy-terminal tail domain.
  • the tail domains may associate to form an ⁇ -helical coiled coil.
  • Conventional myosins such as those found in muscle tissue, are composed of two myosin heavy-chain subunits, each associated with two light-chain subunits that bind at the neck region and play a regulatory role.
  • Unconventional myosins believed to function in intraceUular motion, may contain either one or two heavy chains and associated light chains.
  • Dynein-related Motor Proteins are (-) end-directed motor proteins which act on microtubules. Two classes of dyneins, cytosolic and axonemal, have been identified. CytosoUc dyneins are responsible for translocation of materials along cytoplasmic microtubules, for example, transport from the nerve terminal to the ceU body and transport of endocytic vesicles to lysosomes. As weU, viruses often take advantage of cytoplasmic dyneins to be transported to the nucleus and establish a successful infection (Sodeik, B. et al.
  • Virion proteins of herpes simplex virus 1 interact with the cytoplasmic dynein intermediate chain (Ye, GJ. et al. (2000) J. Virol. 74:1355-1363). Cytoplasmic dyneins are also reported to play a role in mitosis. Axonemal dyneins are responsible for the beating of flageUa and cilia. Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This sliding force produces bending that causes the flageUum 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. Cytoplasmic dynein is the largest and most complex of the motor proteins. Kinesin-related Motor Proteins
  • Ktnesins 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 aU ceU types for the transport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is critical for mamtaining the identity and functionality of these secretory organeUes.
  • 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 ceUular function. (Reviewed in Moore, J.D. and S.A. Endow (1996) Bioessays 18:207-219; and Hoyt, A.M. (1994) Curr. Opin. CeU 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 typicaUy referred to as 'Tdnesin.”
  • KHC is about 1000 amino acids in length
  • KLC is about 550 amino acids in length.
  • Two KHCs dimerize to form a rod-shaped molecule with three distinct regions of secondary structure.
  • At one end of the molecule is a globular motor domain that functions in ATP hydrolysis and microtubule binding.
  • Kinesin motor domains are highly conserved and share over 70% identity. Beyond the motor domain is 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.
  • Members of the more divergent subfamilies of kinesins are caUed kinesin-related proteins
  • KRPs many of which function during mitosis in eukaryotes (Hoyt, supra). Some KRPs are required for assembly of the mitotic spindle. In vivo and in vitro analyses suggest that these KRPs exert force on microtubules that comprise the mitotic spindle, resulting in the separation of spindle poles. Phosphorylation of KRP is required for this activity. Failure to assemble the mitotic spindle results in abortive mitosis and chromosomal aneuploidy, the latter condition being characteristic of cancer ceUs.
  • centromere protein E localizes to the kinetochore of human mitotic chromosomes and may play a role in their segregation to opposite spindle poles.
  • Dvnamin-related Motor Proteins Dynamin is a large GTPase motor protein that functions as a "molecular prnchase,” generating a mechanochemical force used to sever membranes. This activity is important in forming clathrin- coated vesicles from coated pits in endocytosis and in the biogenesis of synaptic vesicles in neurons.
  • dynamin' s self-assembly into spirals that may act to constrict a flat membrane surface into a tubule.
  • GTP hydrolysis induces a change in conformation of the dynamin polymer that pinches the membrane tubule, leading to severing of the membrane tubule and formation of a membrane vesicle.
  • Release of GDP and inorganic phosphate leads to dynamin disassembly.
  • FoUowing disassembly the dynamin may either dissociate from the membrane or remain associated to the vesicle and be transported to another region of the ceU.
  • Three homologous dynamin genes have been discovered, in addition to several dynamin-related proteins.
  • dynamin regions are the N-terminal GTP-binding domain, a central pleckstrin homology domain that binds membranes, a central coiled-coil region that may activate dynamin' s GTPase activity, and a C- terminal proline-rich domain that contains several motifs that bind SH3 domains on other proteins. Some dynamin-related proteins do not contain the pleckstrin homology domain or the proline-rich domain. (See McNiven, M.A. (1998) CeU 94:151-154; Scaife, R.M. and R.L. MargoUs (1997) CeU. Signal. 9:395-401.)
  • Microarrays are analytical tools used in bioanalysis.
  • a microarray has a pluraUty of molecules spatially distributed over, and stably associated with, the surface of a soUd support.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of appHcations, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • One area in particular in which microarrays find use is in gene expression analysis.
  • array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specificaUy related to a particular genetic predisposition, condition, disease, or disorder.
  • Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibriUary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basaUs and locus caeruleus. Early in Alzheimer's pathology, physiological changes are visible in the cingulate cortex (Mmoshima, S. et al. (1997) Annals of Neurology 42:85-94). In subjects with advanced Alzheimer's disease, accumulating plaques damage the neuronal architecture in limbic areas and eventuaUy cripple the memory process.
  • PF 1190 P The potential appUcation of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease.
  • both the levels and sequences expressed in tissues from subjects with breast cancer or prostate cancer may be compared with the levels and sequences expressed in normal tissue.
  • Breast cancer is a genetic disease commonly caused by mutations in breast epitheHal ceUs. Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra). However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to noni herited mutations that occur in breast epitheUal ceUs.
  • EGF has effects on ceU functions related to metastatic potential, such as ceU motiHty, chemotaxis, secretion and differentiation.
  • the abundance of erbB receptors, such as HER-2/neu, HER-3 , and HER-4, and their Ugands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S.S. et al. (1994) Am. J. Clin. Pathol. 102:S13-S24).
  • a human secreted frizzled protein mRNA that is downregulated in breast tumors includes a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix Gla protein which is overexpressed is human breast carcinoma ceUs; Drgl or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S 100 protein family, aU of which are down regulated in mammary carcinoma ceUs relative to normal mammary epitheUal ceUs (Zhou Z. et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395; Ulrix W.
  • Prostate cancer is a common maUgnancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year.
  • cancer ceUs arise in the prostate, they are stimulated by testosterone to a more rapid growth. Thus, removal of the testes can indirectly reduce both rapid growth and metastasis of the cancer.
  • prostatic cancers Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous ceU and transitional ceU carcinomas, both of which arise in the prostatic ducts or other parts of the prostate gland.
  • prostate cancer develops through a multistage progression ultimately resulting in an aggressive, metastatic phenotype.
  • the initial step in tumor progression involves the hyperproUferation of normal luminal and/or basal epitheUal ceUs that become hyperplastic and evolve into early-stage tumors.
  • the early-stage tumors are locaUzed in the prostate but eventuaUy may metastasize, particularly to the bone, brain or lung.
  • About 80% of these tumors remain responsive to androgen treatment, an important hormone controUing the growth of prostate epitheUal ceUs.
  • cancer growth becomes androgen-independent and there is currently no known treatment for this condition.
  • PSA prostate specific antigen
  • PSA is a tissue-specific serine protease almost exclusively produced by prostatic epitheUal ceUs.
  • the quantity of PSA correlates with the number and volume of the prostatic epitheUal ceUs, and consequently, the levels of PSA are an exceUent indicator of abnormal prostate growth.
  • Men with prostate cancer exhibit an early linear increase in PSA levels foUowed by an exponential increase prior to diagnosis.
  • PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting
  • EGF Epidermal Growth Factor
  • FGF Fibroblast Growth Factor
  • TGF ⁇ Tumor Growth Factor alpha
  • TGF- ⁇ family of growth factors are generaUy expressed at increased levels in human cancers and the high expression levels in many cases correlates with advanced stages of maUgnancy and poor survival
  • Various embodiments of the invention provide purified polypeptides, structural and cytoskeleton-associated proteins, referred to coUectively as “SCAP” and individuaUy as "SCAP-1,” “SCAP-2,” “SCAP-3,” “SCAP-4,” “SCAP-5,” “SCAP-6,” “SCAP-7,” “SCAP-8,” “SCAP-9,” “SCAP-10,” “SCAP-11,” “SCAP-12,” “SCAP-13,” “SCAP-14,” “SCAP-15,” “SCAP-16,” “SCAP- 17,” “SCAP-18,” “SCAP-19,” “SCAP-20,” “SCAP-21,” “SCAP-22,” “SCAP-23,” “SCAP-24,”
  • Embodiments also provide methods for utiUzing the purified structural and cytoskeleton-associated proteins and/or their encoding polynucleotides for faciUtating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
  • Related embodiments provide methods for utilizing the purified structural and cytoskeleton-associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical
  • An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l- 37, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 37, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-37.
  • StiU another embodiment provides an isolated polynucleotide encoding a polypeptide selected
  • L5 from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-37.
  • the polynucleotide is selected from the group consisting of SEQ ID NO:38-74.
  • Another embodiment provides a ceU transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.
  • Another embodiment 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: 1-37, b) a polypeptide comprising a naturaUy occurring a ino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
  • the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • Yet another embodiment provides an isolated antibody which specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
  • StiU yet another embodiment 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:38-74, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JJD NO:38-74, 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 can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, 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).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucle
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specificaUy hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said
  • the method can include detecting the amount of the hybridization complex.
  • the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
  • the method comprises a) ampUfying said target polynucleotide or fragment thereof using polymerase chain reaction ampUfication, and b) detecting the presence or absence of said ampUfied target polynucleotide or fragment thereof.
  • the method can include detecting the amount of the ampUfied target polynucleotide or fragment thereof.
  • 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: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologically active fragment 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: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologically active fragment of a
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
  • Other embodiments provide a method of treating a disease or condition
  • composition associated with decreased or abnormal expression of functional SCAP, comprising administering to a patient in need of such treatment the composition.
  • Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional SCAP, comprising administering to a patient in need of such treatment the composition.
  • StiU yet another embodiment 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: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional SCAP, comprising administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
  • 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.
  • Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consistmg of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consistmg of SEQ ID NO: 1-37 , c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
  • 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.
  • StiU yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • Another embodiment 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:38-74, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of n), and v) an RNA equivalent of
  • 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:38-74, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide can comprise a fragment of a polynucleotide 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 fuU length polynucleotide and polypeptide embodiments of the invention.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptide embodiments of the invention. The probabiUty scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide embodiments, 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/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
  • Table 5 shows representative cDNA Kbraries for polynucleotide embodiments.
  • 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 polynucleotides and polypeptides, along with appUcable descriptions, references, and threshold parameters.
  • a host ceU includes a pluraUty of such host ceUs
  • an antibody is a reference to one or more antibodies and equivalents thereof known to those skiUed in the art, and so forth.
  • SCAP refers to the amino acid sequences of substantiaUy purified SCAP 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 SCAP.
  • Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of SCAP either by directly interacting with SCAP or by acting on components of the biological pathway in which SCAP participates.
  • an "aUelic variant” is an alternative form of the gene encoding SCAP.
  • AUeUc 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 aUeUc variants of its naturaUy occu ⁇ ing form.
  • Common mutational changes which give rise to aUeUc variants are generaUy ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding SCAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as SCAP or a polypeptide with at least one functional characteristic of SCAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oUgonucleoti.de probe of the polynucleotide encoding SCAP, and improper or unexpected hybridization to aUeUc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding SCAP.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionaUy equivalent SCAP.
  • DeUberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubiUty, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of SCAP 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 hydrophiUcity 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 can refer to an oUgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occu ⁇ ing or synthetic molecules.
  • amino acid sequence is recited to refer to a sequence of a naturaUy occurring protein molecule
  • amino acid sequence and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • AmpUfication relates to the production of additional copies of a nucleic acid. AmpUfication may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid ampUfication technologies weU known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of SCAP.
  • Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of SCAP either by directly interacting with SCAP or by acting on components of the biological pathway in which SCAP participates.
  • antibody refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind SCAP polypeptides can be prepared using intact polypeptides or using fragments containing smaU 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
  • an animal e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, thyroglobuUn, and keyhole limpet hemocyanin (KLH).
  • the coupled peptide is then used to immunize the animal.
  • the term "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 eUcit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oUgonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial Ubraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer Ufetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers maybe specificaUy cross-linked to their cognate Ugands, e.g., by photo-activation of a cross-Unker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • introduction refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oUgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oUgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oUgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a ceU, the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus'' can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
  • immunologicalaUy active or “immunogenic” refers to the capabiUty of the natural, recombinant, or synthetic SCAP, or of any oUgopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.
  • “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
  • composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides encoding SCAP or fragments of SCAP maybe employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCI
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncaUed bases, extended using the XL-PCR kit (AppUed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVCEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • GELVCEW fragment assembly system GCG, Madison WI
  • Phrap Universality of Washington, Seattle WA
  • Constant a ino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy the function of the protein is conserved and not significantly changed by such 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 generaUy 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 £5 the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemicaUy modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an 30 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 35 measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • “Exon shuffling” refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins maybe assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.
  • a “fragment” is a unique portion of SCAP or a polynucleotide encoding SCAP which can be 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 about 5 to about 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 preferentiaUy selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, maybe encompassed by the present embodiments.
  • a fragment of SEQ ID NO:38-74 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:38-74, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:38-74 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and ampUfication technologies and in analogous methods that distinguish SEQ ID NO:38-74 from related polynucleotides.
  • the precise length of a fragment of SEQ ED NO:38-74 and the region of SEQ ID NO:38-74 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO: 1-37 is encoded by a fragment of SEQ JD NO:38-74.
  • a fragment of SEQ ID NO:l-37 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO: 1-37.
  • a fragment of SEQ ID NO: 1-37 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO:l-37.
  • the precise length of a fragment of SEQ ID NO:l-37 and the region of SEQ ID NO:l-37 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
  • a “full length” polynucleotide is one containing at least a translation initiation codon (e.g., rnethionine) foUowed by an open reading frame and a translation te ⁇ nination codon.
  • a “fuU 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 5 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.
  • Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity 10 can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence aUgnment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989; CABIOS 5:151- 153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191).
  • 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 £5 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 30 compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ED number, or maybe measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.
  • 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 weU-known. Some aUgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
  • Such default parameters may be, for example:
  • Percent identity maybe measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, maybe used to describe a length over which percentage identity may be measured.
  • "Human artificial chromosomes" are linear microchromosomes which may contain
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and stiU retains its original binding abiUty.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in dete ⁇ riining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (T j for the specific sequence at a defined ionic strength and pH.
  • T j thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • An equation for calculating T m and conditions for nucleic acid hybridization are weU known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2 nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specificaUy see volume 2, chapter 9.
  • 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 jtlg/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Useful variations on these wash conditions wiU be readily apparent to those of ordinary skiU in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acids 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 present in solution and another nucleic acid immobilized on a soUd support (e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • soUd support e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect ceUular and systemic defense systems.
  • an "immunogenic fragment” is a polypeptide or oUgopeptide fragment of SCAP 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 SCAP which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an a ⁇ angement of a pluraUty of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microa ⁇ ay.
  • modulate refers to a change in the activity of SCAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of SCAP.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oUgonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • Operably Unked 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 oUgonucleotide 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 preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Ufespan in the ceU.
  • Post-translational modification of an SCAP may involve Upidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic milieu of SCAP.
  • Probe refers to nucleic acids encoding SCAP, their complements, or fragments thereof, which are used to detect identical, aUeUc or related nucleic acids.
  • Probes are isolated oUgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, Ugands, chermluminescent agents, and enzymes.
  • Primmers are short nucleic acids, usuaUy DNA oUgonucleotides, which maybe annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for ampUfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, maybe used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institote 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.
  • the PrimOU primer selection program (available to the pubUc from the Genome Center at University of Texas South West Medical Center, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope.
  • the Primer3 primer selection program (available to the pubUc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows 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 oUgonucleotides for microarrays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the pubUc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aUgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments.
  • oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microa ⁇ ay elements, or specific probes to identify fuUy or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of oUgonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturaUy occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
  • recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
  • Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a ceU.
  • such recombinant nucleic acids maybe part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA 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.
  • An "RNA equivalent,” in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that aU occurrences of the nitrogenous base thy ine 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 SCAP, nucleic acids encoding SCAP, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g. , the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the 5 epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other L0 components with which they are naturaUy 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, L5 microparticles and capiUaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient £0 ceU. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, Upofection, and particle bombardment.
  • transformed 25 ceUs includes stably transformed ceUs in which the inserted DNA is capable of repUcation either as an autonomously repUcating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the ceUs of the organism contains heterologous nucleic acid 30 introduced by way of human intervention, such as by transgenic techniques weU known in the art.
  • the nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the ceU, by way of deUberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants
  • 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
  • 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
  • a variant may be described as, for example, an
  • a spUce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spUcing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the
  • Species variants are polynucleotides 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.
  • 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 (March-07-
  • 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.
  • Various embodiments of the invention include new human structural and cytoskeleton- associated proteins (SCAP), the polynucleotides encoding SCAP, and the use of these compositions for the diagnosis, treatment, or prevention of ceU motiUty disorders, developmental disorders, ceU proUferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders.
  • SCAP new human structural and cytoskeleton- associated proteins
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ED).
  • Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide D) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
  • Column 6 shows the Incyte ED numbers of physical, fuU length clones corresponding to polypeptide and polynucleotide embodiments. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptides shown in column 3.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ED NO:) of the nearest GenBank homolog.
  • Column 4 shows the probabiUty scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank homolog(s) along with relevant citations where appUcable, aU of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention.
  • Columns 1 and 2 show the polypeptide sequence identification number (SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) 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 MOTIF'S program of the GCG sequence analysis software package (Genetics Computer Group, Madison WT).
  • 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:3 is 96% identical, from residue Ml to residue P1638, to Mus musculus Kif21b (GenBank ID g6561829) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.)
  • the BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ID NO:3 also contains a WD domain, G- beta repeat and a kinesin motor domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO:7 is 78% identical, from residue Ml to residue N1196, to mouse formin, isoform IV (GenBank ID g51553) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ID NO:7 also contains a Formin Homology 2 domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and additional BLAST analyses using the PRODOM and DOMO databases provide further corroborative evidence that SEQ ID NO:7 is a formin homology protein.
  • HMM hidden Markov model
  • SEQ ID NO:ll is 99% identical, from residue Ml to residue R744, to a human pinin protein (GenBank ID gl684847) as determined by the Basic Local AUgnment Search
  • BLAST Basic Local AUgnment Search Tool
  • SEQ ED NO: 15 also contains a calponin homology (CH) domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from additional BLAST analyses provide further co ⁇ oborative evidence that SEQ ED NO: 15 is a cytoskeleton-associated protein.
  • CH calponin homology
  • SEQ ID NO:18 is 99% identical, from residue E36 to residue W408, to human nuclear prelamin A recognition factor (GenBank ID g5777952) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 6.4e-220, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ID NO: 18 also contains an an iron only hydrogenase large subunit, C-te domain as determined by
  • HMM hidden Markov model
  • SEQ ED NO:17 is 77% identical, from residue E296 to residue K1526, to
  • BLAST Basic Local AUgnment Search Tool
  • SEQ ED NO: 17 also contains C-2 and PDZ domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein
  • SEQ D NO: 17 is a cytoskeleton-associated protein.
  • SEQ ID NO:26 is 73% identical, from residue A52 to residue E1781, to chicken myosin heavy chain (GenBank ED g7248371) as determined by the Basic Local AUgnment
  • SEQ ID NO:26 is a myosin.
  • SEQ ID NO:29 is 65% identical, from residue K531 to residue E1846, 69% identical, from residue L252 to residue S593, and 69% identical, from residue E12 to residue L253, to GaUus gaUus myosin heavy chain (GenBank ED g7248371) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which
  • SEQ D NO:29 also contains a myosin N-terminal SH3-like domain, a myosin tail, and a myosin head (motor domain) as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO:36 is 90% identical, from residue Ml to residue N450, to the type I keratin intermediate filament E Sal (GenBank ED gl2642308) as determined by the Basic Local AUgnment Search Tool (BLAST).
  • the BLAST probabiUty score is 5.0e-217, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ED NO:36 also contains an intermediate filament protein domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and BLAST analyses provide further co ⁇ oborative evidence that SEQ ED NO:36 is an intermediate filament protein.
  • HMM hidden Markov model
  • SEQ ID NO:l-2, SEQ ID NO:4-6, SEQ ID NO:8-10, SEQ ID NO:12-14, SEQ ID NO:16, SEQ ID NO:19-25, SEQ ID NO:27-28, SEQ ED NO:30-35, and SEQ ID NO:37 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ ID NO: 1-37 are described in Table 7.
  • Table 4 the fuU length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or ampUfication technologies that identify SEQ ID NO:38-74 or that distinguish between SEQ ED NO:38-74 and related polynucleotides.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA Ubraries or from pooled cDNA
  • polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides.
  • polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The S anger Centre, Cambridge, UK) database ⁇ Le., those sequences including the designation "ENST").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
  • the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • a polynucleotide sequence identified as I ⁇ _XXXXXX_N 1 _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was appUed, and YYYYY is the number of the prediction generated by the algorithm, and N 1A3 _ , _, if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stietching" algorithm.
  • a polynucleotide sequence identified as LXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stietching" algorithm was appUed, gBBBBB being the GenBank identification number or ⁇ CBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) may be used in place of the GenBank identifier (z. e. , gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA Ubraries for those full length polynucleotides 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 polynucleotides.
  • 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 SCAP variants.
  • a prefe ⁇ ed SCAP 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 SCAP amino acid sequence, and which contains at least one functional or structural characteristic of SCAP.
  • Various embodiments also encompass polynucleotides which encode SCAP.
  • polynucleotides which encode SCAP In a particular
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:38-74, which encodes SCAP.
  • SEQ ED NO:38-74 which encodes SCAP.
  • the invention also encompasses variants of a polynucleotide encoding SCAP.
  • a variant polynucleotide wiU have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding SCAP.
  • a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:38-74 which has at least about 70%, or alternatively at least
  • any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of SCAP.
  • a polynucleotide variant of the invention is a spUce variant of a
  • a spUce variant may have portions which have significant sequence identity to a polynucleotide encoding SCAP, but wiU generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate spUcing of exons during mRNA processing.
  • a spUce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a
  • portions of the spUce variant wiU have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding SCAP.
  • a polynucleotide comprising a sequence of SEQ ED NO:44 and a polynucleotide comprising a sequence of SEQ ID NO:45 are spUce variants of each other. Any one of the spUce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of SCAP.
  • polynucleotides which encode SCAP and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring SCAP under appropriately selected conditions of stringency, it maybe advantageous to produce polynucleotides encoding SCAP or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy occurring codons. Codons maybe selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utiUzed by the host.
  • RNA transcripts having more desirable properties such as a greater half-Ufe, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of polynucleotides which encode SCAP and SCAP derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide may be inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding SCAP or any fragment thereof.
  • Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ED NO:38-74 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kirnmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.”
  • Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (AppUed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampUfication system (Eavitrogen, Carlsbad CA).
  • 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 earned out using either the ABI 373 or 377 DNA sequencing system (AppUed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).
  • the nucleic acids encoding SCAP maybe extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • one method which maybe employed, restriction-site PCR uses universal and nested primers to ampUfy unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods AppUc. 2:318-322).
  • Another method, inverse PCR uses primers that extend in divergent directions to ampUfy unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (TrigUa, T. et al.
  • a third method, capture PCR involves PCR ampUfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods AppUc. 1:111-119).
  • 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 maybe used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers maybe designed using commerciaUy 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 fuU-length cDNA. Genomic Ubraries may be useful for extension of sequence into 5 'non-transcribed regulatory regions.
  • CapiUary electrophoresis systems which are commerciaUy available maybe used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capiUary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output Ught intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
  • CapiUary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotides or fragments thereof which encode SCAP may be cloned in recombinant DNA molecules that direct expression of SCAP, or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides may be produced and used to express SCAP.
  • the polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter SCAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oUgonucleotides may be used to engineer the nucleotide sequences.
  • oUgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spUce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, EC. 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 SCAP, such as its biological or enzymatic activity or its abiUty to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, EC.
  • DNA shuffling is a process by which a Ubrary of gene variants is produced using PCR-mediated recombination of gene fragments. The Ubrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These prefe ⁇ ed variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations maybe recombined, screened, and then reshuffled until the desired properties are optimized.
  • fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturaUy occurring genes in a directed and controUable manner.
  • polynucleotides encoding SCAP maybe synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
  • SCAP itself or a fragment thereof may be synthesized using chemical methods known in the art.
  • peptide synthesis can be performed using various solution-phase or soUd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; Roberge, J.Y.
  • AdditionaUy the amino acid sequence of SCAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturaUy occurring polypeptide.
  • the peptide may be substantiaUy purified by preparative high performance Uquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (Creighton, supra, pp. 28-53).
  • the polynucleotides encoding SCAP 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 polynucleotides encoding SCAP.
  • Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding SCAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • a variety of expression vector/host systems maybe utiUzed to contain and express polynucleotides encoding SCAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression
  • L5 vectors e.g., baculovirus
  • plant ceU systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems (Sambrook, supra; Ausubel et al., supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945;
  • viral expression vectors e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV
  • bacterial expression vectors e.g., Ti or pBR322 plasmids
  • animal ceU systems Standardbrook, supra;
  • routine cloning, subcloning, and propagation of polynucleotides encoding SCAP can be achieved using a multifunctional E. coli vector such as PBLUESCREPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invitrogen). Ligation of polynucleotides encoding SCAP into the vector's multiple cloning site disrupts the lacZ gene, aUowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • PBLUESCREPT Stratagene, La JoUa CA
  • PSPORT1 plasmid Invitrogen
  • vectors maybe useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509).
  • vectors which direct high level expression of SCAP 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 SCAP.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184).
  • Plant systems may also be used for expression of SCAP. Transcription of polynucleotides encoding SCAP 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. 3:1631). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters maybe used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CeU Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw HiU Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196).
  • viral promoters e.g.
  • a number of viral-based expression systems may be utilized.
  • polynucleotides encoding SCAP may be Ugated into an adeno virus 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 maybe used to obtain infective virus which expresses SCAP in host ceUs (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaUan host ceUs.
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • 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 (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
  • ceU Unes For long term production of recombinant proteins in mammaUan systems, stable expression of SCAP in ceU Unes is prefe ⁇ ed.
  • polynucleotides encoding SCAP can be transformed into ceU 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.
  • ceUs maybe aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfully express the introduced sequences.
  • Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
  • ceU Unes Any number of selection systems may be used to recover transformed ceU Unes. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetaboUte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and par confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
  • trpB and hisD which alter ceUular requirements for metaboUtes
  • Visible markers e.g., anfhocyanins, green fluorescent proteins (GFP; Clontech), ⁇ - glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin maybe 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 (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. For example, if the sequence encoding SCAP is inserted within a marker gene sequence, transformed ceUs containing polynucleotides encoding SCAP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding SCAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU.
  • host ceUs that contain the polynucleotide encoding SCAP and that express SCAP may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR ampUfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
  • Immunological methods for detectmg and measuring the expression of SCAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated ceU sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on SCAP is preferred, but a competitive binding assay may be employed.
  • These and other assays are weU known in the art (Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. TV; CoUgan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding SCAP include oUgolabeUng, nick translation, end-labeling, or PCR ampUfication using a labeled nucleotide.
  • polynucleotides encoding SCAP, or any fragments thereof maybe cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionucUdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host ceUs transformed with polynucleotides encoding SCAP maybe cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
  • the protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode SCAP may be designed to contain signal sequences which direct secretion of SCAP through a prokaryotic or eukaryotic ceU membrane.
  • a host ceU strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited 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 ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and maybe chosen to ensure the co ⁇ ect modification and processing of the foreign protein.
  • natural, modified, or recombinant polynucleotides encoding SCAP may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric SCAP protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may faciUtate the screening of peptide Ubraries for inhibitors of SCAP activity.
  • L5 moieties may also faciUtate purification of fusion proteins using commerciaUy available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins,
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the SCAP encoding sequence and the heterologous protein sequence, so that SCAP may be cleaved away from the heterologous moiety foUowing purification.
  • synthesis of radiolabeled SCAP 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
  • SCAP fragments of SCAP, or variants of SCAP may be used to screen for compounds that specificaUy bind to SCAP.
  • One or more test compounds may be screened for specific binding to SCAP.
  • 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to SCAP.
  • Examples of test compounds can include antibodies, anticalins, oUgonucleotides, proteins (e.g., Ugands or receptors), or smaU molecules.
  • variants of SCAP can be used to screen for binding of test compounds, such as antibodies, to SCAP, a variant of SCAP, or a combination of SCAP and/or one or more variants SCAP.
  • a variant of SCAP can be used to screen for compounds that bind to a variant of SCAP, but not to SCAP having the exact sequence of a sequence of SEQ ED NO: 1-37.
  • SCAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to SCAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to SCAP can be closely related to the natural Ugand of SCAP, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (CoUgan, J.E. et al. (1991) Cu ⁇ ent Protocols in Immunology l(2):Chapter 5).
  • the compound thus identified can be a natural Ugand of a receptor SCAP (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
  • a compound identified in a screen for specific binding to SCAP can be closely related to the natural receptor to which SCAP binds, at least a fragment of the receptor, or a fragment of the receptor including aU or a portion of the Ugand binding site or binding pocket.
  • the compound may be a receptor for SCAP which is capable of propagating a signal, or a decoy receptor for SCAP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. CeU Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336).
  • the compound can be rationaUy designed using known techniques.
  • Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgG j (Taylor, P.C et al. (2001) Curr. Opin. Immunol. 13:611-616).
  • TNF tumor necrosis factor
  • two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to SCAP, fragments of SCAP, or variants of SCAP.
  • the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of SCAP.
  • an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of SCAP.
  • an antibody can be selected such that its binding specificity aUows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of SCAP.
  • anticalins can be screened for specific binding to SCAP, fragments of SCAP, or variants of SCAP.
  • Anticalins are Ugand-binding proteins that have been constructed based on a UpocaUn scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275).
  • the protein architecture of UpocaUns can include a beta-ba ⁇ el having eight antiparaUel beta-strands, which supports four loops at its open end.
  • loops form the natural Ugand-binding site of the UpocaUns, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • the amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
  • screening for compounds which specificaUy bind to, stimulate, or inhibit SCAP involves producing appropriate ceUs which express SCAP, either as a secreted protein or on the ceU membrane.
  • ceUs Prefe ⁇ ed ceUs include ceUs from mammals, yeast, Drosophila, or E. coli. CeUs expressing SCAP or ceU membrane fractions which contain SCAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either SCAP 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 SCAP, either in solution or affixed to a soUd support, and detecting the binding of SCAP to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical Ubraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
  • An assay can be used to assess the abiUty of a compound to bind to its natural Ugand and/or to inhibit the binding of its natural Ugand to its natural receptors.
  • examples of such assays include radio- labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its abiUty to bind to its natural Ugands (Matthews, D.J. and J.A. WeUs. (1994) Chem. Biol. 1:25-30).
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a Ugand) to improve or alter its abiUty to bind to its natural receptors (Cunningham, B.C. and J.A. WeUs (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
  • a polypeptide compound such as a Ugand
  • SCAP, fragments of SCAP, or variants of SCAP maybe used to screen for compounds that modulate the activity of SCAP.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for SCAP activity, wherein SCAP is combined with at least one test compound, and the activity of SCAP in the presence of a test compound is compared with the activity of SCAP in the absence of the test compound. A change in the activity of SCAP in the presence of the test compound is indicative of a compound that modulates the activity of SCAP.
  • a test compound is combined with an in vitro or ceU-free system comprising SCAP under conditions suitable for SCAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of SCAP may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraUty of test compounds maybe screened.
  • polynucleotides encoding SCAP or their mammaUan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • Such techniques are weU known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
  • mouse ES ceUs such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture.
  • the ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgicaUy 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 SCAP may also be manipulated in vitro in ES ceUs derived from human blastocysts.
  • Human ES ceUs have the potential to differentiate into at least eight separate ceU Uneages including endoderm, mesoderm, and ectodermal ceU types. These ceU Uneages differentiate into, for example, neural ceUs, hematopoietic Uneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding SCAP 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 SCAP is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
  • Transformed ceUs 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 SCAP e.g., by secreting SCAP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • CSAP Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of SCAP and structural and cytoskeleton-associated proteins.
  • the expression of CSAP is closely associated with adrenal, bladder, brain, diseased brain, aUocortex, cingulate posterior, dorsal root gangUon, pituitary gland, colon, heart, Uver, Uver tumor, lung, lower thigh, nose, pancreatic islet, penis, prostate tumor, rib bone, synovial membrane, tongue, and uterine tissues, and with breast cancer, bladder cancer, colon cancer, penile cancer, prostate cancer, olfactory neuroblastoma, teratocarcinoma, and rheumatoid arthritis, as weU as with promonocyte ceUs, SH-SY5Y ceUs derived from metastatic bone marrow neuroblastoma, and human umbiUcal vein endotheUal ceUs (HUV-EC- C).
  • examples of tissues expressing SCAP can be found in Table 6 and can also be found in Example XI. Therefore, SCAP appears to play a role in ceU motiUty disorders, developmental disorders, ceU proUferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders. In the treatment of disorders associated with increased SCAP expression or activity, it is desirable to decrease the expression or activity of SCAP. In the treatment of disorders associated with decreased SCAP expression or activity, it is desirable to increase the expression or activity of SCAP.
  • SCAP or a fragment or derivative thereof maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SCAP.
  • disorders include, but are not limited to, a ceU motiUty disorder such as ankylosing spondyUtis, Chediak-Higashi syndrome, Duchenne and Becker muscular dystrophy, intrahepatic cholestasis, myocardial hyperplasia, cardiomyopathy, early onset peridontitis, cancers such as adenocarcinoma, ovarian carcinoma, and chronic myelogenous leukemia, and bacterial and helminthic infections; a developmental disorder such as renal tubular acidosis, anemia, Qishing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormaUties, and mental retardation),
  • a vector capable of expressing SCAP 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 SCAP including, but not limited to, those described above.
  • composition comprising a substantiaUy purified SCAP 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 SCAP including, but not limited to, those provided above.
  • an agonist which modulates the activity of SCAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SCAP including, but not limited to, those Usted above.
  • an antagonist of SCAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SCAP.
  • disorders include, but are not limited to, those ceU motiUty disorders, developmental disorders, ceU proUferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders described above.
  • an antibody which specificaUy binds SCAP may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express SCAP.
  • a vector expressing the complement of the polynucleotide encoding SCAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SCAP including, but not limited to, those described above.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skiU in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one maybe able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of SCAP may be produced using methods which are generaUy known in the art.
  • purified SCAP may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specificaUy bind SCAP.
  • Antibodies to SCAP may also be generated using methods that are weU known in the art. Such antibodies may include, but are not Umited to, polyclonal, monoclonal, cbimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
  • NeutraUzing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others maybe immunized by injection with SCAP or with any fragment or oUgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • Such adjuvants include, but are not Umited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especiaUy preferable.
  • the oUgopeptides, peptides, or fragments used to induce antibodies to SCAP have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oUgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of SCAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the cHmeric molecule may be produced.
  • Monoclonal antibodies to SCAP may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not Umited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique (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; Cole, S.P. et al. (1984) Mol. CeU Biol. 62:109-120).
  • chimeric antibodies such as the spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce SCAP-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin Ubraries (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 the Uterature (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 SCAP may also be generated.
  • fragments include, but are not limited to, F(ab fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfi.de bridges of the F(ab ⁇ )2 fragments.
  • Fab expression Ubraries maybe constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (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 weU known in the art.
  • Such immunoassays typicaUy involve the measurement of complex formation between SCAP and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering SCAP epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
  • K a is defined as the molar concentration of SCAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equiUbrium conditions.
  • K a association constant
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular SCAP epitope, represents a true measure of affinity.
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the SCAP- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are prefe ⁇ ed for use in immunopurification and similar procedures which ultimately require dissociation of SCAP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quaUty and suitabiUty of such preparations for certain downstream appUcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of SCAP-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quaUty and usage in various appUcations, are generaUy available (Catty, supra; CoUgan et al., supra).
  • polynucleotides encoding SCAP 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 SCAP.
  • 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 SCAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).
  • Antisense sequences can be deUvered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein (Slater, J.E. et al. (1998) J. AUergy Clrn. Immunol. 102:469-475; Scanlon, KJ. et al. (1995) 9:1288-1296).
  • Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (MiUer, A.D.
  • polynucleotides encoding SCAP may be used for somatic or germline gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X- ⁇ nked 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
  • conditionaUy lethal gene product e.g., in the case of cancers which result from unregulated ceU proUferation
  • a protein which affords protection against intraceUular parasites e.g., against human retro viruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
  • hepatitis B or C virus HBV, HCV
  • fungal parasites such as Candida albicans and Paracoccidioides bras ⁇ liensis
  • protozoan parasites such as Plasmodiumfalciparum and Trypanosoma cruzi.
  • diseases or disorders caused by deficiencies in SCAP are treated by constructing mammaUan expression vectors encoding SCAP and introducing these vectors by mechanical means into SCAP-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (n) ballistic gold particle deUvery, (in) Uposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
  • Expression vectors that may be effective for the expression of SCAP include, but are not Umited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
  • SCAP 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), (n) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • an inducible promoter e.g., the tetracycline-regulated promoter (Gossen, M. and H.
  • diseases or disorders caused by genetic defects with respect to SCAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding SCAP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (in) a Rev-responsive
  • Retrovirus vectors e.g., PFB and PFBNEO
  • PFB and PFBNEO are commerciaUy available (Stratagene) and are based on pubUshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein.
  • the vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for
  • an adenovirus-based gene therapy deUvery system is used to deUver polynucleotides encoding SCAP to ceUs which have one or more genetic abnormaUti.es with respect to the expression of SCAP.
  • the construction and packaging of adenovirus-based vectors are weU known to those with ordinary skiU in the art.
  • RepUcation defective adenoviras vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al* (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S. Patent No.
  • Adreoviras 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).
  • a herpes-based, gene therapy deUvery system is used to deUver polynucleotides encoding SCAP to target ceUs which have one or more genetic abnormaUties with respect to the expression of SCAP.
  • the use of herpes simplex virus (HSV)-based vectors maybe especiaUy valuable for introducing SCAP to ceUs of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are weU known to those with ordinary skiU 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.
  • HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a ceU under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
  • HSV vectors see also Goins, W.F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al. (1994; Dev. Biol. 163:152-161).
  • the manipulation of cloned herpesviras sequences, the generation of recombinant virus foUowing the transfection of multiple plasmids containing different segments of the large herpesviras genomes, the growth and propagation of herpesviras, and the infection of ceUs with herpesviras are techniques weU known to those of ordinary skiU in the art.
  • an alphaviras (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encoding SCAP to target ceUs.
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • alphavirus RNA repUcation a subgenomic RNA is generated that normaUy encodes the viral capsid proteins.
  • This subgenomic RNA repUcates to higher levels than the fuU length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • inserting the coding sequence for SCAP into the alphaviras genome in place of the capsid-coding region results in the production of a large number of SCAP- coding RNAs and the synthesis of high levels of SCAP in vector transduced ceUs.
  • alphaviras infection is typicaUy associated with ceU lysis within a few days
  • the abiUty to estabUsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repUcation of alphaviruses can be altered to suit the needs of the gene therapy appUcation
  • OUgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple heUx base-pairing methodology.
  • Triple heUx pairing is useful because it causes inhibition of the abiUty of the double heUx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of
  • RNA molecules encoding SCAP The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding SCAP.
  • RNA sequences of between 15 and 20 ribonucleotides, conesponding 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 accessibility to hybridization with complementary
  • RNA molecules may be generated by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing oUgonucleotides such as soUd phase phosphoramidite chemical synthesis.
  • RNA molecules maybe generated by in vitro and in vivo transcription of DNA molecules encoding SCAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU Unes, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular 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 linkages within the backbone of the molecule.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding SCAP.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oUgonucleotides, antisense oUgonucleotides, triple heUx-forming oUgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specificaUy inhibits expression of the polynucleotide encoding SCAP may be therapeuticaUy useful, and in the treatment of disorders associated with decreased SCAP expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding SCAP may be therapeuticaUy useful.
  • At least one, and up to a pluraUty, of 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, commerciaUy-available or proprietary Ubrary of naturaUy-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 Ubrary of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding SCAP is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabiUzed ceU, or an in vitro ceU-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding SCAP are assayed by any method commonly known in the art.
  • TypicaUy the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding SCAP.
  • the amount of hybridization maybe quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be ca ⁇ ied out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU line such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
  • a particular embodiment of the present invention involves screening a combinatorial Ubrary of oUgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Braice, T.W. et al. (2000) US. Patent No. 6,022,691).
  • oUgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. DeUvery by transfection, by Uposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
  • compositions which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
  • 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 SCAP, antibodies to SCAP, and mimetics, agonists, antagonists, or inhibitors of SCAP.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sub ⁇ ngual, or rectal means.
  • compositions for pulmonary administration may be prepared in Uquid or dry powder form. These compositions are generaUy aerosoUzed immediately prior to inhalation by the patient.
  • aerosol deUvery of fast- acting formulations is weU-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • recent developments in the field of pulmonary deUvery via the alveolar region of the lung have enabled the practical deUvery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848).
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is weU within the capabiUty of those skilled in the art.
  • SpeciaUzed forms of compositions maybe prepared for direct intraceUular deUvery of macromolecules comprising SCAP or fragments thereof.
  • Uposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deUvery of the macromolecule.
  • SCAP or a fragment thereof may be joined to a short cationic N- terminal portion from the HJV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, 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 therapeuticaUy effective dose refers to that amount of active ingredient, for example SCAP or fragments thereof, antibodies of SCAP, and agonists, antagonists or inhibitors of SCAP, which ameUorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 5( ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are prefe ⁇ ed.
  • the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with Uttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • the exact dosage wiU be determined by the practitioner, in Ught of factors related to the 5 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, drag combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or 10 biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of deUvery is provided in the Uterature and generaUy available to practitioners in the art.
  • wiU employ different formulations for nucleotides than for proteins or their 15 inhibitors.
  • antibodies which specificaUy bind SCAP may be used for the diagnosis of disorders characterized by expression of SCAP, or in assays to monitor patients being £0 treated with SCAP or agonists, antagonists, or inhibitors of SCAP.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for SCAP include methods which utilize the antibody and a label to detect SCAP in human body fluids or in extracts of ceUs or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of £5 reporter molecules, several of which are described above, are known in the art and may be used.
  • SCAP SCAP-specific kinase kinase kinase
  • ELISAs RIAs
  • FACS fluorescence-activated cell sorting
  • SCAP serum-specific peptide-binding protein
  • normal or standard values for SCAP expression are established by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to SCAP under ⁇ 0 conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of SCAP expressed in subject, control, and disease samples frombiopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
  • polynucleotides encoding SCAP maybe used for diagnostic purposes.
  • the polynucleotides which may be used include oUgonucleotides, 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 SCAP may be co ⁇ elated with disease.
  • diagnostic assay may be used to dete ⁇ nine absence, presence, and excess expression of SCAP, and to monitor regulation of SCAP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding SCAP or closely related molecules maybe used to identify nucleic acid sequences which encode SCAP.
  • LO highly specific region e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampUfication wiU determine whether the probe identifies only naturaUy occurring sequences encoding SCAP, aUeUc variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the SCAP encoding sequences.
  • L5 invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:38-74 or from genomic sequences including promoters, enhancers, and introns of the SCAP gene.
  • Means for producing specific hybridization probes for polynucleotides encoding SCAP include the cloning of polynucleotides encoding SCAP or SCAP derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commerciaUy available, and may be used to
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucUdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotides encoding SCAP maybe used for the diagnosis of disorders associated with
  • Such disorders include, but are not Umited to, a ceU motiUty disorder such as ankylosing spondyUtis, Chediak-Higashi syndrome, Duchenne and Becker muscular dystrophy, intrahepatic cholestasis, myocardial hyperplasia, cardiomyopathy, early onset peridontitis, cancers such as adenocarcinoma, ovarian carcinoma, and chronic myelogenous leukemia, and bacterial and helmmthic infections; a developmental disorder such as renal tubular acidosis, anemia, Oishing's
  • achondroplastic dwarfism Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepitheUal dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a ceU proUferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), mye
  • Polynucleotides encoding SCAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microanays utiUzing fluids or tissues from patients to detect altered SCAP expression. Such qualitative or quantitative methods are weU known in the art.
  • polynucleotides encoding SCAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to sequences encoding SCAP maybe labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding SCAP in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is estabUshed. This may be accompUshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding SCAP, under conditions suitable for hybridization or ampUfication.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner maybe compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to 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 maybe used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development , of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
