WO2002070709A2 - Molecules for disease detection and treatment - Google Patents
Molecules for disease detection and treatment Download PDFInfo
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- WO2002070709A2 WO2002070709A2 PCT/US2002/003709 US0203709W WO02070709A2 WO 2002070709 A2 WO2002070709 A2 WO 2002070709A2 US 0203709 W US0203709 W US 0203709W WO 02070709 A2 WO02070709 A2 WO 02070709A2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to nucleic acid and a ino acid sequences of full-length human molecules for disease detection and treatment and to the use of these sequences i the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, developmental, neurological, and cardiovascular disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of full-length human molecules for disease detection and treatment.
- 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 inhibiting cell proliferation. Mutations which reduce or abrogate the function of tumor-suppressor genes result in aberrant cell proliferation and cancer.
- DNA-based arrays can provide an efficient, Hgh-throughput method to examine gene expression and genetic variability.
- SNPs single nucleotide polymo ⁇ hisms
- DNA-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, hi this case, it is useful to develop a profile, or transcript image, of all the genes that are expressed and the levels at which they are expressed in that particular 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.)
- DMR-N9 myotonic dystrophy
- 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).
- Other genes are identified based upon then expression patterns or association with disease syndromes. For example, autoant ⁇ bodies 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. Autoirnmun. 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. 229:902-909).
- Calponin is an actin-binding protein that may participate in the function and organization the cytoskeleton (Takahashi, K. et al. (1986) Biochem. Biophys. Res. Commun. 141:20-26).
- the N- terminus of calponin can interact with calcium-binding proteins and tropomyosin.
- CH-domain calponin homology domain
- secreted Proteins secreted Proteins
- Protein transport and secretion are essential for cellular function.
- Protem transport is mediated by a signal peptide located at the amino terminus of the protein to be transported or secreted.
- the signal peptide is comprised of about ten to twenty hydrophobic amino acids which target the nascent protein from the ribosome to a particular membrane bound compartment such as the endoplasmic reticulum (ER).
- ER endoplasmic reticulum
- Proteins targeted to the ER may either proceed through the secretory pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or lysosomes. Proteins that transit through the secretory pathway are either secreted into the extracellular space or retained in the plasma membrane.
- Proteins that are retained in the plasma membrane contain one or more transmembrane domains, each comprised of about 20 hydrophobic amino acid residues.
- Secreted proteins are generally synthesized as inactive precursors that are activated by post- translational processing events during transit through the secretory pathway. Such events include glycosylation, proteolysis, and removal of the signal peptide by a signal peptidase. Other events that may occur during protein transport include chaperone-dependent unfolding and folding of the nascent protein and interaction of the protein with a receptor or pore complex. Examples of secreted proteins with amino terminal signal peptides are discussed below and include proteins with important roles in cell-to-cell signaling.
- Such proteins include transmembrane receptors and cell surface markers, extracellular matrix molecules, cytokines, hormones, growth and differentiation factors, enzymes, neuropeptides, vasomediators, cell surface markers, and antigen recognition molecules.
- Cell surface markers include cell surface antigens identified on leukocytic cells of the immune system. These antigens have been identified using systematic, monoclonal antibody (mAb)-based "shot gun” techniques. These techniques have resulted in the production of hundreds of mAbs directed against unknown cell surface leukocytic antigens.
- CD antigens have been grouped into “clusters of differentiation” based on common immunocytochemical localization patterns in various differentiated and undifferentiated leukocytic cell types. Antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a "cluster of differentiation” or "CD” designation. Some of the genes encoding proteins identified by CD antigens have been cloned and verified by standard molecular biology techniques. CD antigens have been characterized as both transmembrane proteins and cell surface proteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI). (Reviewed in Barclay, A.N. et al. (1995) The Leucocyte Antigen Facts Book. Academic Press, San Diego, CA, pp. 17-20.)
- GPI glycosylphosphatidylinositol
- MPs Matrix proteins
- the expression and balance of MPs may be perturbed by biochemical changes that result from congenital, epigenetic, or infectious diseases, ha addition, MPs affect leukocyte migration, proliferation, differentiation, and activation in the immune response.
- MPs are frequently characterized by the presence of one or more domains which may include collagen-like domains, EGF-like domains, irnmunoglobulin-like domains, and fibronectin-like domains.
- MPs may be heavily glycosylated and may contain an Arginine-Glycine-Aspartate (RGD) tripeptide motif which may play a role in adhesive interactions.
- MPs include extracellular proteins such as fibronectin, collagen, galectin, vitronectin and its proteolytic derivative somatomedin B; and cell adhesion receptors such as cell adhesion molecules (CAMs), cadherins, and integrins.
- Mucins are highly glycosylated glycoproteins that are the major structural component of the mucus gel. The physiological functions of mucins are cytoprotection, mechanical protection, maintenance of viscosity in secretions, and cellular recognition.
- MUC6 is a human gastric mucin that is also found in gall bladder, pancreas, seminal vesicles, and female reproductive tract (Toribara, N.W. et al. (1997) J. Biol. Chem. 272:16398-16403). The MUC6 gene has been mapped to human chromosome 11 (Toribara, N.W. et al. (1993) J. Biol. Chem. 268:5879-5885).
- Hemomucin is a novel Drosophila surface mucin that may be involved in the induction of antibacterial effector molecules (Theopold, U. et al. (1996) J. Biol. Chem. 217:12708-12715).
- Tuftelins are one of four different enamel matrix proteins that have been identified so far.
- the other three known enamel matrix proteins are the amelogenrns, enamelin and ameloblastin. Assembly of the enamel extracellular matrix from these component proteins is believed to be critical in producing a matrix competent to undergo mineral replacement.
- Tuftelin mRNA has been found to be expressed in human ameloblastoma tumor, a non-mineralized odontogenic tumor (Deutsch, D. et al. (1998) Connect. Tissue Res. 39:177-184).
- Olfactomedin-related proteins are extracellular matrix, secreted glycoproteins with conserved
- Olfactomedin-related proteins comprise a gene family with at least 5 family members in humans.
- One of the five, ⁇ GR/myocilin protein is expressed in the eye and is associated with the pathogenesis of glaucoma (Kulkarni, N.H. et al. (2000) Genet. Res. 76:41-50). Research by Yokoyama et al.
- AMY 135-amino acid protein
- Mac-2 binding protein is a 90-kD serum protein (90K), a secreted glycoprotein isolated from both the human breast carcinoma cell line SK-BR-3 , and human breast milk. It specifically binds to a human macrophage-associated lectin, Mac-2. Structurally, the mature protein is 567 amino acids in length and is proceeded by an 18-arnino acid leader. There are 16 cysteines and seven potential N- linked glycosylation sites. The first 106 amino acids represent a domain very similar to an ancient protein superfamily defined by a macrophage scavenger receptor cysteine-rich domain (Koths, K. et al. (1993) J. Biol. Chem. 268:14245-14249).
- 90K is elevated in the serum of subpopulations of AIDS patients and is expressed at varying levels in primary tumor samples and tumor cell lines. IJllrich et al. (1994) have demonstrated that 90K stimulates host defense systems and can induce interleukin-2 secretion. This immune stimulation is proposed to be a result of oncogenic transformation, viral infection or pathogenic invasion (Ullrich, A. et al. (1994) J. Biol. Chem. 269:18401-18407).
- Semaphorins are a large group of axonal guidance molecules consisting of at least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the se a domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro. The extracellular region of neuropilins consists of three different domains: CUB, discoidin, and MAM domains. The CUB and the MAM motifs of neuropilin have been suggested to have roles in protein-protein interactions and are thought to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J.A.
