EP1390410A1 - Molecules de detection et de traitement de maladies - Google Patents

Molecules de detection et de traitement de maladies

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Publication number
EP1390410A1
EP1390410A1 EP02739428A EP02739428A EP1390410A1 EP 1390410 A1 EP1390410 A1 EP 1390410A1 EP 02739428 A EP02739428 A EP 02739428A EP 02739428 A EP02739428 A EP 02739428A EP 1390410 A1 EP1390410 A1 EP 1390410A1
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European Patent Office
Prior art keywords
polynucleotide
seq
polypeptide
sequence
amino acid
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EP02739428A
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German (de)
English (en)
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EP1390410A4 (fr
Inventor
Tom Y. Tang
Henry Yue
Mariah R. Baughn
Brendan M. Duggan
Bridget A. Warren
Olga Bandman
Thomas W. Richardson
Neil Burford
Bharati Sanjanwala
Shanya D. Becha
Monique G. Yao
Junming Yang
Uyen K. Tran
April J.A. Hafalia
Jennifer A. Griffin
Anita Swarnakar
Vicki S. Elliott
Shirley A. Recipon
Farrah A. Khan
Ernestine A. Lee
Huibin Yue
Dyung Aina M. Lu
Narinder K. Chawla
Kavitha Thangavelu
Chandra S. Arvizu
Yuming Xu
Craig H. Ison
Jiaqi Huang
Li Ding
Cynthia D. Honchell
Mark L. Borowsky
Brooke M. Emerling
David P. Peterson
Yan Lu
Jayalaxmi Ramkumar
Patricia M. Mason
Yeganeh Zebarjadian
Yalda Azimzai
Laura L. Stuve
Laura L. Kamigaki
Ines Barroso
Sally Lee
Amy E. Kable
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Incyte Corp
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Incyte Genomics Inc
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Publication of EP1390410A1 publication Critical patent/EP1390410A1/fr
Publication of EP1390410A4 publication Critical patent/EP1390410A4/fr
Withdrawn legal-status Critical Current

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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This invention relates to nucleic acid and amino acid sequences of molecules for disease detection and treatment and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, autoiirimune/inflammatory, developmental, and neurological disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and arnino acid sequences of molecules for disease detection and treatment.
  • the human genome is comprised of thousands of genes, many encoding gene products that function in the maintenance and growth of the various cells and tissues in the body. Aberrant expression or mutations in these genes and their products is the cause of, or is associated with, a variety of human diseases such as cancer and other cell proliferative disorders. The identification of these genes and their products is the basis of an ever-expanding effort to find markers for early detection of diseases, and targets for their prevention and treatment.
  • Cancer represents a type of cell proliferative disorder that affects nearly every tissue in the body.
  • a wide variety of molecules, either aberrantly expressed or mutated, can be the cause of, or involved with, various cancers because tissue growth involves complex and ordered patterns of cell proliferation, cell differentiation, and apoptosis.
  • Cell proliferation must be regulated to maintain both the number of cells and their spatial organization. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals such as growth factors and other mitogens, and intracellular cues such as DNA damage or nutrient starvation.
  • Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors.
  • Cancer Aberrant expression or mutations in genes and their products may cause, or increase susceptibility to, a variety of human diseases such as cancer and other cell proliferative disorders.
  • the identification of these genes and their products is the basis of an ever-expanding effort to find markers for early detection of diseases and targets for their prevention and treatment.
  • cancer represents a type of cell proliferative disorder that affects nearly every tissue in the body.
  • the development of cancer, or oncogenesis is often correlated with the conversion of a normal gene into a cancer-causing gene, or oncogene, through abnormal expression or mutation.
  • Oncoproteins the products of oncogenes, include a variety of molecules that influence cell proliferation, such as growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins.
  • tumor-suppressor genes are involved in inMbiting cell proliferation. Mutations which reduce or abrogate the function of tumor-suppressor genes result in aberrant cell proliferation and cancer.
  • Mammalian peripheral blood comprises cells of the erythroid, myeloid, and lymphoid lineages.
  • Each lineage is derived from a pluripotent stem cell which, upon exposure to various molecules and other types of cells, differentiate into effector cells which migrate into the blood and other organs. These cells include red blood cells and platelets (erythroid), macrophages and granulocytes (myeloid), and T and B lymphocytes (lymphoid). Myeloid and lymphoid cells mediate irnmune responses to pathogens such as bacteria, parasites, and viruses.
  • cytokines soluble messenger molecules
  • Both hematopoietic cells and non- hematopoietic cells produce cytokines which stimulate the activation, differentiation and proliferation of T cells, B cells, macrophages, and granulocytes during an active irnmune response.
  • Cytokines bind to specific receptors expressed on cellular membranes and transduce a signal through the cell. Depending on the type of cytokine and the cell to which it binds, this signal initiates activation, differentiation, growth, and/or apoptosis.
  • T cells which respond to and produce a variety of cytokines, are divided into two major groups, CD4 + T helper (Th) cells, and CD8 + cytotoxic T lymphocytes (CTL). Irnmune responses are primarily regulated by CD4 + Th cells which fall into two subclasses based on the kinds of cytokines they secrete. Thl cells secrete primarily IL-2 and EFN- ⁇ ; regulate the responses of CTLs, B cells, and macrophages; and orchestrate the removal of intracellular pathogens. In contrast, Th2 cells secrete primarily IL-4 and IL-10 and promote the development of certain antibody responses such as IgGi, IgA, and IgE.
  • Th T helper
  • CTL cytotoxic T lymphocytes
  • Th2 cells remove extracellular pathogens, which include various bacteria and parasites.
  • IDM insulin-dependent diabetes mellitus
  • MS multiple sclerosis
  • RA rheumatoid artliritis
  • Crohn's disease a Thl response also predominates in acute allograft rejection, eradication of tumors, and unexplained recurrent abortions.
  • Th2 responses predominate in allergy and other atopic disorders, transplantation tolerance, chronic graft versus host disease (GVHD), and systemic autoimmune disease such as systemic lupus erythmatosus (Romangnani et al. (1997) Int. Arch. Allergy Immunol. 113:153-156). Genes affected by these molecules may reasonably be expected to be markers of irnmune cell development, function, and activity.
  • Tumor necrosis factor (TNF) ⁇ is a pleiotropic cytokine that mediates immune regulation and inflammatory responses.
  • TNF- ⁇ -related cytokines generate partially overlapping cellular responses, including differentiation, proliferation, nuclear factor- ⁇ B (NF-kB) activation, and cell death, by triggering the aggregation of receptor monomers (Smith, CA. et al. (1994) Cell 76:959-962).
  • the cellular responses triggered by TNF- ⁇ are initiated through its interaction with distinct cell surface receptors (TNFRs).
  • TNFRs distinct cell surface receptors
  • TNF- ⁇ suppresses the incorporation of [ 3 Hjprolrne into both collagenase-digestible proteins (CDP) and noncollagenous proteins (NCP). Such suppression by TNF- ⁇ is not observed in confluent bovine aortic endothelial cells and human fibroblastic IMR-90 cells. TNF- ⁇ decreases the relative proportion of collagen types IN and V suggesting that T ⁇ F- ⁇ modulates collagen synthesis by SMCs depending on their cell density and therefore may modify formation of atherosclerotic lesions (Hiraga, S. et al. (2000) Life Sci. 66:235-244).
  • HUNECs human umbilical vein endothelial cells
  • D ⁇ A-based arrays can provide an efficient, high-throughput method to examine gene expression and genetic variability.
  • S ⁇ Ps or single nucleotide polymorphisms
  • D ⁇ A-based arrays can dramatically accelerate the discovery of SNPs in hundreds and even thousands of genes.
  • SNP genotyping in which DNA samples from individuals or populations are assayed for the presence of selected SNPs.
  • DNA-based array technology is especially important for the rapid analysis of global gene expression patterns.
  • genetic predisposition, disease, or therapeutic treatment may directly or indirectly affect the expression of a large number of genes in a given tissue.
  • a profile generated from an individual or population affected with a certain disease or undergoing a particular therapy may be compared with a profile generated from a control individual or population.
  • Such analysis does not require knowledge of gene function, as the expression profiles, can be subjected to mathematical analyses which simply treat each gene as a marker.
  • gene expression profiles may help dissect biological pathways by identifying all the genes expressed, for example, at a certain developmental stage, in a particular tissue, or in response to disease or treatment. (See, for example, Lander, E.S. et al. (1996) Science 274:536- 539.)
  • DC Dendritic cells
  • APC antigen presenting cells
  • DC differentiate into separate subsets of mature immune cells that sustain and regulate immune responses following initial contact with antigen.
  • DC subsets include those that preferentially induce particular T helper 1 (Thl) or T helper 2 (Th2) responses and those that regulate B cell responses.
  • DC are being used with increasing frequency to manipulate immune responses, either to downregulate aberrant autoimmune response or to enhance vaccination or tumor-specific response.
  • DC are functionally specialized in correlation with their particular differentiation state.
  • CD34+ myeloid cells found in the bone marrow mature in response to signals into CD 14+ CDllc+ monocytes.
  • An innate or antigen non-specific response takes place initially when monocytes circulate to nonlymphoid tissues and respond to lipopolysaccharide (LPS), a bacterially-derived mitogen, and viruses.
  • LPS lipopolysaccharide
  • Such direct encounters with antigen cause secretion of pro-iriflammatory cytokines that attract and regulate natural killer cells, macrophages, and eosinophils in the first line of defense against invading pathogens.
  • Monocytes then mature into DC, which efficiently capture antigen through endocytosis and antigen-receptor uptake.
  • Antigen processing and presentation trigger activation and differentiation into mature DC that express MHC class II molecules on the cell surface and efficiently activate T-cells, initiating antigen-specific T-cell and B-cell responses.
  • T-cells activate DC through CD40 ligand - CD40 interactions, which stimulate expression of the costimulatory molecules CD80 and CD86, the latter most potent in amptifying T-cell responses.
  • DC interaction via CD40 with T cells also stimulates the production of inflammatory cytokines such as TNF alpha and IL-1.
  • Engagement of RANK, a member of the TNF receptor family by its ligand, TRANCE which is expressed on activated T cells, enhances the survival of DC through inhibition of apoptosis, thereby enhancing T cell activation.
  • the maturation and differentiation of monocytes into matare DC links the antigen non-specific innate immune response to the antigen-specific adaptive immune response.
  • Certain genes are known to be associated with diseases because of their chromosomal location, such as the genes in the myotonic dystrophy (DM) regions of mouse and human.
  • the mutation underlying DM has been localized to a gene encoding the DM-kinase protein, but another active gene, DMR-N9, is in close proximity to the DM-kinase gene (Jansen, G. et al. (1992) Nat. Genet. 1:261-266).
  • DMR-N9 encodes a 650 arnino acid protein that contains WD repeats, motifs found in cell signaling proteins.
  • DMR-N9 is expressed in all neural tissues and in the testis, suggesting a role for DMR-N9 in the manifestation of mental and testicular symptoms in severe cases of DM (Jansen, G. et al. (1995) Hum. Mol. Genet. 4:843-852).
  • genes are identified based upon their expression patterns or association with disease syndromes. For example, autoantibodies to subcellular organelles are found in patients with systemic rheumatic diseases.
  • a recently identified protein, golgin-67 belongs to a family of Golgi autoantigens having alpha-helical coiled-coil domains (Eystathioy, T. et al. (2000) J. Autoimmun. 14:179-187).
  • the Stac gene was identified as a brain specific, developmentally regulated gene.
  • the Stac protein contains an SH3 domain, and is thought to be involved in neuron-specific signal transduction (Suzuki, H. et al. (1996) Biochem. Biophys. Res. Commun.
  • Intracellular Signaling Cell-cell communication is essential for the growth, development, and survival of multicellular organisms. Cells communicate by sending and receiving molecular signals.
  • An example of a molecular signal is a growth factor, which binds and activates a specific transmembrane receptor on the surface of a target cell. The activated receptor transduces the signal intracellularly, thus initiating a cascade of biochemical reactions that ultimately affect gene transcription and cell cycle progression in the target cell.
  • Intracellular signaling is the process by which cells respond to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.) through a cascade of biochemical reactions that begins with the binding of a signaling molecule to a cell membrane receptor and ends with the activation of an intracellular target molecule.
  • Intermediate steps in the process involve the activation of various cytoplasmic proteins by phosphorylation via protein kinases, and their deactivation by protein phosphatases, and the eventual translocation of some of these activated proteins to the cell nucleus where the transcription of specific genes is triggered.
  • the intracellular signaling process regulates all types of cell functions including cell proliferation, cell differentiation, and gene transcription, and involves a diversity of molecules including protein kinases and phosphatases, and second messenger molecules such as cyclic nucleotides, calcium-calmodulin, inositol, and various mitogens that regulate protein phosphorylation.
  • Cells also respond to changing conditions by switching off signals. Many signal transduction proteins are short-lived and rapidly targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Cells also maintain mechanisms to monitor changes in the concentration of denatured or unfolded proteins in membrane-bound extracytoplasmic compartments, including a transmembrane receptor that monitors the concentration of available chaperone molecules in the endoplasmic reticulum and transmits a signal to the cytosol to activate the transcription of nuclear genes encoding chaperones in the endoplasmic reticulum. Certain proteins in intracellular signaling pathways serve to link or cluster other proteins involved in the signaling cascade. These proteins are referred to as scaffold, anchoring, or adaptor proteins.
  • Protein kinases and phosphatases play a key role in the intracellular signaling process by conttolling the phosphorylation and activation of various signaling proteins.
  • the high energy phosphate for this reaction is generally transferred from the adenosine triphosphate molecule (ATP) to a particular protein by a protein kinase and removed from that protein by a protein phosphatase.
  • ATP adenosine triphosphate molecule
  • Protein kinases are roughly divided into two groups: those that phosphorylate serine or threonine residues (serine/t ireonine kinases, STK) and those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK).
  • a few protein kinases have dual specificity for serme/lhreonine and tyrosine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family (Hardie, G. and S. Hanks (1995) The Protein Kinase Facts Books, Vol 1:7-20, Academic Press, San Diego, CA).