  • oUgonucleotides designed from the sequences encoding SCAP may involve the use of PCR. These oUgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. OUgomers wiU preferably contain a fragment of a polynucleotide encoding SCAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding SCAP, and wiU be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oUgonucleotide primers derived from polynucleotides encoding SCAP 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 polynucleotides encoding SCAP are used to ampUfy DNA using the polymerase chain reaction (PCR).
  • the DNA maybe derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
  • SNPs in the DNA cause differences in the secondary and tertiary stractures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oUgonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy, 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 spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs maybe used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insuUn-dependent diabetes meUitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drag, such as Ufe-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drag isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-Upoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11:637-641).
  • Methods which may also be used to quantify the expression of SCAP include radiolabeling or biotinylating nucleotides, coampUfication of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236).
  • the speed of quantitation of multiple samples maybe accelerated by ranning the assay in a high-throughput format where the oUgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oUgonucleotides or longer fragments derived from any of the polynucleotides 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 maybe 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. In particular, 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.
  • SCAP, fragments of SCAP, or antibodies specific for SCAP may be used as elements on a microa ⁇ ay.
  • the microa ⁇ ay may be used to monitor or measure protein-protein interactions, drag-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein).
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totaUty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraUty of elements on a microa ⁇ ay.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, ceU lines, biopsies, or other biological samples.
  • the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceU line.
  • Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and precUnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring environmental compounds.
  • AU 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). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
  • the toxicity of a test compound can be 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 co ⁇ esponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU type.
  • proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
  • a profile of a ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first
  • the proteins are visuaUzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots
  • proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry.
  • the identity of the protein in a spot maybe determined by comparing its partial sequence, preferably of at least 5 contiguous
  • polypeptide sequences of interest 5 amino acid residues, to the polypeptide sequences of interest.
  • further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for SCAP to quantify the levels of SCAP expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microanay to the sample and detecting
  • 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 paraUel with toxicant signatures at the transcript level. There is a poor co ⁇ elation between transcript and protein abundances for some proteins in some tissues (Anderson,
  • proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling maybe more reUable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample
  • LO 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 co ⁇ esponding 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
  • 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
  • £0 by the antibodies is quantified.
  • the amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays maybe prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA
  • nucleic acid sequences encoding SCAP may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence.
  • Either coding or noncoding sequences maybe used, and in some instances, noncoding sequences maybe preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI 5 constractions, or single chromosome cDNA Ubraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI 5 constractions or single chromosome cDNA Ubraries
  • nucleic acid sequences may be used to develop genetic linkage maps, for example, which co ⁇ elate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. 10 Acad. Sci. USA 83:7353-7357).
  • RFLP restriction fragment length polymorphism
  • Fluorescent in situ hybridization maybe correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online MendeUan Inheritance in Man (OMTM) World Wide Web site. Correlation between the location of the gene encoding SCAP on a physical [5 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.
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps.
  • placement of a gene on the chromosome of another mammaUan species, such as mouse, '.0 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.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation 5 (Gatti, R.A. et al. (1988) Nature 336:577-580).
  • the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • SCAP in another embodiment, SCAP, its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries of compounds in any of a variety of drug 0 screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a soUd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between SCAP and the agent being tested may be measured.
  • Another technique for drag screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT appUcation WO84/03564).
  • This method large numbers of different smaU test compounds are synthesized on a soUd substrate.
  • the test compounds are reacted with SCAP, or fragments thereof, and washed.
  • Bound SCAP is then detected by methods weU known in the art.
  • Purified SCAP can also be coated directly onto plates for use in the aforementioned drag screening techniques.
  • non-neutraUzing antibodies can be used to capture the peptide and immobiUze it on a soUd support.
  • nucleotide sequences which encode SCAP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not Umited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs were derived from cDNA Ubraries described in the LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), 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.
  • TRIZOL Invitrogen
  • 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 co ⁇ esponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA Ubraries were constructed with the UNTZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al, supra, ch. 5). Reverse transcription was initiated using oUgo d(T) or random primers. Synthetic oUgonucleotide adapters were Ugated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs were Ugated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCP PT plasmid
  • Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN.
  • Plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophiUzation, at 4°C Alternatively, plasmid DNA was ampUfied from host ceU lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were earned out in a single reaction mixture.
  • Incyte cDNA recovered in plasmids as described in Example II were sequenced as foUows.
  • Sequencing reactions were processed using standard methods or Ingh-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 Biosciences or suppUed in ABI sequencing kits such as the
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (AppUed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art.
  • the polynucleotide sequences derived from Incyte cDNAs were vaUdated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and
  • Incyte cDNA sequences or translations 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; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae,
  • HMM hidden Markov model
  • PFAM PFAM
  • ENCY ENCY
  • TIGRFAM TIGRFAM
  • HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabiUstic approach which analyzes consensus
  • 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 fuU length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated
  • a polypeptide may begin at any of the methionine residues of the full length translated polypeptide.
  • FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM,
  • 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, aU of
  • Genscan gene identification program against pubUc genomic sequence databases e.g., gbpri and gbhtg.
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karlin (1998) Cu ⁇ . Opin. Struct. Biol. 8:346-354).
  • the program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • the output of Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • Genscan The maximum range of sequence for Genscan to analyze at once was set to 30 kb.
  • Genscan predicted cDNA sequences encode structural and cytoskeleton-associated proteins
  • the encoded polypeptides were analyzed by querying against PFAM models for structural and cytoskeleton-associated proteins. Potential structural and cytoskeleton-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as structural and cytoskeleton-associated proteins.
  • 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 e ⁇ ors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis was also used to find any Incyte cDNA or 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.
  • FuU 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 ILL Alternatively, fuU 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 HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic prograrnjming to integrate cDNA and genomic information, generating possible spUce variants that were subsequently confirmed, edited, or extended to create a fuU 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 TV.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • HSPs high-scoring segment pairs
  • GenBank protein homolog the 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 SCAP Encoding Polynucleotides
  • sequences which were used to assemble SEQ ED NO:38-74 were compared with sequences from the Incyte LERESEQ database and pubUc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ED NO:38-74 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from 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.
  • 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.
  • 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) is a unit of measurement based on recombination frequencies between chromosomal markers.
  • 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.
  • Mb megabase
  • 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.
  • L5 search can be modified to determine whether any particular match is categorized as exact or similar.
  • the basis of the search is the product score, which is defined as:
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is
  • the product score represents a balance between fractional overlap and quaUty in a BLAST aUgnment. For example, a product score of 100 is produced only for 100% identity over the
  • 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.
  • polynucleotides encoding SCAP are analyzed with respect to the tissue sources from which they were derived. For example, some fuU length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example HI). Each cDNA sequence is derived from a cDNA Ubrary constructed from a human tissue.
  • Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaUa, female; genitaUa, male; germ ceUs; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding SCAP. VIII. Extension of SCAP Encoding Polynucleotides
  • FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using oUgonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hairpin stractures and primer-primer dimerizations was avoided.
  • Selected human cDNA Ubraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
  • the concentration of DNA in each weU was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE
  • the extended nucleotides were desalted and concentrated, transferred to 384-weU plates, digested with CviJI cholera viras endonuclease (Molecular Biology Research, Madison WT), and sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera viras endonuclease Molecular Biology Research, Madison WT
  • sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Biosciences).
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels,
  • SNPs single nucleotide polymorphisms
  • Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
  • the African population comprised 194 individuals (97 male, 97 female), aU African Americans.
  • the Hispanic population comprised 324 individuals (162 male, 162 female), aU Mexican Hispanic.
  • the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
  • AUele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no aUeUc variance in this population were not further tested in the other three populations.
  • Hybridization probes derived from SEQ ID NO:38-74 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oUgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oUgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oUgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextranbead column (Amersham Biosciences).
  • An aUquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgl 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 (Nytran Plus, Schleicher & SchueU, Durham NH).
  • Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaUzed using autoradiography or an alternative imaging means and compared.
  • the linkage or synthesis of anay elements upon a microarray can be achieved utilizing photoUthography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and soUd with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested substrates include siUcon, siUca, glass sUdes, glass chips, and siUcon 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 weU known to those of ordinary skiU in the art and may contain any appropriate number of elements (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).
  • FuU 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 weU known in the art such as LASERGENE software (DNASTAR).
  • the anay elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each anay element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microa ⁇ ay may be assessed. In one embodiment, microa ⁇ ay preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oUgo-(dT) ceUulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 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 Biosciences).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte).
  • Specific control ⁇ oly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
  • Sequences of the present invention are used to generate array elements.
  • Each array element is ampUfied from bacterial ceUs containing vectors with cloned cDNA inserts.
  • PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are ampUfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. AmpUfied array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified anay elements are immobilized on polymer-coated glass sUdes.
  • Glass microscope sUdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments.
  • Glass sUdes are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distiUed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol.
  • Coated sUdes are cured in a 110°C oven.
  • Anay elements are appUed to the coated glass substrate using a procedure described in U.S.
  • Patent No. 5,807,522 incorporated herein by reference.
  • 1 ⁇ l of the anay element DNA is loaded into the open capiUary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of anay element sample per sUde.
  • Microanays are UV-crossUnked using a STRATALINKER UV-crossUnker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distiUed water.
  • Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2%
  • PBS phosphate buffered saline
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and 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 aUquoted onto the microa ⁇ ay surface and covered with
  • the anays are transfened to a waterproof chamber having a cavity just sUghtly larger than a microscope sUde.
  • the chamber is kept at 100% humidity internaUy by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the anays is incubated for about 6.5 hours at 60° C.
  • the 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.
  • Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser Ught is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the 1.8 cm x 1.8 cm anay used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted Ught is spUt, based on wavelength, into two photomultipUer tube detectors (PMT R1477,
  • the sensitivity of the scans is typicaUy caUbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the anay contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1:100,000.
  • the caUbration is done by labeling samples of the caUbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipUer tube is digitized using a 12-bit RTT-835H analog-to-digital
  • a D conversion board Analog Devices, Inc., Norwood MA
  • instaUed in an IBM-compatible PC computer The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and
  • the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value conesponding to the average intensity of the signal.
  • L5 for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). Anay elements that exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentiaUy expressed using the GEMTOOLS program (Incyte Genomics). Expression
  • SEQ D NO:39 showed differential expression in brain cingulate from a patient with Alzheimer's disease compared to matched microscopicaUy normal tissue from the same donor as determined by microarray analysis.
  • the expression of CSAP-2 was increased at least two-fold in cingulate tissue with Alzheimer's disease. Therefore, SEQ ED NO:39 can be used in diagnostic assays for neurological disorders, including Alzheimer's disease.
  • SEQ ED NO:43 and SEQ ED NO:44 showed differential expression in breast tumor ceU Unes versus normal breast epitheUal ceUs as determined by microa ⁇ ay analysis.
  • the expression of SEQ ID NO:43 and SEQ ID NO:44 was increased by at least two fold in breast tumor ceU Unes from later stages of tumor progression as compared to a primary breast epitheUal ceU line isolated from a normal donor, and a breast mammary gland ceU line isolated from a patient with
  • SEQ ID NO:43 and SEQ ED NO:44 can be used in diagnostic assays for breast cancer.
  • UV treatment an antitumor agent, triggers different ceU cycle regulatory pathways in breast tumor ceUs.
  • PBMCs peripheral blood mononuclear cells
  • IL-4 interleukin 4
  • Human PBMCs can be classified into discrete ceUular populations representing the major ceUular components of the immune system.
  • PBMCs contain about 52% lymphocytes (12 % B lymphocytes, 40% T lymphocytes ⁇ 25% CD4+ and 15% CD8+ ⁇ ), 20% NK ceUs, 25% monocytes, and 3% various ceUs that include dendritic ceUs and
  • EL-4 is a pleiotropic cytokine produced by activated T ceUs, mast ceUs, and basophils. It was initiaUy identified as a B ceU differentiation factor (BCDF) and a B ceU stimulatory factor (BSF1). Subsequent to the molecular cloning and expression of both human and mouse IL-4,
  • EL-4 exhibits anti-tumor effects both in vivo and in vitro. Recently, EL-4 was identified as an important regulator for the CD4+ subset (Thl-Uke vs. Th2- like) development. The biological effects of IL-4 are mediated by the binding of EL-4 to specific ceU surface receptors.
  • the functional Hgh-affinity receptor for IL-4 consists of a Ugand-binding subunit
  • the gamma chain of the IL-2 receptor complex is a functional b chain of the EL-4 receptor complex.
  • PBMCs were coUected from the blood of 6 healthy volunteer donors using standard gradient separation.
  • the PBMCs from each donor were placed in culture for 2 hours in the
  • MLR mixed lymphocyte reaction
  • Cyclosporin A has been a drug of choice for the last 15 years for immunosuppression in organ transplants, GvHD in bone marrow transplants, and in some other inflammatory conditions. CsA forms complexes with cyclopHlin molecules that inhibit the cytoplasmic phosphatase calcineurin. Inhibition of the phosphatase activity of calcineurin prevents the downstream induction of transcription of genes such as EL-2, EL-3 , and EFN- ⁇ .
  • NSCLCs non-smaU ceU lung cancer
  • SCLC smaU ceU lung cancer
  • lung squamous ceU carcinoma tissue (Dn5793, Dn5792, DN5796and Dn5797) was compared to same-donor normal lung tissue; in another experiment, lung adenocarcinoma (Dn5795) was compared to same-donor normal lung tissue.
  • the expression of SEQ ED NO:56 was decreased by at least two-fold in the tumor tissue.
  • SEQ ED NO:56 was also differentiaUy expressed in tissue from a human breast lobular carcinoma in situ (Dn4716) as compared to uninvolved breast tissue from the same donor. A variety of differences occur in the gene expression patterns between normal and cancerous breast tissue. Identifying downstream targets of altered genes in cancerous breast tissue may generate greater understanding of molecular pathways that lead to breast cancer. In this experiment, the expression of SEQ ID NO:56 was decreased by at least two-fold in the breast cancer tissue. SEQ ED NO:59 was differentiaUy expressed in normal brain tissue versus mild Alzheimer's diseased brain tissue based on microarray experimentation.
  • AD Alzheimer's disease
  • a cross- comparison experimental design was used to evaluate the expression of cDNAs from specific dissected regions of human brain (Dn3629 from a 68-year old female with mild AD in the posterior cingulate tissue), as compared to normal human brain tissue from equivalent regions (Dn3625 from a normal 61-year old female).
  • the expression of SEQ ED NO:59 was increased at least two-fold in mild AD tissue from the posterior cingulate region of the brain.
  • SEQ ED NO:56 and SEQ ID NO:59 exhibited significant differential expression patterns using microa ⁇ ay techniques, and further estabUsh the utiUty of SEQ ED NO:56 and SEQ ED NO:59 as diagnostic markers or as therapeutic targets for useful in a variety of conditions and diseases involving cytoskeleton-associated proteins.
  • SEQ ED NO:61 showed differential expression in non-malignant mammary epitheUal ceUs versus various breast carcinoma ceU Unes as determined by microarray analysis.
  • the breast carcinoma ceU Unes include BT20, a breast carcinoma ceU line derived in vitro from ceUs emigrating out of thin sUces of a tumor mass isolated from a 74-year-old female; MCF7, a breast adenocarcinoma ceU Une derived from the pleural effusion of a 69-year-old female; MDA-mb-231, a metastatic breast tumor ceU Une derived from the pleural effusion of a 51- year-old female with metastatic breast carcinoma; SkBR3, a breast adenocarcinoma ceU Une isolated from a maUgnant pleural effusion of a 43-year-old female; and T47D, a breast carcinoma ceU Une derived from a pleural effusion from a 54-year-old female with an infiltrating ductal carcinoma of the breast.
  • SEQ ID NO:61 can be used as a diagnostic marker or as a potential therapeutic target for breast cancer.
  • SEQ ID NO:61 also showed differential expression in normal epitheUal cells versus prostate carcinoma ceU Unes as determined by microarray analysis.
  • the normal epitheUal ceU Une, PZ-HPV-7 was derived from normal tissue from the peripheral zone of the prostate.
  • the prostate carcinoma ceU Unes include LNCaP, a prostate carcinoma ceU Une isolated from a lymph node biopsy of a 50 year old male with metastatic prostate carcinoma; and PC-3, a prostate adenocarcinoma ceU Une isolated from a metastatic site in the bone of a 62 year old male with grade IV prostate adenocarcinoma.
  • SEQ ID NO:61 was decreased by at least two fold in the prostate carcinoma lines relative to normal epitheUal ceUs. Therefore, SEQ ED NO:61 can be used as a diagnostic marker or as a potential therapeutic target for prostate cancer.
  • SEQ ED NO:61 showed differential expression in inflammatory responses as determined by microa ⁇ ay analysis.
  • the expression of SEQ ED NO:61 was increased by at least two fold in human umbilical vein endotheUal ceUs (HUVECs) treated with interferon-gamma (IFN- ⁇ ) and tumor necrosis factor-alpha (TNF- ) relative to untreated HUVECs.
  • Human umbiUcal vein endotheUal ceUs are primary ceUs derived from the endotheUum of the human umbiUcal vein and have been used as an experimental model for investigating the role of the endotheUum in human vascular biology.
  • IFN- ⁇ is a cytokine produced primarily by T-lymphocytes and natural killer ceUs.
  • TNF- ⁇ is produced by activated lymphocytes, macrophages, and other white blood ceUs, and is known to activate endotheUal ceUs.
  • EFN- ⁇ and TNF- ⁇ are pleiotropic cytokines that play important roles in mediation of the inflammatory response through activation of multiple signal transduction pathways. Therefore, SEQ ED NO:61 can be used in diagnostic assays for inflammatory responses.
  • SEQ ED NO:62 showed region-specific gene expression in the human brain as determined by microa ⁇ ay analysis.
  • the expression of SEQ ID NO:62 was decreased by at least two fold in the midbrain segments and the occipital lobe (associative) in the neocortex relative to pooled brain tissues which were constituted from the major regions of the brain from two male brains; a 47 year old and a 48 year old.
  • the tissues from the midbrain segments and the occipital lobe was isolated from a 47 year old male, the same 47 year old donor as in the pooled sample. Therefore, SEQ ED NO:62 can be used as a biomarker for human brains, specifically the midbrain and the occipital lobe region in the neocortex.
  • SEQ ED NO:67 showed differential expression associated with prostate cancer as determined by microarray analysis.
  • the expression of SEQ ED NO:67 showed at least a two-fold decrease in prostate LNCaP and DU-145 carcinoma ceUs compared to prostate PrEC epitheUal ceUs.
  • SEQ ED NO:67 also showed at least a two-fold decrease in expression in prostate LNCaP carcinoma ceUs compared to nontumorigenic prostate PZ-HPV-7 epitheUal ceUs.
  • the expression of SEQ ED NO:67 showed at least a two-fold increase in prostate PC3 carcinoma ceUs compared to either prostate PrEC epitheUal ceUs or PZ-HPV-7 epitheUal ceUs.
  • the LNCaP ceU Une was isolated from a lymph node biopsy of a 50-year old male with metastatic prostate carcinoma.
  • the DU-145 ceU Une was isolated from a 69-year old male with widespread metastatic prostate carcionoma.
  • the PC-3 ceU Une was isolated from a 62-year old male with grade TV prostate adenocarcinoma.
  • the PrEC ceU Une is a prostate epitheUal ceU Une isolated from a normal donor.
  • the PZ-HPV-7 ceU Une was derived from epitheUal ceUs cultured from normal tissue from the peripheral zone of the prostate.
  • SEQ ED NO:67 also showed differential expression associated with breast cancer as determined by microa ⁇ ay analysis.
  • the expression of SEQ ED NO:67 showed at least a two-fold decrease in BT-474, BT-20, Hs578T, and MDA-MB-435S breast carcinoma ceUs compared to human mammary epitheUal ceUs (HMEC).
  • the expression of SEQ ED NO:67 also showed at least a two-fold decrease in MCF7, T-47D, BT-20, Sk-BR-3, MDA-MB-231, and MDA-MB-435S breast carcinoma ceUs compared to nontumorigenic MCF-10A breast mammary gland ceUs.
  • SEQ ID NO:67 showed at least a two-fold increase in expression in MCF-10A, MDA-MB-468 and HMEC ceUs treated with epidermal growth factor compared to untreated ceUs.
  • Epidermal growth factor is highly expressed in breast carcinoma ceUs. Epidermal growth factor promotes proUferation and differentiation of mesenchymal and epitheUal ceUs, angiogenesis, and tumor progression. It is a mitogen for fibroblasts, epitheUal and endotheUal ceUs.
  • BT-474 is a breast ductal carcinoma ceU Une isolated from a soUd, invasive ductal carcinoma of the breast obtained from a 60-year old woman.
  • BT-20 is a breast carcinoma ceU Une derived from the ceUs emigrating out of thin sUces of a tumor mass isolated from a 74-year old female.
  • Hs578T is a breast ductal carcinoma ceU Une that was a isolated from a 74-year old female with breast carcinoma.
  • MDA-MB-435S is spindle shaped strain that evolved from the parent line (435) as isolated from the pleural effusion of a 31-year old female with metastatic, ductal adenocarcinoma of the breast.
  • MDA-MB-468 is a breast adeoncarcinoma ceU Une isolated from the pleural effusion of a 51 -year old female with metastatic adenocarcinoma of the breast.
  • MCF7 is a nonmaUgnant breast adenocarcinoma ceU Une isolated from the pleural effusion of a 69-year old female.
  • T-47D is a breast carcinoma ceU Une isolated from a pleural effusion obtained from a 54-year old female with an infiltrating ductal carcinoma of the breast.
  • Sk-BR-3 is a breast adenocarcinoma ceU Une isolated from a maUgnant pleural effusion of a 43 -year old female.
  • MDA-MB-231 is a breast tumor ceU Une isolated from the pleural effusion of a 51-year old female.
  • MCF-10A is a breast mammary gland ceU Une that was isolated from a 36-year old woman with fibrocystic breast disease.
  • HMEC is a primary breast epitheUal ceU Une isolated from a normal donor. Therefore, SEQ ID NO:67 can be used in disease staging and in diagnostic assays for ceU proUferative disorders, including prostate cancer and breast cancer.
  • SEQ ID NO:67 can be used in disease staging and in diagnostic assays for ceU proUferative disorders, including prostate cancer and breast cancer.
  • Sequences complementary to the SCAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occu ⁇ ing SCAP.
  • oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with s aUer or with larger sequence fragments.
  • Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of SCAP.
  • a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oUgonucleotide is designed to prevent ribosomal binding to the SCAP-encoding transcript.
  • SCAP StaC-lac
  • 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 SCAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).
  • baculoviras Recombinant baculoviras is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculoviras (Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945).
  • SCAP is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crade cell lysates.
  • GST a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobiUzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
  • the GST moiety can be proteolyticaUy cleaved from SCAP at specificaUy engineered sites.
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). Purified SCAP obtained by these methods can be used directly in the assays shown in Examples XVJJ and XVi ⁇ , where appUcable. XIV. Functional Assays
  • SCAP function is assessed by expressing the sequences encoding SCAP at physiologicaUy elevated levels in mammaUan ceU 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 plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Une, for example, an endotheUal or hematopoietic ceU Une, using either Uposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected ceUs and is a reUable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Ught scatter and 90 degree side Ught scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
  • CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG). Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding SCAP and other genes of interest can be analyzed by northern analysis or microarray techniques. XV. Production of SCAP Specific Antibodies
  • the SCAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art.
  • Methods for selection of appropriate epitopes, such as those near the C-tenninus or in hydrophiUc regions are weU described in the art (Ausubel et al., 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- Aldrich, St. Louis MO) by reaction with N-maleii dobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oUgopeptide-KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti-SCAP activity by, for example, binding the peptide or SCAP to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Media containing SCAP are passed over the immunoaf ⁇ nity column, and the column is washed under conditions that aUow the preferential absorbance of SCAP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/SCAP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as ) urea or thiocyanate ion), and SCAP is coUected.
  • SCAP or biologicaUy active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539).
  • Candidate molecules previously arrayed in the weUs of a multi-weU plate are incubated with the labeled SCAP, washed, and any weUs with labeled SCAP complex are assayed. Data obtained using different concentrations of SCAP are used to calculate values for the number, affinity, and association of SCAP with the candidate molecules.
  • molecules interacting with SCAP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • SCAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large Ubraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • a microtubule motiUty assay for SCAP measures motor protein activity.
  • recombinant SCAP is immobilized onto a glass sUde or similar substrate.
  • Taxol-stabiUzed bovine brain microtubules (commerciaUy available) in a solution containing ATP and cytosoUc extract are perfused onto the sUde.
  • Movement of microtubules as driven by SCAP motor activity can be visuaUzed and quantified using video-enhanced Ught microscopy and image analysis techniques.
  • SCAP activity is directly proportional to the frequency and velocity bf microtubule movement.
  • an assay for SCAP measures the formation of protein filaments in vitro.
  • a solution of SCAP at a concentration greater than the "critical concentration" for polymer assembly is appUed 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 SCAP activity.
  • SCAP activity is measured by the binding of SCAP to protein filaments.
  • S-Met labeled SCAP sample is incubated with the appropriate filament protein (actin, tubulin, or intermediate filament protein) and complexed protein is coUected by immunoprecipitation using an antibody against the filament protein.
  • the immunoprecipitate is then run out on SDS-PAGE and the amount of SCAP bound is measured by autoradiography.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Various embodiments of the invention provide human structural and cytoskeleton-associated proteins (SCAP) and polynucleotides which identify and encode SCAP. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of SCAP.