- Plexins are neuronal cell surface molecules that mediate cell adhesion via a homophilic bmding mechanism in the presence of calcium ions. Plexins have been shown to be expressed in the receptors and neurons of particular sensory systems (Ohta, K. et al. (1995) Cell 14:1189-1199). There is evidence that suggests that some plexins function to control motor and CNS axon guidance in the developing nervous system. Plexins, which themselves contain complete semaphorin domains, may be both the ancestors of classical semaphorins and binding partners for semaphorins (Winberg, M.L. et al (1998) Cell 95:903-916).
- PSG Human pregnancy-specific beta 1-glycoprotein
- Autocrine motility factor is one of the motility cytokines regulating tumor cell migration; therefore identification of the signaling pathway coupled with it has critical importance.
- Autocrine motility factor receptor (AMFR) expression has been found to be associated with tumor progression in thymo a (Ohta Y. et al. (2000) Int. J. Oncol. 17:259-264).
- AMFR is a cell surface glycoprotein of molecular weight 78KDa.
- Hormones are secreted molecules that travel through the circulation and bind to specific receptors on the surface of, or within, target cells. Although they have diverse biochemical compositions and mechanisms of action, hormones can be grouped into two categories.
- One category includes small hpophilic hormones that diffuse through the plasma membrane of target cells, bind to cytosolic or nuclear receptors, and form a complex that alters gene expression. Examples of these molecules include retinoic acid, thyroxine, and the cholesterol-derived steroid hormones such as progesterone, estrogen, testosterone, cortisol, and aldosterone.
- the second category includes hydrophilic hormones that function by binding to cell surface receptors that transduce signals across the plasma membrane.
- hormones include amino acid derivatives such as catecholamines (epinephrine, norepmephrine) and l ⁇ starnine, and peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystoltirrin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin.
- catecholamines epinephrine, norepmephrine
- peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystoltirrin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin.
- Pro-opiomelanocortin is the precursor polypeptide of corticotropin (ACTH), a hormone synthesized by the anterior pituitary gland, which functions in the stimulation of the adrenal cortex. POMC is also the precursor polypeptide of the hormone beta-lipotropin (beta-LPH). Each hormone includes smaller peptides with distinct biological activities: alpha-melanotropin (alpha-MSH) and corticotropin-like intermediate lobe peptide (CLJP) are formed from ACTH; gamma-lipotiOpin (gamma-LPH) and beta-endo ⁇ hin are peptide components of beta-LPH; while beta-MSH is contained within gamma-LPH.
- ACTH corticotropin
- beta-LPH beta-lipotropin
- Adrenal insufficiency due to ACTH deficiency results in an endocrine disorder characterized by early- onset obesity, adrenal insufficiency, and red hair pigmentation (Chretien, M. et al. (1979) Can. J. Biochem. 57:1111-1121; Krude, H. et al. (1998) Nat. Genet. 19:155-157; Online Mendelian Inheritance in Man (OMTM) 176830).
- Growth and differentiation factors are secreted proteins which function in intercellular communication. Some factors require oligomerization or association with membrane proteins for activity. Complex interactions among these factors and their receptors trigger intracellular signal transduction pathways that stimulate or inhibit cell division, cell differentiation, cell signaling, and cell motility. Most growth and differentiation factors act on cells in their local environment (paracrine signaling).
- the first class includes the large polypeptide growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor, msulin-like growth factor, and platelet-derived growth factor.
- the second class includes the hematopoietic growth factors such as the colony stimulating factors (CSFs).
- CSFs colony stimulating factors
- Hematopoietic growth factors stimulate the proliferation and differentiation of blood cells such as B- lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and then stem cell precursors.
- the thhd class includes small peptide factors such as bombesin, vasopressin, oxytocin, endothelin, transferrin, angiotensin II, vasoactive intestinal peptide, and bradyldnin, which function as hormones to regulate cellular functions other than proliferation.
- Growth and differentiation factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo.
- Inappropriate expression of growth factors by tumor cells may contribute to vascularization and metastasis of tumors.
- growth factor misregulation can result in anemias, leukemias, and lymphomas.
- Certain growth factors such as interferon are cytotoxic to tumor cells both in vivo and in vitro.
- some growth factors and growth factor receptors are related both structurally and functionally to oncoproteins.
- growth factors affect transcriptional regulation of both proto-oncogenes and oncosuppressor genes. (Reviewed in Pimentel, E. (1994) Handbook of Growth Factors, CRC Press, Ann Arbor, MI, pp.
- the Slit protein first identified in Drosophila, is critical in central nervous system rnidline formation and potentially in nervous tissue histogenesis and axonal pathfinding. Itoh et al. ((1998) Brain Res. Mol. Brain Res. 62:175-186) have identified mammalian homologues of the slit gene (human Slit-1, Slit-2, Slit-3 and rat Slit-1). The encoded proteins are putative secreted proteins containing EGF-like motifs and leucine-rich repeats, both of which are conserved protein-protein interaction domains. Slit-1, -2, and -3 mRNAs are expressed in the brain, spinal cord, and thyroid, respectively (Itoh, A. et al., supra).
- NP/VM neuropeptides and vasomediators
- neuropeptides and neuropeptide hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachy nins, urotensin H and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-borne signaling molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon, cholecystokinin and gastrin.
- neuropeptide hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachy nins, urotensin H and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptid
- NP/VMs can transduce signals directly, modulate the activity or release of other neurotransmitters and hormones, and act as catalytic enzymes in cascades.
- the effects of NP/VMs range from extremely brief to long-lasting. (Reviewed in Martin, CR. et al. (1985) Endocrine Physiology, Oxford University Press, New York, NY, pp. 57-62.)
- NP/VMs are involved in numerous neurological and cardiovascular disorders.
- neuropeptide Y is involved in hypertension, congestive heart failure, affective disorders, and appetite regulation.
- Somatostatin inhibits secretion of growth hormone and prolactin in the anterior pituitary, as well as inMbiting secretion in intestine, pancreatic acinar cells, and pancreatic beta-cells.
- a reduction in somatostatin levels has been reported in Alzheimer's disease and Parkinson's disease.
- Nasopressin acts in the kidney to increase water and sodium abso ⁇ tion, and in higher concentrations stimulates contraction of vascular smooth muscle, platelet activation, and glycogen breakdown in the liver.
- Vasopressin and its analogues are used clinically to treat diabetes insipidus.
- Endothelin and angiotensin are involved in hypertension, and drugs, such as captopril, which reduce plasma levels of angiotensin, are used to reduce blood pressure (Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 194; 252; 284; 55; 111).
- Neuropeptides have also been shown to have roles in nociception (pain). Vasoactive intestinal peptide appears to play an important role in chronic neuropathic pain. Nociceptin, an endogenous ligand for for the opioid receptor-like 1 receptor, is thought to have a predominantly anti-nociceptive effect, and has been shown to have analgesic properties in different animal models of tonic or chronic pain (Dickinson, T. and Fleetwood-Walker, S.M. (1998) Trends Pharmacol. Sci. 19:346-348).
- proteins that contain signal peptides include secreted proteins with enzymatic activity. Such activity includes, for example, oxidoreductase/dehydrogenase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, or ligase activity.