  • STKs include the second messenger dependent protein kinases such as the cyclic- AMP dependent protein kinases (PKA), involved in mediating hormone-induced cellular responses; calcium-calmodulin (CaM) dependent protein kinases, involved in regulation of smooth muscle contraction, glycogen breakdown, and neurotransmission; and the mitogen-activated protein kinases (MAP kinases) which mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades.
  • PKA cyclic- AMP dependent protein kinases
  • CaM calcium-calmodulin dependent protein kinases
  • MAP kinases mitogen-activated protein kinases
  • PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs.
  • Transmembrane PTKs are receptors for most growth factors.
  • Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors.
  • Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes. Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells in which their activation was no longer subject to normal cellular controls.
  • HPK histidine protein kinase family
  • HPKs bear little homology with mammalian STKs or PTKs but have distinctive sequence motifs of their own (Davie, J.R. et al. (1995) J. Biol. Chem. 270:19861-19867).
  • a histidine residue in the N-terminal half of the molecule (region I) is an autophosphorylation site.
  • Three additional motifs located in the C-terminal half of the molecule include an invariant asparagine residue in region II and two glycine-rich loops characteristic of nucleotide binding domains in regions m and IV. Recently a branched chain alpha-ketoacid dehydrogenase kinase has been found with characteristics of HPK in rat (Davie et al., supra).
  • the two principal categories of protein phosphatases are the protein (serme/threonine) phosphatases (PPs) and the protein tyrosine phosphatases (PTPs).
  • PPs dephosphorylate phosphoser e/tlireonrne residues and are important regulators of many cAMP-mediated hormone responses (Cohen, P. (1989) Aram. Rev. Biochem. 58:453-508).
  • PTPs reverse the effects of protein tyrosine kinases and play a significant role in cell cycle and cell signaling processes (Charbonrneau and Tonks, supra).
  • PTPs may prevent or reverse cell transformation and the growth of various cancers by controlling the levels of tyrosine phosphorylation in cells. This hypothesis is supported by studies showing that overexpression of PTPs can suppress transformation in cells, and that specific inhibition of PTPs can enhance cell transformation (Charbonneau and Tonks, supra). Phospholipid and Inositol-phosphate Signaling
  • Inositol phospholipids are involved in an intracellular signaling pathway that begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane. This leads to the phosphorylation of phosphatidylinositol (PI) residues on the inner side of the plasma membrane to the biphosphate state (PIP 2 ) by inositol kinases. Simultaneously, the G-protein linked receptor binding stimulates a tximeric G-protein which in turn activates a phosphoinositide-specific phospholipase C- ⁇ .
  • PI phosphatidylinositol
  • Phospholipase C- ⁇ then cleaves PIP 2 into two products, inositol triphosphate (IP 3 ) and diacylglycerol. These two products act as mediators for separate signaling events.
  • IP 3 inositol triphosphate
  • UP 3 diffuses through the plasma membrane to induce calcium release from the endoplasmic reticulum (ER), while diacylglycerol remains in the membrane and helps activate protein kinase C, a serme-mreonine kinase that phosphorylates selected proteins in the target cell.
  • the calcium response initiated by IP 3 is terminated by the dephosphorylation of JP 3 by specific inositol phosphatases.
  • Cellular responses that are mediated by this pathway are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation.
  • Inositol-phosphate signaling controls tubby, a membrane bound transcriptional regulator that serves as an intracellular messenger of G q -coupled receptors (Santagata et al. (2001) Science 292:2041-2050).
  • Members of the tabby family contain a C-terminal tabby domain of about 260 amino acids that binds to double-stranded DNA and an N-terminal transcriptional activation domain.
  • Tubby binds to phosphatidylinositol 4,5-bisphosphate, which localizes tubby to the plasma membrane.
  • Activation of the G-protein ⁇ q leads to activation of phospholipase C- ⁇ and hydrolysis of phosphoinositide.
  • Loss of phosphatidylinositol 4,5-bisphosphate causes tubby to dissociate from the plasma membrane and to translocate to the nucleus where tabby regulates transcription of its target genes. Defects in the tubby gene are associated with obesity, retinal degeneration, and hearing loss (Boggon, TJ. et al. (1999) Science 286:2119-2125). Cyclic Nucleotide Signaling
  • Cyclic nucleotides function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters.
  • cyclic- AMP dependent protein kinases PKA
  • PKA cyclic- AMP dependent protein kinases
  • Visual excitation and the phototransmission of light signals in the eye is controlled by cyclic-GMP regulated, Ca 2+ -specific channels. Because of the importance of cellular levels of cyclic nucleotides in mediating these various responses, regulating the synthesis and breakdown of cyclic nucleotides is an important matter.
  • adenylyl cyclase which synthesizes cAMP from AMP, is activated to increase cAMP levels in muscle by binding of adrenaline to ⁇ -adrenergic receptors, while activation of guanylate cyclase and increased cGMP levels in photoreceptors leads to reopening of the Ca 2+ -specific channels and recovery ofthe dark state in the eye.
  • transmembrane isoforms of mammalian adenylyl cyclase as well as a soluble form preferentially expressed in testis.
  • Soluble adenylyl cyclase contains a P-loop, or nucleotide binding domain, and may be involved in male fertility (Buck, J. et al. (1999) Proc. Natl. Acad. Sci. USA 96:79-84).
  • PDEs hydrolysis of cyclic nucleotides by cAMP and cGMP-specific phosphodiesterases (PDEs) produces the opposite of these and other effects mediated by increased cyclic nucleotide levels.
  • PDEs appear to be particularly important in the regulation of cyclic nucleotides, considering the diversity found in this family of proteins.
  • PDE1-7 At least seven families of mammalian PDEs (PDE1-7) have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to irhibitory drugs (Beavo, J.A. (1995) Physiol. Rev. 75:725-748).
  • PDE Mubitors have been found to be particularly useful in treating various clinical disorders.
  • Rolipram a specific irihibitor of PDE4, has been used in the treatment of depression, and similar inhibitors are undergoing evaluation as anti-inflamrnatory agents. Ifreophylline is a nonspecific PDE irihibitor used in the treatment of bronchial asthma and other respiratory diseases (Banner, K.H. and C.P. Page (1995) Eur. Respir. J. 8:996-1000).
  • Calcium Signaling Molecules Ca + is another second messenger molecule that is even more widely used as an intracellular mediator than cAMP. Ca 2+ can enter the cytosol by two pathways, in response to extracellular signals.
  • Ca 2+ acts primarily in nerve signal transduction where Ca 2+ enters a nerve terminal through a voltage-gated Ca 2+ channel.
  • the second is a more ubiquitous pathway in which Ca 2+ is released from the ER into the cytosol in response to binding of an extracellular signaling molecule to a receptor.
  • Ca 2+ directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways.
  • Ca 2+ also binds to specific Ca 2+ -binding proteins (CBPs) such as callrnodulin (CaM) which then activate multiple target proteins in the cell including enzymes, membrane transport pumps, and ion channels.
  • CBPs Ca 2+ -binding proteins
  • CaM callrnodulin
  • CaM interactions are involved in a multitude of cellular processes including, but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation (Celio, M.R. et al. (1996) Guidebook to Calcium-binding Proteins, Oxford University Press, Oxford, UK, pp. 15-20).
  • Some Ca 2+ binding proteins are characterized by the presence of one or more EF-hand Ca 2+ binding motifs, which are comprised of 12 arnino acids flanked by ⁇ -helices (Celio, supra).
  • the regulation of CBPs has implications for the control of a variety of disorders.
  • Calcineurin a CaM-regulated protein phosphatase
  • the level of CaM is increased several-fold in tumors and tumor-derived cell lines for various types of cancer (Rasmussen, CD. and A.R. Means (1989) Trends Neurosci. 12:433-438).
  • the annexins are a family of calcium-binding proteins that associate with the cell membrane (Towle, CA. and B.V.
  • Annexins reversiblybind to negatively charged phospholipids (phosphatidylcholine and phosphatidylserine) in a calcium dependent manner.
  • Annexins participate in various processes pertahring to signal transduction at the plasma membrane, including membrane-cytoskeleton interactions, phospholipase irihibition, anticoagulation, and membrane fusion.
  • Annexins contain four to eight repeated segments of about 60 residues. Each repeat folds into five alpha helices wound into a right-handed superhelix.
  • G-proteins are critical mediators of signal transduction between a particular class of extracellular receptors, the G-protein coupled receptors (GPCRs), and intracellular second messengers such as cAMP and Ca 2+ .
  • G-proteins are linked to the cytosolic side of a GPCR such that activation of the GPCR by ligand binding stimulates binding of the G-protein to GTP, inducing an "active" state in the G-protein. In the active state, the G-protein acts as a signal to trigger other events in the cell such as the increase of cAMP levels or the release of Ca 2+ into the, cytosol from the ER, which, in tarn, regulate phosphorylation and activation of other intracellular proteins.
  • G-proteins The superfamily of G-proteins consists of several families which maybe grouped as translational factors, heterotrimeric G-proteins involved in transmembrane signaling processes, and low molecular weight (LMW) G-proteins including the proto- oncogene Ras proteins and products of rab, rap, rho, rac, smg21, smg25, YPT, SEC4, and ARF genes, and tabulins (Kaziro, Y. et al. (1991) Annu. Rev. Biochem. 60:349-400). In all cases, the GTPase activity is regulated through interactions with other proteins.
  • LMW low molecular weight
  • Heterotrimeric G-proteins are composed of 3 subunits, , ⁇ , and ⁇ , which in their inactive conformation associate as a trimer at the inner face of the plasma membrane.
  • G binds GDP or GTP and contains the GTPase activity.
  • the ⁇ complex enhances binding of G ⁇ to a receptor.
  • G ⁇ is necessary for the folding and activity of G ⁇ (Neer, E.J. et al. (1994) Nature 371:297-300). Multiple homologs of each subunit have been identified in mammalian tissues, and different combinations of subunits have specific functions and tissue specificities (Spiegel, A.M. (1997) J. Inher. Metab. Dis. 20:113-121).
  • the alpha subunits of heterotrimeric G-proteins can be divided into four distinct classes.
  • the ⁇ -s class is sensitive to ADP-ribosylationby pertussis toxin which uncouples the receptor: G-protein interaction. This uncoupling blocks signal transduction to receptors that decrease cAMP levels which normally regulate ion channels and activate phospholipases.
  • the inhibitory ⁇ -I class is also susceptible to modification by pertussis toxin which prevents ⁇ -I from lowering cAMP levels.
  • ⁇ -q which activates phospholipase C
  • ⁇ -12 which has sequence homology with the Drosophila gene concertina and may contribute to the regulation of embryomc development (Simon, M.I. (1991) Science 252:802-808).
  • the G ⁇ subunit also called transducin
  • the activity of both subunits maybe regulated by other proteins such as calmodulin and phosducin or the neural protein GAP 43 (Clapham, D. and E.
  • LISl a subunit of the human platelet activating factor acetylhydrolase, cause Miller-Dieker lissencephaly.
  • RACKl binds activated protein kinase C
  • RbAp48 binds retinoblastoma protein.
  • CstF is required for polyadenylation of mammalian pre-mRNA in vitro and associates with subunits of cleavage-stimulating factor. Defects in the regulation of ⁇ -cateruh contribute to the neoplastic transformation of human cells.
  • the WD40 repeats of the human F-box protein bTrCP mediate binding to ⁇ -catenin, thus regulating the targeted degradation of ⁇ -catenin by ubiquitin ligase (Neer, supra; Hart, M. et al. (1999) Curr. Biol. 9:207-210).
  • the ⁇ subunit primary structures are more variable than those ofthe ⁇ subunits. They are often post-translationally modified by isoprenylation and carboxyl-methylation of a cysteine residue four amino acids from the C-terminus; this appears to be necessary for the interaction of the ⁇ subunit with the membrane and with other G-proteins.
  • the ⁇ subunit has been shown to modulate the activity of isoforms of adenylyl cyclase, phospholipase C, and some ion channels. It is involved in receptor phosphorylation via specific kinases, and has been implicated in the p21ras- dependent activation of the MAP kinase cascade and the recognition of specific receptors by G- protei ⁇ s (Clapham and Neer, supra).
  • G-proteins interact with a variety of effectors including adenylyl cyclase (Clapham and Neer, supra).
  • the signaling pathway mediated by cAMP is mitogenic in hormone-dependent endocrine tissues such as adrenal cortex, thyroid, ovary, pituitary, and testes. Cancers in these tissues have been related to a mutationally activated form of a G ⁇ s known as the gsp (Gs protein) oncogene
  • phosducin a retinal phosphoprotein, which forms a specific complex with retinal G ⁇ and G ⁇ (G ⁇ ) and modulates the ability of G ⁇ to interact with retinal G ⁇ (Clapham and Neer, supra).
  • Irregularities in the G-protein signaling cascade may result in abnormal activation of leukocytes and lymphocytes, leading to the tissue damage and destruction seen in many inflammatory and autoimmune diseases such as rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus, and thyroiditis.
  • Abnormal cell proliferation, including cyclic AMP stimulation of brain, thyroid, adrenal, and gonadal tissue proliferation is regulated by G proteins. Mutations in G ⁇ subunits have been found in growth-hormone-secreting pituitary somatotroph tumors, hyperfunctioning thyroid adenomas, and ovarian and adrenal neoplasms (Meij, J.T.A. (1996) Mol. Cell Biochem. 157:31-38; Aussel, C. et al. (1988) J. Immunol. 140:215-220).
  • LMW G-proteins are GTPases which regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. They consist of single polypeptides which, like the alpha subunit of the heterotrimeric G-proteins, are able to bind to and hydrolyze GTP, thus cycling between an inactive and an active state. LMW G-proteins respond to extracellular signals from receptors and activating proteins by transducing mitogenic signals involved in various cell functions. The binding and hydrolysis of GTP regulates the response of LMW G-proteins and acts as an energy source during this process (Bokoch, G.M. and C.J. Der (1993) FASEB J. 7:750-759).
  • At least sixty members ofthe LMW G-profein superfamily have been identified and are currently grouped into the ras, rho, arf, sari, ran, and rab subfamilies.
  • Activated ras genes were initially found in human cancers, and subsequent studies corirfirmed that ras function is critical in determining whether cells continue to grow or become differentiated.
  • Rasl and Ras2 proteins stimulate adenylate cyclase (Kaziro, supra), affecting a broad array of cellular processes. Stimulation of cell surface receptors activates Ras which, in turn, activates cytoplas ic kinases.
  • Rho G-proteins control signal transduction pathways that link growth factor receptors to actin polymerization, which is necessary for normal cellular growth and division.
  • rab, arf, and sari families of proteins control the translocation of vesicles to and from membranes for protein processing, localization, and secretion.
  • Vesicle- and target- specific identifiers bind to each other and dock the vesicle to the acceptor membrane.
  • the budding process is regulated by the closely related ADP ribosylation factors (ARFs) and SAR proteins, while rab proteins allow assembly of SNARE complexes and may play a role in removal of defective complexes (Rothrman, J. and F. Wieland (1996) Science 272:227-234).
  • Ran G-proteins are located in the nucleus of cells and have a key role in nuclear protein import, the control of DNA synthesis, and cell-cycle progression (Hall, A. (1990) Science 249:635-640; Barbacid, M. (1987) Annu. Rev.
  • Rab proteins have a highly variable airiino terminus containing membrane-specific signal information and a prenylated carboxy terminus which determines the target membrane to which the Rab proteins anchor. More than 30 Rab proteins have been identified in a variety of species, and each has a characteristic intracellular location and distinct transport function.
  • Rabl and Rab2 are important in ER-to-Golgi transport; Rab3 transports secretory vesicles to the extracellular membrane; Rab5 is localized to endosomes and regulates the fusion of early endosomes into late endosomes; Rab6 is specific to the Golgi apparatus and regulates intra-Golgi transport events; Rab7 and Rab9 stimulate the fusion of late endosomes and Golgi vesicles with lysosomes, respectively; and Rab 10 mediates vesicle fusion from the medial Golgi to the trans Golgi. Mutant forms of Rab proteins are able to block protein transport along a given pathway or alter the sizes of entire organelles.
  • Rabs play key regulatory roles in membrane ttafficki ⁇ g (Schin ⁇ m ⁇ ller, LS. and S.R. Pfeffer (1998) J. Biol. Chem. 243:22161-22164).
  • the function of Rab proteins in vesicular transport requires the cooperation of many other proteins. Specifically, the membrane-targeting process is assisted by a series of escort proteins (Khosravi-Far, R. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6264-6268).
  • GTP-bound Rab proteins initiate the binding of VAMP-like proteins of the transport vesicle to syntaxin-like proteins on the acceptor membrane, which subsequently triggers a cascade of protein-binding and membrane-fusion events.
  • GAPs GTPase-activating proteins
  • GDI guanine-nucleotide dissociation irihibitor
  • GEFs Guanosuae nucleotide exchange factors
  • GEFs increase the rate of nucleotide dissociation by several orders of magnitude, thus facilitating release of GDP and loading with GTP.
  • the best characterized is the mammalian homolog of the Drosophila Son-of-Sevenless protein.
  • Certain Ras-family proteins are also regulated by guariine nucleotide dissociation irihibitors (GDIs), which inhibit GDP dissociation.
  • GDIs guariine nucleotide dissociation irihibitors
  • the mtrinsic rate of GTP hydrolysis of the LMW G-proteins is typically very slow, but it can be stimulated by several orders of magnitude by GAPs (Geyer, M. and A. Wittinghofer (1997) Curr. Opin. Struct. Biol. 7:786-792).
  • Both GEF and GAP activity maybe controlled in response to extracellular stimuli and modulated by accessory proteins such as RalBPl and POB1.
  • Mutant Ras-family proteins which bind but cannot hydrolyze GTP, are permanently activated, and cause cell proliferation or cancer, as do GEFs that inappropriately activate LMW G-proteins, such as the human oncogene NET1, a Rho-GEF (Drivas, G.T. et al. (1990) Mol. Cell Biol. 10:1793-1798; Alberts, A.S. and R. Treisman (1998) EMBO J. 14:4075-4085).
  • centaurin beta 1 A A member of the ARF family of G-proteins is centaurin beta 1 A, a regulator of membrane traffic and the actin cytoskeleton.
  • the centaurin ⁇ family of GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors contain pleckstrin homology (PH) domains which are activated by phosphoinositides.
  • PH domains bind phosphoinositides, implicating PH domains in signaling processes.
  • Phosphoinositides have a role in converting Arf-GTP to Arf-GDP via the centaurin ⁇ family and a role in Arf activation (Kam, J.L. et al. (2000) J. Biol. Chem.
  • the rho GAP family is also implicated in the regulation of actin polymerization at the plasma membrane and in several cellular processes.
  • the gene ARHGAP6 encodes GTPase-activating protein 6 isoform 4. Mutations in ARHGAP6, seen as a deletion of a 500 kb critical region in Xp22.3, causes the syndrome microphmalmia with linear skin defects (MLS). MLS is an X-linked dominant, male-lethal syndrome (Prakash, S.K. et al. (2000) Hum. Mol. Genet. 9:477-488).
  • a member of the Rho family of G-proteins is CDC42, a regulator of cytoskeletal rearrangements required for cell division.
  • CDC42 is inactivated by a specific GAP (CDC42GAP) that strongly stimulates the GTPase activity of CDC42 while having a much lesser effect on other Rho family members.
  • CDC42GAP also contains an SH3-binding domain that interacts with the SH3 domains of cell signaling proteins such as p85 alpha and c-Src, suggesting that CDC42GAP may serve as a link between CDC42 and other cell si naling pathways (Barfod, E.T. et al. (1993) J. Biol. Chem. 268:26059-26062).
  • the Dbl proteins are a family of GEFs for the Rho and Ras G-proteins (Whitehead, I.P. et al. (1997) Biochim. Biophys. Acta 1332:F1-F23). All Dbl family members contain a Dbl homology (DH) domain of approximately 180 amino acids, as well as a pleckstrin homology (PH) domain located immediately C-terminal to the DH domain. Most Dbl proteins have oncogenic activity, as demonstrated by the ability to transform various cell lines, consistent with roles as regulators of Rho- mediated oncogenic signaling pathways.
  • the kalirin proteins are neuron-specific members of the Dbl family, which are located to distinct subcellular regions of cultured neurons (Johnson, R.C. (2000) J. Cell Biol. 275:19324-19333).
  • RGS G-protein signaling
  • cytokine interleukin
  • the Immuno-associated nucleotide (IAN) family of proteins has GTP-binding activity as indicated by the conserved ATP/GTP-binding site P-loop motif.
  • the LAN family includes IAN-1, IAN-4, LAP38, and IAG-1.
  • IAN-1 is expressed in the immune system, specifically in T cells and thymocytes. Its expression is induced during thymic events (Poirier, G.M.C. et al. (1999) J. hrnmunol. 163:4960-4969).
  • IAP38 is expressed in B cells and macrophages and its expression is induced in splenocytes by pathogens.
  • IAG-1 which is a plant molecule, is induced upon bacterial infection (Krucken, J. et al. (1997) Biochem. Biophys. Res. Commun. 230:167-170).
  • IAN-4 is a mitochondrial membrane protein which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the bcr/abl oncogene.
  • the bcr/abl oncogene is known to be associated with chronic myelogenous leukemia, a clonal myelo-proliferative disorder, which is due to the translocation between the bcr gene on chromosome 22 and the abl gene on chromosome 9.
  • Bcr is the breakpoint cluster region gene and abl is the cellular homolog of the transforming gene of the Abelson murine leukemia virus. Therefore, the IAN family of proteins appears to play a role in cell survival in immune responses and cellular transformation (Daheron, L. et al. (2001) Nucleic Acids Res. 29:1308-1316).
  • Formin-related genes comprise a large family of morphoregulatory genes and have been shown to play important roles in morphogenesis, embryogenesis, cell polarity, cell migration, and cytokinesis through their interaction with Rho family small GTPases.
  • Formin was first identified in mouse limb deformity (Id) mutants where the distal bones and digits of all limbs are fused and reduced in size.
  • FRL contains formin homology domains FH1 , FH2, and FH3. The FH1 domain has been shown to bind the Src homology 3 (SH3) domain, WWP/WW domains, and profilin.
  • the FH2 domain is conserved and was shown to be essential for formin function as disruption at the FH2 domain results in the characteristic Id phenotype.
  • the FH3 domain is located at the N-termi ⁇ us of FRL, and is required for associating with Rac, a Rho family GTPase (Yayoshi-Yamamoto, S. et al. (2000) Mol. Cell. Biol. 20:6872-6881).
  • PSD-95 postsynaptic density 95
  • Dig Dig (Drosophila lethal(l)discs large-1)
  • ZO-1 zonula occludens-1).
  • These proteins play important roles in neuronal synaptic transmission, tumor suppression, and cell junction formation, respectively. Since the discovery of these proteins, over sixty additional PDZ-containing proteins have been identified in diverse prokaryotic and eukaryotic organisms. This domain has been implicated in receptor and ion channel clustering and in the targeting of multiprotein signaling complexes to specialized functional regions of the cytosolic face of the plasma membrane. (For a review of PDZ domam-containing proteins, see Ponting, C.P. et al.
  • PDZ domains are found in the eukaryotic MAGUK (membrane-associated guanylate kinase) protein family, members of which bind to the intracellular domains of receptors and channels.
  • MAGUK membrane-associated guanylate kinase
  • PDZ domains are also found in diverse membrane-localized proteins such as protein tyrosine phosphatases, serme/threonine kinases, G-protein cofactors, and synapse-associated proteins such as syntrophins and neuronal nitric oxide synthase (nNOS).
  • nNOS neuronal nitric oxide synthase
  • the glutamate receptor interacting protein contains seven PDZ domains. GRIP is an adaptor that links certain glutamate receptors to other proteins and may be responsible for the clustering of these receptors at excitatory synapses in the brain (Dong, H. et al. (1997) Nature 386:279-284).
  • the Drosophila scribble (SCRUB) protein contains both multiple PDZ domains and leucine-rich repeats. SCRUB is located at the epithelial septate junction, which is analogous to the vertebrate tight junction, at the boundary of the apical and basolateral cell surface. SCRUB is involved in the distribution of apical proteins and correct placement of adherens junctions to the basolateral cell surface (Bilder, D. and N. Perrimon (2000) Nature 403:676-680).
  • the PX domain is an example of a domain specialized for promoting protein-protein interactions.
  • the PX domain is found in sorting nexins and in a variety of other proteins, including the PhoX components of NADPH oxidase and the Cpk class of phosphatidylinositol 3 -kinase.
  • Most PX domains contain a polyproline motif which is characteristic of SH3 domain-binding proteins (Pouting, C.P. (1996) Protein Sci. 5:2353-2357).
  • SH3 domain-mediated interactions involving the PhoX components of NADPH oxidase play a role in the formation of the NADPH oxidase multi-protein complex (Leto, T.L. et al. (1994) Proc. Natl. Acad. Sci. USA 91:10650-10654; Wilson, L. et al. (1997) Inflamm. Res. 46:265-271).
  • the SH3 domain is defined by homology to a region ofthe proto-oncogene c-Src, a cytoplasmic protein tyrosine kinase.
  • SH3 is a small domain of 50 to 60 amino acids that interacts with proline-rich ligands. SH3 domains are found in a variety of eukaryotic proteins involved in signal transduction, cell polarization, and membrane-cytoskeleton interactions. In some cases, SH3 domain- containing proteins interact directly with receptor tyrosine kinases.
  • the SLAP-130 protein is a substrate of the T-cell receptor (TCR) stimulated protein kinase.
  • SLAP-130 interacts via its SH3 domain with the protein SLP-76 to affect the TCR-induced expression of interleukin-2 (Musci, M.A. et al. (1997) J. Biol. Chem. 272:11674-11677).
  • Another recently identified SH3 domain protein is macrophage actin-associated tyrosine-phosphorylated protein (MAYP) which is phosphorylated during the response of macrophages to colony stimulating factor- 1 (CSF-1) and is likely to play a role in regulating the CSF-1-induced reorganization of the actin cytoskeleton (Yeung, Y.-G. et al. (1998) J. Biol. Chem. 273:30638-30642).
  • the structure of the SH3 domain is characterized by two antiparallel beta sheets packed against each other at right angles. This packing forms a hydrophobic pocket lined with residues that are highly conserved between different SH3 domains. This pocket makes critical hydrophobic contacts wimproline residues in the ligand (Feng, S. et al. (1994) Science 266:1241- 1247).
  • a novel domain resembles the SH3 domain in its ability to bind proline-rich ligands.
  • This domain was originally discovered in dystrophin, a cytoskeletal protein with direct involvement in Duchenne muscular dystrophy (Bork, P. and M. Sudol (1994) Trends Biochem. Sci. 19:531-533).
  • WW domains have since been discovered in a variety of intracellular signaling molecules involved in development, cell differentiation, and cell proliferation.
  • the structure of the WW domain is composed of beta strands grouped around four conserved aromatic residues, generally tryptophan.
  • the SH2 domain is defined by homology to a region of c-Src.
  • SH2 domains interact directly with phospho-tyrosine residues, thus providing an immediate mechanism for the regulation and transduction of receptor tyrosine kinase-mediated signaling pathways.
  • SH2 domains are capable of binding to phosphorylated tyrosine residues in the activated PDGF receptor, thereby providing a highly coordinated and finely tuned response to ligand-mediated receptor activation.
  • the BLNK protein is a linker protein involved in B cell activation, that bridges B cell receptor-associated kinases with SH2 domain effectors that link to various signaling pathways (Fu, C et al.
  • the pleckstrin homology (PH) domain was originally identified in pleckstrin, the predominant substrate for protein kinase C in platelets. Since its discovery, this domain has been identified in over 90 proteins involved in intracellular signaling or cytoskeletal organization. Proteins containing the pleckstrin homology domain include a variety of kinases, phospholipase-C isof rms, guanine nucleotide release factors, and GTPase activating proteins. For example, members ofthe FGD1 family contain both Rho-guanine nucleotide exchange factor (GEF) and PH domains, as well as a FYVE zinc finger domain.
  • GEF Rho-guanine nucleotide exchange factor
  • FGD1 is the gene responsible for faciogenital dysplasia, an inherited skeletal dysplasia (Pasteris, N.G. and J.L. Gorski (1999) Genomics 60:57-66). Many PH domain proteins function in association with the plasma membrane, and this association appears to be mediated by the PH domain itself. PH domains share a common structure composed of two antiparallel beta sheets flanked by an amphipathic alpha helix. Variable loops connecting the component beta strands generally occur within a positively charged environment and may function as ligand binding sites (Lemmon, M . et al. (1996) Cell 85:621-624). Ankyrr ⁇ (ANK) repeats mediate protein-protein interactions associated with diverse intracellular signaling functions.
  • ANK Ankyrr ⁇
  • ANK repeats are found in proteins involved in cell proliferation such as kinases, kinase inhibitors, tumor suppressors, and cell cycle control proteins.
  • proteins involved in cell proliferation such as kinases, kinase inhibitors, tumor suppressors, and cell cycle control proteins.
  • TPR tetratricopeptide repeat
  • CDC16, CDC23, and CDC27 members of the anaphase promoting complex which targets proteins for degradation at the onset of anaphase.
  • Other processes mvolving TPR proteins include cell cycle control, transcription repression, stress response, and protein kinase inhibition (Lamb, J.R. et al. (1995) Trends Biochem. Sci. 20:257-259).
  • the armadiho/beta-catenin repeat is a 42 amino acid motif which forms a superhelix of alpha helices when tandemly repeated.
  • the structure of the armadillo repeat region from beta-catenin revealed a shallow groove of positive charge on one face of the superhelix, which is a potential binding surface.
  • the armadillo repeats of beta-catenin, plakoglobin, and pl20 cas bind the cytoplasmic domains of cadherins.
  • Beta-catenin/cadherin complexes are targets of regulatory signals that govern cell adhesion and mobility (Huber, A.H. et al. (1997) Cell 90:871-882).
  • G-beta beta-transducin
  • alpha, beta, and gamma beta-transducin
  • G proteins guanine nucleotide-binding proteins
  • array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • Breast cancer is a genetic disease commonly caused by mutations in cellular disease. Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and maybe 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 noninherited mutations that occur in breast epithelial cells.
  • EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation.
  • the abundance of erbB receptors, such as HER-2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy ofthe disease (Bacus, SS et al. (1994) Am J Clin Pathol 102-.S13-S24).
  • Prostate cancer is a common malignancy 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 cells 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 cell and transitional cell 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 tamor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells that become hyperplastic and evolve into early-stage tumors.
  • the early-stage tumors are localized in the prostate but eventually may metastasize, particularly to the bone, brain or lung. About 80% of these tumors remain responsive to androgen treatment, an important hormone controlling the growth of prostate epithelial cells. However, in its most advanced state, 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 epithelial cells.
  • the quantity of PSA correlates with the number and volume of the prostatic epithelial cells, and consequently, the levels of PSA are an excellent indicator of abnormal prostate growth.
  • Men with prostate cancer exhibit an early linear increase in PSA levels followed 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 individual cases of prostate cancer.
  • EGF Epidermal Growth Factor
  • FGF Fibroblast Growth Factor
  • TGF ⁇ Tumor Growth Factor alpha
  • TGF- ⁇ family of growth factors are generally expressed at increased levels in human cancers and the high expression levels in many cases correlates with advanced stages of malignancy and poor survival (Gold LI (1999) Crit Rev Oncog 10:303-360).
  • LNCap androgen-dependent stage of prostate cancer
  • PC3 and DU-145 the androgen-independent, hormone refractory stage of the disease
  • the invention features purified polypeptides, molecules for disease detection and treatment, referred to collectively as “MDDT” and individually as “MDDT-1,” “MDDT-2,” “MDDT-3,”
  • MDDT-4 "MDDT-5,” “MDDT-6,” “MDDT-7,” “MDDT-8,” “MDDT-9,” “MDDT-10,” “MDDT- 11,” “MDDT-12,” “MDDT-13,” “MDDT-14,” “MDDT-15,” “MDDT-16,” “MDDT-17,” “MDDT- 18,” “MDDT-19,” “MDDT-20,” “MDDT-21,” “MDDT-22 ,” “MDDT-23,” “MDDT-24,” “MDDT- 25,” “MDDT-26,” “MDDT-27,” “MDDT-28,” “MDDT-29,” “MDDT-30,” “MDDT-31,” “MDDT- 32,” “MDDT-33,” “MDDT-34,” “MDDT-35,” “MDDT-36,” “MDDT-37,” “MDDT-38,” and
  • the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ TD NO:l-39, 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-39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:l-39.
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, 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- 39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-39. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO:40-78.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, 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-39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.
  • the invention provides a cell transformed with the recombinant polynucleotide.
  • the invention provides a transgenic organism comprising the recombinant polynucleotide.
  • the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, b) a polypeptide comprising a naturally occurring arnino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, 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-39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39.
  • the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide comprises at least 60 contiguous nucleotides.
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof, in one alternative, the probe comprises at least 60 contiguous, nucleotides.
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, 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) 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.
  • the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, 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 LD NO:l-39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and a pharmaceutically acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:l-39.
  • the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional MDDT, comprising administering to a patient in need of such treatment the composition.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, 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 LD NO: 1-39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional MDDT, comprising administering to a patient in need of such treatment the composition.
  • the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, 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-39, c) a biologically active fragment of a polypeptide. having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consistmg of SEQ ID NO: 1-39.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample, in one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional MDDT, comprising administering to a patient in need of such treatment the composition.
  • the invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, and d) an irnmunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, 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-39, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.
  • 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 ofthe test compound, and c) comparing the activity of the polypeptide in the presence ofthe test compound with the activity ofthe polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing die 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:40-78, if) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, hi) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of if), 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:40-78, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78, hi) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides ofthe invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide sequences ofthe invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences ofthe invention, along with selected fragments of the polynucleotide sequences.
  • Table 5 shows the representative cDNA library for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
  • Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.
  • MDDT refers to the amino acid sequences of substantially purified MDDT obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of MDDT.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of MDDT either by directly interacting with MDDT or by acting on components of the biological pathway in which MDDT participates.
  • allelic variant is an alternative form of the gene encoding MDDT. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding MDDT include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as MDDT or a polypeptide with at least one functional characteristic of MDDT. Include within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding MDDT, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding MDDT.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent MDDT.
  • Deliberate amino acid substitutions may be made on the basis of sir ilarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the ampMpathic nature of the residues, as long as the biological or immunological activity of MDDT is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threoiiine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucme, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to imrjit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
  • PCR polymerase chain reaction
  • antagonists refers to a molecule which inhibits or attenuates the biological activity of MDDT. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of MDDT either by directly interacting with MDDT or by acting on components of the biological pathway in which MDDT participates.
  • antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind MDDT polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • RNA Ribonucleic acid
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, myroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • KLH keyhole limpet hemocyanin
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
  • Aptamer compositions 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 ribonucleoti.de may be replaced by 2'-F or 2'-NH 2 ) > which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
  • RNA aptamer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • the term “biologically active” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic MDDT, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding MDDT or fragments of MDDT may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCI
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVTEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e. , the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions. Ori inal Residue Conservative Substitution Ala Gly, Ser
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitation, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemically modified polynucleotide or polypeptide.
  • Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an ' . alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins maybe assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
  • a “fragment” is a unique portion of MDDT or the polynucleotide encoding MDDT which is identical in sequence to but shorter in length than the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes maybe at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 arnino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • a fragment of SEQ ID NO:40-78 comprises a region of unique polynucleotide sequence that specifically identifies SEQ LD NO:40-78, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:40-78 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:40-78 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ID NO:40-78 and the region of SEQ ED NO:40-78 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO:l-39 is encoded by a fragment of SEQ LD NO:40-78.
  • a fragment of SEQ LD NO:l-39 comprises a region of unique amino acid sequence that specifically identifies SEQ ED NO:l-39.
  • a fragment of SEQ LD NO:l-39 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ LD NO: 1-39.
  • the precise length of a fragment of SEQ LD NO:l-39 and the region of SEQ LD NO:l-39 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
  • Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • NCBI National Center for Biotechnology information
  • BLAST Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410
  • NCBI National System for Mobile Science
  • BLAST 2 Sequences a tool that is used for direct pahrwise comparison of two nucleotide sequences.
  • BLAST 2 Sequences can be accessed and used interactively at http://www.ncbi.nlm.nm.gov/gorf bl2.htrnl.
  • the "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below).
  • BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy ofthe genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitations. Such conservative substitutions, explained in more detail above, generally preserve the charge and_hydrophobicity at the site of substitation, thus preserving the structure (and therefore function) of the polypeptide.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ LD number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all ofthe elements required for chromosome replication, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determiriing the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions maybe varied among experiments to achieve the desired Stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T 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.
  • High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C maybe used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%. Typically, blocking reagents are used to block non-specific hybridization.
  • Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex maybe formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect .cellular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or oligopeptide fragment of MDDT which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of MDDT which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of MDDT. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or irnmunological properties of MDDT.
  • nucleic acid arid “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine.
  • the terminal lysine confers solubility to the composition.
  • PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and maybe pegylated to extend their lifespan in the cell.
  • "Post-translational modification" of an MDDT may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of MDDT.
  • Probe refers to nucleic acid sequences encoding MDDT, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primmers are short nucleic acids, usually DNA oligonucleotides, which maybe annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides ofthe disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, maybe used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope.
  • the Primer3 primer selection program (available to the public from the Whitehead mstitate Iv ⁇ T Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
  • this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitation, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids maybe part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a "regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chenruluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA sequence, is composed ofthe same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base mymine 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 MDDT, nucleic acids encoding MDDT, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organeUe, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • the terms “specific binding” and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or .synthetic binding composition. The interaction is dependent upon the presence of a particular stiructare of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part ofthe host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant maybe described as, for example, an "allelic” (as defined above), “splice,” “species,” or “polymorphic” variant.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also -may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
  • SNPs single nucleotide polymorphisms
  • the presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of new human molecules for disease detection and treatment (MDDT), the polynucleotides encoding MDDT, and the use of these compositions for the. diagnosis, treatment, or prevention of cell proliferative, autoimmune/mflammatory, developmental, and neurological disorders.
  • MDDT disease detection and treatment
  • the polynucleotides encoding MDDT and the use of these compositions for the. diagnosis, treatment, or prevention of cell proliferative, autoimmune/mflammatory, developmental, and neurological disorders.
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ED). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ LD NO:) and an Incyte polypeptide sequence number (Jncyte Polypeptide LD) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ D NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide LD) as shown.
  • Column 6 shows the Incyte ED numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences 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 LD NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank LD NO:) of the nearest GenBank homolog.
  • Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation ofthe GenBank homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
  • Table 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
  • Column 6 shows airiino acid residues comprising signatare sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
  • SEQ ED NO:2 is 53% identical, from residue G3 to residue G172 and A183 to residue G659, to human mitogen inducible gene mig-2 (GenBank ED g505033) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.2 e-197, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ LD NO:2 also contains a pleckstrin homology (PH) domain as determined by searching for statistically significant matches in the hidden Markov model (LuTVLM)-based PFAM database of conserved protein family domains.
  • PH pleckstrin homology
  • SEQ LD NO: 14 is 91 % identical, from residue Ml to residue V659, to mouse DMR-N9 (GenBank LD g817954) as determined by the Basic Local Alignment Search Tool (BLAST).
  • BLAST Basic Local Alignment Search Tool
  • the BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ LD NO.T4 also contains WD repeats as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO: 14 is a protein associated with myotonic dystrophy.
  • SEQ ED NO:24 is 41% identical, from residue 197 to residue N378, to sponge longevity gene SDLAGL (GenBank LD g9798556) as deterrnined by the Basic Local Alignment Search Tool (BLAST).
  • BLAST probability score is 9.8e-58, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ LD NO:24 also contains a homeobox domain as deteirrnined by searching for statistically 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 corroborative evidence that SEQ LD NO:24 is a longevity assurance gene.
  • HMM hidden Markov model
  • SEQ ED NO:26 is 75% identical, from residue Ml to residue S1273, to a human protein, ORF2, which contains a reverse transcriptase domain (GenBank ED g339777) as deterrriined by the Basic Local Alignment Search Tool (BLAST).
  • BLAST Basic Local Alignment Search Tool
  • the BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ LD NO:26 also contains AP endonuclease family and reverse transcriptase domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO:33 is 90% identical, from residue Ml to residue N1275, to a predicted polypeptide comprising a reverse transcriptase domain (GenBank LD g339771) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO:33 also contains a reverse transcriptase domain and an AP endonuclease domain' as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ LD NO:33 is a reverse transcriptase.
  • SEQ ED NO:l, SEQ ED NO:3-13, SEQ LD NO:15-23, SEQ LD NO:25, SEQ ED NO:27-32 and SEQ LD NO:34-39 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ LD NO:l-39 are described in Table 7.
  • the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ED NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte LD) 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 ofthe cDNA and/or genomic sequences used to assemble the full length polynucleotide sequences of the invention, and of fragments ofthe polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ED NO:40-78 or that distinguish between SEQ LD NO:40-78 and related polynucleotide sequences.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Lncyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotide sequences.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (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. For example, a polynucleotide sequence identified as
  • FL_ZXXXXZ_N i _N 2 _yYYyy_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N ⁇ 3 _, if present, represent specific exons that may have been manually edited during lanalysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as FLXXXXX_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-stretching" algorithm was applied, 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”) maybe used in place of the GenBank identifier (Le., gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • the following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IN and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Lucyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA Hbraries shown in Table 5 are described in Table 6.
  • Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.
  • SNPs single nucleotide polymorphisms
  • Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ED NO:) and the corresponding Incyte project identification number (PLD) for polynucleotides of the invention.
  • Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ED), and column 4 shows the identification number for the SNP (SNP ED).
  • Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full- length polynucleotide sequence (CB1 SNP).
  • Column 7 shows the allele found in the EST sequence.
  • Columns 8 and 9 show the two alleles found at the SNP site.
  • Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST.
  • Columns 11-14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of allele 1 in the population was too low to be detected, while n/a (not available) indicates that the allele frequency was not determined for the population.
  • the invention also encompasses MDDT variants.
  • a preferred MDDT 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 MDDT amino acid sequence, and which contains at least one functional or structural characteristic of MDDT.
  • the invention also encompasses polynucleotides which encode MDDT.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:40-78, which encodes MDDT.
  • the polynucleotide sequences of SEQ ED NO:40-78 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences ofthe nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses a variant of a polynucleotide sequence encoding MDDT.
  • a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding MDDT.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:40- 78 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ LD NO:40-78.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of MDDT.
  • a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding MDDT.
  • a splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding MDDT, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
  • a splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to the polynucleotide sequence encoding MDDT over its entire length; however, portions of the splice variant will 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 sequence encoding MDDT.
  • a polynucleotide comprising a sequence of SEQ LD NO:78 is a splice variant of a polynucleotide comprising a sequence of SEQ ED NO:47. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of MDDT.
  • nucleotide sequences which encode MDDT and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring MDDT under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding MDDT or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-natarally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode MDDT and
  • MDDT derivatives, or fragments thereof, entirely by synthetic chemistry After production, the synthetic sequence maybe inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce ⁇ mutations into a sequence encoding MDDT or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ TD NO:40-78 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R.
  • Hybridization conditions including annealing and wash conditions, are described in "Definitions.”
  • Methods for DNA sequencing are well known in the art and maybe used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, RA.
  • the nucleic acid sequences encoding MDDT may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • one method which maybe employed, restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
  • Another method uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences inhuman and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic.
  • primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • commercially available software such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or comxrm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display maybe computer controlled.
  • Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode MDDT maybe cloned in recombinant DNA molecules that direct expression of MDDT, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express MDDT.
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter MDDT-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences.
  • oligonucleotide- mediated site-directed mutagenesis maybe used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • the nucleotides of the present invention maybe subjected to DNA shuffling techniques such as MOLECULARBREEDLNG (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of MDDT, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDLNG Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C.
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify fliose gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. 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 naturally occurring genes in a directed and controllable manner.
  • sequences encoding MDDT maybe synthesized, in whole or in part, using chemical methods well known in the art.
  • chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
  • MDDT itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or solid-phase techniques.
  • the nucleotide sequences encoding MDDT or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and.translational control of the inserted coding sequence in a suitable host.
  • These elements include regulatory sequences, such as enhancers, constitutive and- • . inducible promoters, and 5' and 3 'untranslated regions in the vector and in polynucleotide sequences encoding MDDT. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding MDDT. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
  • a variety of expression vector/host systems maybe utilized to contain and express sequences encoding MDDT. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g., baculovirus)
  • plant cell systems transformed with viral expression vectors e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus
  • Expression vectors derived from retroviruses, adenoviruses, or herpes, or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding MDDT. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding MDDT can be achieved using a multifunctional E. coli vector such as PBLUESCPJPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCPJPT Stratagene, La Jolla CA
  • PSPORT1 plasmid Invitrogen
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of MDDT.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, maybe used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • promoters such as alpha factor, alcohol oxidase, and PGH promoters
  • yeast Saccharomyces cerevisiae or Pichia pastoris may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of MDDT. Transcription of sequences encoding MDDT may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters maybe used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J.
  • constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection See, e.g., The McGraw HiU Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196.
  • ha mammalian ceUs a number of viral-based expression systems may be utilized.
  • sequences encoding MDDT may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
  • Insertion in' a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses MDDT in host ceUs.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian 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 deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355.)
  • sequences encoding MDDT can be transformed into ceU lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. 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 ofthe 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 cells may be propagated using tissue culture techniques appropriate to the ceU type.
  • any number of selection systems may be used to recover transformed ceU lines. These include, but are not limited to, the herpes simplex virus fliymidine kinase and adenine phosphoribosyltransferase genes, for use in tt and apr ceUs, respectively. (See, e.g., Wigler, M. et al. (1977) CeU 11 :223-232; Lowy, I. et al. (1980) CeU 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to cMorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUular requirements for metabolites.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.)
  • the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression ofthe gene may need to be coirfirmed.
  • the sequence encoding MDDT is inserted within a marker gene sequence, transformed ceUs containing sequences encoding MDDT can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding MDDT 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 nucleic acid sequence encoding MDDT and that express MDDT 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 amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of MDDT 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 MDDT is preferred, but a competitive binding assay may be employed.
  • assays are weU known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Lnterscience, New York NY; and Pound, J.D.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding MDDT include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding MDDT, 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 an appropriate RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host ceUs transformed with nucleotide may be 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 MDDT may be designed to contain signal sequences which direct secretion of MDDT through a prokaryotic or eukaryotic ceU membrane.
  • a host ceU strain may be chosen for its ability to modulate expression ofthe inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro” form ofthe 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 WD 8) are available from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture CoUection
  • Manassas VA American Type Culture CoUection
  • natural, modified, or recombinant nucleic acid sequences encoding MDDT may be ligated to a heterologous sequence resulting in translation of a fusion protein in any ofthe aforementioned host systems.
  • a chimeric MDDT protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may facilitate the screening of peptide libraries for inhibitors of MDDT activity.
  • Heterologous protein and peptide moieties may also facilitate 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, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaf ⁇ inity 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 MDDT encoding sequence and the heterologous protein sequence, so that MDDT may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
  • a variety of commerciaUy available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled MDDT may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-metHonine.
  • MDDT of the present invention or fragments thereof may be used to screen for compounds that specificaUy bind to MDDT. At least one and up to a plurality of test compounds may be screened for specific binding to MDDT.
  • test compounds include antibodies, ougonucleotides, proteins (e.g., ligands or receptors), or smaU molecules.
  • the compound thus identified is closely related to the natural ligand of MDDT, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • the compound thus identified is a natural ligand of a receptor MDDT.
  • the compound can be closely related to the natural receptor to which MDDT 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 MDDT which is capable of propagating a signal, or a decoy receptor for MDDT 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 tamor 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 tamor necrosis factor
  • MDDT involves producing appropriate ceUs which express MDDT, either as a secreted protein or on the ceU membrane.
  • Preferred ceUs include ceUs from mammals, yeast, Drosophila, or E. coli.
  • CeUs expressing MDDT or ceU membrane fractions which contain MDDT are then contacted with a test compound and binding, stimulation, or inhibition of activity of either MDDT 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 MDDT, either in solution or affixed to a solid support, and detecting the binding of MDDT 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 libraries, or nataral product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
  • An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural Hgand to its natural receptors.
  • examples of such assays include radio- labeling assays such as fliose described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands. (See, e.g., Matthews, D.J. and J.A. WeUs. (1994) Chem. Biol.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a Hgand) to improve or alter its ability to bind to its natural receptors.
  • a polypeptide compound such as a Hgand
  • MDDT of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of MDDT.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for MDDT activity, wherein MDDT is combined with at least one test compound, and the activity of MDDT in the presence of a test compound is compared with the activity of MDDT in the absence of the test compound. A change in the activity of MDDT in the presence ofthe test compound is indicative of a compound that modulates the activity of MDDT.
  • a test compound is combined with an in vitro or ceh-free system comprising MDDT under conditions suitable for MDDT activity, and the assay is performed.
  • a test compound which modulates the activity of MDDT may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraHty of test compounds maybe screened.
  • polynucleotides encoding MDDT or their mammaHan 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.
  • mouse ES ceUs such as the mouse 129/SvJ ceU line
  • 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.
  • 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 MDDT 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 lineages including endoderm, mesoderm, and ectodermal ceU types. These ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147). Polynucleotides encoding MDDT can also be used to create 'rmockin" humanized animals
  • pigs pigs
  • transgenic animals mice or rats
  • a region of a polynucleotide encoding MDDT is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
  • Transformed ceUs are injected into blastulae, and the blastalae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress MDDT e.g., by secreting MDDT in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • MDDT appears to play a role in ceU proHferative, autoimmune/inflammatory, developmental, and neurological disorders.
  • MDDT 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 MDDT.
  • disorders include, but are not limited to, a ceU proHferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal • gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangHa, gastrointestinal tract, heart, kidney, Hver
  • a vector capable of expressing MDDT 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 MDDT including, but not limited to, those described above.
  • compositions comprising a substantially purified MDDT 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 MDDT including, but not Hunted to, those provided above.
  • an agonist which modulates the activity of MDDT maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of MDDT including, but not Hmited to, those listed above.
  • an antagonist of MDDT may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of MDDT.
  • disorders include, but are not Hmited to, those ceU proHferative, autoimmune/inflammatory, developmental, and neurological disorders described above.
  • an antibody which specificaUy binds MDDT maybe used directly as an antagonist or indirectly as a targeting or deHvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express MDDT.
  • a vector expressing the complement of the polynucleotide encoding MDDT maybe administered to a subject to treat or prevent a disorder associated with increased expression or activity of MDDT including, but not Hmited to, those described above.
  • any ofthe proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention maybe 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 may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of MDDT maybe produced using methods which are generaUy known in the art.
  • purified MDDT may be used to produce antibodies or to screen Hbraries. of pharmaceutical agents to identify those which specificaUy bind MDDT.
  • Antibodies to MDDT may also be generated using methods that are weU known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Hbrary.
  • NeutraHzing antibodies i.e., those which inhibit dinner formation) are generaUy preferred for therapeutic use.
  • 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 may be immunized by injection with MDDT or with any fragment or oHgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not Hmited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecifliin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG BaciUi Calmette-Guerin
  • Corynebacterium parvum are especiaUy preferable.
  • the oHgopeptides, peptides, or fragments used to induce antibodies to MDDT 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 oHgopeptides, peptides, or fragments are identical to a portion ofthe amino acid sequence of the nataral protein. Short stretches of MDDT amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to MDDT maybe prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not Hmited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
  • chimeric antibodies such as the spHcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used.
  • spHcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin Hbraries or panels of highly specific binding reagents as disclosed in the Hteratare. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter,
  • Antibody fragments which contain specific binding sites for MDDT may also be generated.
  • fragments include, but are not Hmited to, F(ab') 2 fragments produced by pepsin digestion ofthe antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression Hbraries maybe constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, WD. et al. (1989) Science 246:1275-1281.)
  • immunoassays maybe used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are weU known in the art.
  • Such immunoassays typicaUy involve the measurement of complex formation between MDDT and its specific antibody.
  • a two-site, monoclonal-based immunoassay utiHzing monoclonal antibodies reactive to two non-interfering MDDT epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
  • Various methods such as Scatchard analysis in conjunction with radioimmuno assay techniques may be used to assess the affinity of antibodies for MDDT.
  • K a is defined as the molar concentration of MDDT-antibody complex divided by the molar concentrations of free antigen and free antibody under equiHbrium conditions.
  • the K a deteirmined for a preparation of monoclonal antibodies, which are monospecific for a particular MDDT epitope, represents a true measure of affinity.
  • Lui ⁇ -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 MDDT- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of MDDT, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, LRL 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 quaHty and suitability of such preparations for certain downstream appHcations.
  • a polyclonal antibody preparation containing at least 1-2 g specific antibody/rnl, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of MDDT-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quaHty and usage in various appHcations, are generaUy available. (See, e.g., Catty, supra, and CoHgan et al. supra.)
  • the polynucleotides encoding MDDT 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 oHgonucleotides) to the coding or regulatory regions of the gene encoding MDDT.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oHgonucleotides
  • antisense oHgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding MDDT. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., 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 Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al.
  • Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
  • viral vectors such as retrovirus and adeno-associated virus vectors.
  • Other gene deHvery mechanisms include Hposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • Rossi J.J. (1995) Br. Med. BuU. 51(l):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736.
  • polynucleotides encoding MDDT maybe 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 (SCED)-Xl disease characterized by X- Hnked 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
  • SCED severe combined immunodeficiency
  • ADA adenosine de
  • HJBV hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodiumfalciparum and Trypanosoma cruz ⁇ .
  • the expression of MDDT from an appropriate population of transduced ceUs may aUeviate the clinical manifestations caused by the genetic deficiency.
  • diseases or disorders caused by deficiencies in MDDT are treated by constructing mammaHan expression vectors encoding MDDT and introducing these vectors by mechanical means into MDDT-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ii) ballistic gold particle deHvery, (iii) Hposome-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 MDDT include, but are not Hmited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRJPT, PCMV-TAG, PEGSH7PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • MDDT maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalo virus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. 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) Curr. Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalo virus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • Hposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from InvitiOgen
  • aUow.one with ordinary skiU in the art to deHver polynucleotides to target ceUs in culture and require rriiriimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A J. Eb (1973) Virology 52:456-467), ox by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
  • the introduction of DNAto primary ceUs requires modification of these standardized mammaHan transfection protocols.
  • diseases or disorders caused by genetic defects with respect to MDDT expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding MDDT under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are cosorptionauy available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • the vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for 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. Mffler (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol 72:8463-8471; Zufferey, R. et al.
  • VSVg vector producing ceU line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines 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 skiUed 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.
  • an adenovirus-based gene therapy deHvery system is used to deHver polynucleotides encoding MDDT to ceUs which have one or more genetic abnormaHties with respect to the expression of MDDT.
  • the construction and packaging of adenovirus-based vectors are weU known to those with ordinary skiU in the art.
  • RepHcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adeno viral vectors are described in U.S. Patent No.
  • Addenovirus vectors for gene therapy hereby incorporated by reference.
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both ' incorporated by reference herein.
  • a herpes-based, gene therapy deHvery system is used to deHver polynucleotides encoding MDDT to target ceUs which have one or more genetic abnormaHties with respect to the expression of MDDT.
  • herpes simplex virus (HSV)-based vectors may be especiaUy valuable for introducing MDDT 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 repHcation-competent herpes simplex virus (HSV) type 1-based vector has been used to deHver 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.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deHver polynucleotides encoding MDDT to target ceUs.
  • SFV SemHki Forest Virus
  • SFV SemHki Forest Virus
  • alphavirus RNA repHcation a subgenomic RNA is generated that normaUy encodes the viral capsid proteins.
  • This subgenomic RNA repHcates 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).
  • enzymatic activity e.g., protease and polymerase.
  • inserting the coding sequence for MDDT into the alphavirus genome in place ofthe capsid-coding region results in the production of a large number of MDDT-coding RNAs and the synthesis of high levels of MDDT in vector transduced ceUs.
  • alphavirus infection is typicaUy associated with ceU lysis within a few days
  • the abiHty to establish a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SEN) indicates that the lytic repHcation of alphaviruses can be altered to suit the needs of the gene therapy appHcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses will' aUow the introduction of MDDT 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 alphavirus cDNA and RNA transfections, and performing alphavirus infections, are weU known to those with ordinary skiU in the art.
  • OHgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple heHx base-pairing methodology.
  • Triple heHx pairing is useful because it causes inhibition of the abiHty of the double heHx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the Hteratare. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura PubHshing, 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 RNA.
  • 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 sequences encoding MDDT.
  • RNA target Specific ribozyme cleavage sites within any potential RNA target are initiaUy identified by 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, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oHgonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oHgonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing oHgonucleotides such as soHd phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding MDDT. 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, constitatively or inducibly, can be introduced into ceU lines, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3 ' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase Hhkages 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 MDDT.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oHgonucleotides, antisense oHgonucleotides, triple heHx-forming oHgonucleotides, 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 MDDT may be therapeuticaUy useful, and in the treatment of disorders associated with decreased MDDT expression or activity, a compound which specificaUy promotes expression ofthe polynucleotide encoding MDDT may be therapeuticaUy useful.
  • At least one, and up to a pluraHty, of test compounds maybe 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 Hbrary 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 Hbrary of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding MDDT is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabiHzed ceU, or an in vitro ceU-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding MDDT 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 MDDT.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human 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 Hbrary of oHgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oHgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
  • oHgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oHgonucleotides
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. DeHvery by transfection, by Hposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
  • any of the therapeutic methods described above may be appHed 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 ofthe invention relates to the adrninistration 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 PubHshing, Easton PA).
  • Such compositions may consist of MDDT, antibodies to MDDT, and mimetics, agonists, antagonists, or inhibitors of MDDT.
  • compositions utilized in this invention may be administered by any number of routes including, but not Hmited to, oral, intravenous, intramuscular, intra-arterial, mtrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • compositions for pulmonary administration may be prepared in Hquid or dry powder form. These compositions are generaUy aerosoHzed immediately prior to inhalation by the patient.
  • aerosol deHvery of fast- acting formulations is weU-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • Pulmonary deHvery has the advantage of administration without needle injection, and obviates the need for 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 capability of those skiUed in the art.
  • SpeciaHzed forms of compositions may be prepared for direct intraceUular deHvery of macromolecules comprising MDDT or fragments thereof.
  • Hposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deHvery of the macromolecule.
  • MDDT or a fragment thereof may be joined to a short cationic N- terminal portion from the FiJN 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 MDDT or fragments thereof, antibodies of MDDT, and agonists, antagonists or inhibitors of MDDT, which ameHorates the symptoms or condition.
  • Therapeutic efficacy and toxicity maybe deteirmined 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 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • 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 Httle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient,
  • the exact dosage wiU be determined by the practitioner, in Hght of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response, to therapy. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending on the half-Hfe 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 deHvery is provided in the Hteratare and generaUy available to practitioners in the art. Those skiUed in the art wiU employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, deHvery of polynucleotides or polypeptides wiUbe specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specificaUy bind MDDT may be used for the diagnosis of disorders characterized by expression of MDDT, or in assays to monitor patients being treated with MDDT or agonists, antagonists, or inhibitors of MDDT.
  • Antibodies useful for diagnostic purposes maybe prepared in the same manner as described above for therapeutics. Diagnostic assays for MDDT include methods which utiHze the antibody and a label to detect MDDT 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 reporter molecules, several of which are described above, are known in the art and may be used.
  • a variety of protocols for measuring MDDT including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of MDDT expression.
  • Normal or standard values for MDDT expression are estabHshed by combining body fluids or ceU extracts taken from normal mammaHan subjects, for example, human subjects, with antibodies to MDDT under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of MDDT expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabHshes the parameters for diagnosing disease.
  • the polynucleotides encoding MDDT may be used for diagnostic purposes.
  • the polynucleotides which may be used include oHgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of MDDT may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of MDDT, and to monitor regulation of MDDT levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding MDDT or closely related molecules may be used to identify nucleic acid sequences which encode MDDT.
  • the specificity ofthe probe whether it is made from a highly specific region, e.g., the 5 'regulatory region, or from a less specific region, e.g., a' conserved motif, and the stringency of the hybridization or ampHfication wiU determine whether the probe identifies only nataraUy occurring sequences encoding MDDT, aUeHc 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 MDDT encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ED NO:40-78 or from genomic sequences including promoters, enhancers, and introns of the MDDT gene.
  • Means for producing specific hybridization probes for DNAs encoding MDDT include the cloning of polynucleotide sequences encoding MDDT or MDDT derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commerciaUy available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucHdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding MDDT may be used for the diagnosis of disorders associated with expression of MDDT.
  • disorders include, but are not Hmited to, a ceU proHferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangHa, gastrointestinal tract, heart, kidney, Hver, lung, muscle, ovary
  • the polynucleotide sequences encoding MDDT may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-Hke assays; and in microarrays utiHzing fluids or tissues from patients to detect altered MDDT expression.
  • Such quaHtative or quantitative methods are weU known in the art.
  • the nucleotide sequences encoding MDDT may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide - sequences encoding MDDT may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding MDDT in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is estabHshed. This maybe accompHshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding MDDT, under conditions suitable for hybridization or ampHfication.
  • 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 may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabHsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to deteirmine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earHer thereby preventing the development or further progression of the cancer.
  • oHgonucleotides designed from the sequences encoding MDDT may involve the use of PCR. These oHgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. OHgomers wiU preferably contain a fragment of a polynucleotide encoding MDDT, or a fragment of a polynucleotide complementary to the polynucleotide encoding MDDT, and wiU.be employed under optimized conditions for identification of a specific gene or condition. OHgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oHgonucleotide primers derived from the polynucleotide sequences • encoding MDDT may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oHgonucleotide primers derived from the polynucleotide sequences encoding MDDT are used to ampHfy DNA using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oHgonucleotide primers are fluorescently labeled, which aUows detection ofthe amplimers in high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy sequence database analysis methods, termed in siHco SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs maybe 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-insuHn-dependent diabetes melHtas. SNPs are also useful for exarmning 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 utiHty in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as Hfe-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-Hpoxygenase 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.
  • Methods which may also be used to quantify the expression of MDDT include radiolabeling or biotinylating nucleotides, coampHfication of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C et al. (1993) Anal. Biochem.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oHgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oHgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a 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 deteirmine 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, hn 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.
  • MDDT, fragments of MDDT, or antibodies specific for MDDT may be used as elements on a microarray.
  • the microarray maybe used to monitor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or 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. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No.
  • a transcript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totaHty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in Hgh-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraHty of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, 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 preclinical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and nataraUy-occurring env ⁇ onmental 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, expressly incorporated by reference herein).
  • a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
  • These fmgerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene famines.
  • IdeaUy a genome- wide measurement of expression provides the highest quaHty signatare. 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 normaHze the rest ofthe expression data. The normaHzation procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant 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.
  • the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU type.
  • proteome can be subjected individuaUy 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, hn 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 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 visuaHzed 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 generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry.
  • the identity of the protein in a spot may be dete ⁇ riined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention, hn some cases, further sequence data maybe obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for MDDT to quantify the levels of MDDT expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatares at the transcript level.
  • There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling maybe more reHable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
  • the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT appHcation WO95/251116; Shalon, D. et al. (1995) PCT appHcation WO95/35505; HeUer, RA. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and HeUer, M.J.
  • nucleic acid sequences encoding MDDT maybe 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 may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal • mapping.
  • the sequences maybe mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA Hbraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA Hbraries.
  • the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
  • RFLP restriction fragment length polymorphism
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online MendeHan Inheritarice in Man (OMLM) World Wide Web site. Correlation between the location of the gene encoding MDDT on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the ⁇ region of DNA associated with that disorder and thus may further positional cloning efforts.
  • OMLM Online MendeHan Inheritarice in Man
  • nucleotide sequence ofthe instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • MDDT its catalytic or immunogenic fragments, or oHgopeptides thereof can be used for screening Hbraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a soHd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between MDDT and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different smaU test compounds are synthesized on a soHd substrate. The test compounds are reacted with MDDT, or fragments thereof, and washed. Bound MDDT is then detected by methods weU known in the art. Purified MDDT can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutraHzing antibodies can be used to capture the peptide and immobiHze it on a soHd support.
  • the nucleotide sequences which encode MDDT 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 Hmited to, such v properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs were derived from cDNA Hbraries described in the LLFESEQ 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 denatarants, 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 oHgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • Stratagene was provided with RNA and constructed the corresponding cDNA Hbraries. Otherwise, cDNA was synthesized and cDNA Hbraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Ihvitrogen), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oHgo d(T) or random primers. Synthetic oHgonucleotide adapters were Hgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • the cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CUB column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs were Hgated into compatible restriction enzyme sites ofthe polylinker of a suitable plasmid, e.g., PBLUESCRLPT 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 pLNCY (Incyte Genomics), or derivatives thereof.
  • PBLUESCRLPT plasmid (Stratagene)
  • PSPORT1 plasmid Invitrogen
  • PCDNA2.1 plasmid Invitrogen, Carlsbad CA
  • PBK-CMV plasmid PCR2-TOPOTA plasmid
  • coH ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invitrogen.
  • 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 distiUed water and stored, with or without lyophiHzation, at 4°C
  • plasmid DNA was ampHfied 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 carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of ampHfied plasmid DNA was quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN LI fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
  • Incyte cDNA recovered in plasmids as described in Example II were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (AppHed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MTCROLAB 2200 (Hamilton) Hquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or suppHed in ABI sequencing kits such as the ABI PRISM BIGDYE Teirrninator cycle sequencing ready reaction kit (AppHed 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 (AppHed Biosystems) in conjunction with standard i ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in. . Ausubel, 1997, supra, unit 7.7). Some of the.cDNA sequences were selected for extension using the techniques disclosed in Example VTJI.
  • the polynucleotide sequences derived from Incyte cDNAs were vaHdated by removing vector, Hfrker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of pubHc databases such as the GenBank primate, rodent, mammaHan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, LNCY, and TIGRFAM (Haft, D.H.
  • pubHc databases such as the GenBank primate, rodent, mammaHan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM
  • HMM-based protein domain databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letanic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene famiHes. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce fuU length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to fuU length. 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 fuU length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences.
  • a polypeptide of the invention may begin at any of the mefl ⁇ oiriine residues of the fuU length translated polypeptide.
  • FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, LNCY, and TIGRFAM; and HMM-based protein domain databases such as SMART.
  • GenBank protein databases Genpept
  • PROTEOME databases
  • BLOCKS BLOCKS
  • PRINTS DOMO
  • PRODOM hidden Markov model
  • Prosite Prosite
  • HMM-based protein family databases such as PFAM, LNCY, and TIGRFAM
  • HMM-based protein domain databases such as SMART.
  • FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering
  • Polynucleotide and polypeptide sequence aHgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence aHgnment program (DNASTAR), which also calculates the percent identity between aHgned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides appHcable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where appHcable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
  • Genscan gene identification program against pubHc 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 (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354).
  • the program concatenates predicted exons to form an assembled cDNA sequence extending from a metMonine 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.
  • the encoded polypeptides were analyzed by querying against PFAM models for molecules for disease detection and treatment. Potential molecules for disease detection and treatment were also identified by homology to Incyte cDNA sequences that had been annotated as molecules for disease detection and treatment. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubHc databases.
  • Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis was also used to find any Incyte cDNA or pubHc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
  • Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • FuU length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubHc cDNA sequences using the assembly process described in Example LU.
  • Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example LV. 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 prograrnming to integrate cDNA and genomic information, generating possible spHce 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 fuU length with an algorithm based on BLAST analysis.
  • the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example LV.
  • a chimeric protein was generated by using the resultant Mgh-scoring segment pahs (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 Mgh-scoring segment pahs
  • GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubHc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of MDDT Encoding Polynucleotides The sequences which were used to assemble SEQ ED NO:40-78 were compared with sequences from the Incyte LEFESEQ database and pubHc domain databases using BLAST and other implementations ofthe Smith-Waterman algorithm.
  • pubHc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of aU sequences of that cluster, including its particular SEQ 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 tine teirminus of the chromosome's p- arm.
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceU type or tissue have been bound.
  • a membrane on which RNAs from a particular ceU type or tissue have been bound See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.
  • Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LLFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to deteirmine 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 normaHzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipHed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter ofthe two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quaHty in a BLAST aHgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter ofthe two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotide sequences encoding MDDT 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 JJI). Each cDNA sequence is derived from a cDNA Hbrary 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; genitaHa, female; genitaHa, male; germ ceUs; hemic and immune system; Hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognatbic system; unclassified/mixed; or urinary tract.
  • the number of Hbraries in each category is counted and divided by the total number of Hbraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Hbraries in each category is counted and divided by the total number of Hbraries across aU categories.
  • the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding MDDT.
  • cDNA sequences and cDNA Hbrary/tissue information are found in the LLFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of MDDT Encoding Polynucleotides
  • FuU length polynucleotide sequences were also produced by extension of an appropriate fragment of the fuU length molecule using oHgonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5 ' extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about,50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • Selected human cDNA Hbraries 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 and 0.5 ⁇ l of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan LT (Labsystems Oy, Helsinki, Finland) to measure the fluorescence ofthe sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aHquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to detemr ne which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WT), and sonicated or sheared prior to reHgation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WT
  • sonicated or sheared prior to reHgation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were reHgated using T4 Hgase (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. coH cells. 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 carb Hquid media..
  • Step 1 94 °C, 3 in; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72 °C, 5 min; Step 7: storage at 4 °C.
  • DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reampHfied using the same conditions as described above.
  • fuU length polynucleotide sequences are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with oHgonucleotides designed for such extension, and an appropriate genomic Hbrary.
  • SNPs single nucleotide polymorphisms
  • LIFESEQ database Incyte Genomics
  • Sequences from the same gene were clustered together and assembled as described in Example LTJ, 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 errors by requiring a minimum Phred quaHty score of 15, and removed sequence aHgnment errors and errors resulting from improper trimming of vector sequences, chimeras, and spHce variants.
  • Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze aUele 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 Annish 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 aUeHc variance in this population were not further tested in the other three populations.
  • Hybridization probes derived from SEQ ED NO:40-78 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oHgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • OHgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oHgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oHgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences).
  • An aHquot 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 ofthe foUowing endonucleases: Ase I, Bgl TJ, Eco RI, Pst I, Xba I, or Pvu TJ (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 visuaHzed using autoradiography or an alternative imaging means and compared.
  • the linkage or synthesis of array elements upon a microarray can be achieved utiHzing photoHthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each ofthe aforementioned technologies should be uniform and soHd with a non-porous surface (Schena (1999), supra).
  • Suggested substrates include siHcon, siHca, glass sHdes, glass chips, and siHcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechamcal 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. (See, e.g., Schena, M. et al. (1995) Science
  • FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oHgomers thereof may • , comprise the elements of the microarray. Fragments or oHgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry ma be used for detection of hybridization.
  • RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oHgo-(dT) cehulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oHgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits
  • RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPLN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
  • reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol.
  • the sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
  • Sequences of the present invention are used to generate array elements.
  • Each array element is ampHfied from bacterial ceUs containing vectors with cloned cDNA inserts.
  • PCR ampHfication uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are ampHfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • AmpHfied array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified array elements are immobilized on polymer-coated glass sHdes.
  • Glass microscope sHdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
  • Glass sHdes are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distiUed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol.
  • Coated sHdes are cured in a 110°C oven.
  • Array elements are appHed to the coated glass substrate using a procedure described in U.S.
  • Patent No. 5,807,522 incorporated herein by reference.
  • 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of array element sample per sHde.
  • Microarrays are UV-crosslinked using a STRATALINKER UV-crosslrhker (Stratagene).
  • Microarrays 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
  • 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 Hues at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser Hght is focused on the array using a 20X microscope objective (Nikon, Inc., MelviUe NY).
  • the sHde 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 array used in the present example is scanned with a resolution of 20 micrometers. hn two separate scans, a mixed gas multiline laser excites the two fluorophores sequentiaUy.
  • Emitted Hght is spHt, based on wavelength, into two photomultipHer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores.
  • PMT R1477 Hamamatsu Photonics Systems, Bridgewater NJ
  • filters positioned between the array and the photomultipHer tabes are used to filter the signals.
  • the .,. emission maxima ofthe fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typicaUy caHbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
  • the caHbration is done by labeling samples of the caHbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipHer tube is digitized using a 12-bit RTI-835H analog-to-digital
  • a D conversion board Analog Devices, Inc., Norwood MA
  • instaUed in an IBM-compatible PC computer The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). Array elements that exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentiaUy expressed using the GEMTOOLS program (Incyte Genomics). Expression
  • SEQ ID NO:40 expression of SEQ ID NO:40 was upregulated in PBMC ceHs stimulated with Hpopolysaccharide (LPS), a component of the bacterial ceU waU which induces an inflammatory response.
  • LPS Hpopolysaccharide
  • PBMCs coUected from the blood of four healthy donors was stimulated with 1 ⁇ g/ml LPS for 4, 24, and 72 hours.
  • the PBMCs contained about 52% lymphocytes (12% B-ceUs and 40% T- ceUs), 20% nataral kiUer ceUs, 25% monocytes, and 3% various ceUs including dendritic ceUs.
  • Stimulated ceUs were then compared to untreated, time-matched controls. Similarly, expression of SEQ JD NO:40 was upregulated in vascular tissue stimulated with the inflammatory cytokine TNF ⁇ or a combination ofthe protein kinase C activator, PMA, and ionomycin.
  • CeUs were then stimulated with either 10 ng/ml TNF ⁇ or 1 ⁇ M PMA, 1 ⁇ g/ml ionomycin over a defined time course. Upregulation of SEQ JD NO:40 in treated ceUs relative to untreated, time-matched controls was seen within 1-4 hours foUowing treatment.
  • SEQ LD NO:42 was downregulated in ovarian adenocarcinoma and abreast adenocarcinoma ceU Hue, BT-20, relative to normal ovary and breast, respectively.
  • Ovarian tumor tissue obtained from a 79-year-old female was compared to normal ovary obtained from the same donor.
  • BT-20 is a breast adenocarcinoma line derived in vitro from ceUs emigrating out of thin sHces of a tamor mass isolated form a 74-year-old female.
  • BT-20 ceUs were compared to primary mammary epitheHal ceUs (HMEC) and a breast mammary gland ceU line (MCF-10A) isolated from a 36-year-old woman with fibrocystic disease.
  • the breast ceU lines were grown in basal medium in the absence of growth factors and hormones for 24 hours prior to the comparison.
  • THP-1 is a promonocyte ceU line isolated from the peripheral blood of a 1-year-old male with acute monocytic leukemia. Upon stimulation with PMA, THP-1 differentiates into a macrophage-like ceU that displays many characteristics of peripheral human macrophages. THP-1 ceUs stimulated in vitro with 0.1 ⁇ M PMA and 1 ⁇ g/ml ionomycin for 0.5, 1, 2, 4, and 8 hours were compared to untreated, time-matched control ceUs. Expression of SEQ LD NO:45 was downregulated in several breast ceU cancer lines relative to HMECs.
  • CeU lines included BT-20, BT474, BT483, Hs578T, MCF-7, and MD-AMB-468.
  • Expression of SEQ JD NO:48 was upregulated in HUVECs stimulated with TNF ⁇ foUowing pre-treatment with either PMA or a low dose of TNF ⁇ .
  • HUVECs were pre-treated with either 100 nM PMA or 0.1 ng/ml TNF ⁇ for 24 hours, washed, and then stimulated with TNF ⁇ for an additional 1, 4, and 24 hours.
  • HUVECs were cultured in JJvLDM, 10% fetal calf serum at 37°C, 5% CO 2 . Treated ceUs were compared to untreated, time-matched controls.
  • SEQ ID NO:68 is upregulated 3.4 fold in matare DC versus monocytes, suggesting that SEQ JD NO:68, encoding SEQ ED NO:29, could be used for example, to understand the process by which monocytes differentiate into immature dendritic ceUs and eventuaUy aUow manipulation of the immune system leading to potential immunotherapies for diseases such as cancer, AIDS, and infectious diseases; and enhancing vaccine efficacy.
  • SEQ ID NO 8 showed differential expression in inflammatory responses as determined by microarray analysis.
  • The. expression of SEQ ID NO:78 was increased by at least two fold in THP-1 human promonocyte line which had been stimulated for 26 hours with 1 ⁇ M PMA (phorbol 12-myristate 13-acetate) when compared to untreated THP-1 ceUs.
  • PMA is abroad activator ofthe protein kinase C-dependent pathways.
  • THP-1 is promonocyte Hue derived from peripheral blood of a 1 year old male with acute monocytic leukemia.
  • the ceU line acquires monocytic characteristics upon stimulation with PMA.
  • Monocytes play a critical role in the initiation and maintenance of inflammatory immune responses.
  • SEQ JD NO78 is useful in diagnostic assays for inflammatory responses. Further, as deteirmined by microarray analysis, SEQ ID NO78 showed differential expression in SKBr3 breast carcinoma ceU line versus HMEC primary mammary epitheHal ceUs and MCF10A breast mammary gland ceUs.
  • SkBR3 is a breast adenocarcinoma ceU line isolated from a maHgnant pleural effusion of a 43 -year-old female.
  • HMEC a primary mammary epitheHal ceU Hue was derived from normal human mammary tissue (Clonetics, San Diego, CA).
  • MCF10A a breast mammary gland (luminal ductal characteristics) ceU Hue was isolated from a 36 year old woman with fibrocystic breast disease.
  • the microarray experiments showed that the expression of SEQ ID NO 8 was increased by at least two fold in SKBr3 breast adenocarcinoma line relative to ceUs from the primary mammary epitheHal ceU Hue, HMEC and the breast mammary gland ceU Hue, MCF10A. Therefore, SEQ JD NO:78 is useful as diagnostic markers or as potential therapeutic targets for breast cancer.
  • SEQ 3D NO:78 showed differential expression in MDAPCa2b prostate adenocarcinoma ceU line versus PrEC normal prostate epitheHal cells as determined by microarray analysis.
  • MDAPCa2b is a prostate adenocarcinoma ceU Hue isolated from a metastatic site in the bone of a 63-year-old male.
  • MDAPCa2b ceU line expresses prostate specific antigen (PSA) and androgen receptor, grows in vitro and vivo, and is androgen sensitive.
  • PSA prostate specific antigen
  • PrEC is a primary prostate epitheHal ceU line isolated from a normal donor.
  • SEQ ID NO78 is useful as a diagnostic marker or as a potential therapeutic target for prostate cancer.
  • Sequences complementary to the MDDT-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naruraUy occurring MDDT.
  • oligonucleotides comprising from about 15 to 30 base pairs is described, essentiaHy the same procedure is used with s aUer or with larger sequence fragments.
  • oHgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of MDDT.
  • a complementary oHgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to th coding sequence.
  • a complementary oHgonucleotide is designed to prevent ribosomal binding to the MDDT-encoding transcript.
  • Expression and purification of MDDT is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the ti ⁇ -l ⁇ c (toe) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the l ⁇ c operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express MDDT upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • IPTG isopropyl beta-D- thiogalactopyranoside
  • Expression of MDDT in eukaryotic ceUs is achieved by infecting insect or mammaHan ceU lines with recombinant Autographica caHfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica caHfornica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of bacu ⁇ ovirus is replaced with cDNA encoding MDDT by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates.
  • Viral infectivity is maintained an the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection ofthe latter requires additional genetic modifications to baculovirus.
  • Spodoptera frugiperda Sf9 insect ceUs in most cases, or human hepatocytes, in some cases. Infection ofthe latter requires additional genetic modifications to baculovirus.
  • MDDT is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU lysates.
  • GST a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
  • the GST moiety can be proteolyticaUy cleaved from MDDT 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 (1995, supra, ch. 10 and 16). Purified MDDT obtained by these methods can be used directly in the assays shown in Examples XVJJ and XVLO, where appHcable. XIV. Functional Assays
  • MDDT function is assessed by expressing the sequences encoding MDDT at physiologicaUy elevated levels in mammaHan ceU culture systems.
  • cDNA is subcloned into a mammaHan 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 line, for example, an endotheHal or hematopoietic ceU Hue, using either Hposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected ceUs and is a reHable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein
  • FCM Flow cytometry
  • CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human irrimunoglobulin 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 MDDT and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • PAGE polyacrylamide gel electrophoresis
  • the MDDT amino acid sequence is analyzed using LASERGENE software
  • oHgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppHed Biosystems) using EMOC chemistry and coupled to KLH (Sigma- Aldrich, St.
  • NataraUy occurring or recombinant MDDT is substantiaUy purified by iirnmunoaffmity chromatography using antibodies specific for MDDT.
  • An iimmunoaffinity column is constructed by covalently coupling anti-MDDT 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 MDDT are passed over the irnmunoafnnity column, and the column is washed under conditions that aUow the preferential absorbance of MDDT (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/MDDT 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 MDDT is coUected.
  • aUow the preferential absorbance of MDDT e.g., high ionic strength buffers in the presence of detergent.
  • the column is eluted under conditions that disrupt antibody/MDDT 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 MDDT is coUected.
  • a chaotrope such as urea
  • MDDT or biologicaUy active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • Bolton-Hunter reagent See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
  • Candidate molecules previously arrayed in the weUs of a multi-weU plate are incubated with the labeled MDDT, washed, and any weUs with labeled MDDT complex are assayed. Data obtained using different concentrations of MDDT are used to calculate values for the number, affinity, and association of MDDT with the candidate molecules.
  • MDDT molecules interacting with MDDT 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). MDDT may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a Hgh-tbroughput manner to determine aU interactions between the proteins encoded by two large Hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101). XVIII.
  • Phorbol ester binding activity of MDDT is measured using an assay based on the fluorescent phorbol ester sapinotoxin-D (SAPD). Binding of SAPD to MDDT is quantified by measuring the resonance energy transfer from MDDT tryptophans to the 2-(N-methylamino)berizoyl fluorophore of the phorbol ester, as described by Slater et al. (((1996) J. Biol. Chem. 271:4627-4631).
  • MDDT activity is associated with its abiHty to form protein-protein complexes and is measured by its abiHty to regulate growth characteristics of NIH3T3 mouse fibroblast ceUs.
  • a cDNA encoding MDDT is subcloned into an appropriate eukaryotic expression vector. This vector is transfected into NLH3T3 ceUs using methods known in the art. Transfected ceUs are compared with non-transfected ceUs for the foUowing quantifiable properties: growth in culture to high density, reduced attachment of ceUs to the substrate, altered ceU morphology, and abiHty to induce tumors when injected into iimnnunodeficient mice.
  • the activity of MDDT is proportional to the extent of increased growth or frequency of altered ceU morphology in NLH3T3 ceUs transfected with MDDT.
  • MDDT activity is measured by binding of MDDT to radiolabeled foirmdn polypeptides containing the proline-rich region that specificaUy binds to SH3 containing proteins (Chan, D.C et al. (1996) EMBO J. 15:1045-1054).
  • Samples of MDDT are run on SDS-PAGE gels, and transferred onto nitrocehulose by electtoblotting. The blots are blocked for 1 hr at room temperature in TBST (137 mM NaCI, 2.7 mM KCl, 25 mM Tris (pH 8.0) and 0.1% Tween-20) containing non-fat dry milk.
  • Blots are then incubated with TBST containing the radioactive forrnin polypeptide for 4 hrs to overnight. After washing the blots four times with TBST, the blots are exposed to autoradiographic film. Radioactivity is quantitated by cutting out the radioactive spots and counting them in a radioisotope counter. The amount of radioactivity recovered is proportional to the activity of MDDT in the assay.
  • MDDT protein kinase activity is measured by quantifying the phosphorylation of an appropriate substrate in the presence of gamma-labeled 3Z P-ATP. MDDT is incubated with the substrate, 32 P-ATP, and an appropriate kinase buffer. The 32 P incorporated into the product is separated from free 32 P-ATP by electrophoresis, and the incorporated 3 P is quantified using a beta radioisotope counter. The amount of incorporated 32 P is proportional to the protein kinase activity of MDDT in the assay. A determination ofthe specific amino acid residue phosphorylated by protein kinase activity is made by phosphoamino acid analysis of the hydrolyzed protein.
  • an assay for MDDT protein phosphatase activity measures the hydrolysis of para-nitrophenyl phosphate (PNPP).
  • MDDT is incubated together with PNPP in HEPES buffer pH 7.5, in the presence of 0.1% ⁇ -mercaptoethanol at 37°C for 60 min.
  • the reaction is stopped by tine addition of 6 ml of 10 N NaOH, and the increase in Hght absorbance of the reaction mixtare at 410 nm resulting from the hydrolysis of PNPP is measured using a spectrophotometer.
  • the increase in Hght absorbance is proportional to the activity of MDDT in the assay (Diamond, R.H. et al. (1994) Mol. CeU Biol. 14:3752-3762).
  • adenylyl cylcase activity of MDDT is demonstrated by the abiHty to convert ATP to cAMP (Mittal, C.K. (1986) Meth. Enzymol. 132:422-428).
  • MDDT is incubated with the substrate [ ⁇ - 32 P]ATP, foUowing which the excess substrate is separated from the product cycHc [ 32 P] AMP.
  • MDDT activity is deteirrnhned in 12 x 75 mm disposable cultare tubes containing 5 ⁇ l of 0.6 M Tris-HCl, pH 7.5, 5 ⁇ l of 0.2 M MgCl 2 , 5 ⁇ l of 150 mM creature phosphate containing 3 units of creatine phosphokinase, 5 ⁇ l of 4.0 mM l-methyl-3-isobutykanthine, 5 ⁇ l of 20 mM cAMP, 5 ⁇ l 20 mM dithiothreitol, 5 ⁇ l of 10 mM ATP, 10 ⁇ l [ ⁇ - 32 P]ATP (2-4 x 10 6 cpm), and water in a total volume of 100 ⁇ l.
  • the reaction mixtare is prewarmed to 30 °C
  • the reaction is initiated by adding MDDT to the prewarmed reaction mixtare. After 10-15 minutes of incubation at 30 °C, the reaction is terminated by adding 25 ⁇ l of 30% ice-cold trichloroacetic acid (TCA). Zero-time incubations and reactions incubated in the absence of MDDT are used as negative controls. Products are separated by ion exchange chromatography, and cycHc [ 32 P] AMP is quantified using a ⁇ -radioisotope counter. The MDDT activity is proportional to the amount of cycHc [ 32 P] AMP formed in the reaction.
  • An alternative assay measures MDDT-mediated G-protein signaling activity by monitoring the mobiUzation of Ca 2+ as an indicator ofthe signal transduction pathway stimulation.
  • the assay requires preloading neutrophils or T ceUs with a fluorescent dye such as FURA-2 or BCECF (Universal Imaging Corp, Westchester PA) whose emission characteristics are altered by Ca 2+ binding.
  • Ca 2+ flux takes place. This flux can be observed and quantified by assaying the ceUs in a fluorometer or fluorescent activated ceU sorter. Measurements of Ca 2+ flux are compared between ceUs in their normal state and those transfected with MDDT. Increased Ca 2+ mobilization attributable to increased MDDT concentration is proportional to MDDT activity.
  • activating stimuH artificiaUy e.g., anti-CD3 antibody Hgation of the T ceU receptor
  • physiologicaUy e.g., by aUogeneic stimulation
  • GTP-binding activity of MDDT is determined in an assay that measures the binding of MDDT to [ ⁇ - 32 P]-labeled GTP.
  • Purified MDDT is first blotted onto filters and rinsed in a suitable buffer. The filters are then incubated in buffer containing radiolabeled [ ⁇ - 32 P]-GTP. The filters are washed in buffer to remove unbound GTP and counted in a radioisotope counter.
  • Nonspecific binding is determined in an assay that contains a 100-fold excess of unlabeled GTP. The amount of specific binding is proportional to the activity of MDDT.
  • GTPase activity of MDDT is determined in an assay that measures the conversion of [ ⁇ - 32 P]-GTP to [ ⁇ - 32 P]-GDP.
  • MDDT is incubated with [ ⁇ - 32 P]-GTP in buffer for an appropriate period of time, and the reaction is ternninated by heating or acid precipitation foUowed by centrifugation.
  • An aHquot of the supernatant is subjected to polyacrylamide gel electrophoresis (PAGE) to separate GDP and GTP together with unlabeled standards.
  • the GDP spot is cut out and counted in a radioisotope counter.
  • the amount of radioactivity recovered in GDP is proportional to the GTPase activity of MDDT.
  • MDDT activity is measured by quantifying the amount of a non-hydrolyzable
  • GTP analogue GTPyS
  • Varying amounts of MDDT are incubated at 30°C in 50 mM Tris buffer, pH 7.5, containing 1 mM dithiothreitol, 1 mM EDTA and 1 ⁇ M [ 35 S]GTP ⁇ S.
  • Samples are passed through nitroceUulose filters and washed twice with a buffer consisting of 50 mM Tris-HCl, pH 7.8, 1 mM NaN 3 , 10 mM MgCl 2 , 1 mM EDTA, 0.5 mM dithiothreitol, 0.01 mM PMSF, and 200 mM NaCI.
  • the filter-bound counts are measured by Hquid scintiUation to quantify the amount of bound [ 35 S]GTP ⁇ S.
  • MDDT activity may also be measured as the amount of GTP hydrolysed over a 10 minute incubation period at 37 °C. MDDT is incubated in 50mM Tris-HCl buffer, pH 7.8, containing lmM dithiothreitol, 2mM EDTA, 10/ Vl [ ⁇ - 32 P]GTP, and 1 ⁇ M H-rab protein.
  • GTPase activity is initiated by adding MgClh, to a final concentration of 10 mM. Samples are removed at various time points, mixed with an equal volume of ice-cold 0.5mM EDTA, and frozen. AHquots are spotted onto polyethyleneimine-ceUulose thin layer chromatography plates, which are developed in 1M LiCl, dried, and autoradiographed. The signal detected is proportional to MDDT activity.
  • MDDT activity may be demonstrated as the abiHty to interact with its associated low molecular weight (LMW) GTPase in an in vitro binding assay.
  • LMW GTPases are expressed as fusion proteins with glutathione S-transferase (GST), and purified by affinity chromatography on glutathione-Sepharose.
  • GST glutathione S-transferase
  • the LMW GTPases are loaded with GDP by incubating 20 mM Tris buffer, pH 8.0, containing 100 mM NaCI, 2 mM EDTA, 5 mM MgCh., 0.2 mM DTT, 100 ⁇ M AMP-PNP and 10 ⁇ M GDP at 30°C for 20 minutes.
  • MDDT is expressed as a FLAG fusion protein in a baculovirus system. Extracts of these baculovirus ceUs containing MDDT-FLAG fusion proteins are precleared with GST beads, then incubated with GST-GTPase fusion proteins. The complexes formed are precipitated by glutathione-Sepharose and separated by SDS- polyacrylamide gel electrophoresis. The separated proteins are blotted onto mtroceUulose membranes and probed with commerciaUy available anti-FLAG antibodies. MDDT activity is proportional to the amount of MDDT-FLAG fusion protein detected in the complex.
  • yeast two-hybrid system Zalcman, G. et al. (1996) J. Biol. Chem. 271:30366-30374.
  • a plasmid such as pGAD1318 which may contain the coding region of MDDT can be used to transform reporter L40 yeast ceUs which contain the reporter genes LacZ and HIS 3 downstream from the binding sequences for LexA.
  • yeast ceUs have been previously transformed with a pLexA-Rab6-GDP (mouse) plasmid or with a plasmid which contains pLexA-lamin C.
  • the pLEXA-lamin C ceUs serve as a negative control.
  • the transformed ceUs are plated on a histidine-free medium and incubated at 30 °C for 3 days. His + colonies are subsequently patched on selective plates and assayed for ⁇ - galactosidase activity by a filter assay. MDDT binding with Rab6-GDP is indicated by positive His + /lacZ + activity for the ceUs transformed with the plasmid containing the mouse Rab6-GDP and negative His + /lacZ + activity for those transformed with the plasmid containing lamin C.
  • MDDT activity is measured by binding of MDDT to a substrate which recognizes WD-40 repeats, such as ElongihB, by coimmunoprecipitation (Kamura, T. et al. (1998) Genes Dev. 12:3872-3881). Briefly, epitope tagged substrate and MDDT are mixed and immunoprecipitated with commercial antibody against the substrate tag. The reaction solution is run on SDS-PAGE and the presence of MDDT visuaHzed using an antibody to the MDDT tag. Substrate binding is proportional to MDDT activity. Alternatively, MDDT activity is measured by its inclusion in coated vesicles.
  • MDDT can be expressed by ttansfoirming a mammaHan ceU line such as COS7, HeLa, or CHO with a eukaryotic expression vector encoding MDDT.
  • Eukaryotic expression vectors are commerciaUy available, and the techniques to introduce them into ceUs are weU known to those skiUed in the art.
  • ceUs are incubated for 48-72 hours after transformation under conditions appropriate for the ceU line to aUow expression and accumulation of MDDT and ⁇ - galactosidase.
  • MDDT activity is measured by its abiHty to alter vesicle trafficking pathways.
  • Vesicle trafficking in ceUs transformed with MDDT is examined using fluorescence microscopy.
  • Antibodies specific for vesicle coat proteins or typical vesicle trafficking substrates such as transferrin or the mannose-6-phosphate receptor are commerciaUy available.
  • Various ceUular components such as ER, Golgi bodies, peroxisomes, endosomes, lysosomes, and the plasmalemma are examined.
  • Transport vesicles are salt-released from the Golgi membranes, loaded under a sucrose gradient, centrifuged, and fractions are coUected and analyzed by SDS-PAGE.
  • Co-localization of MDDT with clathrin or COP coatamer is indicative of MDDT activity in vesicle formation.
  • the contribution of MDDT in vesicle formation can be confirmed by mcubating lysates with antibodies specific for MDDT prior to GTP ⁇ S addition.
  • the antibody wiU bind to MDDT and interfere with its activity, thus preventing vesicle formation.
  • ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
  • ABI/PARACEL FDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
  • ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
  • fastx score 100 or greater
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
  • TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
  • HMM hidden Markov model

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Abstract

L'invention concerne des molécules humaines de détection et de traitement de maladies (MDDT) et des polynucléotides qui identifient et codent ces MDDT. L'invention concerne également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. L'invention concerne enfin des méthodes de diagnostic, de traitement et de prévention de troubles liés à une expression aberrante de MDDT.
EP02739428A 2001-05-25 2002-05-24 Molecules de detection et de traitement de maladies Withdrawn EP1390410A4 (fr)

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US29372301P 2001-05-25 2001-05-25
US293723P 2001-05-25
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US295257P 2001-06-01
US29722001P 2001-06-08 2001-06-08
US297220P 2001-06-08
US30052601P 2001-06-21 2001-06-21
US300526P 2001-06-21
US30187401P 2001-06-29 2001-06-29
US301874P 2001-06-29
US35941302P 2002-02-22 2002-02-22
US359413P 2002-02-22
PCT/US2002/016676 WO2002096951A1 (fr) 2001-05-25 2002-05-24 Molecules de detection et de traitement de maladies

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AU2002226912A1 (en) 2000-11-16 2002-05-27 Cedars-Sinai Medical Center Profiling tumor specific markers for the diagnosis and treatment of neoplastic disease
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WO2000075298A2 (fr) * 1999-06-03 2000-12-14 Incyte Genomics, Inc. Molecules pour la detection et le traitement de maladies

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WO2000075298A2 (fr) * 1999-06-03 2000-12-14 Incyte Genomics, Inc. Molecules pour la detection et le traitement de maladies

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 1 October 2000 (2000-10-01), "Interleukin-6 precursor (IL-6) (B-cell stimulatory factor 2) (BSF-2) (Interferon beta-2) (Hybridoma growth factor) (CTL differentiation factor) (CDF)" XP002308417 Database accession no. P05231 -& YASUKAWA K ET AL: "STRUCTURE AND EXPRESSION OF HUMAN B CELL STIMULATORY FACTOR-2 (BSF-2/IL-6) GENE" EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 6, no. 10, 1987, pages 2939-2945, XP002950930 ISSN: 0261-4189 *
PAPANICOLAOU D A ET AL: "The pathophysiologic roles of interleukin-6 in human disease." ANNALS OF INTERNAL MEDICINE. 15 JAN 1998, [Online] vol. 128, no. 2, 15 January 1998 (1998-01-15), pages 127-137, XP002308416 ISSN: 0003-4819 Retrieved from the Internet: URL:http://www.annals.org/cgi/content/full /128/2/127> [retrieved on 2004-12-01] *
SAVINO R ET AL: "GENERATION OF INTERLEUKIN-6 RECPETOR ANTAGONISTS BY MOLECULAR-MODELING GUIDED MUTAGENESIS OF RESIDUES IMPORTANT FOR GP130 ACTIVATION" EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 13, no. 6, 15 March 1994 (1994-03-15), pages 1357-1367, XP000565719 ISSN: 0261-4189 *
See also references of WO02096951A1 *

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WO2002096951A1 (fr) 2002-12-05

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