Description

STRUCTURAL AND CYTOSKELETON-ASSOCIATED PROTEINS
TECHNICAL FIELD
The invention relates to novel nucleic acids, structural and cytoskeleton-associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of cell motility disorders, developmental disorders, cell proliferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders. The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and structural and cytoskeleton-associated proteins.
BACKGROUND OF THE INVENTION
The cytoskeleton is a cytoplas ic network of protein fibers that mediate 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. Major cytoskeletal fibers include the microtubules, the microfilaments, and the intermediate filaments. Motor proteins, including myosin, dynein, and kinesin, drive movement of or along the fibers. The motor protein dynamin drives the formation of membrane vesicles. Accessory or associated proteins modify the structure or activity of the fibers while cytoskeletal membrane anchors connect the fibers to the cell membrane. Microtubules and Associated Proteins Tubulins
Microtubules, cytoskeletal fibers with a diameter of about 24 nm, have multiple roles in the cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing 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 are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
Microtubules are polymers of GTP-binding tubulin protein subunits. Each subunit is a heterodimer of α- and β- tubulin, multiple isoforms of which exist. 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 α-tubulin and the other with β-tubulin, and the two ends differ in their rates of assembly. Generally, each microtubule is 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. Gamma tubulin present in the MTOC is important for nucleating the polymerization of α- and β- 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
Microtubule-associated proteins (MAPs) have roles in the assembly and stabilization of microtubules. One major family of MAPs, 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 π. Type I MAPs, which include MAP1A and MAP1B, are large, filamentous molecules that co-purify with microtubules and are abundantly expressed in brain and testes. Type I MAPs 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 MAP1B 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 MAP1A, MAP1B, and microtubules. It is suggested that LC3 is synthesized from a source other than the MAPIA or MAP1B transcripts, and that the expression of LC3 maybe important in regulating the microtubule binding activity of MAPIA and MAP1B during cell proliferation (Mann, S.S. et al. (1994) J. Biol. Chem. 269:11492-11497). Type π 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
5 intraneuronal Tau aggregates (Spillantini, M.G. and M. Goedert (1998) Trends Neurosci. 21:428-433). Another microtubule associated protein, STOP (stable tubule only polypeptide), is a calmodulin-regulated protein that regulates stability (Denarier, E. et al. (1998) Biochem. Biophys. Res. Commun. 24:791-796). In order for neurons to maintain conductive connections over great distances, they rely upon axodendritic extensions, which in turn are supported by microtubules. STOP proteins
[0 function to stabilize the microtubular network. STOP proteins are associated with axonal microtubules, and are also abundant in neurons (Guillaud, L. et al. (1998) J. Cell Biol. 142:167-179). STOP proteins are necessary for normal neurite formation, and have been observed to stabilize microtubules, in vitro, against cold-, calcium-, or drug-induced disassembly (Margolis, R.L. et al. (1990) EMBO 9:4095-502).
5 The protein caldendrin is a calcium-binding protein selectively localized to the somatodendritic compartment of neurons and is highly enriched at postsynaptic structures. At least one isoform of caldendrin is found to be associated with neuronal cytoskeleton. Sequence alignment studies showed that caldendrin displays significant similarity with calmodulin and contains two EF-hand motifs (Seidenbecher, C. I. et al. (1998) J. Biol. Chem. 273:21324-21331).
0 Microfilaments and Associated Proteins Actins
Microfilaments, cytoskeletal filaments with a diameter of about 7-9 nm, 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
5 form of actin, the most abundant intracellular protein in the eukaryotic cell. Human cells contain six isoforms of actin. The three α-actins are found in different kinds of muscle, nonmuscle β-actin and nonmuscle γ-actin are found in nonmuscle cells, and another γ-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. In muscle cells, thin filaments containing actin slide past thick filaments containing the motor protein myosin during contraction. A family of actin-related proteins exist that are not part of the actin cytoskeleton, but rather associate with microtubules and dynein. Actin- Associated Proteins
Actin-associated proteins have roles in cross-linking, severing, and stabilization of actin filaments and in sequestering actin monomers. Several of the 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. Actin-interacting proteins (AJPs) participate in the regulation of actin filament organization. Other actin-associated proteins such as TARA, a novel F-actin binding protein, function in a similar capacity by regulating actin cytoskeletal organization. Calmodulin-like calcium-binding domains in actin cross-linking proteins allow calcium regulation of cross-linking. Group I cross-linking proteins have unique actin-binding domains and include the 30 kD protein, EF-la, fascin, and scruin. Group II cross-linking proteins have a 7,000-MW actin-binding domain and include villin and dematin. Group HI cross-linking proteins have pairs of a 26,000-MW actin-binding domain and include fimbrin, spectrin, dystrophin, ABP 120, and filamin.
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 and tropomodulin. The proteins thymosin and profilin sequester actin monomers in the cytosol, allowing a pool of unpolymerized actin to exist. The actin-associated proteins tropomyosin, troponin, and caldesmon regulate muscle contraction in response to calcium.
Microtubule and actin filament networks cooperate in processes such as vesicle and organelle transport, cleavage furrow placement, directed cell migration, spindle rotation, and nuclear migration. Microtubules and actin may coordinate to transport vesicles, organelles, and cell fate determinants, or transport may involve targeting and capture of microtubule ends at cortical actin sites. These cytoskeletal systems maybe bridged by myosin-kinesin complexes, myosin-CLIP170 complexes, formin-homology (FH) proteins, dynein, the dynactin complex, Kar9ρ, coronin, ERM proteins, and kelch repeat-containing proteins (for a review, see Goode, B.L. et al. (2000) Curr. Opin. Cell Biol. 12:63-71). The kelch repeat is a motif originally observed in the kelch protein, which is involved in formation of cytoplasmic bridges called ring canals. A variety of mammalian and other kelch family proteins have been identified. The kelch repeat domain is believed to mediate interaction with actin (Robinson, D.N. and L. Cooley (1997) J. CeU Biol. 138:799-810).
The formin homology (FH) proteins are nuclear phosphoproteins which contain proline-rich sequences similar to Src homology 3 (SH3)-binding domains. The SH3 region is a small protein domain present in a large group of proteins, including cytoskeletal elements and signaling proteins. FH proteins are linked to cell signaling pathways by interactions with members of the Rho family of GTP- binding proteins. The FH proteins appear to play a crucial role in coodinating cytokinesis with nuclear division, as well as participating in actin-mediated processes affecting cell polarity and shape (Wasserman, S. (1998) Trends CellBiol. 8:111-115). The formins are encoded by alternatively spliced transcripts arising from the limb deformity (Id) locus of the mouse. Mutations in this locus disrupt embryonic pattern formation, leading to reduction and fusion of the distal bones and digits of all limbs and to variable incidence of renal aplasia (Woychik, R.P. et al. (1990) Nature 346:850-853; Jackson-Grusby, L. et al. (1992) Genes Dev. 6:29-37). A mutation in the human FH protein hDIAl was recently found to be the cause of an autosomal dominant hearing disorder (Wasserman, supra). ADF/cofilins are a family of conserved 15-18 kDa actin-binding proteins that play a role in cytokinesis, endocytosis, and in development of embryonic tissues, as well as in tissue regeneration and in pathologies such as ischemia, oxidative or osmotic stress. LBVI kinase 1 downregulates ADF (Carlier, M.F. et al. (1999) J. Biol. Chem. 274:33827-33830).
LIM is an acronym of three transcription factors, Lin-11, Isl-1, and Mec-3, in which the motif was first identified. The LIM domain is a double zinc-finger motif that mediates the protein- protein interactions of transcription factors, signaling, and cytoskeleton-associated proteins (Roof, D.J. et al. (1997) J. Cell Biol. 138:575-588). These proteins are distributed in the nucleus, cytoplasm, or both (Brown, S. et al. (1999) J. Biol. Chem. 274:27083-27091). Recently, ALP (actinin-associated LIM protein) has been shown to bind alpha-actinin-2 (Bouju, S. et al. (1999) Neuromuscul. Disord. 9:3-10).
The Frabin protein is another example of an actin-filament binding protein (Obaishi, H. et al. (1998) J. Biol. Chem. 273:18697-18700). Frabin (FGDl-related F-actin-binding protein) possesses one actin-filament binding (FAB) domain, one Dbl homology (DH) domain, two pleckstrin homology (PH) domains, and a single cysteine-rich FYVE ( Fablp, YOTB, Vaclp, and EEA1 (early endosomal antigen 1)) domain. Frabin has shown GDP/GTP exchange activity for Cdc42 small G protein (Cdc42), and indirectly induces activation of Rac small G protein (Rac) in intact cells. Through the activation of Cdc42 and Rac, Frabin is able to induce formation of both filopodia- and lamellipodia-like processes (Ono, Y. et al. (2000) Oncogene 19:3050-3058).
The Rho family of small GTP-binding proteins are important regulators of actin-dependent cell functions including cell shape change, adhesion, and motility. The Rho family consists of three major subfamilies: Cdc42, Rac, and Rho. Rho family members cycle between GDP-bound inactive and GTP-bound active forms by means of a GDP/GTP exchange factor (GEF) (Umikawa, M. et al. (1999) J. Biol. Chem. 274:25197-25200). The Rho GEF family is crucial for microfilament organization.
Interaptin is an actin-binding protein of the α-actinin superfamily (Rivero, F. et al. (1998) J. Cell Biol. 142:735-750). The protein from Dictyostelium discoideum is 1737 amino acids in length and contains three domains, an N-terminal F-actin-binding domain of about 250 amino acids, a central rod domain of 1180 amino acids, which is predicted to have a coiled-coil oc-helical structure, and a C- terminal membrane-association domain of about 120 amino acids. Interaptin localizes to the nuclear envelope and the endoplasmic reticulum. The expression level of interaptin in Dictyostelium is developmentally regulated. The highest expression occurs at the stage in which prespore and prestalk cell populations differentiate and sort into multicellular structures. Cyclic AMP, which is known to regulate the expression of many developmentally controlled genes in Dictyostelium discoideum, stimulates the expression of interaptin. The presence of actin binding and membrane association domains indicates that interaptin may provide a link between the intracellular membrane and actin cytoskeleton and play a role in membrane trafficking.
Titin may serve as a scaffold protein for the assembly of sarcomeric proteins during the development of striated muscle and also has a role in muscle elasticity (Trinick, J. and Tskhovrebova, L. (1999) Trends Cell Biol. 9:377-380). It is found exclusively in skeletal and cardiac muscle, and not in smooth muscle. It binds to actin and myosin and other sarcomeric proteins, including actinin, T-cap, M-protein and myomesin. Drosophila melangaster D-titin contains fifty-three immunoglobulin C2 domains, six fibronectin HI domains, two PEVK (rich in proline, glutamic acid, valine, and lysine residues) domains, and a kinase domain (Zhang, Y. et al. (2000) J. Cell Sci. 3101-3115). During muscle development, titin is required for the formation of multi-nucleate syncitia and the organization of actin-myosin filaments into striated arrays. Nebulin-related Proteins
Nebulin is a large sarcomeric protein that interacts with actin filaments in skeletal muscle (Wang, K. et al. (1996) J. Biol. Chem. 271:4304-4314). Nebulin contains 185 or more copies of a 35- residue module that has a consensus sequence and a predicted oc-helical structure. The 35-residue module comprises an actin-binding domain. In the central region of nebulin, the 35-residue modules exhibit a seven module super-repeat pattern. This super-repeat pattern is not present in the C-terminal 100 kDa region of nebulin. The N-teiminal region of nebulin contains 8 linker modules and an 8 kDa acidic domain. The C-terminal region is distinct and contains an SH3 domain. Within the sarcomere, nebulin is oriented with its C-terminus located at the Z-line, and its N-terminus at the pointed slow- growing end of thin filaments in the acto-myosin overlap region.
Nebulin exists as different isoforms which range in size from 600-900 kDa (Kruger, M. et al. (1991) J. Cell. Biol. 115:97-107). The size of nebulin is tissue- and species-specific and is developmentally regulated. Based on the observation that isoform size correlates with the length of Ihin filaments in skeletal muscle, nebulin is proposed to play a role as a molecular ruler that regulates the length of thin filaments. Each nebulin 35-residue module may associate with one actin monomer; thus, isoforms with different numbers of modules could determine the length of thin filaments. The N- terrninal region of nebulin interacts with tropomodulin, which may assist in this function (McElhinny, A.S. et al. (2001) J. Biol. Chem. 276:583-592). Tropomodulin caps actin at the pointed end of thin filaments and maintains filament length by preventing actin monomer dissociation or addition.
Nebulin is absent from cardiac muscle, but related proteins with nebulin-like modules may provide similar functions. Nebulette, for example, is specifically expressed in heart and has a C- terminal region containing twenty-three 35-residue nebulin-like modules (Moncman, C.L. and K. Wang (2000) J. Muscle Res. Cell Motil. 21:153-169; Millevoi, S. et al. (1998) J. Mol. Biol. 282:111- 123). The domain structure of nebulette is similar to nebulin, though it is a smaller protein of only 107 kDa. It has an acidic N-terminal domain, a repeat domain containing nebulin-like modules, a linker domain, and an SH3 domain. The repeat domain of nebulette is about one-tenth the size of that of nebulin. The 35-residue modules of nebulette have a consensus motif, and a subfamily of modules 15- 22 share a conserved motif. Unlike nebulin, nebulette modules do not display a super-repeat pattern. Nebulette binds to actin as well as other sarcomeric proteins including myosin, calmodulin, tropomyosin, troponin, and α-actinin (Moncman, C.L. and K. Wang (1999) Cell Motil. Cytoskeleton 44:1-22). The orientation of nebulette in the sarcomere is analogous to that of nebulin with its C- terminus at the Z-line and its N-terminus in the I-band.
Nebulin-related anchoring protein (N-RAP) is expressed in cardiac and skeletal muscle (Luo, G. et al. (1997) Cell Motil. Cytoskeleton 38:75-90). It is a 133 kDa protein found at the ends of myofibrils at muscle myotendon junctions and intercalated disks. The C-terminal region of N-RAP has 27 copies of the 35-residue nebulin-like modules. Seventeen of the modules are organized in a super- repeat pattern. The N-terminal region contains a cysteine-rich LIM domain. LIM domains bind two zinc ions in two adjacent zinc finger-like structures and are known to mediate protein-protein interactions. N-RAP may mediate interactions between actin filaments of myofibrils and other sarcomeric proteins. N-RAP binds to actin, talin, and vinculin (Luo, G. et al. (1999) Biochemistry 38:6135-6143). It interacts with actin and vinculin through its super-repeat region and with talin through its LIM domain. Talin and vinculin are also located at myotendon junctions and together with N-RAP may provide a link between actin filaments of the myofibril and the sarcolemma and transmit tension from the myofibril to the extracellular matrix.
Mutations of sarcomeric proteins are associated with muscle weakness and disease (Laing, N.G. (1999) Curr. Opin. Neurol. 12:513-518). Autosomal recessive nemaline myopathy in some cases is caused by nebulin deficiency (Pelin, K. et al. (1999) Proc. Natl. Acad. Sci. 96:2305-2310). The disease is characterized by the presence of nemaline bodies in muscle fibers. The nemaline bodies contain proteins normally associated with the Z disc and thin filament. Defects in nebulin apparently perturb interactions among sarcomeric proteins and result in the pathological aggregation of proteins in nemaline bodies. Intermediate Filaments and Associated Proteins
Intermediate filaments (IFs) are cytoskeletal fibers with a diameter of about 10 nm, intermediate between that of microfilaments and microtubules. IPs 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.
Five types of IF proteins are known in mammals. Type I and Type II proteins are the acidic and basic keratins, respectively. Heterodimers of the acidic and basic keratins are the building blocks of keratin IFs. Keratins are abundant in soft epithelia such as skin and cornea, hard epifhelia such as nails and hair, and in epithelia that line internal body cavities. Mutations in keratin genes lead to epithelial diseases including epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white sponge nevus. Some of these diseases result in severe skin blistering. (See, e.g., Wawersik, M. et al. (1997) J. Biol. Chem. 272:32557-32565; and Corden L.D. and W.H. McLean (1996) Exp. Dermatol. 5:297-307.) Type HI JF proteins include desmin, glial fibrillary acidic protein, vimentin, and peripherin.
Desmin filaments in muscle cells link myofibrils into bundles and stabilize sarcomeres in contracting muscle. M-Protein is a structural constituent of myofibrillar M-band in striated muscle, and is a member of the cytoskeletal protein superfamily along with immunoglobulin/fibronectin repeats (Noguchi, J. et al. (1992) J. Biol. Chem. 267:20302-20310). Glial fibrillary acidic protein filaments are found in the glial cells that surround neurons and astrocytes. Vimentin filaments are found in blood vessel endothelial cells, some epithelial cells, and mesenchymal cells such as fibroblasts, and are commonly associated with microtubules. Vimentin filaments may have roles in keeping the nucleus and other organelles in place in the cell. Type IN IFs include the neurofilaments and nestin. Neurofilaments, composed of three polypeptides, NF-L, NF-M, and NF-H, are frequently associated with microtubules in axons. Neurofilaments are responsible for the radial growth and diameter of an axon, and ultimately for the speed of nerve impulse transmission. Changes in phosphorylation and metabolism of neurofilaments are observed in neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease (Julien, J.P. and W.E. Mushynski
(1998) Prog. Nucleic Acid Res. Mol. Biol. 61:1-23). Type V IFs, the lamins, are found in the nucleus where they support the nuclear membrane.
The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. Lamins are type V intermediate filament (IF) proteins. Prelamin A is a prenylated and methylated nuclear protein (Barton, R.M. and Worman, H.J. (1999) J. Biol. Chem.
274:30008-30018). Prenylation is a post-translational modification that is necessary for membrane attachment.
IFs have a central α-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 (IFAPs) 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 are particularly closely associated. IFAPs include BPAG1, plakoglobin, desmoplakin I, desmoplakin II, plectin, ankyrin, filaggrin, and lamin B receptor.
Trichohyalin (THH) is a major structural protein in the inner root sheath cells and medulla layer of hair follicles (Tarcsa, E. et al. (1997) J. Biol. Chem. 272:27893-27901). It is also present in other specialized epithelia such as human foreskin epidermis, hard palate, and nail bed. In hair follicle cells, THH is a large insoluble protein that forms droplets mixed with keratin intermediate filaments.
The protein undergoes post-translational modification by transglutaminases and peptidyl-arginine deirninases that convert THH to a more soluble form. THH may serve as a keratin intermediate filament matrix protein and/or as a constituent of the cell envelope and may serve barrier functions in hardened epithelia.
Cytoskeletal-Membrane Anchors
Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These attachments are important for mamtaining cell shape and for muscle contraction. In erythrocytes, the spectrin-actin cytoskeleton is attached to the 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. In platelets, the spectrin-actin cytoskeleton is also linked to the membrane by ankyrin; a second actin network is anchored to the membrane by filamin. In muscle cells the protein dystrophin links actin filaments to the plasma membrane; mutations in the dystrophin gene lead to Duchenne muscular dystrophy. Focal adhesions
Focal adhesions are specialized structures in the plasma membrane involved in the adhesion of a cell to a substrate, such as the extracellular matrix (ECM). Focal adhesions form the connection between an extracellular substrate and the cytoskeleton, and affect such functions as cell shape, cell motility and cell proliferation. Transmembrane integrin molecules form the basis of focal adhesions. Upon ligand binding, integrins cluster in the plane of the plasma membrane. Cytoskeletal linker proteins such as the actin binding proteins α-actinin, talin, tensin, vinculin, paxillin, and filamin are recruited to the clustering site. Key regulatory proteins, such as Rho and Ras family proteins, focal adhesion kinase, and Src family members are also recruited. These events lead to the reorganization of actin filaments and the formation of stress fibers. These intracellular rearrangements promote further integrin-ECM interactions and integrin clustering. Thus, integrins mediate aggregation of protein complexes on both the cytosolic and extracellular faces of the plasma membrane, leading to the assembly of the focal adhesion. Many signal transduction responses are mediated via various adhesion complex proteins, including Src, FAK, paxillin, and tensin. (For a review, see Yamada, K.M. and B. Geiger, (1997) Curr. Opin. CeU Biol. 9:76-85.)
IFs are also attached to membranes by cytoskeletal-membrane anchors. The nuclear lamina is attached to the inner surface of the nuclear membrane by the lamin B receptor. Vimentin IFs are attached to the plasma membrane by ankyrin and plectin. Desmosome and hemidesmosome membrane junctions hold together epithelial ceUs of organs and skin. These membrane junctions aUow shear forces to be distributed across the entire epithelial ceU layer, thus providing strength and rigidity to the epitlielium. IFs in epithelial ceUs are attached to the desmosome by plakoglobin and desmoplakins. The proteins that link IFs to hemidesmosomes are not known. Desmin IFs surround the sarcomere in muscle and are linked to the plasma membrane by paranemin, synemin, and ankyrin. Smoothelin proteins are smooth muscle-specific cytoskeleton-associated proteins that have been shown to be co-distributed with filamin and desmin (Wehrens, X.H. et al. (1997) FEBS Lett. 405:315-320). Two types of smoothelin have been identified: smoothelin-A, expressed in visceral tissues, and smoothelin-B, expressed in vascular tissues (Rensen, S. et al. (2000) Cytogenet. CeU Genet. 89:225-229).
Pinin is a ceU adhesion-associated and nuclear protein that localizes near intermediate filament /desmosomal junctures. These proteins are associated with mature desmosomes of the epithelia (Ouyang, P. and Sugrue, S.P. (1992) J. CeU Biol. 118:1477-1488), and may function to reinforce the interaction between intermediate filaments with desmosomal membranes (Ouyang, P. and Sugrue, S.P. (1996) J. CeUBiol. 135:1027-1042). Motor Proteins Mvosin-related Motor Proteins
Myosins are actin-activated ATPases, found in eukaryotic ceUs, that couple hydrolysis of ATP with motion. Myosin provides the motor function for muscle contraction and intraceUular movements such as phagocytosis and rearrangement of ceU contents during mitotic ceU division (cytokinesis). The contractile unit of skeletal muscle, termed the sarcomere, consists of highly ordered arrays of thin actm-containing filaments and thick myos -containing filaments. Crossbridges form between the thick and thin filaments, and the ATP-dependent movement of myosin heads within the thick filaments puUs the thin filaments, shortening the sarcomere and thus the muscle fiber.
Myosins are composed of one or two heavy chains and associated light chains. Myosin heavy chains contain an ammo-terminal motor or head domain, a neck that is the site of light-chain binding, and a carboxy-terminal tail domain. The tail domains may associate to form an α-helical coiled coil. Conventional myosins, such as those found in muscle tissue, are composed of two myosin heavy-chain subunits, each associated with two light-chain subunits that bind at the neck region and play a regulatory role. Unconventional myosins, believed to function in intraceUular motion, may contain either one or two heavy chains and associated light chains. There is evidence for about 25 myosin heavy chain genes in vertebrates, more than half of them unconventional. Dynein-related Motor Proteins Dyneins are (-) end-directed motor proteins which act on microtubules. Two classes of dyneins, cytosolic and axonemal, have been identified. CytosoUc dyneins are responsible for translocation of materials along cytoplasmic microtubules, for example, transport from the nerve terminal to the ceU body and transport of endocytic vesicles to lysosomes. As weU, viruses often take advantage of cytoplasmic dyneins to be transported to the nucleus and establish a successful infection (Sodeik, B. et al. (1997) J. CeU Biol. 136:1007-1021). Virion proteins of herpes simplex virus 1, for example, interact with the cytoplasmic dynein intermediate chain (Ye, GJ. et al. (2000) J. Virol. 74:1355-1363). Cytoplasmic dyneins are also reported to play a role in mitosis. Axonemal dyneins are responsible for the beating of flageUa and cilia. Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This sliding force produces bending that causes the flageUum 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. Cytoplasmic dynein is the largest and most complex of the motor proteins. Kinesin-related Motor Proteins
Ktnesins 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 aU ceU types for the transport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is critical for mamtaining the identity and functionality of these secretory organeUes.
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 ceUular function. (Reviewed in Moore, J.D. and S.A. Endow (1996) Bioessays 18:207-219; and Hoyt, A.M. (1994) Curr. Opin. CeU Biol. 6:63-68.) The prototypical kinesin molecule is a heterotetramer comprised of two heavy polypeptide chains (KHCs) and two light polypeptide chains (KLCs). The KHC subunits are typicaUy referred to as 'Tdnesin." 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. At one end of the molecule is a globular motor domain that functions in ATP hydrolysis and microtubule binding. Kinesin motor domains are highly conserved and share over 70% identity. Beyond the motor domain is an α-helical coiled-coil region which mediates dimerization. At the other end of the molecule is 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. Members of the more divergent subfamilies of kinesins are caUed kinesin-related proteins
(KRPs), many of which function during mitosis in eukaryotes (Hoyt, supra). Some KRPs are required for assembly of the mitotic spindle. In vivo and in vitro analyses suggest that these KRPs exert force on microtubules that comprise the mitotic spindle, resulting in the separation of spindle poles. Phosphorylation of KRP is required for this activity. Failure to assemble the mitotic spindle results in abortive mitosis and chromosomal aneuploidy, the latter condition being characteristic of cancer ceUs. In addition, a unique KRP, centromere protein E, localizes to the kinetochore of human mitotic chromosomes and may play a role in their segregation to opposite spindle poles. Dvnamin-related Motor Proteins Dynamin is a large GTPase motor protein that functions as a "molecular prnchase," generating a mechanochemical force used to sever membranes. This activity is important in forming clathrin- coated vesicles from coated pits in endocytosis and in the biogenesis of synaptic vesicles in neurons. Binding of dynamin to a membrane leads to dynamin' s self-assembly into spirals that may act to constrict a flat membrane surface into a tubule. GTP hydrolysis induces a change in conformation of the dynamin polymer that pinches the membrane tubule, leading to severing of the membrane tubule and formation of a membrane vesicle. Release of GDP and inorganic phosphate leads to dynamin disassembly. FoUowing disassembly the dynamin may either dissociate from the membrane or remain associated to the vesicle and be transported to another region of the ceU. Three homologous dynamin genes have been discovered, in addition to several dynamin-related proteins. Conserved dynamin regions are the N-terminal GTP-binding domain, a central pleckstrin homology domain that binds membranes, a central coiled-coil region that may activate dynamin' s GTPase activity, and a C- terminal proline-rich domain that contains several motifs that bind SH3 domains on other proteins. Some dynamin-related proteins do not contain the pleckstrin homology domain or the proline-rich domain. (See McNiven, M.A. (1998) CeU 94:151-154; Scaife, R.M. and R.L. MargoUs (1997) CeU. Signal. 9:395-401.)
The cytoskeleton is reviewed in Lodish, H. et al. (1995) Molecular CeU Biology, Scientific American Books, New York NY. Expression profiling Microarrays are analytical tools used in bioanalysis. A microarray has a pluraUty of molecules spatially distributed over, and stably associated with, the surface of a soUd support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of appHcations, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specificaUy related to a particular genetic predisposition, condition, disease, or disorder.
From PF-1086 P Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibriUary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basaUs and locus caeruleus. Early in Alzheimer's pathology, physiological changes are visible in the cingulate cortex (Mmoshima, S. et al. (1997) Annals of Neurology 42:85-94). In subjects with advanced Alzheimer's disease, accumulating plaques damage the neuronal architecture in limbic areas and eventuaUy cripple the memory process.
From PF 1190 P The potential appUcation of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with breast cancer or prostate cancer may be compared with the levels and sequences expressed in normal tissue.
There are more than 180,000 new cases of breast cancer diagnosed each year, and the mortality rate for breast cancer approaches 10% of aU deaths in females between the ages of 45-54 (K. Gish (1999) AWIS Magazine 28:7-10). However the survival rate based on early diagnosis of localized breast cancer is extremely high (97%), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). Current procedures for clinical breast examination are lacking in sensitivity and specificity, and efforts are underway to develop comprehensive gene expression profiles for breast cancer that maybe used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Perou CM. et al. (2000) Nature 406:747-752).
Breast cancer is a genetic disease commonly caused by mutations in breast epitheHal ceUs. Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra). However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to noni herited mutations that occur in breast epitheUal ceUs.
A good deal is already known about the expression of specific genes associated with breast cancer. For example, the relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly weU studied. (See Khazaie et al., supra, and references cited therein for a review of this area.) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on ceU functions related to metastatic potential, such as ceU motiHty, chemotaxis, secretion and differentiation. Changes in expression of other members of the erbB receptor family, of which EGFR is one, have also been impUcated in breast cancer. The abundance of erbB receptors, such as HER-2/neu, HER-3 , and HER-4, and their Ugands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S.S. et al. (1994) Am. J. Clin. Pathol. 102:S13-S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix Gla protein which is overexpressed is human breast carcinoma ceUs; Drgl or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S 100 protein family, aU of which are down regulated in mammary carcinoma ceUs relative to normal mammary epitheUal ceUs (Zhou Z. et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395; Ulrix W. et al (1999) FEBS Lett. 455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Immunol. 213:51-64; and Lee, S.W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508). Prostate cancer is a common maUgnancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year.
Once cancer ceUs arise in the prostate, they are stimulated by testosterone to a more rapid growth. Thus, removal of the testes can indirectly reduce both rapid growth and metastasis of the cancer. Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous ceU and transitional ceU carcinomas, both of which arise in the prostatic ducts or other parts of the prostate gland.
As with most cancers, prostate cancer develops through a multistage progression ultimately resulting in an aggressive, metastatic phenotype. The initial step in tumor progression involves the hyperproUferation of normal luminal and/or basal epitheUal ceUs that become hyperplastic and evolve into early-stage tumors. The early-stage tumors are locaUzed in the prostate but eventuaUy may metastasize, particularly to the bone, brain or lung. About 80% of these tumors remain responsive to androgen treatment, an important hormone controUing the growth of prostate epitheUal ceUs. However, in its most advanced state, cancer growth becomes androgen-independent and there is currently no known treatment for this condition.
A primary diagnostic marker for prostate cancer is prostate specific antigen (PSA). PSA is a tissue-specific serine protease almost exclusively produced by prostatic epitheUal ceUs. The quantity of PSA correlates with the number and volume of the prostatic epitheUal ceUs, and consequently, the levels of PSA are an exceUent indicator of abnormal prostate growth. Men with prostate cancer exhibit an early linear increase in PSA levels foUowed by an exponential increase prior to diagnosis. However, since PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting
5 individual cases of prostate cancer.
Current areas of cancer research provide additional prospects for markers as weU as potential therapeutic targets for prostate cancer. Several growth factors have been shown to play a critical role in tumor development, growth, and progression. The growth factors Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Tumor Growth Factor alpha (TGFα) are important in the growth
LO of normal as weU as hyperproUferative prostate epitheUal ceUs, particularly at early stages of tumor development and progression, and affect signaling pathways in these ceUs in various ways (Lin J et al. (1999) Cancer Res. 59:2891-2897; Putz T et al (1999) Cancer Res 59:227-233). The TGF-β family of growth factors are generaUy expressed at increased levels in human cancers and the high expression levels in many cases correlates with advanced stages of maUgnancy and poor survival
L5 (Gold LI (1999) Crit Rev Oncog 10:303-360). FinaUy, there are human ceU lines representing both the androgen-dependent stage of prostate cancer (LNCap) as weU as the androgen-independent, hormone refractory stage of the disease (PC3 and DU-145) that have proven useful in studying gene expression patterns associated with the progression of prostate cancer, and the effects of ceU treatments on these expressed genes (Chung TD (1999) Prostate 15:199-207).
£0 There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of ceU motiUty disorders, developmental disorders, ceU proUferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders.
SUMMARY OF THE INVENTION
£5 Various embodiments of the invention provide purified polypeptides, structural and cytoskeleton-associated proteins, referred to coUectively as "SCAP" and individuaUy as "SCAP-1," "SCAP-2," "SCAP-3," "SCAP-4," "SCAP-5," "SCAP-6," "SCAP-7," "SCAP-8," "SCAP-9," "SCAP-10," "SCAP-11," "SCAP-12," "SCAP-13," "SCAP-14," "SCAP-15," "SCAP-16," "SCAP- 17," "SCAP-18," "SCAP-19," "SCAP-20," "SCAP-21," "SCAP-22," "SCAP-23," "SCAP-24,"
30 "SCAP-25," "SCAP-26," "SCAP-27," "SCAP-28," "SCAP-29," "SCAP-30," "SCAP-31," "SCAP- 32," "SCAP-33," "SCAP-34," "SCAP-35," "SCAP-36," and "SCAP-37," and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utiUzing the purified structural and cytoskeleton-associated proteins and/or their encoding polynucleotides for faciUtating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified structural and cytoskeleton-associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical
5 conditions.
An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l- 37, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID
[0 NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-37. StiU another embodiment provides an isolated polynucleotide encoding a polypeptide selected
L5 from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an
£0 immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-37. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:38-74.
StiU another embodiment provides a recombinant polynucleotide comprising a promoter
£5 sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid
30 sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37. Another embodiment provides a ceU transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment 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: 1-37, b) a polypeptide comprising a naturaUy occurring a ino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37. The method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Yet another embodiment provides an isolated antibody which specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37. StiU yet another embodiment 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:38-74, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JJD NO:38-74, 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). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specificaUy hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said
5 probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In stiU other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
StiU yet another embodiment provides a method for detecting a target polynucleotide in a
10 sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
15 complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) ampUfying said target polynucleotide or fragment thereof using polymerase chain reaction ampUfication, and b) detecting the presence or absence of said ampUfied target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the ampUfied target polynucleotide or fragment thereof.
£0 Another embodiment 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: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologically active fragment of a
£5 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and a pharmaceuticaUy acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37. Other embodiments provide a method of treating a disease or condition
30 associated with decreased or abnormal expression of functional SCAP, comprising administering to a patient in need of such treatment the composition.
Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceuticaUy acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional SCAP, comprising administering to a patient in need of such treatment the composition.
StiU yet another embodiment 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: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional SCAP, comprising administering to a patient in need of such treatment the composition. Another embodiment provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37. 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.
Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consistmg of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consistmg of SEQ ID NO: 1-37 , c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37. 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.
StiU yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
Another embodiment 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:38-74, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of n), 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:38-74, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide 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.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for fuU length polynucleotide and polypeptide embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptide embodiments of the invention. The probabiUty scores for the matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide embodiments, 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/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
Table 5 shows representative cDNA Kbraries for polynucleotide embodiments. 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 polynucleotides and polypeptides, along with appUcable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, materials, and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host ceU" includes a pluraUty of such host ceUs, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skiUed in the art, and so forth.
Unless defined otherwise, aU technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. AU pubUcations mentioned herein are cited for the purpose of describing and disclosing the ceU lines, protocols, reagents and vectors which are reported in the pubUcations and which might be used in connection with various embodiments of the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. DEFINITIONS
"SCAP" refers to the amino acid sequences of substantiaUy purified SCAP 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.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of SCAP. Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of SCAP either by directly interacting with SCAP or by acting on components of the biological pathway in which SCAP participates.
An "aUelic variant" is an alternative form of the gene encoding SCAP. AUeUc 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 aUeUc variants of its naturaUy occuπing form. Common mutational changes which give rise to aUeUc variants are generaUy ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. "Altered" nucleic acid sequences encoding SCAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as SCAP or a polypeptide with at least one functional characteristic of SCAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oUgonucleoti.de probe of the polynucleotide encoding SCAP, and improper or unexpected hybridization to aUeUc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding SCAP. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionaUy equivalent SCAP. DeUberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubiUty, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of SCAP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophiUcity 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.
The terms "amino acid" and "amino acid sequence" can refer to an oUgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occuπing or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturaUy occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"AmpUfication" relates to the production of additional copies of a nucleic acid. AmpUfication may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid ampUfication technologies weU known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of SCAP. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of SCAP either by directly interacting with SCAP or by acting on components of the biological pathway in which SCAP participates.
The term "antibody" refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind SCAP polypeptides can be prepared using intact polypeptides or using fragments containing smaU 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) can be derived from the translation of RNA, or synthesized chemicaUy, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, thyroglobuUn, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal. The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specificaUy to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e. , the immunogen used to eUcit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oUgonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial Ubraries. Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH2), which may improve a desired property, e.g., resistance to nucleases or longer Ufetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers maybe specificaUy cross-linked to their cognate Ugands, e.g., by photo-activation of a cross-Unker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence. Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oUgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oUgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oUgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a ceU, the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation. The designation "negative" or "minus'' can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule. The term "biologicaUy active" refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule. Likewise, "immunologicaUy active" or "immunogenic" refers to the capabiUty of the natural, recombinant, or synthetic SCAP, or of any oUgopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies. "Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide" and a "composition comprising a given polypeptide" can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotides encoding SCAP or fragments of SCAP maybe employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncaUed bases, extended using the XL-PCR kit (AppUed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVCEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative a ino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy the function of the protein is conserved and not significantly changed by such 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. Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gin, His
5 Asp Asn, Glu
Cys Ala, Ser
Gin Asn, Glu, His
Glu Asp, Gin, His
Gly Ala
LO His Asn, Arg, Gin, Glu lie Leu, Val
Leu Tie, Val
Lys Arg, Gin, Glu
Met Leu, lie
L5 Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
£0 Val He, Leu, Thr
Conservative amino acid substitutions generaUy 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 £5 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.
The term "derivative" refers to a chemicaUy modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an 30 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 35 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. 10 "Exon shuffling" refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins maybe assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.
A "fragment" is a unique portion of SCAP or a polynucleotide encoding SCAP which can be 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. For example, a fragment may comprise from about 5 to about 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 preferentiaUy selected from certain regions of a molecule. For example, 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. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, maybe encompassed by the present embodiments.
A fragment of SEQ ID NO:38-74 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:38-74, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO:38-74 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and ampUfication technologies and in analogous methods that distinguish SEQ ID NO:38-74 from related polynucleotides. The precise length of a fragment of SEQ ED NO:38-74 and the region of SEQ ID NO:38-74 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO: 1-37 is encoded by a fragment of SEQ JD NO:38-74. A fragment of SEQ ID NO:l-37 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO: 1-37. For example, a fragment of SEQ ID NO: 1-37 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO:l-37. The precise length of a fragment of SEQ ID NO:l-37 and the region of SEQ ID NO:l-37 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide is one containing at least a translation initiation codon (e.g., rnethionine) foUowed by an open reading frame and a translation teπnination codon. A "fuU length" polynucleotide sequence encodes a "full length" polypeptide sequence. "Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as appUed to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aUgned using a 5 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.
Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity 10 can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence aUgnment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989; CABIOS 5:151- 153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191). For pairwise aUgnments of 15 polynucleotide sequences, the default parameters are set as foUows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms £0 which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local AUgnment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to aUgn a known polynucleotide sequence with other £5 polynucleotide sequences from a variety of databases. Also available is a tool caUed "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nm.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to 30 compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example:
Matrix: BLOSUM62
Reward for match: 1 Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: 10 Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ED number, or maybe measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.
The phrases "percent identity" and "% identity," as appUed to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aUgned using a standardized algorithm. Methods of polypeptide sequence aUgnment are weU-known. Some aUgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence aUgnment program (described and referenced above). For pairwise aUgnments of polypeptide sequences using CLUSTAL V, the default parameters are set as foUows: Ktuple=l, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide aUgnments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50 Expect: 10
Word Size: 3
Filter: on
Percent identity maybe measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, maybe used to describe a length over which percentage identity may be measured. "Human artificial chromosomes" (HACs) are linear microchromosomes which may contain
DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome repUcation, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and stiU retains its original binding abiUty.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in deteπriining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 μg/ml sheared, denatured salmon sperm DNA.
GeneraUy, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (Tj for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are weU known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specificaUy see volume 2, chapter 9.
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%. TypicaUy, 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 jtlg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions wiU be readily apparent to those of ordinary skiU in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C0t or R0t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a soUd support (e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed). The words "insertion" and "addition" refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively. "Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect ceUular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oUgopeptide fragment of SCAP 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 SCAP which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarray" refers to an aπangement of a pluraUty of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate. The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microaπay.
The term "modulate" refers to a change in the activity of SCAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of SCAP. The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oUgonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably Unked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, 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.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oUgonucleotide 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 preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Ufespan in the ceU.
"Post-translational modification" of an SCAP may involve Upidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic milieu of SCAP.
"Probe" refers to nucleic acids encoding SCAP, their complements, or fragments thereof, which are used to detect identical, aUeUc or related nucleic acids. Probes are isolated oUgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, Ugands, chermluminescent agents, and enzymes. "Primers" are short nucleic acids, usuaUy DNA oUgonucleotides, which maybe annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for ampUfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, maybe used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989; Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY), Ausubel, F.M. et al. (1999) Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons, New York NY), and Innis, M. et al. (1990; PCR Protocols, A Guide to Methods and AppUcations, Academic Press, San Diego CA). 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 Institote 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, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope. The Primer3 primer selection program (available to the pubUc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows 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 oUgonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the pubUc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aUgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments. The oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microaπay elements, or specific probes to identify fuUy or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of oUgonucleotide selection are not limited to those described above. A "recombinant nucleic acid" is a nucleic acid that is not naturaUy 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. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a ceU.
Alternatively, such recombinant nucleic acids maybe part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA 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. An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that aU occurrences of the nitrogenous base thy ine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing SCAP, nucleic acids encoding SCAP, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specificaUy binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g. , the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the 5 epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
The term "substantiaUy 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 about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other L0 components with which they are naturaUy associated.
A "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, L5 microparticles and capiUaries. The substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" or "expression profile" refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient £0 ceU. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, Upofection, and particle bombardment. The term "transformed 25 ceUs" includes stably transformed ceUs in which the inserted DNA is capable of repUcation either as an autonomously repUcating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the ceUs of the organism contains heterologous nucleic acid 30 introduced by way of human intervention, such as by transgenic techniques weU known in the art. The nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the ceU, by way of deUberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants
5 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
.0 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
15 sequence identity over a certain defined length. A variant may be described as, for example, an
"aUeUc" (as defined above), "spUce," "species," or "polymorphic" variant. A spUce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spUcing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the
£0 reference molecule. Species variants are polynucleotides 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
£5 presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-
30 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
Various embodiments of the invention include new human structural and cytoskeleton- associated proteins (SCAP), the polynucleotides encoding SCAP, and the use of these compositions for the diagnosis, treatment, or prevention of ceU motiUty disorders, developmental disorders, ceU proUferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders. Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ED). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide D) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown. Column 6 shows the Incyte ED numbers of physical, fuU length clones corresponding to polypeptide and polynucleotide embodiments. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptides shown in column 3.
Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ED NO:) of the nearest GenBank homolog.
Column 4 shows the probabiUty scores for the matches between each polypeptide and its homolog(s). Column 5 shows the annotation of the GenBank homolog(s) along with relevant citations where appUcable, aU of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) 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 MOTIF'S program of the GCG sequence analysis software package (Genetics Computer Group, Madison WT). 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.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties estabUsh that the claimed polypeptides are cytoskeleton-associated proteins. For example, SEQ ID NO:3 is 96% identical, from residue Ml to residue P1638, to Mus musculus Kif21b (GenBank ID g6561829) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ID NO:3 also contains a WD domain, G- beta repeat and a kinesin motor domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFTLESCAN analyses, and BLAST analyses of the PRODOM and DOMO databases provide further coπoborative evidence that SEQ ID NO:3 is a kinesin-related protein.
As another example, SEQ ED NO:7 is 78% identical, from residue Ml to residue N1196, to mouse formin, isoform IV (GenBank ID g51553) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ID NO:7 also contains a Formin Homology 2 domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and additional BLAST analyses using the PRODOM and DOMO databases provide further corroborative evidence that SEQ ID NO:7 is a formin homology protein.
As another example, SEQ ID NO:ll is 99% identical, from residue Ml to residue R744, to a human pinin protein (GenBank ID gl684847) as determined by the Basic Local AUgnment Search
Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence alignment by chance. Data from BLAST-PRODOM and BLAST-DOMO analyses provide further coπoborative evidence that SEQ ID NO: 11 is a cytoskeleton-associated protein. As another example, SEQ ID NO:15 is 99% identical, from residue Ml to residue D621, to human smoothelin-B (GenBank ID g6103358) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ED NO: 15 also contains a calponin homology (CH) domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from additional BLAST analyses provide further coπoborative evidence that SEQ ED NO: 15 is a cytoskeleton-associated protein.
As another example, SEQ ID NO:18 is 99% identical, from residue E36 to residue W408, to human nuclear prelamin A recognition factor (GenBank ID g5777952) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 6.4e-220, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ID NO: 18 also contains an an iron only hydrogenase large subunit, C-te domain as determined by
5 searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLAST-PRODOM, BLAST-DOMO, and MOTIFS analyses provide further coπoborative evidence that SEQ ID NO: 18 is a cytoskeleton-associated protein with hydrogenase activity.
As another example, SEQ ED NO:17 is 77% identical, from residue E296 to residue K1526, to
10 rat multidomain presynaptic cytomatrix protein Piccolo (GenBank ED g7493836) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ED NO: 17 also contains C-2 and PDZ domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein
15 family domains. (See Table 3.) Data from BLIMPS, PROFTLESCAN, BLAST-PRODOM,
BLAST-DOMO, and MOTIFS analyses provide further coπoborative evidence that SEQ D NO: 17 is a cytoskeleton-associated protein.
As another example, SEQ ID NO:26 is 73% identical, from residue A52 to residue E1781, to chicken myosin heavy chain (GenBank ED g7248371) as determined by the Basic Local AUgnment
£0 Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ED NO:26 also contains myosin head (motor domain) and myosin tail domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTJFS analyses
£5 provide further corroborative evidence that SEQ ID NO:26 is a myosin.
As another example, SEQ ID NO:29 is 65% identical, from residue K531 to residue E1846, 69% identical, from residue L252 to residue S593, and 69% identical, from residue E12 to residue L253, to GaUus gaUus myosin heavy chain (GenBank ED g7248371) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which
30 indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ D NO:29 also contains a myosin N-terminal SH3-like domain, a myosin tail, and a myosin head (motor domain) as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses and BLAST analyses of the PRODOM and DOMO databases provide further coπoborative evidence that SEQ ED NO:29 is a myosin protein.
As another example, SEQ ED NO:36 is 90% identical, from residue Ml to residue N450, to the type I keratin intermediate filament E Sal (GenBank ED gl2642308) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 5.0e-217, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ED NO:36 also contains an intermediate filament protein domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and BLAST analyses provide further coπoborative evidence that SEQ ED NO:36 is an intermediate filament protein.
SEQ ID NO:l-2, SEQ ID NO:4-6, SEQ ID NO:8-10, SEQ ID NO:12-14, SEQ ID NO:16, SEQ ID NO:19-25, SEQ ID NO:27-28, SEQ ED NO:30-35, and SEQ ID NO:37 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO: 1-37 are described in Table 7. As shown in Table 4, the fuU length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 Usts the polynucleotide sequence identification number (Polynucleotide SEQ ED NO:), the coπesponding Incyte polynucleotide consensus sequence number (Incyte ED) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or ampUfication technologies that identify SEQ ID NO:38-74 or that distinguish between SEQ ED NO:38-74 and related polynucleotides. The polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA Ubraries or from pooled cDNA
Ubraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The S anger Centre, Cambridge, UK) database {Le., those sequences including the designation "ENST"). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, a polynucleotide sequence identified as I^_XXXXXX_N1_N2_YYYYY_N3_N4 represents a "stitched" sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was appUed, and YYYYY is the number of the prediction generated by the algorithm, and N1A3_,_, if present, represent specific exons that may have been manuaUy edited during analysis (See Example V). Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stietching" algorithm. For example, a polynucleotide sequence identified as LXXXXXX_gAAAAA_gBBBBB_l_Nis a "stretched" sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stietching" algorithm was appUed, gBBBBB being the GenBank identification number or ΝCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "ΝM," "ΝP," or "NT") may be used in place of the GenBank identifier (z. e. , gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The foUowing Table Usts examples of component sequence prefixes and coπesponding sequence analysis methods associated with the prefixes (see Example TV and Example V).
Figure imgf000044_0001
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown. Table 5 shows the representative cDNA Ubraries for those full length polynucleotides 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 polynucleotides. The tissues and vectors which were used to construct the cDNA Ubraries shown in Table 5 are described in Table 6.
5 The invention also encompasses SCAP variants. A prefeπed SCAP 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 SCAP amino acid sequence, and which contains at least one functional or structural characteristic of SCAP.
Various embodiments also encompass polynucleotides which encode SCAP. In a particular
L0 embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:38-74, which encodes SCAP. The polynucleotide sequences of SEQ ED NO:38-74, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base mymine are replaced withuracil, and the sugar backbone is composed of ribose instead of deoxyribose.
L5 The invention also encompasses variants of a polynucleotide encoding SCAP. In particular, such a variant polynucleotide wiUhave at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding SCAP. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:38-74 which has at least about 70%, or alternatively at least
£0 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:38-74. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of SCAP.
In addition, or in the alternative, a polynucleotide variant of the invention is a spUce variant of a
£5 polynucleotide encoding SCAP. A spUce variant may have portions which have significant sequence identity to a polynucleotide encoding SCAP, but wiU generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate spUcing of exons during mRNA processing. A spUce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a
SO polynucleotide encoding SCAP over its entire length; however, portions of the spUce variant wiU have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding SCAP. For example, a polynucleotide comprising a sequence of SEQ ED NO:44 and a polynucleotide comprising a sequence of SEQ ID NO:45 are spUce variants of each other. Any one of the spUce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of SCAP.
It wiU be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding SCAP, some bearing minimal similarity to the polynucleotide sequences of any known and naturaUy occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as appUed to the polynucleotide sequence of naturaUy occurring SCAP, and aU such variations are to be considered as being specificaUy disclosed.
Although polynucleotides which encode SCAP and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring SCAP under appropriately selected conditions of stringency, it maybe advantageous to produce polynucleotides encoding SCAP or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy occurring codons. Codons maybe selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utiUzed by the host. Other reasons for substantiaUy altering the nucleotide sequence encoding SCAP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-Ufe, than transcripts produced from the naturaUy occurring sequence.
The invention also encompasses production of polynucleotides which encode SCAP and SCAP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide may be inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a polynucleotide encoding SCAP or any fragment thereof.
Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ED NO:38-74 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kirnmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (AppUed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampUfication system (Eavitrogen, Carlsbad CA). Preferably, 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 earned out using either the ABI 373 or 377 DNA sequencing system (AppUed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding SCAP maybe extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which maybe employed, restriction-site PCR, uses universal and nested primers to ampUfy unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods AppUc. 2:318-322). Another method, inverse PCR, uses primers that extend in divergent directions to ampUfy unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (TrigUa, T. et al. (1988) Nucleic Acids Res. 16:8186). A third method, capture PCR, involves PCR ampUfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods AppUc. 1:111-119). In this method, 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 maybe used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060). AdditionaUy, one mayuse PCR, nested primers, and PROMOTERFINDER Ubraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen Ubraries and is useful in finding intron exon junctions. For aU PCR-based methods, primers maybe designed using commerciaUy 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.
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 fuU-length cDNA. Genomic Ubraries may be useful for extension of sequence into 5 'non-transcribed regulatory regions.
CapiUary electrophoresis systems which are commerciaUy available maybe used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capiUary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output Ught intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed. CapiUary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotides or fragments thereof which encode SCAP may be cloned in recombinant DNA molecules that direct expression of SCAP, or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides may be produced and used to express SCAP.
The polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter SCAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oUgonucleotides may be used to engineer the nucleotide sequences. For example, 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 No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, EC. 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 SCAP, such as its biological or enzymatic activity or its abiUty to bind to other molecules or compounds. DNA shuffling is a process by which a Ubrary of gene variants is produced using PCR-mediated recombination of gene fragments. The Ubrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These prefeπed variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations maybe recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturaUy occurring genes in a directed and controUable manner.
In another embodiment, polynucleotides encoding SCAP maybe synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232). Alternatively, SCAP itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or soUd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis maybe achieved using the ABI 43 IA peptide synthesizer (AppUed Biosystems). AdditionaUy, the amino acid sequence of SCAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturaUy occurring polypeptide.
The peptide may be substantiaUy purified by preparative high performance Uquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (Creighton, supra, pp. 28-53).
In order to express a biologicaUy active SCAP, the polynucleotides encoding SCAP 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 polynucleotides encoding SCAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding SCAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding SCAP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals maybe needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host ceU system used (Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162).
5 Methods which are weU known to those skiUed in the art may be used to construct expression vectors containing polynucleotides encoding SCAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel et al., supra, ch. 1, 3,
.0 and 15).
A variety of expression vector/host systems maybe utiUzed to contain and express polynucleotides encoding SCAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression
L5 vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems (Sambrook, supra; Ausubel et al., supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945;
£0 Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw HiU Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355). Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for deUvery of polynucleotides to the targeted organ, tissue, or ceU population
£5 (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; BuUer, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I.M. and N. Somia (1997) Nature 389:239-242). The invention is not limited by the host ceU employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending
30 upon the use intended for polynucleotides encoding SCAP. For example, routine cloning, subcloning, and propagation of polynucleotides encoding SCAP can be achieved using a multifunctional E. coli vector such as PBLUESCREPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invitrogen). Ligation of polynucleotides encoding SCAP into the vector's multiple cloning site disrupts the lacZ gene, aUowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors maybe useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509). When large quantities of SCAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of SCAP may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of SCAP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184). Plant systems may also be used for expression of SCAP. Transcription of polynucleotides encoding SCAP 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. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters maybe used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CeU Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw HiU Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196).
In mammaUan ceUs, a number of viral-based expression systems may be utilized. In cases where an adenoviras is used as an expression vector, polynucleotides encoding SCAP may be Ugated into an adeno virus 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 maybe used to obtain infective virus which expresses SCAP in host ceUs (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaUan host ceUs. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to 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 (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
For long term production of recombinant proteins in mammaUan systems, stable expression of SCAP in ceU Unes is prefeπed. For example, polynucleotides encoding SCAP can be transformed into ceU 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. FoUowing the introduction of the vector, ceUs maybe aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfully express the introduced sequences. Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
Any number of selection systems may be used to recover transformed ceU Unes. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetaboUte, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and par confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14). Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUular requirements for metaboUtes (Hartman, S.C and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051). Visible markers, e.g., anfhocyanins, green fluorescent proteins (GFP; Clontech), β- glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin maybe 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 (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131).
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding SCAP is inserted within a marker gene sequence, transformed ceUs containing polynucleotides encoding SCAP can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding SCAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU. In general, host ceUs that contain the polynucleotide encoding SCAP and that express SCAP may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR ampUfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detectmg and measuring the expression of SCAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated ceU sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on SCAP is preferred, but a competitive binding assay may be employed. These and other assays are weU known in the art (Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. TV; CoUgan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley- Interscience, New York NY; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ).
A wide variety of labels and conjugation techniques are known by those skiUed in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding SCAP include oUgolabeUng, nick translation, end-labeling, or PCR ampUfication using a labeled nucleotide. Alternatively, polynucleotides encoding SCAP, or any fragments thereof, maybe cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commerciaUy available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures maybe conducted using a variety of commerciaUy available kits, such as those provided by Amersham Biosciences, Promega (Madison WT), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionucUdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host ceUs transformed with polynucleotides encoding SCAP maybe cultured under conditions suitable for the expression and recovery of the protein from ceU culture. The protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used. As wiU be understood by those of skill in the art, expression vectors containing polynucleotides which encode SCAP may be designed to contain signal sequences which direct secretion of SCAP through a prokaryotic or eukaryotic ceU membrane. In addition, a host ceU strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, Upidation, and acylation. Post-translational processing which cleaves a "prepro" or
5 "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and maybe chosen to ensure the coπect modification and processing of the foreign protein.
10 In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding SCAP may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric SCAP protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may faciUtate the screening of peptide Ubraries for inhibitors of SCAP activity. Heterologous protein and peptide
L5 moieties may also faciUtate purification of fusion proteins using commerciaUy available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins,
£0 respectively. FLAG, c-myc, and hemagglutinin (HA) enable irrrmunoaffinity purification of fusion proteins using commerciaUy available monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the SCAP encoding sequence and the heterologous protein sequence, so that SCAP may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein
£5 expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). A variety of commerciaUy available kits may also be used to faciUtate expression and purification of fusion proteins.
In another embodiment, synthesis of radiolabeled SCAP 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
30 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
SCAP, fragments of SCAP, or variants of SCAP may be used to screen for compounds that specificaUy bind to SCAP. One or more test compounds may be screened for specific binding to SCAP. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to SCAP. Examples of test compounds can include antibodies, anticalins, oUgonucleotides, proteins (e.g., Ugands or receptors), or smaU molecules.