- matrix metalloproteinases are secreted hydrolytic enzymes that degrade the extracellular matrix and thus play an important role in tumor metastasis, tissue morphogenesis, and arthritis (Reponen, P. et al. (1995) Dev. Dyn. 202:388-396; Firestein, G.S. (1992) Curr. Opin. Rheumatol. 4:348-354; Ray, J.M.
- acetyl-CoA synthetase activity is the formation of acetyl-CoA from acetate and CoA.
- Acetyl-CoA sythetases share a region of sequence similarity identified as the AMP-binding domain signature.
- Acetyl-CoA synthetase has been shown to be associated with hypertension (Toh, H. (1991) Protein Seq. Data Anal. 4:111-117; and Iwai, N. et al. (1994) Hypertension 23 :375-380).
- PPIases peptidyl-prolyl cis-trans isomerases
- FKBPs FK506 binding proteins
- CyPs cyclopMlins
- FKBPs the PPIase activity of FKBPs is inhibited by binding of FK506 or rapamycin.
- FKBP12, FKBP13, FKBP25, FKBP52, and FKBP65 the members of the FKBP family which are named according to their calculated molecular masses (FKBP12, FKBP13, FKBP25, FKBP52, and FKBP65), and localized to different regions of the cell where they associate with different protein complexes (Coss, M. et al. (1995) J. Biol. Chem. 270:29336-29341; Schreiber, S.L. (1991) Science 251:283-287).
- CyP The peptidyl-prolyl isomerase activity of CyP may be part of the signaling pathway that leads to T-cell activation. CyP isomerase activity is associated with protein folding and protein ixafficking, and may also be involved in assembly/disassembly of protein complexes and regulation of protein activity. For example, in Drosophila, the CyP NinaA is required for correct localization of rhodopsins, while a mammalian CyP (Cyp40) is part of the Hsp90/Hsc70 complex that binds steroid receptors.
- the mammalian CypA has been shown to bind the gag protein from human immunodeficiency virus 1 (HJV-1), an interaction that can be inhibited by cyclosporin. Since cyclosporin has potent anti-HJN-1 activity, CypA may play an essential function in HJN-1 replication. Finally, Cyp40 has been shown to bind and inactivate the transcription factor c-Myb, an effect that is reversed by cyclosporin. This effect implicates CyPs in the regulation of transcription, transformation, and differentiation (Bergsma, D.J. et al (1991) J. Biol. Chem. 266:23204-23214; Hunter, T. (1998) Cell 92:141-143; and Leverson, J.D.
- Gamma-carboxygluta ic acid (Gla) proteins rich in proline are members of a family of vitamin K-dependent single-pass integral membrane proteins. These proteins are characterized by an extracellular amino terminal domain of approximately 45 amino acids rich in Gla. The intracellular carboxyl terminal region contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover (Kulman, J.D. et al. (2001) Proc. ⁇ atl. Acad. Sci. USA 98:1370- 1375).
- Gla The process of post-translational modification of glutamic residues to form Gla is Vitamin K- dependent carboxylation.
- Proteins which contain Gla include plasma proteins involved in blood coagulation. These proteins are profhrombin, proteins C, S, and Z, and coagulation factors VII, LX, and X.
- Osteocalcin bone-Gla protein, BGP
- matrix Gla-protein MGP
- Gla Friedman, P.A. and CT. Przysiecki (1987) Int. J. Biochem. 19:1-7; C Vermeer (1990) Biochem. J. 266:625-636).
- the invention features purified polypeptides, full-length human 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 ,” and “MDDT-20.”
- the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, 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 JD NO: 1-20, c) a biologically active fragment of a polypeptide having an amino
- the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-20.
- 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 JD NO: 1-20, 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- 20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
- the polynucleotide encodes a polypeptide selected from the group consisting of SEQ JD NO: 1-20.
- the polynucleotide is selected from the group consisting of SEQ ID NO:21-40.
- the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consistmg of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, 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: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1
- the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, 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: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
- 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 JD NO: 1-20, 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 JD NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.
- 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 JD NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, 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 JD NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
- the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
- the probe comprises at least 60 contiguous nucleotides.
- the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, 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:21-40, 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 JD NO: 1-20, 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 JD NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and a pharmaceutically acceptable excipient.
- the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
- 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: 1-20, 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:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:1-20.
- 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 a ino acid sequence selected from the group consisting of SEQ JD NO: 1-20, 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:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.
- the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
- the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
- the invention provides a method of treating a disease or condition associated with overexpression of functional 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 JD NO: 1-20, 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: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
- 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 JD NO:1-20, 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:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
- the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
- the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, 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 the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv
- Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:21-40, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
- the target polynucleotide 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, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptides of the 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 of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
- Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
- Table 5 shows the representative cDNA library for polynucleotides of the invention.
- Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
- Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
- 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 rnimics the biological activity of
- 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.
- allehc variant is an alternative form of the gene encoding MDDT. Allehc 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. Included 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 similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic 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 threonine.
- Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
- amino acid and amino acid sequence refer to an 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 limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
- Amphfication relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
- PCR polymerase chain reaction
- Antagonist refers to a molecule which inhibits or attenuates the biological activity of 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 e.g., a mouse, a rat, or a rabbit
- antigenic deteiminant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
- an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
- 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 maybe 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 j ), 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 then cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J.
- the term "intramer” 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 phosphorofhioates, mefhylphosphonates, 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-pahs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
- the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
- biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
- immunologically active or “immunogenic” refers to the capabihty of the natural, recombinant, or synthetic MDDT, or of any ohgopeptide 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 maybe employed as hybridization probes.
- the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
- the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
- salts e.g., NaCl
- 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 GELVfEW 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 arnino acid substitutions.
- Conservative arnino 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 substitution, and/or (c) the bulk of the side chain.
- a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the • absence of one or more amino acid residues or nucleotides.
- derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or arnino 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 maybe carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
- “Exon shuffling” refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
- a “fragment” is a unique portion of 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 maybe preferentially selected from certain regions of a molecule.
- a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 arnino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
- these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
- a fragment of SEQ ID NO:21-40 comprises a region of unique polynucleotide sequence that specifically identifies SEQ JD NO:21-40, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
- a fragment of SEQ JD NO:21-40 is useful, for example, in hybridization and amphfication technologies and in analogous methods that distinguish SEQ JD NO.-21-40 from related polynucleotide sequences.
- the precise length of a fragment of SEQ JD NO:21-40 and the region of SEQ ID NO.-21-40 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 JD NO: 1-20 is encoded by a fragment of SEQ ID NO:21-40.
- a fragment of SEQ ID NO: 1-20 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-20.
- a fragment of SEQ JD NO:1-20 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-20.
- the precise length of a fragment of SEQ JD NO:1-20 and the region of SEQ ID NO:1-20 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., mefhionine) 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 ahgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
- NCBI National Center for Biotechnology Information
- BLAST Basic Local Ahgnment Search Tool
- NCBI National Center for Biotechnology Information
- BLAST Basic Local Ahgnment Search Tool
- the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
- BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences” tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example: Matrix: BLOSUM62
- Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ JD 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, maybe used to describe a length over which percentage identity maybe measured.
- nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar arnino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
- percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence ahgnment are well-known. Some alignment methods take into account conservative arnino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge andjiydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
- NCBI BLAST software suite may be used.
- BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
- Such default parameters maybe, for example:
- Gap x drop-off 50
- Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
- Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity maybe measured.
- HACs Human artificial chromosomes
- HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
- humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
- Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in detem ⁇ ring the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pahs of nucleic acid strands that are not perfectly matched.
- Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
- wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T Rule) for the specific sequence at a defined ionic strength and pH.
- T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
- High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
- blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
- Organic solvent such as formamide at a concentration of about 35-50% v/v
- RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
- Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
- hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
- a hybridization complex 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 arnino 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., cytoMnes, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
- an "immunogenic fragment” is a polypeptide or ohgopeptide fragment of MDDT which is capable of ehciting an immune response when introduced into a living organism, for example, a mammal.
- the term ''immunogenic fragment” also includes any polypeptide or ohgopeptide 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.
- array element refers 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 immunological properties of MDDT.
- nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
- 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 preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
- Post-translational modification of an 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, che ⁇ riluminescent agents, and enzymes.
- Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pahs 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 of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used. Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2 nd ed., vol.
- PCR primer pahs 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.
- OLIGO 4.06 software is useful for the selection of PCR primer pahs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
- Similar primer selection programs have incorporated additional features for expanded capabilities.
- the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope.
- Primer3 primer selection program (available to the public from the Whitehead stirate MTT Center for Genome Research, Cambridge MA) allows the user to input a "nhsprirning library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of ohgonucleotides 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 ahgnments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of ahgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved ohgonucleotides and polynucleotide fragments.
- ohgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oHgonucleotide selection are not limited to those described above.
- a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
- the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
- a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid maybe part of a vector that is used, for example, to transform a ceh.
- such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
- a "regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
- Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chenhluminescent, or chromogenic agents; substrates; cof actors; inhibitors; magnetic particles; and other moieties known in the art.
- RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base ymine 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 ceh, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
- specific bmding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g. , the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
- substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturaUy associated.
- a “substitution” refers to the replacement of one or more arnino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
- “Substrate” refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
- the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
- a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
- Transformation describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
- transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
- a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the ceUs of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art.
- the nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the ceU, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
- the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
- the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
- a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
- Such a pah of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
- a variant may be described as, for example, an "aUehc" (as defined above), "splice,” “species,” or “polymorphic” variant.
- a splice variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
- the conesponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
- Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides wiU generaUy have significant amino acid identity relative to each other.
- a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
- Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
- SNPs single nucleotide polymorphisms
- the presence of SNPs maybe 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 pah 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 fuU-lengtli 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 ceU prohferative, autoirnmune/inflammatory, developmental, neurological, and cardiovascular disorders.
- Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its conesponding polypeptide are correlated to a single Incyte project identification number (lhcyte Project JD). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
- Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
- Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
- Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide JD) for polypeptides of the invention.
- Column 3 shows the GenBank identification number (GenBank JD NO:) of the nearest GenBank ho olog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs.
- Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
- Column 5 shows the annotation of the GenBank and PROTEOME database homolog along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
- Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and
- FIG. 3 shows the number of amino acid residues in each polypeptide.
- Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as detemiined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
- Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
- Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
- SEQ ID NO:3 is 96% identical, from residue Ml to residue V725, to rat corneal wound healing related protein (GenBank ID g8926320) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.)
- the BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance.
- SEQ ID NO:3 is a human fuU- length molecule for disease detection and treatment, i an alternative example, SEQ ID NO:7 is 24% identical, from residue E214 to residue T735, to corn cahnodulin-binding protein MPCBP (GenBank ID gl0086260) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.)
- the BLAST probability score is 1.2e-21, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance.
- SEQ ID NO:7 also contains TPR domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ID NO: 7 is a fuU-length human molecule for disease detection and treatment.
- SEQ ID NO: 10 is 63% identical, from residue P239 to residue V1461, to rat periaxin (GenBank ID g505297) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance.
- SEQ ID NO: 10 also contains a PDZ domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ID NO: 10 is a periaxin.
- SEQ ID NO: 14 is 36% identical, from residue Y20 to residue V203, to a putative phosphatidylinositol-4-phosphate 5-kinase from thale cress (GenBank JD g2739367) as determined by the Basic Local Ahgnment Search Tool (BLAST).
- BLAST Basic Local Ahgnment Search Tool
- the BLAST probabihty score is 2.0e-25, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance.
- SEQ ID NO: 14 also contains a MORN motif as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains. (See Table 3.) Data from BLAST analyses provide further conoborative evidence that SEQ JD NO:14 is a kinase.
- SEQ ID NO:l-2, SEQ ID NO:4-6, SEQ JD NO:8-9, SEQ JD NO:ll-13, and SEQ JD NO:15-20 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ JD NO: 1-20 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 JD NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
- Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amphfication technologies that identify SEQ JD NO:21-40 or that distinguish between SEQ JD NO:21-40 and related polynucleotide sequences.
- the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte 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 fuU length polynucleotide sequences.
- the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (ie., those sequences including the designation "ENST").
- the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
- the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
- a polynucleotide sequence identified as FL_XXXXX_N 1 _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was apphed, and YYYYY is the number of the prediction generated by the algorithm, and N 1 ⁇ 3 _ disturb , if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
- the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-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 apphed, gfiBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and Nrefening to specific exons (See Example V).
- a RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).
- a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
- the foUowing Table hsts examples of component sequence prefixes and conesponding sequence analysis methods associated with the prefixes (see Example TV 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 fuU length polynucleotide sequences which were assembled using Incyte cDNA sequences.
- the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
- the tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
- the invention also encompasses MDDT variants.
- a prefened 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 ID NO:21-40, which encodes MDDT.
- the polynucleotide sequences of SEQ JD NO:21-40, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base mymine 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 ID NO:21- 40 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 JD NO:21-40.
- 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 wiU generaUy 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 wiUhave 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 ID NO:21 is a splice variant of a polynucleotide comprising a sequence of SEQ ID NO:39 and a polynucleotide comprising a sequence of SEQ ID NO:34 is a splice variant of a polynucleotide comprising a sequence of SEQ ID NO:40.
- 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 generaUy capable of hybridizing to the nucleotide sequence of the naturaUy occurring MDDT under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding MDDT or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy occuning 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 deshable properties such as a greater half-hfe, than transcripts produced from the naturaUy occurring sequence.
- the invention also encompasses production of DNA sequences which encode MDDT and MDDT derivatives, or fragments thereof, entirely by synthetic chemistry.
- the synthetic sequence maybe inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art.
- synthetic chemistry may be used to introduce mutations into a 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 ID NO:21-40 and fragments thereof under various conditions of stringency.
- Hybridization conditions including annealing and wash conditions, are described in "Definitions.” Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
- the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Apphed Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amphfication system (Life Technologies, Gaithersburg MD).
- sequence preparation is automated with machines such as the MICROLAB 2200 hquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems).
- Sequencing is then canied out using either the ABI 373 or 377 DNA sequencing system (Apphed Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
- the resulting sequences are analyzed using a variety of algorithms which are weU known in the art. (See, e.g., Ausubel, F.M. ' (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 1.1; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
- 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.
- restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
- Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a chcularized template.
- the template is derived from restriction fragments comprising a known genomic locus and sunounding sequences.
- a third method, capture PCR involves PCR amphfication of DNA fragments adjacent to known sequences inhuman and yeast artificial chromosome DNA.
- multiple restriction enzyme digestions and hgations maybe used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
- Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al.
- primers may be designed using commerciaUy available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
- oligo d(T) library When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a fuU-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
- Capillary electrophoresis systems which are commerciaUy available maybe used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
- capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
- Output/hght intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Apphed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
- CapiUary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
- 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 ceUs. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantiaUy the same or a functionaUy equivalent amino acid sequence maybe produced and used to express MDDT.
- nucleotide sequences of the present invention can be engineered using methods generaUy 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 ohgonucleotides may be used to engineer the nucleotide sequences.
- ohgonucleotide- mediated site-directed mutagenesis maybe used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce variants, and so forth.
- nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDTNG (Maxygen Inc. , Santa Clara CA; described in U.S. Patent No.
- DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties.
- sequences encoding MDDT maybe synthesized, in whole or in part, using chemical methods weU known in the art.
- chemical methods See, e.g., Carufhers, 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 sohd-phase techniques.
- Automated synthesis maybe achieved using the ABI 431 A peptide synthesizer (Apphed Biosystems).
- AdditionaUy the amino acid sequence of MDDT, or any part thereof, maybe altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturaUy occurring polypeptide.
- the peptide may be substantiaUy purified by preparative high performance hquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol.
- the composition of the synthetic peptides maybe confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
- an appropriate expression vector i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
- 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. In cases where sequences encoding MDDT and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons maybe of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host ceU system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162.)
- a variety of expression vector/host systems may be 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 ceh systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or withbacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceh systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or
- 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 ceU population.
- the invention is not limited by the host ceh employed.
- cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding MDDT.
- routine cloning, subcloning, and propagation of polynucleotide sequences encoding MDDT can be achieved using a multifunctional E. coh vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Life Technologies).
- PBLUESCRIPT Stratagene, La JoUa CA
- PSPORT1 plasmid Life Technologies.
- these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
- vectors which direct high level expression of MDDT may be used.
- vectors containing the strong, inducible SP6 or T7 bacteriophage promoter maybe used.
- Yeast expression systems may be used for production of MDDT.
- a number of vectors containing constitutive or inducible promoters may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris.
- constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
- such vectors direct either the secretion or intraceUular 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 smaU subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
- 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. 3:1311).
- plant promoters such
- CeU Differ. 17:85-105. These constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hih Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196.)
- a number of viral-based expression systems may be utilized.
- sequences encoding MDDT may be hgated 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.
- S V40 or EBV- based vectors may also be used for high-level protein expression.
- HACs Human artificial chromosomes
- HACs Human artificial chromosomes
- HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (hposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
- hposomes polycationic amino polymers, or vesicles
- 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 may be aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
- the purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfully express the introduced sequences.
- Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
- any number of selection systems may be used to recover transformed ceU lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr ceUs, respectively. (See, e.g., Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823.) Also, antimetabohte, 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 chlorsulfuron and phosphinotricin acetyltransferase, respectively.
- Additional selectable genes have been described, e.g., tipB 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.)
- marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
- sequence encoding 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 amphfication, 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 ceh sorting (FACS).
- ELISAs enzyme-linked immunosorbent assays
- RIAs radioimmunoassays
- FACS fluorescence activated ceh sorting
- a two-site, monoclonal-based immunoassay utihzing 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. TV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.)
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding MDDT include ohgolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- 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 radionuchdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host ceUs transformed with nucleotide sequences encoding MDDT maybe cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
- the protein produced by a transformed ceU may be secreted or retained mtraceUularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode MDDT maybe 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 of the 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, hpidation, and acylation.
- Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
- Different host ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g. , CHO, HeLa, MDCK, HEK293 , and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the conect modification and processing of the foreign protein.
- 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 of the 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), calmoduhn 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 immobihzed glutathione, maltose, phenylarsine oxide, calmoduhn, and metal-chelate resins, respectively.
- FLAG, c-myc, and hemagglutinin (HA) enable i ⁇ nnunoaf hity 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 maybe achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
- 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 plurahty of test compounds may be screened for specific binding to MDDT.
- test compounds include antibodies, ohgonucleotides, proteins (e.g., 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 can be closely related to the natural receptor to which MDDT binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationaUy designed using known techniques.
- screening for these compounds involves producing appropriate ceUs which express MDDT, either as a secreted protein or on the ceU membrane.
- Prefened 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 sohd 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 natural product mixtures, and the test compound(s) maybe free in solution or affixed to a sohd support.
- MDDT of the present invention or fragments thereof maybe 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 of the test compound is indicative of a compound that modulates the activity of MDDT.
- a test compound is combined with an in vitro or ceU-free system comprising MDDT under conditions suitable for MDDT activity, and the assay is performed. In either of these assays, 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 plurality of test compounds maybe screened.
- polynucleotides encoding MDDT or their mammalian homologs may be "knocked out" in an arrimal model system using homologous recombination in embryonic stem (ES) ceUs.
- ES embryonic stem
- Such techniques are weU known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.)
- mouse ES ceUs such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture.
- the ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
- the vector integrates into the corresponding region of the host genome by homologous recombination.
- homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
- Transformed ES ceUs are identified and microinjected into mouse ceUblastocysts such as those from the C57BL/6 mouse strain.
- the blastocysts are surgicaUy transfened to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
- Transgenic animals thus generated maybe 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 "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
- knockin technology 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 blastulae 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).
- THERAPEUTICS e.g., by secreting MDDT in its milk.
- MDDT appears to play a role in ceU prohferative, autoimmune/inflammatory, developmental, neurological, and cardiovascular disorders. In the treatment of disorders associated with increased MDDT expression or activity, it is deshable to decrease the expression or activity of MDDT. In the treatment of disorders associated with decreased MDDT expression or activity, it is deshable to increase the expression or activity of MDDT.
- 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, ganglia, 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.
- composition comprising a substantiaUy 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 limited 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 limited 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 limited to, those ceU proliferative, autoimmune/inflammatory, developmental, neurological, and cardiovascular disorders described above.
- an antibody which specificaUy binds MDDT maybe used directly as an antagonist or indirectly as a targeting or delivery 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 limited to, those described above.
- any of the 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 may be produced using methods which are generaUy known in the art.
- purified MDDT maybe used to produce antibodies or to screen libraries 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 library.
- Neutrahzing antibodies i.e., those which inhibit dimer formation
- Single chain antibodies may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
- various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others 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 limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
- BCG Bacilli Calmette-Guerin
- Corynebacterium parvum are especiaUy preferable. It is preferred that the oligopeptides, 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 arnino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of MDDT amino acids maybe fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
- Monoclonal antibodies to MDDT may be prepared using any technique which provides for the production of antibody molecules by continuous ceh lines in culture. These include, but are not limited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique.
- the hybridoma technique the human B-ceU hybridoma technique
- EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, RJ. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. CeU Biol. 62:109-120.
- Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial irrrmunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
- Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the hterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
- Antibody fragments which contain specific binding sites for MDDT may also be generated.
- fragments include, but are not limited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab ⁇ )2 fragments.
- Fab expression libraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
- immunoassays 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 utilizing 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 radioimmunoassay 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 determined for a preparation of monoclonal antibodies, which are monospecific for a particular MDDT epitope, represents a true measure of affinity.
- High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are prefened for use in immunoassays in which the MDDT- antibody complex must withstand rigorous manipulations.
- Low-afhhity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are prefened 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, IRL Press, Washington DC; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
- polyclonal antibody preparations may be further evaluated to determine the quality and suitabihty of such preparations for certain downstream applications.
- a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is 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 applications, are generaUy available. (See, e.g., Catty, supra, and Coligan 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 delivered mtraceUularly 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, KJ. et al.
- Antisense sequences can also be introduced mtraceUularly through the use of vhal vectors, such as retrovirus and adeno-associated virus vectors.
- vhal vectors such as retrovirus and adeno-associated virus vectors.
- Other gene delivery mechanisms include liposome-derived systems, artificial vhal envelopes, and other systems known in the art.
- polynucleotides encoding MDDT maybe used for somatic or germline gene therapy.
- Gene therapy maybe performed to (i) conect a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al.
- MDDT are treated by constructing mammalian 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) dhect DNA microinjection into individual ceUs, (ii) baUistic gold particle dehvery, (hi) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J-L. and H. Recipon (1998) Cun. Opin. Biotechnol. 9:445-450).
- Expression vectors that maybe effective for the expression of MDDT include, but are not limited to, the PCDNA 3.1, EPJTAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
- MDDT ma be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the 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) Cun. Opin. Biotechnol.
- a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
- CommerciaUy available hposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
- hposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
- transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
- the introduction of DNA to primary ceUs requires modification of these standardized mammalian 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 commerciaUy 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. Vhol. 61:1647-1650; Bender, M.A. et al. (1987) J. Vhol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Vhol. 62:3802-3806; DuU, T. et al. (1998) J. Vhol. 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 skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Vhol. 71:7020-7029; Bauer, G. et al.
- an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding MDDT to ceUs which have one or more genetic abnormalities 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 adenovrral vectors are described in U.S. Patent No.
- a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding MDDT to target ceUs which have one or more genetic abnormalities with respect to the expression of MDDT.
- the use of 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 skuT in the art.
- a reputation-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res.
- HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
- U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transfened to a ceU under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
- HSV vectors see also Goins, W.F. et al. (1999) J.
- an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding MDDT to target ceUs.
- SFV Semliki Forest Virus
- This subgenomic RNA rephcates to higher levels than the fuU length genomic RNA, resulting in the overproduction of capsid proteins relative to the vhal 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 of the 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 typically associated with ceU lysis within a few days, the ability to estabhsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic rephcation of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
- the 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 ability 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 hterature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Can, 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 die 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 sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, maybe 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 che icaUy 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, constitutively or inducibly, can be introduced into ceU lines, ceUs, or tissues.
- RNA molecules may be modified to increase intraceUular stabihty and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3 ' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
- An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding 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 of the polynucleotide encoding MDDT maybe 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 libra y 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 library 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 permeabilized ceU, or an in vitro cell-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 Schizosaccharomvces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human 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 library 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 arnino polymers maybe 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 of the invention relates to the administration of a composition which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
- Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
- Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack 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 limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- compositions for pulmonary administration maybe prepared in hquid or dry powder form. These compositions are generaUy aerosolized 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 capabihty of those skilled in the art.
- Specialized forms of compositions may be prepared for dhect 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 HTV 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.
- ceU culture assays e.g., of neoplastic ceUs
- 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 ameliorates the symptoms or condition.
- Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
- the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD SO /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 chculating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
- the exact dosage wiU be determined by the practitioner, in light 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, drag 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 literature and generaUy available to practitioners in the art. Those skilled in the art whl 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 In another embodiment, 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 may be prepared in the same manner as described above for therapeutics. Diagnostic assays for MDDT include methods which utilize 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 maybe labeled by covalent or non-covalent attachment of a reporter molecule.
- reporter molecules 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 established 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 establishes the parameters for diagnosing disease.
- the polynucleotides encoding MDDT maybe 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 maybe 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 of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amphfication wiU determine whether the probe identifies only naturaUy 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 LD NO :21 -40 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.
- 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 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 manow, brain, breast, cervix, gaU bladder, gangha, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
- the polynucleotide sequences encoding MDDT maybe used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microanays utilizing fluids or tissues from patients to detect altered MDDT expression. Such qualitative 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.
- 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.
- assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with expression of
- MDDT a normal or standard profile for expression is estabhshed. This may be accomphshed by combining body fluids or ceh extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding MDDT, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a 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 determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
- the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
- the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
- a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
- Additional diagnostic uses for ohgonucleotides designed from the sequences encoding MDDT may involve the use of PCR. These oligomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro.
- Oligomers wiU preferably contain a fragment of a polynucleotide encoding MDDT, or a fragment of a polynucleotide complementary to the polynucleotide encoding MDDT, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers 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 amplify 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 of the ampHmers 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 may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
- SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insuHn-dependent diabetes melhtus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceh anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be conelated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a 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. Jmmunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.
- ohgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microanay.
- the microanay can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
- the microanay may also be used to identify genetic variants, mutations, and polymorphisms.
- This information maybe used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
- 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 microanay.
- the microanay maybe used to monitor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above.
- a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or 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 of the 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 high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurahty of elements on a microanay.
- the resultant transcript image would provide a profile of gene activity.
- Transcript images maybe generated using transcripts isolated from tissues, ceU Hues, 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 prechnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring envhonmental 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.
- Jf a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene famiHes. IdeaUy, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as weU, as the levels of expression of these genes are used to normahze the rest of the expression data. The normahzation procedure is useful for comparison of expression data after treatment with different compounds.
- the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
- Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels conesponding to the polynucleotides of the present invention maybe quantified.
- the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
- proteome refers to the global pattern of protein expression in a particular tissue or ceU type.
- proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
- a profile of a ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceh type.
- the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
- the proteins are 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 determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data 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 microanay, and protein expression levels are quantified by exposing the microanay to the sample and detecting the levels of protein bound to each anay element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788).
- Detection maybe 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 anay element.
- Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parahel with toxicant signatures at the transcript level.
- There is a poor conelation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
- the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be 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 conesponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
- Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
- the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
- 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, R.A. 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 may be used, and in some instances, noncoding sequences maybe preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy cause undesired cross hybridization during chromosomal mapping.
- sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
- HACs human artificial chromosomes
- YACs yeast artificial chromosomes
- BACs bacterial artificial chromosomes
- PI constructions or single chromosome cDNA libraries.
- the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which conelate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
- RFLP restriction fragment length polymorphism
- FISH Fluorescent in situ hybridization
- Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMLM) World Wide Web site. Conelation 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 Mendelian Inheritance in Man
- In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps.
- placement of a gene on the chromosome of another mammahan species, such as mouse may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques.
- Once the gene or genes responsible for a disease or syndrome have been crudely locahzed by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to llq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
- nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
- 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 mtraceUularly. 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-neutralizing antibodies can be used to capture the peptide and immobilize it on a sohd support.
- the nucleotide sequences which encode MDDT maybe used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are cunently known, including, but not limited to, such properties as the triplet genetic code and specific base pah interactions.
- Incyte cDNAs were derived from cDNA Hbraries described in the LJEESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
- poly(A)+ RNA was isolated using ohgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
- RNA was provided with RNA and constructed the conesponding cDNA Hbraries. Otherwise, cDNA was synthesized and cDNA hbraries were constructed with the
- UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using 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 CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
- cDNAs were Hgated into compatible restriction enzyme sites of the polyhnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (InvitiOgen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pEMCY (Incyte Genomics), or derivatives thereof.
- Recombinant plasmids were transformed into competent E. coh ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies.
- Plasmids obtained as described in. Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E. A.L. PREP 96 plasmid purification kit from QIAGEN.
- Plasmids were resuspended in 0.1 ml of distiHed water and stored, with or without lyophihzation, at 4°C Alternatively, plasmid DNA was ampHfied from host ceh lysates using dhect 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.
- Incyte cDNA recovered in plasmids as described in Example II were sequenced as foUows. Sequencing reactions were processed using standard methods or Mgh-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
- cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supphed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were earned out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Apphed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 1.1). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VTXT.
- the polynucleotide sequences derived from Incyte cDNAs were vahdated by removing vector, linker, 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, Saccharomvces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART (Schultz et al.
- HMM is a probabihstic 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 (see Examples TV and V) were used to extend Incyte cDNA assemblages to full 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 conesponding fuU length polypeptide sequences.
- a polypeptide of the invention may begin at any of the methionine residues of the fuU length translated polypeptide. FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS,
- HMM-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART.
- FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence 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, probabihty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabihty value, the greater the identity between two sequences).
- Genscan is a general-purpose gene identification program which analyzes genomic D ⁇ A sequences from a variety of organisms (See Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C and S. Karlin (1998) Cun. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cD ⁇ A sequence extending from a methionine to a stop codon.
- Genscan is a FASTA database of polynucleotide and polypeptide sequences.
- the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
- the encoded polypeptides were analyzed by querying against PFAM models for fuU-length human molecules for disease detection and treatment.
- Potential fuU-length human molecules for disease detection and treatment were also identified by homology to Incyte cD ⁇ A sequences that had been annotated as fuh-lengfh human molecules for disease detection and treatment.
- Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubhc databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to conect enors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to conect or confirm the Genscan predicted sequence.
- FuU length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example HI. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences. V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences
- Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example TV. Partial cDNAs assembled as described in Example in were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible sphce variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
- Partial DNA sequences were extended to fuU length with an algorithm based on BLAST analysis.
- the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
- a chimeric protein was generated by using the resultant high-scoring segment 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 high-scoring segment pahs
- 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
- sequences which were used to assemble SEQ JD NO:21-40 were compared with sequences from the Incyte LIFESEQ database and pubhc domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID NO :21-40 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Gehethon were used to detemiine if any of the clustered sequences had been previously mapped.
- pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Gehethon were used to detemiine if any of the clustered sequences had been previously mapped.
- Map locations are represented by ranges, or intervals, of human chromosomes.
- the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
- centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers.
- cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
- the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
- Human genome maps and other resources available to the pubhc such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nhn.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
- 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. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)
- the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
- the product score is a 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 die length of the shorter of the two sequences).
- the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pah (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 pah 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 of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
- polynucleotide sequences encoding MDDT are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example HI). Each cDNA sequence is derived from a cDNA kbrary 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; stomatognathic system; unclassified/mixed; or urinary tract.
- the number of Hbraries in each category is counted and divided by the total number of Hbraries across aU categories.
- each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU Hue, 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 ah categories.
- the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding MDDT.
- cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
- 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 well 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 H (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the 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 determine 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 WI), and sonicated or sheared prior to rehgation into pUC 18 vector (Amersham Pharmacia Biotech).
- CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
- 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 religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed ceUs were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384- weU plates in LB/2x carb Hquid media.
- the ceUs were lysed, and DNA was ampHfied by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the foUowing parameters: Step 1: 94 °C, 3 min; Step 2: 94 °C, 15 sec; Step 3: 60 °C, 1 min; Step 4: 72 °C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamphfied using the same conditions as described above.
- SNPs single nucleotide polymorphisms
- LIFESEQ database Incyte Genomics
- Sequences from the same gene were clustered together and assembled as described in Example HI, aUowing the identification of aU sequence variants in the gene.
- An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecaU enors by requiring a rninimum Phred quality score of 15, and removed sequence ahgnment enors and enors 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 Amish individuals.
- the African population comprised 194 individuals (97 male, 97 female), aU African Americans.
- the Hispanic population comprised 324 individuals (162 male, 162 female), aU Mexican Hispanic.
- the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
- AUele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no aUehc variance in this population were not further tested in the other three populations.
- Hybridization probes derived from SEQ ID NO:21-40 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pahs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
- Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
- the labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). 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 of the foUowing endonucleases: Ase I, Bgl H, Eco RI, Pst I, Xba I, or Pvu H (DuPont NEN).
- the DNA from each digest is fractionated on a 0.7% agarose gel and transfened 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 anay elements upon a microanay can be achieved utilizing photohthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
- the substrate in each of the aforementioned technologies should be uniform and sohd with a non-porous surface (Schena (1999), supra).
- Suggested substrates include sihcon, silica, glass shdes, glass chips, and siHcon wafers.
- a procedure analogous to a dot or slot blot may also be used to anange and link elements to the surface of a substrate using thermal, UN, chemical, or mechanical bonding procedures.
- a typical anay may be produced using available methods and machines weU known to those of ordinary skiU in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
- FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oHgomers thereof may comprise the elements of the 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 anay element.
- laser desorbtion and mass spectrometry may be used for detection of hybridization.
- the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microanay may be assessed.
- microanay preparation and usage is described in detail below.
- RNA is isolated from tissue samples using the guanidinium thiocyanate method and ⁇ oly(A) + RNA is purified using the oligo-(dT) ceUulose method.
- Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l ohgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
- the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte).
- Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
- Sequences of the present invention are used to generate array elements.
- Each anay element is ampHfied from bacterial ceUs containing vectors with cloned cDNA inserts.
- PCR amphfication uses primers complementary to the vector sequences flanking the cDNA insert.
- Anay elements are 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 Pharmacia Biotech). Purified anay elements are immobilized on polymer-coated glass shdes. Glass microscope shdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments.
- 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 capiUary printing element by a high-speed robotic apparatus.
- the apparatus then deposits about 5 nl of anay element sample per shde.
- Microanays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distiUed water. Non-specific binding sites are blocked by incubation of microanays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2% SDS and distiUed water as before.
- PBS phosphate buffered saline
- Hybridization Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and
- the chamber containing the anays is incubated for about 6.5 hours at 60° C
- the anays are washed for 10 min at 45° C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried. Detection
- Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
- the excitation laser Hght is focused on the anay 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.
- 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) conesponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipHer tubes are used to filter the signals.
- the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
- Each array is typicaUy scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
- the sensitivity of the scans is typicaUy cahbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
- a specific location on the anay contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1:100,000.
- the caHbration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
- the output of the photomultipHer tube is digitized using a 12-bit RTT-835H analog-to-digital (AID) conversion board (Analog Devices, Inc., Norwood MA) instaUed in an IBM-compatible PC computer.
- the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
- the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first conected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
- 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). XII. Complementary Polynucleotides
- Sequences complementary to the MDDT-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring MDDT.
- ohgonucleotides comprising from about 15 to 30 base pahs
- essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
- Appropriate oHgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of MDDT.
- a complementary oHgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
- a complementary oHgonucleotide is designed to prevent ribosomal binding to the MDDT-encoding transcript.
- MDDT 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 tip-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
- Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
- Antibiotic resistant bacteria express MDDT upon induction with isopropyl beta-D- thiogalactopyranoside (LPTG).
- MDDT in eukaryotic ceUs
- baculovirus recombinant Autographica cahfornica nuclear polyhedrosis virus
- AcMNPV Autographica cahfornica nuclear polyhedrosis virus
- the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding MDDT by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Vhal infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
- Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases.
- 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, penrntting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU lysates.
- GST glutathione S- transferase
- a peptide epitope tag such as FLAG or 6-His
- FLAG an 8-amino acid peptide
- 6- His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified MDDT obtained by these methods can be used directly in the assays shown in Examples XVLT and XVLTI, 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 (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endothehal or hematopoietic ceU line, using either Hposome formulations or electroporation.
- 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
- Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected ceUs and is a rehable predictor of cDNA expression from the recombinant vector.
- Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
- Flow cytometry (FCM) an automated, laser optics- based technique, is used to identify transfected ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties.
- FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward hght scatter and 90 degree side Hght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G.
- CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG).
- Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
- mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding MDDT and other genes of interest can be analyzed by northern analysis or microanay techniques.
- MDDT substantiaUy purified using polyacrylamide gel electrophoresis (PAGE; see, e.g.,
- the MDDT amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
- LASERGENE software DNASTAR
- Methods for selection of appropriate epitopes, such as those near the C-tenninus or in hydrophihc regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
- ohgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Apphed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-malein ⁇ dobenzoyl-N-hy ⁇ hoxysuccinimide ester (MBS) to increase immunogenicity.
- MBS N-malein ⁇ dobenzoyl-N-hy ⁇ hoxysuccinimide ester
- Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
- Resulting antisera are tested for antipeptide and anti-MDDT activity by, for example, binding the peptide or MDDT to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
- Media containing MDDT are passed over the irrrmunoaffinity 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.
- Candidate molecules previously anayed 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.
- 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 PATHCALLLNG process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large Hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
- An assay for growth stimulating or inhibiting activity of MDDT measures the amount of DNA synthesis in Swiss mouse 3T3 ceUs (McKay, I. and Leigh, I., eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York, NY).
- varying amounts of MDDT are added to quiescent 3T3 cultured ceUs in the presence of [ 3 H]thymidine, a radioactive DNA precursor.
- MDDT for this assay can be obtained by recombinant means or from biochemical preparations. Incorporation of [ 3 H]thynhdine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is directly proportional to the amount of newly synthesized DNA.
- a linear dose-response curve over at least a hundred-fold MDDT concentration range is indicative of growth modulating activity.
- One unit of activity per milHHter is defined as the concentration of MDDT producing a 50% response level, where 100% represents maximal incorporation of [ 3 H]thymidine into acid-precipitable DNA .
- an assay for MDDT activity measures the stimulation or inlhbition of neurotransmission in cultured ceUs. Cultured CHO fibroblasts are exposed to MDDT. FoUowing endocytic uptake of MDDT, the ceUs are washed with fresh culture medium, and a whole ceU voltage- clamped Xenopus myocyte is manipulated into contact with one of the fibroblasts in MDDT-free medium. Membrane currents are recorded from the myocyte. Increased or decreased cunent relative to control values are indicative of neuromodulatory effects of MDDT (Morimoto, T. et al. (1995) Neuron 15:689-696).
- an assay for MDDT activity measures the amount of MDDT in secretory, membrane-bound organeUes.
- Transfected ceUs as described above are harvested and lysed.
- the lysate is fractionated using methods known to those of skiU in the art, for example, sucrose gradient ultracentrifugation. Such methods aUow the isolation of subceUular components such as the Golgi apparatus, ER, smaU membrane-bound vesicles, and other secretory organeUes.
- Immunoprecipitations from fractionated and total ceU lysates are performed using MDDT-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques.
- the concentration of MDDT in secretory organeUes relative to MDDT in total ceU lysate is proportional to the amount of MDDT in transit through the secretory pathway.
- AMP binding activity is measured by combining MDDT with 32 P-labeled AMP.
- the reaction is incubated at 37°C and terminated by addition of trichloroacetic acid.
- the acid extract is neutraHzed and subjected to gel electrophoresis to remove unbound label.
- the radioactivity retained in the gel is proportional to MDDT activity.
- Library Vector Library Description subtraction was constructed by pooling equal numbers of cDNA clones from 3 prostate tissue libraries derived from prostate tissue, prostate epithelial cells, and fibroblasts from prostate stroma from 3 different donors. Subtractive hybridization conditions were based on the methodologies of Swaroop et al., NAR 19 (1991):1954 and Bonaldo, et al. Genome Research 6 (1996) :791.
- PROSTUT20 pINCY The library was constructed using RNA isolated from prostatetumor tissue removed from a 58-year-old Caucasian male during radical prostatectomy, regional lymph node excision, and prostate needle biopsy. Pathology indicatedadenocarcinoma (Gleason grade 3+2) of the prostate, which formed a predominant massinvolving primarily the right side and focally involved the left side, peripherallyand anteriorly. The patient presented with elevated prostate specific antigen (PSA) and induration. Family history included breast cancer .
- PSA prostate specific antigen
- SINIDME01 PCDNA2.1 This 5' biased random primed library was constructed using RNA isolated from diseased ileum tissue removed from a 29-year-old Caucasian female during jejunostomy. Pathology indicated mild chronic inflammation. The patient presented with ulcerative colitis. Patient history included a benign neoplasm of the large bowel. Patient medications included Asacol, Rowasa, Clomid and Pergonol. Family history included benign hypertension in the mother, and colon cancer and cerebrovascular accident in the grandparent (s) .
- SPLNFET02 pINCY Library was constructed using RNA isolated from spleen tissue removed from a Caucasian male fetus, who died at 23 weeks' gestation.
- THP1NOB01 PBLUESCRIPT "Library was constructed using RNA isolated from cultured, unstimulated THP-1 cells.
- THP-1 is a human promonocyte line derived from the peripheral blood of a 1- year-old Caucasian male with acute monocytic leukemia (ref: Int. J. Cancer (1980) 26:171) . "
- URETTU 01 pINCY Library was constructed using RNA isolated from right ureter tumor tissue of a 69- year-old Caucasian male during ureterectomy and lymph node excision. Pathology indicated invasive grade 3 transitional cell carcinoma. Patient history included benign colon neoplasm, tobacco use, asthma, emphysema, acute duodenal ulcer, and hyperplasia of the prostate. Family history included atherosclerotic coronary artery disease, congestive heart failure, and malignant lung neoplasm.
- 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. Pa Ac Inc., Pasadena, CA.
- ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
- HMM hidden Markov model
- Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
- TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
- HMM hidden Markov model
Abstract
Description
Claims
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JP2002570734A JP2004535159A (en) | 2001-02-09 | 2002-02-08 | Disease detection and therapeutic molecules |
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US7709206B2 (en) | 2002-12-31 | 2010-05-04 | Metamorphix, Inc. | Compositions, methods and systems for inferring bovine breed or trait |
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Cited By (9)
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EP1581542A2 (en) * | 2002-12-17 | 2005-10-05 | Sagres Discovery, Inc. | Novel compositions and methods in cancer |
EP1581542A4 (en) * | 2002-12-17 | 2008-06-11 | Sagres Discovery Inc | Novel compositions and methods in cancer |
US7709206B2 (en) | 2002-12-31 | 2010-05-04 | Metamorphix, Inc. | Compositions, methods and systems for inferring bovine breed or trait |
US8026064B2 (en) | 2002-12-31 | 2011-09-27 | Metamorphix, Inc. | Compositions, methods and systems for inferring bovine breed |
US8450064B2 (en) | 2002-12-31 | 2013-05-28 | Cargill Incorporated | Methods and systems for inferring bovine traits |
US8669056B2 (en) | 2002-12-31 | 2014-03-11 | Cargill Incorporated | Compositions, methods, and systems for inferring bovine breed |
US9982311B2 (en) | 2002-12-31 | 2018-05-29 | Branhaven LLC | Compositions, methods, and systems for inferring bovine breed |
US10190167B2 (en) | 2002-12-31 | 2019-01-29 | Branhaven LLC | Methods and systems for inferring bovine traits |
US11053547B2 (en) | 2002-12-31 | 2021-07-06 | Branhaven LLC | Methods and systems for inferring bovine traits |
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