In related embodiments, variants of SCAP can be used to screen for binding of test compounds, such as antibodies, to SCAP, a variant of SCAP, or a combination of SCAP and/or one or more variants SCAP. In an embodiment, a variant of SCAP can be used to screen for compounds that bind to a variant of SCAP, but not to SCAP having the exact sequence of a sequence of SEQ ED NO: 1-37. SCAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to SCAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to SCAP can be closely related to the natural Ugand of SCAP, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (CoUgan, J.E. et al. (1991) Cuπent Protocols in Immunology l(2):Chapter 5). In another embodiment, the compound thus identified can be a natural Ugand of a receptor SCAP (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
In other embodiments, a compound identified in a screen for specific binding to SCAP can be closely related to the natural receptor to which SCAP binds, at least a fragment of the receptor, or a fragment of the receptor including aU or a portion of the Ugand binding site or binding pocket. For example, the compound may be a receptor for SCAP which is capable of propagating a signal, or a decoy receptor for SCAP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. CeU Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336). The compound can be rationaUy designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL; Immunex Corp., Seattle WA), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGj (Taylor, P.C et al. (2001) Curr. Opin. Immunol. 13:611-616).
In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to SCAP, fragments of SCAP, or variants of SCAP. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of SCAP. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of SCAP. In another embodiment, an antibody can be selected such that its binding specificity aUows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of SCAP.
In an embodiment, anticalins can be screened for specific binding to SCAP, fragments of SCAP, or variants of SCAP. Anticalins are Ugand-binding proteins that have been constructed based on a UpocaUn scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of UpocaUns can include a beta-baπel having eight antiparaUel beta-strands, which supports four loops at its open end. These loops form the natural Ugand-binding site of the UpocaUns, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
In one embodiment, screening for compounds which specificaUy bind to, stimulate, or inhibit SCAP involves producing appropriate ceUs which express SCAP, either as a secreted protein or on the ceU membrane. Prefeπed ceUs include ceUs from mammals, yeast, Drosophila, or E. coli. CeUs expressing SCAP or ceU membrane fractions which contain SCAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either SCAP 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. For example, the assay may comprise the steps of combining at least one test compound with SCAP, either in solution or affixed to a soUd support, and detecting the binding of SCAP to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical Ubraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
An assay can be used to assess the abiUty of a compound to bind to its natural Ugand and/or to inhibit the binding of its natural Ugand to its natural receptors. Examples of such assays include radio- labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724. In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its abiUty to bind to its natural Ugands (Matthews, D.J. and J.A. WeUs. (1994) Chem. Biol. 1:25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a Ugand) to improve or alter its abiUty to bind to its natural receptors (Cunningham, B.C. and J.A. WeUs (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
SCAP, fragments of SCAP, or variants of SCAP maybe used to screen for compounds that modulate the activity of SCAP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for SCAP activity, wherein SCAP is combined with at least one test compound, and the activity of SCAP in the presence of a test compound is compared with the activity of SCAP in the absence of the test compound. A change in the activity of SCAP in the presence of the test compound is indicative of a compound that modulates the activity of SCAP. Alternatively, a test compound is combined with an in vitro or ceU-free system comprising SCAP under conditions suitable for SCAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of SCAP may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraUty of test compounds maybe screened.
In another embodiment, polynucleotides encoding SCAP or their mammaUan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs. Such techniques are weU known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337). For example, mouse ES ceUs, such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture. The ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgicaUy 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 SCAP may also be manipulated in vitro in ES ceUs derived from human blastocysts. Human ES ceUs have the potential to differentiate into at least eight separate ceU Uneages including endoderm, mesoderm, and ectodermal ceU types. These ceU Uneages differentiate into, for example, neural ceUs, hematopoietic Uneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
Polynucleotides encoding SCAP can also be used to create "knockin" humanized animals ' (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding SCAP is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome. Transformed ceUs 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. Alternatively, a mammal inbred to overexpress SCAP, e.g., by secreting SCAP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of SCAP and structural and cytoskeleton-associated proteins. In addition, the expression of CSAP is closely associated with adrenal, bladder, brain, diseased brain, aUocortex, cingulate posterior, dorsal root gangUon, pituitary gland, colon, heart, Uver, Uver tumor, lung, lower thigh, nose, pancreatic islet, penis, prostate tumor, rib bone, synovial membrane, tongue, and uterine tissues, and with breast cancer, bladder cancer, colon cancer, penile cancer, prostate cancer, olfactory neuroblastoma, teratocarcinoma, and rheumatoid arthritis, as weU as with promonocyte ceUs, SH-SY5Y ceUs derived from metastatic bone marrow neuroblastoma, and human umbiUcal vein endotheUal ceUs (HUV-EC- C). In addition, examples of tissues expressing SCAP can be found in Table 6 and can also be found in Example XI. Therefore, SCAP appears to play a role in ceU motiUty disorders, developmental disorders, ceU proUferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders. In the treatment of disorders associated with increased SCAP expression or activity, it is desirable to decrease the expression or activity of SCAP. In the treatment of disorders associated with decreased SCAP expression or activity, it is desirable to increase the expression or activity of SCAP.
Therefore, in one embodiment, SCAP or a fragment or derivative thereof maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SCAP. Examples of such disorders include, but are not limited to, a ceU motiUty disorder such as ankylosing spondyUtis, Chediak-Higashi syndrome, Duchenne and Becker muscular dystrophy, intrahepatic cholestasis, myocardial hyperplasia, cardiomyopathy, early onset peridontitis, cancers such as adenocarcinoma, ovarian carcinoma, and chronic myelogenous leukemia, and bacterial and helminthic infections; a developmental disorder such as renal tubular acidosis, anemia, Qishing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormaUties, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepitheUal dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a ceU 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 a cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangUa, gastrointestinal tract, heart, kidney, Uver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, saUvary glands, skin, spleen, testis, thymus, thyroid, and uterus; a viral infection such as those caused by adenovirases (acute respiratory disease, pneumonia), arenaviruses (lymphocytic choriomeningitis), bunyaviruses (Hantavirus), coronaviruses (pneumonia, chronic bronchitis), hepadnaviruses (hepatitis), herpesviruses (herpes simplex virus, variceUa-zoster virus, Epstein-Barr virus, cytomegalo virus), flavivirases (yeUow fever), orthomyxoviruses (influenza), papiUomaviruses (cancer), paramyxoviruses (measles, mumps), picornoviruses (rhinoviras, poUoviras, coxsac e-virus), polyomavirases (BK virus, JC virus), poxviruses (smaUpox), reoviras (Colorado tick fever), retroviruses (human immunodeficiency virus, human T lymphotropic virus), rhabdoviruses (rabies), rotaviruses (gastroenteritis), and togaviruses (encephaUtis, rabeUa); a heart and skeletal muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, Upid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy; and a neurological disorder such as epilepsy, ischemic cerebro vascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myeUtis and radicuUtis, viral central nervous system disease, a prion disease including kuru, Creutzfeldt- Jakob disease, and Gerslmarm-Sttaussler-Scheinker syndrome, fatal familial insomnia, nutritional and metaboUc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metaboUc, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder.
In another embodiment, a vector capable of expressing SCAP 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 SCAP including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantiaUy purified SCAP 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 SCAP including, but not limited to, those provided above.
In stiU another embodiment, an agonist which modulates the activity of SCAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SCAP including, but not limited to, those Usted above.
In a further embodiment, an antagonist of SCAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SCAP. Examples of such disorders include, but are not limited to, those ceU motiUty disorders, developmental disorders, ceU proUferative disorders, viral infections, heart and skeletal muscle disorders, and neurological disorders described above. In one aspect, an antibody which specificaUy binds SCAP may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express SCAP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding SCAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SCAP including, but not limited to, those described above.
In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skiU in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one maybe able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of SCAP may be produced using methods which are generaUy known in the art. In particular, purified SCAP may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specificaUy bind SCAP. Antibodies to SCAP may also be generated using methods that are weU known in the art. Such antibodies may include, but are not Umited to, polyclonal, monoclonal, cbimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary. NeutraUzing antibodies (i.e., those which inhibit dimer formation) are generaUy preferred for therapeutic use. Single chain antibodies (e.g., from camels or Uamas) may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302). For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others maybe immunized by injection with SCAP or with any fragment or oUgopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not Umited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especiaUy preferable.
It is prefeπed that the oUgopeptides, peptides, or fragments used to induce antibodies to SCAP have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oUgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of SCAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the cHmeric molecule may be produced.
Monoclonal antibodies to SCAP may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not Umited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique (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; Cole, S.P. et al. (1984) Mol. CeU Biol. 62:109-120). In addition, techniques developed for the production of "chimeric antibodies," such as the spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce SCAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin Ubraries (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 the Uterature (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 SCAP may also be generated.
For example, such fragments include, but are not limited to, F(ab fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfi.de bridges of the F(ab^)2 fragments. Alternatively, Fab expression Ubraries maybe constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281).
Various 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 weU known in the art. Such immunoassays typicaUy involve the measurement of complex formation between SCAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering SCAP epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for SCAP. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of SCAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equiUbrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple SCAP epitopes, represents the average affinity, or avidity, of the antibodies for SCAP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular SCAP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the SCAP- antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 107 L/mole are prefeπed for use in immunopurification and similar procedures which ultimately require dissociation of SCAP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY). The liter and avidity of polyclonal antibody preparations may be further evaluated to determine the quaUty and suitabiUty of such preparations for certain downstream appUcations. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generaUy employed in procedures requiring precipitation of SCAP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quaUty and usage in various appUcations, are generaUy available (Catty, supra; CoUgan et al., supra).
In another embodiment of the invention, polynucleotides encoding SCAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, 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 SCAP. Such technology is weU known in the art, and antisense oUgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding SCAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).
In therapeutic use, any gene deUvery system suitable for introduction of the antisense sequences into appropriate target ceUs can be used. Antisense sequences can be deUvered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein (Slater, J.E. et al. (1998) J. AUergy Clrn. Immunol. 102:469-475; Scanlon, KJ. et al. (1995) 9:1288-1296). Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (MiUer, A.D. (1990) Blood 76:271; Ausubel et al., supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene deUvery mechanisms include Uposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J.J. (1995) Br. Med. BuU. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87:1308-1315; Morris, M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736). In another embodiment of the invention, polynucleotides encoding SCAP may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X- ϋnked 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. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) CeU 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, famiUal hypercholesterolemia, and hemophiUa resulting from Factor Vm or Factor C deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, I.M. and N. Somia (1997) Nature 389:239-242)), (n) express a conditionaUy lethal gene product (e.g., in the case of cancers which result from unregulated ceU proUferation), or (in) express a protein which affords protection against intraceUular parasites (e.g., against human retro viruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasϊliensis; and protozoan parasites such as Plasmodiumfalciparum and Trypanosoma cruzi). In the case where a genetic deficiency in SCAP expression or regulation causes disease, the expression of SCAP from an appropriate population of transduced ceUs may aUeviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in SCAP are treated by constructing mammaUan expression vectors encoding SCAP and introducing these vectors by mechanical means into SCAP-deficient ceUs. Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (n) ballistic gold particle deUvery, (in) Uposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of SCAP include, but are not Umited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCR PT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). SCAP maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (n) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Cuπ. Opin. Biotechnol. 9:451-456), commerciaUy available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PEMD; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (in) a tissue-specific promoter or the native promoter of the endogenous gene encoding SCAP from a normal individual.
CommerciaUy available Uposome transformation kits (e.g., the PERFECT LEPED
5 TRANSFECTION KIT, available from Invitrogen) aUow one with ordinary skiU in the art to deUver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A. J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845). The introduction of DNA to primary ceUs requires modification of these
LO standardized mammaUan transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to SCAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding SCAP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (in) a Rev-responsive
L5 element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commerciaUy available (Stratagene) and are based on pubUshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for
£0 receptors on the target ceUs or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. MiUer (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging ceU Unes producing high transducing efficiency retroviral supernatant") discloses
£5 a method for obtaining retrovirus packaging ceU Unes and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4+ T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716; Ranga, U. et al. (1998)
30 Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In an embodiment, an adenovirus-based gene therapy deUvery system is used to deUver polynucleotides encoding SCAP to ceUs which have one or more genetic abnormaUti.es with respect to the expression of SCAP. The construction and packaging of adenovirus-based vectors are weU known to those with ordinary skiU in the art. RepUcation defective adenoviras vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al* (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenoviras vectors for gene therapy"), hereby incorporated by reference. For 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).
In another embodiment, a herpes-based, gene therapy deUvery system is used to deUver polynucleotides encoding SCAP to target ceUs which have one or more genetic abnormaUties with respect to the expression of SCAP. The use of herpes simplex virus (HSV)-based vectors maybe especiaUy valuable for introducing SCAP to ceUs of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are weU known to those with ordinary skiU 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. (1999) Exp. Eye Res. 169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a ceU under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al. (1994; Dev. Biol. 163:152-161). The manipulation of cloned herpesviras sequences, the generation of recombinant virus foUowing the transfection of multiple plasmids containing different segments of the large herpesviras genomes, the growth and propagation of herpesviras, and the infection of ceUs with herpesviras are techniques weU known to those of ordinary skiU in the art. In another embodiment, an alphaviras (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encoding SCAP to target ceUs. The biology of the prototypic alphaviras, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Cuπ. Opin. Biotechnol. 9:464-469). During alphavirus RNA repUcation, a subgenomic RNA is generated that normaUy encodes the viral capsid proteins. This subgenomic RNA repUcates to higher levels than the fuU length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for SCAP into the alphaviras genome in place of the capsid-coding region results in the production of a large number of SCAP- coding RNAs and the synthesis of high levels of SCAP in vector transduced ceUs. While alphaviras infection is typicaUy associated with ceU lysis within a few days, the abiUty to estabUsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repUcation of alphaviruses can be altered to suit the needs of the gene therapy appUcation
5 (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses wiU aUow the introduction of SCAP into a variety of ceU types. The specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphaviras cDNA and RNA transfections, and performing alphaviras infections, are weU known to those with ordinary skill in the art.
10 OUgonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple heUx base-pairing methodology. Triple heUx pairing is useful because it causes inhibition of the abiUty of the double heUx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have
L5 been described in the Uterature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches, Futura PubUshing, Mt. Kisco NY, pp. 163-177). A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of
£0 RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding SCAP.
Specific ribozyme cleavage sites within any potential RNA target are initiaUy identified by
£5 scanning the target molecule for ribozyme cleavage sites, including the foUowing sequences: GUA, GUU, and GUC Once identified, short RNA sequences of between 15 and 20 ribonucleotides, conesponding 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 accessibility to hybridization with complementary
30 oUgonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing oUgonucleotides such as soUd phase phosphoramidite chemical synthesis. Alternatively, RNA molecules maybe generated by in vitro and in vivo transcription of DNA molecules encoding SCAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU Unes, ceUs, or tissues. RNA molecules may be modified to increase intraceUular 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 linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in aU of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding SCAP. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oUgonucleotides, antisense oUgonucleotides, triple heUx-forming oUgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased SCAP expression or activity, a compound which specificaUy inhibits expression of the polynucleotide encoding SCAP may be therapeuticaUy useful, and in the treatment of disorders associated with decreased SCAP expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding SCAP may be therapeuticaUy useful.
At least one, and up to a pluraUty, of 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, commerciaUy-available or proprietary Ubrary of naturaUy-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 Ubrary of chemical compounds created combinatoriaUy or randomly. A sample comprising a polynucleotide encoding SCAP is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabiUzed ceU, or an in vitro ceU-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding SCAP are assayed by any method commonly known in the art. TypicaUy, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding SCAP. The amount of hybridization maybe quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be caπied out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU line such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial Ubrary of oUgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Braice, T.W. et al. (2000) US. Patent No. 6,022,691).
Many methods for introducing vectors into ceUs or tissues are available and equaUy suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors maybe introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. DeUvery by transfection, by Uposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
Any of the therapeutic methods described above may be appUed to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys. An additional embodiment of the invention relates to the administration of a composition which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient. Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, Easton PA). Such compositions may consist of SCAP, antibodies to SCAP, and mimetics, agonists, antagonists, or inhibitors of SCAP.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, subϋngual, or rectal means.
Compositions for pulmonary administration may be prepared in Uquid or dry powder form. These compositions are generaUy aerosoUzed immediately prior to inhalation by the patient. In the case of smaU molecules (e.g. traditional low molecular weight organic drugs), aerosol deUvery of fast- acting formulations is weU-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary deUvery via the alveolar region of the lung have enabled the practical deUvery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary deUvery has the advantage of administration without needle injection, and obviates the need for potentiaUy toxic penetration enhancers. Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is weU within the capabiUty of those skilled in the art.
SpeciaUzed forms of compositions maybe prepared for direct intraceUular deUvery of macromolecules comprising SCAP or fragments thereof. For example, Uposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deUvery of the macromolecule. Alternatively, SCAP or a fragment thereof may be joined to a short cationic N- terminal portion from the HJV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572). For any compound, the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, 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 therapeuticaUy effective dose refers to that amount of active ingredient, for example SCAP or fragments thereof, antibodies of SCAP, and agonists, antagonists or inhibitors of SCAP, which ameUorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED50 (the dose therapeuticaUy effective in 50% of the population) or LD50 (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 LD5( ED50 ratio. Compositions which exhibit large therapeutic indices are prefeπed. The data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED50 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 wiU be determined by the practitioner, in Ught of factors related to the 5 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, drag combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or 10 biweekly depending on the half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, 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 generaUy available to practitioners in the art. Those skilled in the art wiU employ different formulations for nucleotides than for proteins or their 15 inhibitors. Similarly, deUvery of polynucleotides or polypeptides wiUbe specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
In another embodiment, antibodies which specificaUy bind SCAP may be used for the diagnosis of disorders characterized by expression of SCAP, or in assays to monitor patients being £0 treated with SCAP or agonists, antagonists, or inhibitors of SCAP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for SCAP include methods which utilize the antibody and a label to detect SCAP in human body fluids or in extracts of ceUs or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of £5 reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring SCAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of SCAP expression. Normal or standard values for SCAP expression are established by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to SCAP under ι0 conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of SCAP expressed in subject, control, and disease samples frombiopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease. In another embodiment of the invention, polynucleotides encoding SCAP maybe used for diagnostic purposes. The polynucleotides which may be used include oUgonucleotides, 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 SCAP may be coπelated with disease. The
5 diagnostic assay may be used to deteπnine absence, presence, and excess expression of SCAP, and to monitor regulation of SCAP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding SCAP or closely related molecules maybe used to identify nucleic acid sequences which encode SCAP. The specificity of the probe, whether it is made from a
LO highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampUfication wiU determine whether the probe identifies only naturaUy occurring sequences encoding SCAP, aUeUc variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the SCAP encoding sequences. The hybridization probes of the subject
L5 invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:38-74 or from genomic sequences including promoters, enhancers, and introns of the SCAP gene.
Means for producing specific hybridization probes for polynucleotides encoding SCAP include the cloning of polynucleotides encoding SCAP or SCAP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commerciaUy available, and may be used to
£0 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 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotides encoding SCAP maybe used for the diagnosis of disorders associated with
£5 expression of SCAP. Examples of such disorders include, but are not Umited to, a ceU motiUty disorder such as ankylosing spondyUtis, Chediak-Higashi syndrome, Duchenne and Becker muscular dystrophy, intrahepatic cholestasis, myocardial hyperplasia, cardiomyopathy, early onset peridontitis, cancers such as adenocarcinoma, ovarian carcinoma, and chronic myelogenous leukemia, and bacterial and helmmthic infections; a developmental disorder such as renal tubular acidosis, anemia, Oishing's
30 syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepitheUal dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a ceU 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 a cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcrnoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangUa, gastrointestinal tract, heart, kidney, Uver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, saUvary glands, skin, spleen, testis, thymus, thyroid, and uterus; a viral infection such as those caused by adenovirases (acute respiratory disease, pneumonia), arenaviruses (lymphocytic choriomeningitis), bunyaviruses (Hantaviras), coronaviruses (pneumonia, chronic bronchitis), hepadnavirases (hepatitis), herpesviruses (herpes simplex viras, variceUa-zoster viras, Epstein-Ban viras, cytomegalo viras), flaviviruses (yeUow fever), orthomyxovirases (influenza), papiUomaviruses (cancer), paramyxovirases (measles, mumps), picornoviruses (rhinoviras, poUoviras, coxsackie-virus), polyomaviruses (BK virus, JC virus), poxvirases (smaUpox), reovirus (Colorado tick fever), retroviruses (human immunodeficiency virus, human T lymphotropic viras), rhabdovirases (rabies), rotavirases (gastroenteritis), and togaviruses (encephaUtis, rubeUa); a heart and skeletal muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, Upid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy; and a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myeUtis and radicuUtis, viral central nervous system disease, a prion disease including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metaboUc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoretinal hemangioblastomatosis, encephalotiigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metaboUc, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder. Polynucleotides encoding SCAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microanays utiUzing fluids or tissues from patients to detect altered SCAP expression. Such qualitative or quantitative methods are weU known in the art.
In a particular aspect, polynucleotides encoding SCAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding SCAP maybe labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding SCAP in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of SCAP, a normal or standard profile for expression is estabUshed. This may be accompUshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding SCAP, under conditions suitable for hybridization or ampUfication. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner maybe compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabUsh the presence of a disorder.
Once the presence of a disorder is estabUshed and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays maybe used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development , of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oUgonucleotides designed from the sequences encoding SCAP may involve the use of PCR. These oUgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. OUgomers wiU preferably contain a fragment of a polynucleotide encoding SCAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding SCAP, and wiU be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oUgonucleotide primers derived from polynucleotides encoding SCAP 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. In SSCP, oUgonucleotide primers derived from polynucleotides encoding SCAP are used to ampUfy DNA using the polymerase chain reaction (PCR). The DNA maybe derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary stractures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oUgonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. AdditionaUy, 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. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
SNPs maybe used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insuUn-dependent diabetes meUitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drag, such as Ufe-threatening toxicity. For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drag isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-Upoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of SCAP include radiolabeling or biotinylating nucleotides, coampUfication of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples maybe accelerated by ranning the assay in a high-throughput format where the oUgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oUgonucleotides or longer fragments derived from any of the polynucleotides 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 maybe 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. In particular, 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. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile. In another embodiment, SCAP, fragments of SCAP, or antibodies specific for SCAP may be used as elements on a microaπay. The microaπay may be used to monitor or measure protein-protein interactions, drag-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type. A transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein). Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totaUty of transcripts or reverse transcripts of a particular tissue or ceU type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraUty of elements on a microaπay. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, ceU lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceU line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and precUnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring environmental compounds. AU 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). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. IdeaUy, a genome-wide measurement of expression provides the highest quaUty signature. Even genes whose expression is not altered by any tested compounds are important as weU, as the levels of expression of these genes are used to normaUze the rest of the expression data. The normaUzation procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (see, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and desirable in toxicological screening using toxicant signatures to include aU expressed gene sequences.
In an embodiment, the toxicity of a test compound can be 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 coπesponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in
5 an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or ceU type. The term proteome refers to the global pattern of protein expression in a particular tissue or ceU type. Each protein component of a proteome can be subjected individuaUy 0 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 ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first
L5 dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visuaUzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots
,0 from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry. The identity of the protein in a spot maybe determined by comparing its partial sequence, preferably of at least 5 contiguous
5 amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for SCAP to quantify the levels of SCAP expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microanay to the sample and detecting
3 the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103- lll; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by 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 paraUel with toxicant signatures at the transcript level. There is a poor coπelation between transcript and protein abundances for some proteins in some tissues (Anderson,
5 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 transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling maybe more reUable and informative in such cases. In another embodiment, the toxicity of a test compound is assessed by treating a biological
LO 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 coπesponding 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
L5 residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, 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
£0 by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays maybe prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA
£5 93:10614-10619; Baldeschweiler et al. (1995) PCT appUcation W095/251116; SUalon, D. et al. (1995) PCT appUcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of microaπays are weU known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A Practical Approach, Oxford University Press, London).
30 In another embodiment of the invention, nucleic acid sequences encoding SCAP may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence. Either coding or noncoding sequences maybe used, and in some instances, noncoding sequences maybe preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy cause undesired cross hybridization during chromosomal mapping. The 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 5 constractions, or single chromosome cDNA Ubraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acid sequences ma be used to develop genetic linkage maps, for example, which coπelate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. 10 Acad. Sci. USA 83:7353-7357).
Fluorescent in situ hybridization (FISH) maybe correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online MendeUan Inheritance in Man (OMTM) World Wide Web site. Correlation between the location of the gene encoding SCAP on a physical [5 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.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage 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, '.0 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 linkage 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 5 (Gatti, R.A. et al. (1988) Nature 336:577-580). The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, SCAP, its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries of compounds in any of a variety of drug 0 screening techniques. The fragment employed in such screening may be free in solution, affixed to a soUd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between SCAP and the agent being tested may be measured.
Another technique for drag screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT appUcation WO84/03564). In this method, large numbers of different smaU test compounds are synthesized on a soUd substrate. The test compounds are reacted with SCAP, or fragments thereof, and washed. Bound SCAP is then detected by methods weU known in the art. Purified SCAP can also be coated directly onto plates for use in the aforementioned drag screening techniques. Alternatively, non-neutraUzing antibodies can be used to capture the peptide and immobiUze it on a soUd support.
In another embodiment, one may use competitive drag screening assays in which neutraUzing antibodies capable of binding SCAP specificaUy compete with a test compound for binding SCAP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with SCAP.
In additional embodiments, the nucleotide sequences which encode SCAP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not Umited to, such properties as the triplet genetic code and specific base pair interactions. Without further elaboration, it is beUeved that one skilled in the art can, using the preceding description, utiUze the present invention to its fuUest extent. The foUowing preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of aU patents, appUcations, and pubUcations mentioned above and below, including U.S. Ser. No. 60/306,810, U.S. Ser. No. 60/308,338, U.S. Ser. No. 60/310,980, U.S. Ser. No. 60/313,098, U.S. Ser. No. 60/316,796, U.S. Ser. No. 60/317,899, U.S. Ser. No. 60/322,183, and U.S. Ser. No. 60/326,101, are hereby expressly incorporated by reference.
EXAMPLES I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA Ubraries described in the LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), 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.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase. For most Ubraries, 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). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the coπesponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA Ubraries were constructed with the UNTZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al, supra, ch. 5). 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. For most Ubraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs were Ugated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCP PT plasmid
(Stratagene), PSPORT1 plasmid (Invitrogen), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK- CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pESfCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Invitrogen. II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophiUzation, at 4°C Alternatively, plasmid DNA was ampUfied from host ceU lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were earned out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of ampUfied plasmid DNA was quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as foUows.
5 Sequencing reactions were processed using standard methods or Ingh-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 Biosciences or suppUed in ABI sequencing kits such as the
L0 ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppUed Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); 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.
5 Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VUI.
The polynucleotide sequences derived from Incyte cDNAs were vaUdated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and
0 programs based on BLAST, dynamic prograrnming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations 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; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae,
5 Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, ENCY, and TIGRFAM (Haft, D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is a probabiUstic approach which analyzes consensus ) primary stractures of gene families; see, for example, Eddy, S.R. (1996) Cuπ. 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. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples TV and V) were used to extend Incyte cDNA assemblages to fuU length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated
5 to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the full length translated polypeptide. FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM,
LO INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence aUgnment program
L5 (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, aU of
£0 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).
The programs described above for the assembly and analysis of fuU length polynucleotide and
£5 polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ED NO:38-74. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and ampUfication technologies are described in Table 4, column 2. IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative structural and cytoskeleton-associated proteins were initiaUy identified by running the
30 Genscan gene identification program against pubUc genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karlin (1998) Cuπ. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode structural and cytoskeleton-associated proteins, the encoded polypeptides were analyzed by querying against PFAM models for structural and cytoskeleton-associated proteins. Potential structural and cytoskeleton-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as structural and cytoskeleton-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubUc databases. Where necessary, the Genscan- predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct eπors 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. FuU 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 ILL Alternatively, fuU 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 HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic prograrnjming to integrate cDNA and genomic information, generating possible spUce variants that were subsequently confirmed, edited, or extended to create a fuU 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. For example, if an interval was present on a cDNA and two genomic sequences, then aU three intervals were considered to be equivalent. This process aUows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as weU as sequence variants. Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri pubUc databases. Incoπect exons predicted by Genscan were conected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary. "Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example HI were queried against pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example TV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. 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 SCAP Encoding Polynucleotides
The sequences which were used to assemble SEQ ED NO:38-74 were compared with sequences from the Incyte LERESEQ database and pubUc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ED NO:38-74 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from 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 aU sequences of that cluster, including its particular SEQ ED 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. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the pubUc, such as the NCBI "GeneMap'99" World Wide Web site
5 (http://www.ncbi.rum.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above. VII. Analysis of Polynucleotide Expression
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
LO from a particular ceU type or tissue have been bound (Sambrook, supra, ch. 7; Ausubel et al., supra, ch. 4).
Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LEFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer
L5 search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
£0
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is
£5 calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score. The product score represents a balance between fractional overlap and quaUty in a BLAST aUgnment. For example, a product score of 100 is produced only for 100% identity over the
10 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. Alternatively, polynucleotides encoding SCAP are analyzed with respect to the tissue sources from which they were derived. For example, some fuU length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example HI). Each cDNA sequence is derived from a cDNA Ubrary constructed from a human tissue. Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaUa, female; genitaUa, male; germ ceUs; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories. Similarly, each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding SCAP. VIII. Extension of SCAP Encoding Polynucleotides
FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using oUgonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hairpin stractures and primer-primer dimerizations was avoided.
Selected human cDNA Ubraries 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 weU known in the art. PCR was performed in 96-weU plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, ( EL^SO^ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the foUowing parameters for primer pair PCI A and PCI B: Step 1: 94 °C, 3 min; Step 2: 94 °C, 15 sec; Step 3: 60 °C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4 °C In the alternative, the parameters for primer pair T7 and SK+ were as foUows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C
The concentration of DNA in each weU was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE
5 and 0.5 μl of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan EL (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aUquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the
0 sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-weU plates, digested with CviJI cholera viras endonuclease (Molecular Biology Research, Madison WT), and sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels,
.5 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 Biosciences), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed ceUs were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-weU plates in LB/2x to carb Uquid media.
The ceUs were lysed, and DNA was ampUfied by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the foUowing 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
£5 PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries . were reampUfied using the same conditions as described above. Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppUed Biosystems). iO In like manner, full length polynucleotides are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with oUgonucleotides designed for such extension, and an appropriate genomic Ubrary. DX. Identification of Single Nucleotide Polymorphisms in SCAP Encoding Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ED NO:38-74 using the LB ESEQ database (Incyte Genomics). Sequences from the same gene were clustered together and assembled as described in Example JJJ, aUowing the identification of aU sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecaU enors by requiring a minimum Phred quaUty score of 15, and removed sequence aUgnment eπors and errors resulting from improper trimming of vector sequences, chimeras, and spUce variants. An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statisticaUy generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statisticaUy generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed dupUcates and SNPs found in immunoglobuUns or T-ceU receptors .
Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), aU African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), aU Mexican Hispanic. The Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. AUele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no aUeUc variance in this population were not further tested in the other three populations. X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO:38-74 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oUgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments. OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oUgomer, 250 μCi of [γ-32P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oUgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextranbead column (Amersham Biosciences). An aUquot containing 107 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgl 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 (Nytran Plus, Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaUzed using autoradiography or an alternative imaging means and compared.
XI. Microarrays
The linkage or synthesis of anay elements upon a microarray can be achieved utilizing photoUthography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and soUd with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested substrates include siUcon, siUca, glass sUdes, glass chips, and siUcon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines weU known to those of ordinary skiU in the art and may contain any appropriate number of elements (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).
FuU 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 weU known in the art such as LASERGENE software (DNASTAR). The anay elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection. After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each anay element. Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization. The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microaπay may be assessed. In one embodiment, microaπay preparation and usage is described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)+ RNA is purified using the oUgo-(dT) ceUulose method. Each poly(A)+ RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl oUgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control ρoly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 μl 5X SSC/0.2% SDS. Microarray Preparation
Sequences of the present invention are used to generate array elements. Each array element is ampUfied from bacterial ceUs containing vectors with cloned cDNA inserts. PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are ampUfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg. AmpUfied array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
Purified anay elements are immobilized on polymer-coated glass sUdes. Glass microscope sUdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments. Glass sUdes are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distiUed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated sUdes are cured in a 110°C oven. Anay elements are appUed to the coated glass substrate using a procedure described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 μl of the anay element DNA, at an average concentration of 100 ng/μl, is loaded into the open capiUary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of anay element sample per sUde. Microanays are UV-crossUnked using a STRATALINKER UV-crossUnker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distiUed water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2%
5 SDS and distiUed water as before. Hybridization
Hybridization reactions contain 9 μl of sample mixture consisting of 0.2 μg 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 aUquoted onto the microaπay surface and covered with
L0 an 1.8 cm2 coversUp. The anays are transfened to a waterproof chamber having a cavity just sUghtly larger than a microscope sUde. The chamber is kept at 100% humidity internaUy by the addition of 140 μl of 5X SSC in a corner of the chamber. The chamber containing the anays is incubated for about 6.5 hours at 60° C. The 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.
15 Detection
Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser Ught is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The sUde
20 containing the array is placed on a computer-controUed X-Y stage on the microscope and raster- scanned past the objective. The 1.8 cm x 1.8 cm anay used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted Ught is spUt, based on wavelength, into two photomultipUer tube detectors (PMT R1477,
25 Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the anay 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 anay is typicaUy scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
30 The sensitivity of the scans is typicaUy caUbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A specific location on the anay contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control ceUs), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentiaUy expressed, the caUbration is done by labeling samples of the caUbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
5 The output of the photomultipUer tube is digitized using a 12-bit RTT-835H analog-to-digital
(A D) conversion board (Analog Devices, Inc., Norwood MA) instaUed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and
LO measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value conesponding to the average intensity of the signal. The software used
L5 for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). Anay elements that exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentiaUy expressed using the GEMTOOLS program (Incyte Genomics). Expression
£0 For example, SEQ D NO:39 showed differential expression in brain cingulate from a patient with Alzheimer's disease compared to matched microscopicaUy normal tissue from the same donor as determined by microarray analysis. The expression of CSAP-2 was increased at least two-fold in cingulate tissue with Alzheimer's disease. Therefore, SEQ ED NO:39 can be used in diagnostic assays for neurological disorders, including Alzheimer's disease.
£5 For example, SEQ ED NO:43 and SEQ ED NO:44 showed differential expression in breast tumor ceU Unes versus normal breast epitheUal ceUs as determined by microaπay analysis. The expression of SEQ ID NO:43 and SEQ ID NO:44 was increased by at least two fold in breast tumor ceU Unes from later stages of tumor progression as compared to a primary breast epitheUal ceU line isolated from a normal donor, and a breast mammary gland ceU line isolated from a patient with
30 fibrocystic breast disease. Therefore, SEQ ID NO:43 and SEQ ED NO:44 can be used in diagnostic assays for breast cancer. UV treatment, an antitumor agent, triggers different ceU cycle regulatory pathways in breast tumor ceUs. One of the ceU Unes in which showed increased expression of SEQ ED NO:43 and SEQ ID NO:44, MDA-mb-231, is a breast tumor ceU line that forms poorly differentiated adenocarcinoma in nude mice and ALS treated BALB/c mice. When this ceU line was treated with 5 mJ/cm2 UV-C (254 nm) radiation for 24 hours, expression of SEQ ED NO:43 and SEQ ID NO:44 was decreased by at least two fold. Therefore, SEQ ID NO:43 and SEQ ID NO:44 can be used in studying the ceU cycle checkpoint control mechanisms involved in tumorigenesis.
5 SEQ ED NO:56 was differentiaUy expressed in human peripheral blood mononuclear ceUs
(PBMCs) treated with 10 ng/ml interleukin 4 (IL-4). Human PBMCs can be classified into discrete ceUular populations representing the major ceUular components of the immune system. PBMCs contain about 52% lymphocytes (12 % B lymphocytes, 40% T lymphocytes {25% CD4+ and 15% CD8+}), 20% NK ceUs, 25% monocytes, and 3% various ceUs that include dendritic ceUs and
10 progenitor ceUs. The proportions, as weU as the biology of these ceUular components tend to vary ' sUghtly between healthy individuals, depending on factors such as age, gender, past medical history, and genetic background. EL-4 is a pleiotropic cytokine produced by activated T ceUs, mast ceUs, and basophils. It was initiaUy identified as a B ceU differentiation factor (BCDF) and a B ceU stimulatory factor (BSF1). Subsequent to the molecular cloning and expression of both human and mouse IL-4,
15 numerous other functions have been ascribed to B cells and other hematopoietic and non- hematopoietic ceUs including endotheUal ceUs, etc. EL-4 exhibits anti-tumor effects both in vivo and in vitro. Recently, EL-4 was identified as an important regulator for the CD4+ subset (Thl-Uke vs. Th2- like) development. The biological effects of IL-4 are mediated by the binding of EL-4 to specific ceU surface receptors. The functional Hgh-affinity receptor for IL-4 consists of a Ugand-binding subunit
£0 (EL-4 R) and a second subunit (b chain) that can modulate the Ugand binding affinity of the receptor complex. In certain ceU types, the gamma chain of the IL-2 receptor complex is a functional b chain of the EL-4 receptor complex.
In one experiment, PBMCs were coUected from the blood of 6 healthy volunteer donors using standard gradient separation. The PBMCs from each donor were placed in culture for 2 hours in the
£5 presence or absence of recombinant EL-4. Treated PBMCs and untreated control PBMCs from the different donors were pooled according to their respective treatment. The expression of SEQ ED NO:56 was significantly increased by at least two-fold in the PBMCs treated with IL-4.
In another experiment, human PBMC ceUs were used to compare the anti-inflammatory action of cyclosporin A in conjunction with a mixed lymphocyte reaction (MLR). MLR is a
0 recognized model of T ceU activation and is relevant for investigating a number of syndromes where such activation takes place, including graft- versus-host disease and transplant rejection (host-versus- graft disease). Cyclosporin A (CsA) has been a drug of choice for the last 15 years for immunosuppression in organ transplants, GvHD in bone marrow transplants, and in some other inflammatory conditions. CsA forms complexes with cyclopHlin molecules that inhibit the cytoplasmic phosphatase calcineurin. Inhibition of the phosphatase activity of calcineurin prevents the downstream induction of transcription of genes such as EL-2, EL-3 , and EFN-γ. In the absence of these terminal mediators, T ceU activation is aborted and the development of the immune response is dramaticaUy impaired. In this experiment, the expression of SEQ ED NO:56 was significantly decreased by at least two-fold in PBMC ceUs treated with cyclosporin (1 μM), and MLR in conjunction with cyclosporin (1 μM), as compared to untreated ceUs.
Another group of experiments were designed to compare the expression of SEQ ID NO:56 in human lung tumor tissue to equivalent normal lung tissue. Lung cancers are divided into four histopathologicaUy distinct groups. Three groups (squamous ceU carcinoma, adenocarcinoma, and large ceU carcinoma) are classified as non-smaU ceU lung cancers (NSCLCs). The fourth group of cancer is referred to as smaU ceU lung cancer (SCLC). CoUectively, NSCLCs account for approximately 70% of cases while SCLCs account for approximately 18% of aU cases. Pair comparisons were performed in which normal lung tissue and lung tumor tissue from the same donor were examined. In four of the experiments, lung squamous ceU carcinoma tissue (Dn5793, Dn5792, DN5796and Dn5797) was compared to same-donor normal lung tissue; in another experiment, lung adenocarcinoma (Dn5795) was compared to same-donor normal lung tissue. In aU cases, the expression of SEQ ED NO:56 was decreased by at least two-fold in the tumor tissue.
SEQ ED NO:56 was also differentiaUy expressed in tissue from a human breast lobular carcinoma in situ (Dn4716) as compared to uninvolved breast tissue from the same donor. A variety of differences occur in the gene expression patterns between normal and cancerous breast tissue. Identifying downstream targets of altered genes in cancerous breast tissue may generate greater understanding of molecular pathways that lead to breast cancer. In this experiment, the expression of SEQ ID NO:56 was decreased by at least two-fold in the breast cancer tissue. SEQ ED NO:59 was differentiaUy expressed in normal brain tissue versus mild Alzheimer's diseased brain tissue based on microarray experimentation. Alzheimer's disease (AD) is a progressive dementia characterized neuropathologicaUy by the presence of amyloid β-peptide- containing plaques and neurofibriUary tangles in specific brain regions. In addition, neurons and synapses are lost and inflammatory responses are activated in microgUa and astrocytes. A cross- comparison experimental design was used to evaluate the expression of cDNAs from specific dissected regions of human brain (Dn3629 from a 68-year old female with mild AD in the posterior cingulate tissue), as compared to normal human brain tissue from equivalent regions (Dn3625 from a normal 61-year old female). The expression of SEQ ED NO:59 was increased at least two-fold in mild AD tissue from the posterior cingulate region of the brain.
These experiments indicate that SEQ ED NO:56 and SEQ ID NO:59 exhibited significant differential expression patterns using microaπay techniques, and further estabUsh the utiUty of SEQ ED NO:56 and SEQ ED NO:59 as diagnostic markers or as therapeutic targets for useful in a variety of conditions and diseases involving cytoskeleton-associated proteins.
For example, SEQ ED NO:61 showed differential expression in non-malignant mammary epitheUal ceUs versus various breast carcinoma ceU Unes as determined by microarray analysis. The non-maUgnant mammary epitheUal ceU line, MCF10A, was isolated from a 36-year-old female with fibrocystic breast disease. The breast carcinoma ceU Unes include BT20, a breast carcinoma ceU line derived in vitro from ceUs emigrating out of thin sUces of a tumor mass isolated from a 74-year-old female; MCF7, a breast adenocarcinoma ceU Une derived from the pleural effusion of a 69-year-old female; MDA-mb-231, a metastatic breast tumor ceU Une derived from the pleural effusion of a 51- year-old female with metastatic breast carcinoma; SkBR3, a breast adenocarcinoma ceU Une isolated from a maUgnant pleural effusion of a 43-year-old female; and T47D, a breast carcinoma ceU Une derived from a pleural effusion from a 54-year-old female with an infiltrating ductal carcinoma of the breast. AU ceU cultures were propagated in a chemicaUy-defined, serum-free medium, H14, according to the suppUer's recommendations and grown to 70-80% confluence prior to RNA isolation. The microaπay experiments showed that the expression of SEQ ED NO:61 was decreased by at least two fold in the five breast carcinoma lines relative to non-maUgnant mammary epitheUal ceUs. Therefore, SEQ ID NO:61 can be used as a diagnostic marker or as a potential therapeutic target for breast cancer.
SEQ ID NO:61 also showed differential expression in normal epitheUal cells versus prostate carcinoma ceU Unes as determined by microarray analysis. The normal epitheUal ceU Une, PZ-HPV-7, was derived from normal tissue from the peripheral zone of the prostate. The prostate carcinoma ceU Unes include LNCaP, a prostate carcinoma ceU Une isolated from a lymph node biopsy of a 50 year old male with metastatic prostate carcinoma; and PC-3, a prostate adenocarcinoma ceU Une isolated from a metastatic site in the bone of a 62 year old male with grade IV prostate adenocarcinoma. AU ceU Unes were cultured in a defined serum-free medium, TCH, according to the suppUer's recommendations and grown to 70-80% confluence prior to RNA isolation. The experiments showed that the expression of SEQ ID NO:61 was decreased by at least two fold in the prostate carcinoma lines relative to normal epitheUal ceUs. Therefore, SEQ ED NO:61 can be used as a diagnostic marker or as a potential therapeutic target for prostate cancer.
In addition, SEQ ED NO:61 showed differential expression in inflammatory responses as determined by microaπay analysis. The expression of SEQ ED NO:61 was increased by at least two fold in human umbilical vein endotheUal ceUs (HUVECs) treated with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF- ) relative to untreated HUVECs. Human umbiUcal vein endotheUal ceUs are primary ceUs derived from the endotheUum of the human umbiUcal vein and have been used as an experimental model for investigating the role of the endotheUum in human vascular biology. IFN-γ is a cytokine produced primarily by T-lymphocytes and natural killer ceUs. TNF-α is produced by activated lymphocytes, macrophages, and other white blood ceUs, and is known to activate endotheUal ceUs. Both EFN-γ and TNF-α are pleiotropic cytokines that play important roles in mediation of the inflammatory response through activation of multiple signal transduction pathways. Therefore, SEQ ED NO:61 can be used in diagnostic assays for inflammatory responses.
In another example, SEQ ED NO:62 showed region-specific gene expression in the human brain as determined by microaπay analysis. The expression of SEQ ID NO:62 was decreased by at least two fold in the midbrain segments and the occipital lobe (associative) in the neocortex relative to pooled brain tissues which were constituted from the major regions of the brain from two male brains; a 47 year old and a 48 year old. The tissues from the midbrain segments and the occipital lobe was isolated from a 47 year old male, the same 47 year old donor as in the pooled sample. Therefore, SEQ ED NO:62 can be used as a biomarker for human brains, specifically the midbrain and the occipital lobe region in the neocortex.
SEQ ED NO:67 showed differential expression associated with prostate cancer as determined by microarray analysis. The expression of SEQ ED NO:67 showed at least a two-fold decrease in prostate LNCaP and DU-145 carcinoma ceUs compared to prostate PrEC epitheUal ceUs. SEQ ED NO:67 also showed at least a two-fold decrease in expression in prostate LNCaP carcinoma ceUs compared to nontumorigenic prostate PZ-HPV-7 epitheUal ceUs. The expression of SEQ ED NO:67 showed at least a two-fold increase in prostate PC3 carcinoma ceUs compared to either prostate PrEC epitheUal ceUs or PZ-HPV-7 epitheUal ceUs. The LNCaP ceU Une was isolated from a lymph node biopsy of a 50-year old male with metastatic prostate carcinoma. The DU-145 ceU Une was isolated from a 69-year old male with widespread metastatic prostate carcionoma. The PC-3 ceU Une was isolated from a 62-year old male with grade TV prostate adenocarcinoma. The PrEC ceU Une is a prostate epitheUal ceU Une isolated from a normal donor. The PZ-HPV-7 ceU Une was derived from epitheUal ceUs cultured from normal tissue from the peripheral zone of the prostate.
SEQ ED NO:67 also showed differential expression associated with breast cancer as determined by microaπay analysis. The expression of SEQ ED NO:67 showed at least a two-fold decrease in BT-474, BT-20, Hs578T, and MDA-MB-435S breast carcinoma ceUs compared to human mammary epitheUal ceUs (HMEC). The expression of SEQ ED NO:67 also showed at least a two-fold decrease in MCF7, T-47D, BT-20, Sk-BR-3, MDA-MB-231, and MDA-MB-435S breast carcinoma ceUs compared to nontumorigenic MCF-10A breast mammary gland ceUs. SEQ ID NO:67 showed at least a two-fold increase in expression in MCF-10A, MDA-MB-468 and HMEC ceUs treated with epidermal growth factor compared to untreated ceUs. Epidermal growth factor is highly expressed in breast carcinoma ceUs. Epidermal growth factor promotes proUferation and differentiation of mesenchymal and epitheUal ceUs, angiogenesis, and tumor progression. It is a mitogen for fibroblasts, epitheUal and endotheUal ceUs. BT-474 is a breast ductal carcinoma ceU Une isolated from a soUd, invasive ductal carcinoma of the breast obtained from a 60-year old woman. BT-20 is a breast carcinoma ceU Une derived from the ceUs emigrating out of thin sUces of a tumor mass isolated from a 74-year old female. Hs578T is a breast ductal carcinoma ceU Une that was a isolated from a 74-year old female with breast carcinoma. MDA-MB-435S is spindle shaped strain that evolved from the parent line (435) as isolated from the pleural effusion of a 31-year old female with metastatic, ductal adenocarcinoma of the breast. MDA-MB-468 is a breast adeoncarcinoma ceU Une isolated from the pleural effusion of a 51 -year old female with metastatic adenocarcinoma of the breast. MCF7 is a nonmaUgnant breast adenocarcinoma ceU Une isolated from the pleural effusion of a 69-year old female. T-47D is a breast carcinoma ceU Une isolated from a pleural effusion obtained from a 54-year old female with an infiltrating ductal carcinoma of the breast. Sk-BR-3 is a breast adenocarcinoma ceU Une isolated from a maUgnant pleural effusion of a 43 -year old female. MDA-MB-231 is a breast tumor ceU Une isolated from the pleural effusion of a 51-year old female. MCF-10A is a breast mammary gland ceU Une that was isolated from a 36-year old woman with fibrocystic breast disease. HMEC is a primary breast epitheUal ceU Une isolated from a normal donor. Therefore, SEQ ID NO:67 can be used in disease staging and in diagnostic assays for ceU proUferative disorders, including prostate cancer and breast cancer. XII. Complementary Polynucleotides
Sequences complementary to the SCAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occuπing SCAP. Although use of oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with s aUer or with larger sequence fragments. Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of SCAP. To inhibit transcription, a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oUgonucleotide is designed to prevent ribosomal binding to the SCAP-encoding transcript. XIII. Expression of SCAP
Expression and purification of SCAP is achieved using bacterial or virus-based expression systems. For expression of SCAP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited 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 SCAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of SCAP in eukaryotic ceUs is achieved by infecting insect or mammaUan ceU Unes with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculo viras. The nonessential polyhedrin gene of baculoviras is replaced with cDNA encoding SCAP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculoviras is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculoviras (Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945).
In most expression systems, SCAP is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crade cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobiUzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). FoUowing purification, the GST moiety can be proteolyticaUy cleaved from SCAP at specificaUy engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commerciaUy available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). Purified SCAP obtained by these methods can be used directly in the assays shown in Examples XVJJ and XViπ, where appUcable. XIV. Functional Assays
SCAP function is assessed by expressing the sequences encoding SCAP at physiologicaUy elevated levels in mammaUan ceU 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 plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human ceU Une, for example, an endotheUal or hematopoietic ceU Une, using either Uposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected ceUs and is a reUable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Ught scatter and 90 degree side Ught scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
The influence of SCAP on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding SCAP and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG). Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding SCAP and other genes of interest can be analyzed by northern analysis or microarray techniques. XV. Production of SCAP Specific Antibodies
SCAP substantiaUy purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols. Alternatively, the SCAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art. Methods for selection of appropriate epitopes, such as those near the C-tenninus or in hydrophiUc regions are weU described in the art (Ausubel et al., supra, ch. 11).
TypicaUy, oUgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppUed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleii dobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oUgopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-SCAP activity by, for example, binding the peptide or SCAP to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. XVI. Purification of Naturally Occurring SCAP Using Specific Antibodies
NaturaUy occurring or recombinant SCAP is substantiaUy purified by immunoafrϊnity chromatography using antibodies specific for SCAP. An iiumunoafftnity column is constructed by covalently coupling anti-SCAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing SCAP are passed over the immunoafϊϊnity column, and the column is washed under conditions that aUow the preferential absorbance of SCAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/SCAP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as ) urea or thiocyanate ion), and SCAP is coUected.
XVII. Identification of Molecules Which Interact with SCAP
SCAP, or biologicaUy active fragments thereof, are labeled with 125I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539). Candidate molecules previously arrayed in the weUs of a multi-weU plate are incubated with the labeled SCAP, washed, and any weUs with labeled SCAP complex are assayed. Data obtained using different concentrations of SCAP are used to calculate values for the number, affinity, and association of SCAP with the candidate molecules.
Alternatively, molecules interacting with SCAP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
SCAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large Ubraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
XVIII. Demonstration of SCAP Activity
A microtubule motiUty assay for SCAP measures motor protein activity. In this assay, recombinant SCAP is immobilized onto a glass sUde or similar substrate. Taxol-stabiUzed bovine brain microtubules (commerciaUy available) in a solution containing ATP and cytosoUc extract are perfused onto the sUde. Movement of microtubules as driven by SCAP motor activity can be visuaUzed and quantified using video-enhanced Ught microscopy and image analysis techniques. SCAP activity is directly proportional to the frequency and velocity bf microtubule movement.
Alternatively, an assay for SCAP measures the formation of protein filaments in vitro. A solution of SCAP at a concentration greater than the "critical concentration" for polymer assembly is appUed 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 SCAP activity. In another alternative, SCAP activity is measured by the binding of SCAP to protein filaments. 35S-Met labeled SCAP sample is incubated with the appropriate filament protein (actin, tubulin, or intermediate filament protein) and complexed protein is coUected by immunoprecipitation using an antibody against the filament protein. The immunoprecipitate is then run out on SDS-PAGE and the amount of SCAP bound is measured by autoradiography.
Various modifications and variations of the described compositions, methods, and systems of the invention wiU be apparent to those skilled in the art without departing from the scope and spirit of the invention. It wiU be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drag discovery process, as weU as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly Umited to such specific embodiments. Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the foUowing claims and their equivalents. Table 1
Figure imgf000104_0001
Table 1
Figure imgf000105_0001
Table 2
- op.
Figure imgf000106_0001
Figure imgf000106_0002
Table 2
Figure imgf000107_0001
Table 2
Figure imgf000108_0002
Figure imgf000108_0001
Table 3
Figure imgf000109_0002
Figure imgf000109_0001
Table 3
Figure imgf000110_0001
Table 3
Figure imgf000111_0001
Table 3
Figure imgf000112_0001
Table 3
Figure imgf000113_0001
Figure imgf000113_0002
Table 3
Figure imgf000114_0001
Figure imgf000114_0003
Figure imgf000114_0002
Table 3
Figure imgf000115_0001
Table 3
Figure imgf000116_0001
Table 3
Figure imgf000117_0001
Table 3
Figure imgf000118_0001
Table 3
Figure imgf000119_0001
Table 3
Figure imgf000120_0001
Table 3
Figure imgf000121_0001
Table 3
Figure imgf000122_0001
Table 3
Figure imgf000123_0001
Table 4
Figure imgf000124_0001
Table 4
Figure imgf000125_0001
Table 4
Figure imgf000126_0001
Table 4
Figure imgf000127_0002
Figure imgf000127_0001
Table 4
Figure imgf000128_0001
Table 4
Figure imgf000129_0001
Table 4
Figure imgf000130_0001
Table 4
S3
Figure imgf000131_0001
Table 4
Figure imgf000132_0001
Table 5
Figure imgf000133_0001
Table 6
Figure imgf000134_0001
Table 6
Figure imgf000135_0001
Table 6
Figure imgf000136_0001
Table 6
Figure imgf000137_0001
Table 6
Figure imgf000138_0001
Table 6
Figure imgf000139_0001
Table 6
Figure imgf000140_0001
Table 7
Figure imgf000141_0001
Table 7
Figure imgf000142_0001
Table 7
Figure imgf000143_0001

Claims

What is claimed is:
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-2, SEQ ID NO:5-10, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19- 25, SEQ ID NO:28-29, SEQ ID NOS 1, and SEQ ID NO:33-35, c) a polypeptide comprising a naturally occurring amino acid sequence at least 97% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:3-4, d) a polypeptide comprising a naturally occurring amino acid sequence at least 93% identical to the amino acid sequence of SEQ ID NO: 12, e) a polypeptide comprising a naturally occurring amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO:26, f) a polypeptide comprising a naturally occurring amino acid sequence at least 92% identical to the amino acid sequence of SEQ ID NO: 18, g) a polypeptide comprising a naturally occurring amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:32, h) a polypeptide comprising a naturally occurring amino acid sequence at least 91 % identical to the amino acid sequence of SEQ ID NO:36, i) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, and j) an immunogenic fragment of a polypeptide having an a ino acid sequence selected from the group consistmg of SEQ ID NO: 1-37.
2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
3. An isolated polynucleotide encoding a polypeptide of claim 1.
4. An isolated polynucleotide encoding a polypeptide of claim 2.
5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:38-74.
6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim 6.
8. A transgenic organism comprising a recombinant polynucleotide of claim 6.
9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:l-37.
11. An isolated antibody which specifically binds to a polypeptide of claim 1.
12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consistmg of SEQ ID NO:38-74, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ LD NO:38-40, SEQ ID NO:42-60, and SEQ ID NO:62-74, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 96% identical to the polynucleotide sequence of SEQ ID NO:41, d) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 97% identical to the polynucleotide sequence of SEQ ID NO:61, e) a polynucleotide complementary to a polynucleotide of a), f) a polynucleotide complementary to a polynucleotide of b), g) a polynucleotide complementary to a polynucleotide of c), h) a polynucleotide complementary to a polynucleotide of d), and i) an RNA equivalent of a)-h).
13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: 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.
15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.
16. A method of detecting a target polynucleotide in a sample, said target polynucleotide ' having a sequence of a polynucleotide of claim 12, the method comprising: a) ampHfying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
19. A method for treating a disease or condition associated with decreased expression of functional SCAP, comprising administering to a patient in need of such treatment the composition of claim 17.
20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with decreased expression of functional SCAP, comprising administering to a patient in need of such treatment a composition of claim 21.
23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with overexpression of functional SCAP, comprising administering to a patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to the polypeptide of claim
1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the 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 of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, 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.
30. A diagnostic test for a condition or disease associated with the expression of SCAP in a biological sample, the method comprising: a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detectmg the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
31. The antibody of claim 11, wherein the antibody is: a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab')2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an acceptable excipient.
33. A method of diagnosing a condition or disease associated with the expression of SCAP in a subject, comprising administering to said subject an effective amount of the composition of claim 32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with the expression of SCAP in a subject, comprising administering to said subject an effective amount of the composition of claim 34.
36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim
11, the method comprising: a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:l-37, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from the animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37 in a sample, the method comprising: a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37 in the sample.
45. A method of purifying a polypeptide comprising an a ino acid sequence selected from the group consisting of SEQ ID NO:l-37 from a sample, the method comprising: a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-37.
46. A microarray wherein at least one element of the microaπay is a polynucleotide of claim
13.
47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising: a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.
48. An anay comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim 12.
49. An anay of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.
50. An anay of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.
51. An anay of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.
52. An array of claim 48, which is a microarray.
53. An anay of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.
54. An anay of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:l.
57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:2.
58. A polypeptide of claim 1, comprising the a ino acid sequence of SEQ ID NO:3.
59. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:4.
60. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:5.
61. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:6.
62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:7.
63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:8.
64. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:9.
65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:10.
66. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 11.
67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:12.
68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 13.
69. A polypeptide of claim 1, comprismg me amino acid sequence of SEQ ID NO: 14.
70. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:15.
71. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:16.
72. A polypeptide of claim 1, comprising the arnino acid sequence of SEQ ID NO:17.
73. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 18.
74. A polypeptide of claim 1, comprising the arnino acid sequence of SEQ ID NO:19.
75. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:20.
76. A polypeptide of claim 1, comprising the arnino acid sequence of SEQ ID NO:21.
77. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:22.
78. A polypeptide of claim 1, comprising the arnino acid sequence of SEQ ID NO:23.
79. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:24.
80. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:25.
81. A polypeptide of claim 1, comprising the arnino acid sequence of SEQ ID NO:26.
82. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:27.
83. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:28.
84. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:29.
85. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:30.
86. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:31.
87. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:32.
88. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:33.
89. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:34.
90. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:35.
91. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:36.
92. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:37.
93. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:38.
94. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:39.
95. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:40.
96. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:41.
97. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:42.
98. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:43.
99. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:44.
100. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:45.
101. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:46.
102. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:47.
103. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:48.
104. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:49.
105. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:50.
106. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:51.
107. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:52.
108. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:53.
109. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:54.
110. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:55.
111. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:56.
112. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:57.
113. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:58.
114. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ 3D NO:59.
115. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:60.
116. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:61.
117. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:62.
118. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:63.
119. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:64.
120. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:65.
121. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ LD NO:66.
122. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:67.
123. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:68.
124. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:69.
125. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:70.
126. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:71.
127. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:72.
128. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:73.
129. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ LD
NO:74.
PCT/US2002/022866 2001-07-20 2002-07-19 Structural and cytoskeleton-associated proteins WO2003008625A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002320621A AU2002320621A1 (en) 2001-07-20 2002-07-19 Structural and cytoskeleton-associated proteins

Applications Claiming Priority (16)

Application Number Priority Date Filing Date Title
US30681001P 2001-07-20 2001-07-20
US60/306,810 2001-07-20
US30833801P 2001-07-27 2001-07-27
US60/308,338 2001-07-27
US31098001P 2001-08-07 2001-08-07
US60/310,980 2001-08-07
US31309801P 2001-08-17 2001-08-17
US60/313,098 2001-08-17
US31679601P 2001-08-31 2001-08-31
US60/316,796 2001-08-31
US31789901P 2001-09-07 2001-09-07
US60/317,899 2001-09-07
US32218301P 2001-09-14 2001-09-14
US60/322,183 2001-09-14
US32610101P 2001-09-28 2001-09-28
US60/326,101 2001-09-28

Publications (2)

Publication Number Publication Date
WO2003008625A2 true WO2003008625A2 (en) 2003-01-30
WO2003008625A3 WO2003008625A3 (en) 2003-06-12

Family

ID=27575373

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/022866 WO2003008625A2 (en) 2001-07-20 2002-07-19 Structural and cytoskeleton-associated proteins

Country Status (2)

Country Link
AU (1) AU2002320621A1 (en)
WO (1) WO2003008625A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007060899A1 (en) * 2005-11-22 2007-05-31 National University Corporation Nagoya University Application of actin-binding protein to disease associated with cell motility
US11504390B2 (en) 2019-03-20 2022-11-22 Regeneron Pharmaceuticals, Inc. Treatment of increased lipid levels with sterol regulatory element binding transcription factor 1 (SREBF1) inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MESSER ET AL.: 'Molecular cloning and sequencing the chicken smooth muscle myosin regulatory light chain' FEBS LETTERS vol. 234, no. 1, 1988, pages 49 - 52, XP002961626 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007060899A1 (en) * 2005-11-22 2007-05-31 National University Corporation Nagoya University Application of actin-binding protein to disease associated with cell motility
US9206242B2 (en) 2005-11-22 2015-12-08 National University Corporation Nagoya University Application of actin-binding protein to disease associated with cell motility
US11504390B2 (en) 2019-03-20 2022-11-22 Regeneron Pharmaceuticals, Inc. Treatment of increased lipid levels with sterol regulatory element binding transcription factor 1 (SREBF1) inhibitors

Also Published As

Publication number Publication date
AU2002320621A1 (en) 2003-03-03
WO2003008625A3 (en) 2003-06-12

Similar Documents

Publication Publication Date Title
EP1402000A2 (en) Structural and cytoskeleton-associated proteins
WO2004048550A2 (en) Immune response associated proteins
WO2001085942A2 (en) Cytoskeleton-associated proteins
WO2002053719A2 (en) Cytoskeleton-associated proteins
WO2003063769A2 (en) Vesicle-associated proteins
US6962799B2 (en) Microtubule-associated proteins and tubulins
WO2002042330A2 (en) Cystoskeleton-associated proteins
WO2004048529A2 (en) Cell adhesion and extracellular matrix proteins
WO2003031595A2 (en) Molecules for disease detection and treatment
WO2004015396A2 (en) Cell adhesion and extracellular matrix proteins
CA2409392A1 (en) Intracellular signaling proteins
EP1294884A2 (en) Secretion and trafficking molecules
WO2003008625A2 (en) Structural and cytoskeleton-associated proteins
WO2002088322A2 (en) Cell adhesion and extracellular matrix proteins
WO2003094843A2 (en) Cell adhesion and extracellular matrix proteins
WO2004094623A2 (en) Cell adhesion and extracellular matrix proteins
WO2003051902A1 (en) Neurotransmission-associated proteins
EP1451212A2 (en) Structural and cytoskeleton-associated proteins
JP2004533227A (en) Cytoskeletal binding protein
WO2003027230A2 (en) Cell adhesion and extracellular matrix proteins
WO2004111202A2 (en) Neurotransmission-association proteins
EP1385954A2 (en) Proteins associated with cell growth, differentiation, and death
WO2003025129A2 (en) Neurotransmission-associated proteins
WO2002092759A2 (en) Molecules for disease detection and treatment
WO2002074913A2 (en) Nucleic acid-associated proteins

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP