WO2002060942A2 - Molecules de modification et de maintenance proteiques - Google Patents
Molecules de modification et de maintenance proteiques Download PDFInfo
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- WO2002060942A2 WO2002060942A2 PCT/US2002/002813 US0202813W WO02060942A2 WO 2002060942 A2 WO2002060942 A2 WO 2002060942A2 US 0202813 W US0202813 W US 0202813W WO 02060942 A2 WO02060942 A2 WO 02060942A2
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to nucleic acid and amino acid sequences of protein modification and maintenance molecules and to the use of these sequences in the diagnosis, treatment, and prevention of gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of protein modification and maintenance molecules.
- Proteases cleave proteins and peptides at the peptide bond that forms the backbone of the protein or peptide chain.
- Proteolysis is one of the most important and frequent enzymatic reactions that occurs both within and outside of cells. Proteolysis is responsible for the activation and maturation of nascent polypeptides, the degradation of misfolded and damaged proteins, and the controlled turnover of peptides within the cell.
- Proteases participate in digestion, endocrine function, and tissue remodeling during embryonic development, wound healing, and normal growth. Proteases can play a role in regulatory processes by affecting the half life of regulatory proteins. Proteases are involved in the etiology or progression of disease states such as inflammation, angiogenesis, tumor dispersion and metastasis, cardiovascular disease, neurological disease, and bacterial, parasitic, and viral infections.
- Proteases can be categorized on the basis of where they cleave their substrates.
- Exopeptidases which include aminopeptidases, dipeptidyl peptidases, tripeptidases, carboxypeptidases, peptidyl-di-peptidases, dipeptidases, and omega peptidases, cleave residues at the termini of their substrates.
- Endopeptidases including serine proteases, cysteine proteases, and metalloproteases, cleave at residues within the peptide.
- Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three- dimensional structure. (See Beynon, R.J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York NY, pp. 1-5.) Serine Proteases
- SPs serine proteases
- the serine proteases are a large, widespread family of proteolytic enzymes that include the digestive enzymes trypsin and chymotrypsin, components of the complement and blood-clotting cascades, and enzymes that control the degradation and turnover of macromolecules within the cell and in the extracellular matrix.
- Most of the more than 20 subfamilies can be grouped into six clans, each with a common ancestor. These six clans are hypothesized to have descended from at least four evolutionarily distinct ancestors.
- SPs are named for the presence of a serine residue found in the active catalytic site of most families.
- the active site is defined by the catalytic triad, a set of conserved asparagine, histidine, and serine residues critical for catalysis. These residues form a charge relay network that facilitates substrate binding. Other residues outside the active site form an oxyanion hole that stabilizes the tetrahedral transition intermediate formed during catalysis. SPs have a wide range of substrates and can be subdivided into subfamilies on the basis of their substrate specificity.
- the main subfamilies are named for the residue(s) after which they cleave: trypases (after arginine or lysine), aspases (after aspartate), chymases (after phenylalanine or leucine), metases (methionine), and serases (after serine) (Rawlings, N.D. and AJ. Barrett (1994) Meth. Enzymol. 244:19-61).
- zymogens inactive precursors that are activated by proteolysis.
- trypsinogen is converted to its active form, trypsin, by enteropeptidase.
- Enteropeptidase is an intestinal protease that removes an N-terminal fragment from trypsinogen. The remaining active fragment is trypsin, which in turn activates the precursors of the other pancreatic enzymes.
- proteolysis of prothrombin the precursor of thrombin, generates three separate polypeptide fragments. The N-terminal fragment is released while the other two fragments, which comprise active thrombin, remain associated through disulfide bonds.
- the two largest SP subfamilies are the chymotrypsin (SI) and subtilisin (S8) families. Some members of the chymotrypsin family contain two structural domains unique to this family. Kringle domains are triple-looped, disulfide cross-linked domains found in varying copy number. Kringles are thought to play a role in binding mediators such as membranes, other proteins or phospholipids, and in the regulation of proteolytic activity (PROSITE PDOC00020). Apple domains are 90 amino- acid repeated domains, each containing six conserved cysteines. Three disulfide bonds link the first and sixth, second and fifth, and third and fourth cysteines (PROSITE PDOC00376). Apple domains are involved in protein-protein interactions.
- SI family members include trypsin, chymotrypsin, coagulation factors IX-XII, complement factors B, C, and D, granzymes, kallikrein, and tissue- and urokinase-plasminogen activators.
- the subtilisin family has members found in the eubacteria, archaebacteria, eukaryotes, and viruses. Subtilisins include the proprotein-processing endopeptidases kexin and furin and the pituitary prohormone convertases PCI, PC2, PC3, PC6, and PACE4 (Rawlings and Barrett, supra).
- SPs have functions in many normal processes and some have been implicated in the etiology or treatment of disease.
- Enterokinase the initiator of intestinal digestion, is found in the intestinal brush border, where it cleaves the acidic propeptide from trypsinogen to yield active trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7588-7592).
- Prolylcarboxypeptidase a lysosomal serine peptidase that cleaves peptides such as angiotensin II and in and [des-Arg9] bradykinin, shares sequence homology with members of both the serine carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268: 16631-16638).
- the protease neuropsin may influence synapse formation and neuronal connectivity in the hippocampus in response to neural signaling (Chen, Z.-L. et al. (1995) J Neurosci 15:5088-5097).
- Tissue plasminogen activator is useful for acute management of stroke (Zivin, J.A. (1999) Neurology 53: 14- 19) and myocardial infarction (Ross, A.M. (1999) Clin. Cardiol. 22: 165-171).
- Some receptors PAR, for proteinase-activated receptor
- PARs highly expressed throughout the digestive tract, are activated by proteolytic cleavage of an extracellular domain.
- the major agonists for PARs, thrombin, trypsin, and mast cell tryptase are released in allergy and inflammatory conditions. Control of PAR activation by proteases has been suggested as a promising therapeutic target (Vergnolle, N. (2000) Aliment.
- Tryptases the predominant proteins of human mast cells, have been implicated as pathogenetic mediators of allergic and inflammatory conditions, most notably asthma. Properties that distinguish tryptases among the serine proteinases include their activity as heparin-stabilized tetramers, their resistance to many proteinaceous inhibitors, and their preference for peptidergic over macromolecular substrates (Sommerhoff, CP. et al. (2000) Biochim. Biophys. Acta 1477:75-89).
- Prostate-specific antigen is a kallikrein-like serine protease synthesized and secreted exclusively by epithelial cells in the prostate gland. Serum PSA is elevated in prostate cancer and is the most sensitive physiological marker for monitoring cancer progression and response to therapy. PSA can also identify the prostate as the origin of a metastatic tumor (Brawer, M.K. and P.H. Lange (1989) Urology 33: 11-16).
- the signal peptidase is a specialized class of SP found in all prokaryotic and eukaryotic cell types that serves in the processing of signal peptides from certain proteins.
- Signal peptides are amino-terminal domains of a protein which direct the protein from its ribosomal assembly site to a particular cellular or extracellular location. Once the protein has been exported, removal of the signal sequence by a signal peptidase and posttranslational processing, e.g., glycosylation or phosphorylation, activate the protein.
- Signal peptidases exist as multi-subunit complexes in both yeast and mammals.
- the canine signal peptidase complex is composed of five subunits, all associated with the microsomal membrane and containing hydrophobic regions that span the membrane one or more times (Shelness, G.S. and G. Blobel (1990) J. Biol. Chem. 265:9512-9519). Some of these subunits serve to fix the complex in its proper position on the membrane while others contain the actual catalytic activity.
- proteases which have a serine in their active site are dependent on the hydrolysis of ATP for their activity. These proteases contain proteolytic core domains and regulatory ATPase domains which can be identified by the presence of the P-loop, an ATP/GTP-binding motif (PROSITE PDOC00803). Members of this family include the eukaryotic mitochondrial matrix proteases, Clp protease and the proteasome. Clp protease was originally found in plant chloroplasts but is believed to be widespread in both prokaryotic and eukaryotic cells. The gene for early-onset torsion dystonia encodes a protein related to Clp protease (Ozelius, L.J. et al. (1998) Adv. Neurol. 78:93-105).
- the proteasome is an intracellular protease complex found in some bacteria and in all eukaryotic cells, and plays an important role in cellular physiology.
- Proteasomes are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins of all types, including proteins that function to activate or repress cellular processes such as transcription and cell cycle progression (Ciechanover, A. (1994) Cell 79: 13-21).
- UCS ubiquitin conjugation system
- proteins targeted for degradation are conjugated to ubiquitin, a small heat stable protein. The ubiquitinated protein is then recognized and degraded by the proteasome.
- the resultant ubiquitin-peptide complex is hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and free ubiquitin is released for reutilization by the UCS.
- Ubiquitin-proteasome systems are implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra). This pathway has been implicated in a number of diseases, including cystic fibrosis, Angelman's syndrome, and Liddle syndrome (reviewed in Schwartz, A.L. and A. Ciechanover (1999) Annu. Rev. Med. 50:57-74).
- a murine proto-oncogene, Unp encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells.
- the human homologue of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D.A.
- Ubiquitin carboxyl terminal hydrolase is involved in the differentiation of a lymphoblastic leukemia cell line to a non-dividing mature state (Maki, A. et al.
- ubiquitin carboxyl terminal hydrolase (1996) Differentiation 60:59-66).
- PGP 9.5 ubiquitin carboxyl terminal hydrolase
- the proteasome is a large (-2000 kDa) multisubunit complex composed of a central catalytic core containing a variety of proteases arranged in four seven- membered rings with the active sites facing inwards into the central cavity, and terminal ATPase subunits covering the outer port of the cavity and regulating substrate entry (for review, see Schmidt, M. et al. (1999) Curr. Opin. Chem. Biol. 3:584-591). Cysteine Proteases
- Cysteine proteases are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Nearly half of the CPs known are present only in viruses. CPs have a cysteine as the major catalytic residue at the active site where catalysis proceeds via a thioester intermediate and is facilitated by nearby histidine and asparagine residues. A glutamine residue is also important, as it helps to form an oxyanion hole.
- Two important CP families include the papain-like enzymes (Cl) and the calpains (C2). Papain-like family members are generally lysosomal or secreted and therefore are synthesized with signal peptides as well as propeptides.
- Papains include cathepsins B, C, H, L, and S, certain plant allergens and dipeptidyl peptidase (for a review, see Rawlings, N.D. and A.J. Barrett (1994) Meth. Enzymol. 244:461-486).
- Some CPs are expressed ubiquitously, while others are produced only by cells of the immune system.
- CPs are produced by monocytes, macrophages and other cells which migrate to sites of inflammation and secrete molecules involved in tissue repair. Overabundance of these repair molecules plays a role in certain disorders.
- autoimmune diseases such as rheumatoid arthritis
- cysteine peptidase cathepsin C degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones. Bone weakened by such degradation is also more susceptible to tumor invasion and metastasis.
- Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbates the destruction of the rheumatoid synovium (Keyszer, G.M. (1995) Arthritis Rheum. 38:976-984).
- Calpains are calcium-dependent cytosolic endopeptidases which contain both an N-terminal catalytic domain and a C-terminal calcium-binding domain. Calpain is expressed as a proenzyme heterodimer consisting of a catalytic subunit unique to each isoform and a regulatory subunit common to different isoforms. Each subunit bears a calcium-binding EF-hand domain. The regulatory subunit also contains a hydrophobic glycine-rich domain that allows the enzyme to associate with cell membranes. Calpains are activated by increased intracellular calcium concentration, which induces a change in conformation and limited autolysis. The resultant active molecule requires a lower calcium concentration for its activity (Chan, S.L. and M.P.
- Calpain expression is predominantly neuronal, although it is present in other tissues.
- Several chronic neurodegenerative disorders, including ALS, Parkinson's disease and Alzheimer's disease are associated with increased calpain expression (Chan and Mattson, supra).
- Calpain-mediated breakdown of the cytoskeleton has been proposed to contribute to brain damage resulting from head injury (McCracken, E. et al. (1999) J. Neurotrauma 16:749-761).
- Calpain-3 is predominantly expressed in skeletal muscle, and is responsible for limb-girdle muscular dystrophy type 2A (Minami, N. et al. (1999) J. Neurol. Sci. 171:31-37).
- thiol proteases Another family of thiol proteases is the caspases, which are involved in the initiation and execution phases of apoptosis.
- a pro-apoptotic signal can activate initiator caspases that trigger a proteolytic caspase cascade, leading to the hydrolysis of target proteins and the classic apoptotic death of the cell.
- Two active site residues, a cysteine and a histidine, have been implicated in the catalytic mechanism.
- Caspases are among the most specific endopeptidases, cleaving after aspartate residues.
- Caspases are synthesized as inactive zymogens consisting of one large (p20) and one small (plO) subunit separated by a small spacer region, and a variable N-terminal prodomain. This prodomain interacts with cofactors that can positively or negatively affect apoptosis.
- An activating signal causes autoproteolytic cleavage of a specific aspartate residue (D297 in the caspase-1 numbering convention) and removal of the spacer and prodomain, leaving a pl0/p20 heterodimer. Two of these heterodimers interact via their small subunits to form the catalytically active tetramer.
- caspases contain a "death effector domain" in their prodomain by which they can be recruited into self-activating complexes with other caspases and FADD protein associated death receptors or the TNF receptor complex.
- two dimers from different caspase family members can associate, changing the substrate specificity of the resultant tetramer.
- Endogenous caspase inhibitors inhibitor of apoptosis proteins, or IAPs also exist. All these interactions have clear effects on the control of apoptosis (reviewed in Chan and Mattson, supra: Salveson, G.S. and V.M. Dixit (1999) Proc. Natl. Acad. Sci. USA 96: 10964-10967).
- Caspases have been implicated in a number of diseases. Mice lacking some caspases have severe nervous system defects due to failed apoptosis in the neuroepithelium and suffer early lethality. Others show severe defects in the inflammatory response, as caspases are responsible for processing IL-lb and possibly other inflammatory cytokines (Chan and Mattson, supra). Cowpox virus and baculoviruses target caspases to avoid the death of their host cell and promote successful infection. In addition, increases in inappropriate apoptosis have been reported in AIDS, neurodegenerative diseases and ischemic injury, while a decrease in cell death is associated with cancer (Salveson and Dixit, supra: Thompson, C.B. (1995) Science 267: 1456-1462). Aspartyl proteases
- Aspartyl proteases include the lysosomal proteases cathepsins D and E, as well as chymosin, renin, and the gastric pepsins. Most retroviruses encode an AP, usually as part of the pol polyprotein. APs, also called acid proteases, are monomeric enzymes consisting of two domains, each domain containing one half of the active site with its own catalytic aspartic acid residue. APs are most active in the range of pH 2-3, at which one of the aspartate residues is ionized and the other neutral. The pepsin family of APs contains many secreted enzymes, and all are likely to be synthesized with signal peptides and propeptides.
- Retropepsins are analogous to a single domain of pepsin, and become active as homodimers with each retropepsin monomer contributing one half of the active site. Retropepsins are required for processing the viral polyproteins.
- APs have roles in various tissues, and some have been associated with disease. Renin mediates the first step in processing the hormone angiotensin, which is responsible for regulating electrolyte balance and blood pressure (reviewed in Crews, D.E. and S.R. Williams (1999) Hum. Biol. 71:475-503). Abnormal regulation and expression of cathepsins are evident in various inflammatory disease states. Expression of cathepsin D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis. The increased expression and differential regulation of the cathepsins are linked to the metastatic potential of a variety of cancers (Chambers, A.F. et al. (1993) Crit. Rev. Oncol. 4:95-114). Metalloproteases
- Metalloproteases require a metal ion for activity, usually manganese or zinc.
- manganese metalloenzymes include aminopeptidase P and human proline dipeptidase (PEPD).
- Aminopeptidase P can degrade bradykinin, a nonapeptide activated in a variety of inflammatory responses. Aminopeptidase P has been implicated in coronary ischemia reperfusion injury. Administration of aminopeptidase P inhibitors has been shown to have a cardioprotective effect in rats (Ersahin, C. et al (1999) J. Cardiovasc. Pharmacol. 34:604-611).
- the active site is made up of two histidines which act as zinc ligands and a catalytic glutamic acid C- terminal to the first histidine.
- Proteins containing this signature sequence are known as the metzincins and include aminopeptidase N, angiotensin-converting enzyme, neurolysin, the matrix metalloproteases and the adamalysins (ADAMS).
- ADAMS adamalysins
- a number of the neutral metalloendopeptidases are involved in the metabolism of peptide hormones.
- High aminopeptidase B activity for example, is found in the adrenal glands and neurohypophyses of hypertensive rats (Prieto, I. et al. (1998) Horm. Metab. Res. 30:246-248).
- Oligopeptidase M/neurolysin can hydrolyze bradykinin as well as neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem 270:2092-2098).
- Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the brain, where it has been implicated in limiting food intake (Tritos, N.A. et al. (1999) Neuropeptides 33:339-349).
- MMPs matrix metalloproteases
- ECM extracellular matrix
- TLMPs tissue inhibitor of metalloprotease
- fibronectin-like repeats, transmembrane domains, or C-terminal hemopexinase-like domains can be used to separate MMPs into collagenase, gelatinase, stromelysin and membrane-type MMP subfamilies.
- the Zn +2 ion in the active site interacts with a cysteine in the pro-sequence.
- Activating factors disrupt the Zn +2 -cysteine interaction, or "cysteine switch,” exposing the active site. This partially activates the enzyme, which then cleaves off its propeptide and becomes fully active.
- MMPs are often activated by the serine proteases plasmin and furin.
- MMPs are often regulated by stoichiometric, noncovalent interactions with inhibitors; the balance of protease to inhibitor, then, is very important in tissue homeostasis (reviewed in Yong, V.W. et al. (1998) Trends Neurosci. 21:75).
- MMPs are implicated in a number of diseases including osteoarthritis (Mitchell, P. et al. (1996) J. Clin. Invest. 97:761), atherosclerotic plaque rupture (Sukhova, G.K. et al. (1999) Circulation 99:2503), aortic aneurysm (Schneiderman, J. et al. (1998) Am. J. Path. 152:703), non-healing wounds (Saarialho-Kere, U.K. et al. (1994) J. Clin. Invest. 94:79), bone reso ⁇ tion
- MMP inhibitors prevent metastasis of mammary carcinoma and experimental tumors in rat, and Lewis lung carcinoma, hemangioma, and human ovarian carcinoma xenografts in mice (Eccles, S.A. et al. (1996) Cancer Res. 56:2815; Anderson et al. (1996) Cancer Res. 56:715-718; Volpert, O.V. et al. (1996) J. Clin. Invest. 98:671; Taraboletti, G. et al. (1995) J. NCI 87:293; Davies, B. et al. (1993) Cancer Res. 53:2087). MMPs may be active in Alzheimer's disease. A number of MMPs are implicated in multiple sclerosis, and administration of MMP inhibitors can relieve some of its symptoms (reviewed in Yong, supra).
- ADAMs Another family of metalloproteases is the ADAMs, for A Disintegrin and Metalloprotease Domain, which they share with their close relatives the adamalysins, snake venom metalloproteases (SVMPs).
- ADAMs combine features of both cell surface adhesion molecules and proteases, containing a prodomain, a protease domain, a disintegrin domain, a cysteine rich domain, an epidermal growth factor repeat, a transmembrane domain, and a cytoplasmic tail. The first three domains listed above are also found in the SVMPs.
- the ADAMs possess four potential functions: proteolysis, adhesion, signaling and fusion.
- the ADAMs share the metzincin zinc binding sequence and are inhibited by some MMP antagonists such as TIMP-L ADAMs are implicated in such processes as sperm-egg binding and fusion, myoblast fusion, and protein-ectodomain processing or shedding of cytokines, cytokine receptors, adhesion proteins and other extracellular protein domains (Schlondorff, J. and CP. Blobel (1999) J. Cell. Sci. 112:3603-3617).
- the Kuzbanian protein cleaves a substrate in the NOTCH pathway (possibly NOTCH itself), activating the program for lateral inhibition in Drosophila neural development.
- TACE amyloid precursor protein
- ADAM ADAM 10
- TACE has also been identified as the TNF activating enzyme (Black, R.A. et al. (1997) Nature 385:729).
- TNF is a pleiotropic cytokine that is important in mobilizing host defenses in response to infection or trauma, but can cause severe damage in excess and is often overproduced in autoimmune disease.
- TACE cleaves membrane-bound pro-TNF to release a soluble form.
- Other ADAMs may be involved in a similar type of processing of other membrane-bound molecules.
- the ADAMTS sub-family has all of the features of ADAM family metalloproteases and contain an additional thrombospondin domain (TS).
- TS thrombospondin domain
- the prototypic ADAMTS was identified in mouse, found to be expressed in heart and kidney and upregulated by proinflammatory stimuli (Kuno, K. et al. (1997) J. Biol. Chem. 272:556). To date eleven members are recognized by the Human Genome Organization (HUGO; http://www.gene.ucl.ac.Uk/users/hester/adamts.html#Approved). Members of this family have the ability to degrade aggrecan, a high molecular weight proteoglycan which provides cartilage with important mechanical properties including compressibility, and which is lost during the development of arthritis.
- Enzymes which degrade aggrecan are thus considered attractive targets to prevent and slow the degradation of articular cartilage (See, e.g., Tortorella, M.D. (1999) Science 284: 1664; Abbaszade, I. (1999) J. Biol. Chem. 274:23443). Other members are reported to have antiangiogenic potential (Kuno et al., supra) and/or procollagen processing (Colige, A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2374).
- Protease inhibitors are reported to have antiangiogenic potential (Kuno et al., supra) and/or procollagen processing (Colige, A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2374).
- Protease inhibitors and other regulators of protease activity control the activity and effects of proteases.
- Protease inhibitors have been shown to control pathogenesis in animal models of proteolytic disorders (Mu ⁇ hy, G. (1991) Agents Actions Suppl. 35:69-76).
- Low levels of the cystatins, low molecular weight inhibitors of the cysteine proteases correlate with malignant progression of tumors (Calkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler 376:71-80).
- Serpins are inhibitors of mammalian plasma serine proteases. Many se ⁇ ins serve to regulate the blood clotting cascade and/or the complement cascade in mammals.
- Sp32 is a positive regulator of the mammalian acrosomal protease, acrosin, that binds the proenzyme, proacrosin, and thereby aides in packaging the enzyme into the acrosomal matrix (Baba, T. et al. (1994) J. Biol. Chem. 269: 10133-10140).
- the Kunitz family of serine protease inhibitors are characterized by one or more "Kunitz domains" containing a series of cysteine residues that are regularly spaced over approximately 50 amino acid residues and form three intrachain disulfide bonds.
- TFPI-1 and TFPI-2 tissue factor pathway inhibitor
- bikunin inter- ⁇ -trypsin inhibitor
- aprotinin tissue factor pathway inhibitor
- TFPI-1 and TFPI-2 tissue factor pathway inhibitor
- inter- ⁇ -trypsin inhibitor inter- ⁇ -trypsin inhibitor
- bikunin bikunin.
- the invention features purified polypeptides, protein modification and maintenance molecules, referred to collectively as “PMMM” and individually as “PMMM-1,” “PMMM-2,” “PMMM-3,” “PMMM-4,” “PMMM-5,” “PMMM-6,” “PMMM-7,” “PMMM-8,” “PMMM-9,” “PMMM-10,” “PMMM-11,” “PMMM-12,” “PMMM-13,” “PMMM-14,” “PMMM-15,” and “PMMM-16.”
- 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: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from
- the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.
- polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-16. In another alternative, the polynucleotide is selected from the group consisting of SEQ ED NO: 17-32.
- the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.
- the invention provides a cell transformed with the recombinant polynucleotide.
- the invention provides a transgenic organism comprising the recombinant polynucleotide.
- the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.
- the method comprises a) cuituring a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
- the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.
- the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32, 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: 17-32, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
- the polynucleotide comprises at least 60 contiguous nucleotides.
- the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 17-32, 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 ED NO: 17-32, 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: 17-32, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
- the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
- the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and a pharmaceutically acceptable excipient.
- the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1- 16.
- the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional PMMM, 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: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.
- 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 PMMM, comprising administering to a patient in need of such treatment the composition.
- the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.
- 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 PMMM, comprising administering to a patient in need of such treatment the composition.
- the invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-16.
- the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
- the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, 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: 1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.
- 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 ID NO: 17-32, 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 ID NO: 17-32, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32, 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: 17-32, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32, 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 2 shows the GenBank identification number and annotation of the nearest GenBank homolog 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.
- PMMM refers to the amino acid sequences of substantially purified PMMM obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
- agonist refers to a molecule which intensifies or mimics the biological activity of
- PMMM may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PMMM either by directly interacting with PMMM or by acting on components of the biological pathway in which PMMM participates.
- allelic variant is an alternative form of the gene encoding PMMM. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
- altered nucleic acid sequences encoding PMMM include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as PMMM or a polypeptide with at least one functional characteristic of PMMM. Included within this definition are polymo ⁇ hisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding PMMM, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding PMMM.
- 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 PMMM.
- Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of PMMM 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 hydrophihcity values may include: asparagine and glutamine; and serine and threonine.
- Amino acids with uncharged side chains having similar hydrophihcity 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 occu ⁇ ing 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.
- Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
- PCR polymerase chain reaction
- Antagonist refers to a molecule which inhibits or attenuates the biological activity ⁇ of PMMM.
- Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PMMM either by directly interacting with PMMM or by acting on components of the biological pathway in which PMMM 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 PMMM 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 determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
- an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
- aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
- Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
- Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
- the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
- Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
- Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
- introduction refers to an aptamer which is expressed in vivo.
- a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96:3606-3610).
- spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
- antisense refers to any composition capable of base-pairing with the "sense"
- Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
- Antisense molecules may be produced by any method including chemical synthesis or transcription.
- the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
- the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
- biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
- immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic PMMM, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
- “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement,
- 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 PMMM or fragments of PMMM may be employed as hybridization probes.
- the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
- the probe may be deployed in an aqueous solution containing salts (e.g., 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
- other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
- Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated
- Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
- the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
- Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha 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 amino group.
- a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
- a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
- a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
- “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
- Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins 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 PMMM or the polynucleotide encoding PMMM 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. For example, 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 pu ⁇ oses may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
- a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
- these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
- a fragment of SEQ ID NO: 17-32 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: 17-32, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
- a fragment of SEQ ID NO: 17-32 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO: 17-32 from related polynucleotide sequences.
- the precise length of a fragment of SEQ ED NO: 17-32 and the region of SEQ ID NO: 17-32 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended pu ⁇ ose for the fragment.
- a fragment of SEQ ID NO: 1-16 is encoded by a fragment of SEQ ID NO: 17-32.
- a fragment of SEQ ED NO: 1-16 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO: 1-16.
- a fragment of SEQ ID NO: 1-16 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO: 1-16.
- the precise length of a fragment of SEQ ID NO: 1-16 and the region of SEQ ED NO: 1-16 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended pu ⁇ ose for the fragment.
- a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
- a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
- Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
- percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
- NCBI National Center for Biotechnology Information
- BLAST Basic Local Alignment Search Tool
- NCBI National Center for Biotechnology Information
- BLAST Basic Local Alignment 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 may be, 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 ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
- Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
- Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
- Percent identity and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid 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. Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as inco ⁇ orated into the MEG ALIGN version 3.12e sequence alignment program (described and referenced above).
- NCBI BLAST software suite may be used.
- BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
- Such default parameters may be, for example:
- Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
- Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
- HACs Human artificial chromosomes
- HACs are 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 determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
- Permissive conditions for 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 m ) for the specific sequence at a defined ionic strength and pH.
- T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
- High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
- blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
- Organic solvent such as formamide at a concentration of about 35-50% v/v
- RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
- Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
- hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
- a hybridization complex may be formed in solution (e.g., C 0 t or R ⁇ ,t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
- insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
- Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
- an “immunogenic fragment” is a polypeptide or oligopeptide fragment of PMMM which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
- the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of PMMM 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 PMMM.
- modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PMMM.
- 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 may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
- “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. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
- PNA protein nucleic acid
- PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
- PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
- Post-translational modification of an PMMM 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 PMMM.
- Probe refers to nucleic acid sequences encoding PMMM, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences.
- Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
- Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for 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.
- PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that pu ⁇ ose 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 pu ⁇ ose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have inco ⁇ orated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
- the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
- the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
- this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
- the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
- a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
- the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
- a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
- such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
- a "regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
- Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
- RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
- sample is used in its broadest sense.
- a sample suspected of containing PMMM, nucleic acids encoding PMMM, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
- binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
- substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
- substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
- Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
- the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
- a “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
- Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
- transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
- a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
- the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
- the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
- the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
- a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
- Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
- a variant may be described as, for example, an "allelic” (as defined above), “splice,” “species,” or “polymo ⁇ hic” variant.
- a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
- the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
- Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
- a polymo ⁇ hic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
- Polymo ⁇ hic variants also may encompass "single nucleotide polymo ⁇ hisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
- a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
- Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
- the invention is based on the discovery of new human protein modification and maintenance molecules (PMMM), the polynucleotides encoding PMMM, and the use of these compositions for the diagnosis, treatment, or prevention of gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders.
- PMMM human protein modification and maintenance molecules
- 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 (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ED NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
- polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
- Table 2 shows sequences with homology to the polypeptides of the invention as identified by
- Table 3 shows various structural features of the polypeptides of the invention.
- Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the conesponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for each polypeptide of the invention.
- Column 3 shows the number of amino acid residues in each polypeptide.
- Column 4 shows potential phosphorylation sites and potential glycosylation sites as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison Wl), and amino acid residues comprising signature sequences, domains, and motifs.
- Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
- SEQ ID NO: 1 is 56% identical from residue Ml to residue A16, 60% identical from residue C24 to residue Q76, and 53% identical, from residue G60 to residue A268, to Mus musculus tryptase 4 (GenBank ED g 10947096) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.1e-78, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ ED NO: 1 also contains a trypsin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO: 1 is a serine protease.
- HMM hidden Markov model
- SEQ ED NO:2 is 73% identical, from residue Ml to residue V379, to monkey prochymosin (GenBank ID g7008025) as determined by the Basic Local Alignment Search Tool (BLAST).
- BLAST Basic Local Alignment Search Tool
- the BLAST probability score is 4.3e-142, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ ID NO: 2 also contains an eukaryotic aspartyl protease domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further conoborative evidence that SEQ ED NO:2 is an aspartic protease.
- SEQ ED NO:6 is 60% identical, from residue S31 to residue HI 120, to human zinc metalloendopeptidase ADAMTS10 (GenBank ID gl 1493589) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ED NO:6 also contains a reprolysin family propeptide, a reprolysin (M12B) family zinc metallopeptidase domain, and thrombospondin type 1 domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further corroborative evidence that SEQ ID NO: 6 is a zinc metalloprotease.
- HMM hidden Markov model
- SEQ ID NO:7 is 41% identical, from residue L10 to residue N298, to an epidermis specific serine protease from Xenopus laevis (GenBank ID g6009515) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 8.7e- 57, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:7 also contains a trypsin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO:7 is a serine protease.
- HMM hidden Markov model
- SEQ ED NO: 8 is 44% identical, from residue R20 to residue M425, to human serine protease (GenBank ID g6137097) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.2e-87, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO: 8 also contains a SEA domain and a Trypsin site as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ID NO: 8 is a serine protease (note that the "SEA domain” is found in enterokinase, a protease which cleaves the acidic propeptide from trypsinogen to yield active trypsin, (Kitamoto, Y. et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91:7588-7592) and serine proteases from the trypsin family provide catalytic activity).
- SEQ ID NO: 11 is 32% identical, from residue C588 to residue S903, to
- Mus musculus bone mo ⁇ hogenetic protein (GenBank ID g439607) as determined by the Basic Local Alignment Search Tool (BLAST).
- BLAST Basic Local Alignment Search Tool
- the BLAST probability score is l.le-62, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ ED NO:l 1 also contains a CUB domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from MOTIFS, and additional BLAST analyses provide further conoborative evidence that SEQ ID NO: 11 is a developmentally regulated protease.
- SEQ ED NO: 12 is 43% identical (over 204 amino acid residues) to a murine thrombospondin type 1 domain (GenBank ID g4519541), characteristic of the ADAMTS metalloproteinases family, as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 9.4e-49, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO: 12 also shares 30% identity (over 183 amino acid residues) with a Spodoptera frugiperda endoprotease (GenBank ID gl 167860), with a BLAST probability score of 7.3e-10.
- BLAST Basic Local Alignment Search Tool
- SEQ ID NO: 13 is 37% identical (over 457 amino acid residues) to a human zinc metallopeptidase (GenBank ID gl 1493589), as determined by BLAST analysis, with a probability score is 4.5e-75.
- SEQ ED NO: 13 also shares 34% identity (over 475 amino acid residues) with murine papilin (GenBank ED gl 1935122), a protease with homology to the ADAMTS metalloprotease family. The BLAST probability score is 5.9e-74.
- SEQ ID NO: 13 also contains a thrombospondin type 1 domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
- SEQ ID NO: 16 is 100% identical, from residue Pl 19 to residue S365, to human bK57G9.1 (novel Kringle and CUB domain protein) (GenBank ID g6572252) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.2e-135, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ ED NO: 16 also contains a CUB, a WSC, and a Kringle domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ID NO: 16 is a protease.
- SEQ ID NO:3-5, SEQ ID NO:9-10, and SEQ ID NO: 14-15 were analyzed and annotated in a similar manner.
- the algorithms and parameters for the analysis of SEQ ED NO: 1-16 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 ID NO:), the conesponding 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 amplification technologies that identify SEQ ID NO: 17-32 or that distinguish between SEQ ID NO: 17-32 and related polynucleotide sequences.
- the polynucleotide fragments described in Column 2 of Table 4 may refer specifically, 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 full length polynucleotide sequences.
- the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
- the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
- the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
- a polynucleotide sequence identified as FL_XXXXX_N,_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 applied, and YYYYY is the number of the prediction generated by the algorithm, and N I 2 3 , if present, represent specific exons that may have been manually 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 FLXXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAA ⁇ being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N refening to specific exons (See Example V).
- a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).
- 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 following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
- Incyte cDNA coverage redundant with the sequence coverage shown in Table
- Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences.
- the representative cDNA library is the Incyte cDNA library which is most frequently represented by the 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 PMMM variants.
- a prefened PMMM 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 PMMM amino acid sequence, and which contains at least one functional or structural characteristic of PMMM.
- the invention also encompasses polynucleotides which encode PMMM.
- the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 17-32, which encodes PMMM.
- the polynucleotide sequences of SEQ ID NO: 17-32, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occunences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
- the invention also encompasses a variant of a polynucleotide sequence encoding PMMM.
- 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 PMMM.
- a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO: 17-32 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 ED NO: 17-32.
- Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of PMMM.
- a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding PMMM.
- a splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding PMMM, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
- a splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to the polynucleotide sequence encoding PMMM over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide sequence encoding PMMM. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of PMMM.
- nucleotide sequences which encode PMMM and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurcing PMMM under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding PMMM or its derivatives possessing a substantially different codon usage, e.g., inclusion of non- naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
- RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occuning sequence.
- the invention also encompasses production of DNA sequences which encode PMMM and PMMM derivatives, or fragments thereof, entirely by synthetic chemistry.
- the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
- synthetic chemistry may be used to introduce mutations into a sequence encoding PMMM 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: 17-32 and fragments thereof under various conditions of stringency.
- Hybridization conditions including annealing and wash conditions, are described in "Definitions.”
- Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
- the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NI), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
- sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, lohn Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.)
- the nucleic acid sequences encoding PMMM 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.
- various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
- restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
- Another method uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
- the template is derived from restriction fragments comprising a known genomic locus and sunounding sequences.
- a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic.
- primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
- commercially available software such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
- Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
- Capillary electrophoresis systems which are commercially available may be used to analyze the size or 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/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
- Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
- polynucleotide sequences or fragments thereof which encode PMMM may be cloned in recombinant DNA molecules that direct expression of PMMM, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express PMMM.
- nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter PMMM-encoding sequences for a variety of pu ⁇ oses including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
- oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
- the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of PMMM, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
- MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. e
- DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These prefened variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
- genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
- sequences encoding PMMM may be synthesized, in whole or in part, using chemical methods well known in the art.
- chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
- PMMM itself or a fragment thereof may be synthesized using chemical methods.
- peptide synthesis can be performed using various solution-phase or solid-phase techniques.
- the nucleotide sequences encoding PMMM or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
- these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding PMMM.
- Such elements may vary in their strength and specificity.
- Specific initiation signals may also be used to achieve more efficient translation of sequences encoding PMMM. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
- a variety of expression vector/host systems may be utilized to contain and express sequences encoding PMMM. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
- yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g., baculovirus)
- plant cell systems transformed with viral expression vectors e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic
- Expression vectors derived from retroviruses, adenoviruses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
- the invention is not limited by the host cell employed.
- a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding PMMM.
- routine cloning, subcloning, and propagation of polynucleotide sequences encoding PMMM can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies).
- Yeast expression systems may be used for production of PMMM.
- a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
- such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
- Plant systems may also be used for expression of PMMM. Transcription of sequences encoding PMMM may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.
- 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
- constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
- pathogen-mediated transfection See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.
- a number of viral-based expression systems may be utilized.
- sequences encoding PMMM may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses PMMM in host cells.
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
- SV40 or EBV- based vectors may also be used for high-level protein expression.
- HACs Human artificial chromosomes
- HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
- HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic pu ⁇ oses. (See, e.g., Ha ington, J.J. et al. (1997) Nat. Genet. 15:345-355.)
- sequences encoding PMMM can be transformed into cell 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. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
- the pu ⁇ ose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
- Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.. Any number of selection systems may be used to recover transformed cell lines.
- he ⁇ es simplex virus thymidine kinase and adenine phosphoribosyltransferase genes for use in tk ⁇ and apr cells, respectively.
- adenine phosphoribosyltransferase genes for use in tk ⁇ and apr cells, respectively.
- antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
- dhfr confers resistance to methotrexate
- neo confers resistance to the aminoglycosides neomycin and G-418
- als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
- Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular 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.
- 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 PMMM is inserted within a marker gene sequence, transformed cells containing sequences encoding PMMM can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding PMMM under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
- host cells that contain the nucleic acid sequence encoding PMMM and that express PMMM may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of PMMM 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 cell sorting (FACS).
- ELISAs enzyme-linked immunosorbent assays
- RIAs radioimmunoassays
- FACS fluorescence activated cell sorting
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on PMMM is preferred, but a competitive binding assay may be employed.
- assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Cunent 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 PMMM include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- sequences encoding PMMM, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
- RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
- T7, T3, or SP6 RNA polymerase
- reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host cells transformed with nucleotide sequences encoding PMMM may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
- the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode PMMM may be designed to contain signal sequences which direct secretion of PMMM through a prokaryotic or eukaryotic cell membrane.
- a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
- Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
- Post-translational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
- Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the conect modification and processing of the foreign protein.
- ATCC American Type Culture Collection
- natural, modified, or recombinant nucleic acid sequences encoding PMMM may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
- a chimeric PMMM protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of PMMM activity.
- Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
- Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
- GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
- FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
- a fusion protein may also be engineered to contain a proteolytic cleavage site located between the PMMM encoding sequence and the heterologous protein sequence, so that PMMM may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
- synthesis of radiolabeled PMMM may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
- PMMM of the present invention or fragments thereof may be used to screen for compounds that specifically bind to PMMM.
- At least one and up to a plurality of test compounds may be screened for specific binding to PMMM.
- test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
- the compound thus identified is closely related to the natural ligand of PMMM, 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 PMMM binds, or to at least a fragment of the receptor, e.g., the ligand binding site.
- the compound can be rationally designed using known techniques.
- screening for these compounds involves producing appropriate cells which express PMMM, either as a secreted protein or on the cell membrane.
- Prefened cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing PMMM or cell membrane fractions which contain PMMM are then contacted with a test compound and binding, stimulation, or inhibition of activity of either PMMM 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 PMMM, either in solution or affixed to a solid support, and detecting the binding of PMMM to the compound.
- the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
- the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
- PMMM of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of PMMM.
- Such compounds may include agonists, antagonists, or partial or inverse agonists.
- an assay is performed under conditions permissive for PMMM activity, wherein PMMM is combined with at least one test compound, and the activity of PMMM in the presence of a test compound is compared with the activity of PMMM in the absence of the test compound. A change in the activity of PMMM in the presence of the test compound is indicative of a compound that modulates the activity of PMMM.
- a test compound is combined with an in vitro or cell-free system comprising PMMM under conditions suitable for PMMM activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of PMMM 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 may be screened.
- polynucleotides encoding PMMM or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
- ES embryonic stem
- Such techniques are well known in the art and are useful for the generation of animal models of human disease.
- mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
- the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244: 1288-1292).
- a marker gene e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244: 1288-1292).
- the vector integrates into the conesponding 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 cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
- the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
- Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
- Polynucleotides encoding PMMM may also be manipulated in vitro in ES cells derived from human blastocysts.
- Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
- Polynucleotides encoding PMMM 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 PMMM is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
- Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
- Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
- a mammal inbred to overexpress PMMM e.g., by secreting PMMM in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55- 74).
- PMMM Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of PMMM and protein modification and maintenance molecules.
- the expression of PMMM is closely associated with bone tumor, kidney, ovarian tumor, gastrointestinal, diseased prostate, uterus tumor, and brain tissue, including posterior cingulate tissue, as well as fibroblasts. Therefore, PMMM appears to play a role in gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders.
- PMMM 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 PMMM.
- disorders include, but are not limited to, a gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, chrhosis, passive congestion of the liver, hepatoma,
- a vector capable of expressing PMMM 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 PMMM including, but not limited to, those described above.
- composition comprising a substantially purified PMMM in conjunction with a suitable pharmaceutical canier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PMMM including, but not limited to, those provided above.
- an agonist which modulates the activity of PMMM may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PMMM including, but not limited to, those listed above.
- an antagonist of PMMM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PMMM.
- disorders include, but are not limited to, those gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders described above.
- an antibody which specifically binds PMMM may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express PMMM.
- a vector expressing the complement of the polynucleotide encoding PMMM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PMMM including, but not limited to, those described above.
- any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
- the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
- An antagonist of PMMM may be produced using methods which are generally known in the art.
- purified PMMM may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind PMMM.
- Antibodies to PMMM may also be generated using methods that are well 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.
- Neutralizing 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, llamas, humans, and others may be immunized by injection with PMMM or with any fragment or oligopeptide 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
- Corvnebacterium parvum are especially preferable.
- the oligopeptides, peptides, or fragments used to induce antibodies to PMMM have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of PMMM amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
- Monoclonal antibodies to PMMM may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
- chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
- techniques developed for the production of single chain antibodies may be adapted, using methods known in the art, to produce PMMM-specific single chain antibodies.
- Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin 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 literature. (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 PMMM 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 allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
- immunoassays may be used for screening to identify antibodies having the desired specificity.
- Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
- Such immunoassays typically involve the measurement of complex formation between PMMM and its specific antibody.
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering PMMM epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
- K a is defined as the molar concentration of PMMM-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
- K a association constant
- the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular PMMM 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 PMMM-antibody complex must withstand rigorous manipulations.
- Low-affinity 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 PMMM, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
- polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
- a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of PMMM-antibody complexes.
- Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)
- the polynucleotides encoding PMMM may be used for therapeutic pu ⁇ oses.
- modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding PMMM.
- complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
- antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding PMMM. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.)
- Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein.
- Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
- polynucleotides encoding PMMM may be used for somatic or germline gene therapy.
- Gene therapy may be 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. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al.
- SCID severe combined immunodeficiency
- ADA adenosine deaminase
- PMMM are treated by constructing mammalian expression vectors encoding PMMM and introducing these vectors by mechanical means into PMMM-deficient cells.
- Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62: 191-217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Cun. Opin. Biotechnol. 9:445-450).
- Expression vectors that may be effective for the expression of PMMM include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
- PMMM may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the 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) OUT. Opin. Biotechnol.
- a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
- liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
- 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 cells requires modification of these standardized mammalian transfection protocols.
- diseases or disorders caused by genetic defects with respect to PMMM expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding PMMM 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 cis-acting RNA sequences and coding sequences required for efficient vector propagation.
- Retrovirus vectors e.g., PFB and PFBNEO
- Retrovirus vectors are commercially 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 cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61: 1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al.
- VSVg vector producing cell line
- U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby inco ⁇ orated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T- cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020- 7029; Bauer, G. et al.
- an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding PMMM to cells which have one or more genetic abnormalities with respect to the expression of PMMM.
- the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No.
- Addenovirus vectors for gene therapy hereby inco ⁇ orated by reference.
- adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both inco ⁇ orated by reference herein.
- a he ⁇ es-based, gene therapy delivery system is used to deliver polynucleotides encoding PMMM to target cells which have one or more genetic abnormalities with respect to the expression of PMMM.
- the use of he ⁇ es simplex virus (HSV)-based vectors may be especially valuable for introducing PMMM to cells of the central nervous system, for which HSV has a tropism.
- the construction and packaging of he ⁇ es-based vectors are well known to those with ordinary skill in the art.
- a replication-competent he ⁇ es 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 ("He ⁇ es simplex virus strains for gene transfer"), which is hereby inco ⁇ orated 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 cell under the control of the appropriate promoter for pu ⁇ oses including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
- HSV vectors see also Goins, W.F. et al.
- an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding PMMM to target cells.
- SFV Semliki Forest Virus
- This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the ove ⁇ roduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
- enzymatic activity e.g., protease and polymerase.
- inserting the coding sequence for PMMM into the alphavirus genome in place of the capsid-coding region results in the production of a large number of PMMM-coding RNAs and the synthesis of high levels of PMMM in vector transduced cells.
- alphavirus infection is typically associated with cell lysis within a few days
- the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SEN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
- the wide host range of alphaviruses will allow the introduction of PMMM into a variety of cell types.
- the specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
- the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
- Oligonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163- 177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
- Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
- the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
- engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding PMMM.
- RNA sequences within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC
- short RNA sequences of between 15 and 20 ribonucleotides, conesponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
- the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
- RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
- RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding PMMM. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
- these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
- RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase 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 PMMM.
- Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non- macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
- a compound which specifically inhibits expression of the polynucleotide encoding PMMM may be therapeutically useful, and in the treatment of disorders associated with decreased PMMM expression or activity, a compound which specifically promotes expression of the polynucleotide encoding PMMM may be therapeutically useful.
- test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
- a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly.
- a sample comprising a polynucleotide encoding PMMM is exposed to at least one test compound thus obtained.
- the sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system.
- Alterations in the expression of a polynucleotide encoding PMMM are assayed by any method commonly known in the art.
- the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding PMMM.
- the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
- a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Amdt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun.
- a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
- oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
- vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
- compositions which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
- Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
- Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
- Such compositions may consist of PMMM, antibodies to PMMM, and mimetics, agonists, antagonists, or inhibitors of PMMM.
- compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g.
- Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
- compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
- the determination of an effective dose is well within the capability of those skilled in the art.
- compositions may be prepared for direct intracellular delivery of macromolecules comprising PMMM or fragments thereof.
- liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule.
- PMMM or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285: 1569-1572).
- the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
- An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- a therapeutically effective dose refers to that amount of active ingredient, for example PMMM or fragments thereof, antibodies of PMMM, and agonists, antagonists or inhibitors of PMMM, which ameliorates the symptoms or condition.
- Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
- the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
- Compositions which exhibit large therapeutic indices are prefened. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
- the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with 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 will 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, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
- Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
- Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
- DIAGNOSTICS In another embodiment, antibodies which specifically bind PMMM may be used for the diagnosis of disorders characterized by expression of PMMM, or in assays to monitor patients being treated with PMMM or agonists, antagonists, or inhibitors of PMMM.
- Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for PMMM include methods which utilize the antibody and a label to detect PMMM in human body fluids or in extracts of cells or tissues.
- the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
- a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
- PMMM ELISAs, RIAs, and FACS
- ELISAs ELISAs
- RIAs RIAs
- FACS fluorescence-activated cell sorting
- normal or standard values for PMMM expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to PMMM under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of PMMM 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 PMMM may be used for diagnostic pu ⁇ oses.
- the polynucleotides which may be used include oligonucleotide 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 PMMM may be correlated with disease.
- the diagnostic assay may be used to determine absence, presence, and excess expression of PMMM, and to monitor regulation of PMMM levels during therapeutic intervention.
- hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding PMMM or closely related molecules may be used to identify nucleic acid sequences which encode PMMM.
- 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 amplification will determine whether the probe identifies only naturally occurring sequences encoding PMMM, allelic 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 PMMM encoding sequences.
- the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO: 17-32 or from genomic sequences including promoters, enhancers, and introns of the PMMM gene.
- Means for producing specific hybridization probes for DNAs encoding PMMM include the cloning of polynucleotide sequences encoding PMMM or PMMM derivatives into vectors for the production of mRNA probes.
- Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides 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 PMMM may be used for the diagnosis of disorders associated with expression of PMMM.
- disorders include, but are not limited to, a gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cinhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcer
- the polynucleotide sequences encoding PMMM may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microanays utilizing fluids or tissues from patients to detect altered PMMM expression. Such qualitative or quantitative methods are well known in the art.
- the nucleotide sequences encoding PMMM may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
- the nucleotide sequences encoding PMMM may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding PMMM in the sample indicates the presence of the associated disorder.
- Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
- a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding PMMM, 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 substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish 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 allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
- oligonucleotides designed from the sequences encoding PMMM may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding PMMM, or a fragment of a polynucleotide complementary to the polynucleotide encoding PMMM, 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.
- oligonucleotide primers derived from the polynucleotide sequences encoding PMMM may be used to detect single nucleotide polymo ⁇ hisms (SNPs).
- SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
- Methods of SNP detection include, but are not limited to, single-stranded conformation polymo ⁇ hism (SSCP) and fluorescent SSCP (fSSCP) methods.
- SSCP single-stranded conformation polymo ⁇ hism
- fSSCP fluorescent SSCP
- oligonucleotide primers derived from the polynucleotide sequences encoding PMMM are used to amplify DNA using the polymerase chain reaction (PCR).
- the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
- SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
- the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high- throughput equipment such as DNA sequencing machines.
- sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymo ⁇ hisms by comparing the 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-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease.
- variants in the mannose-binding lectin, MBL2 have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis.
- SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity.
- a variation in 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-lipoxygenase pathway.
- Methods which may also be used to quantify the expression of PMMM include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
- radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
- the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
- oligonucleotides 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 polymo ⁇ hisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
- this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
- therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
- PMMM, fragments of PMMM, or antibodies specific for PMMM may be used as elements on a microanay.
- the microanay may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
- a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type.
- a transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, expressly inco ⁇ orated by reference herein.)
- a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type.
- the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microanay.
- the resultant transcript image would provide a profile of gene activity.
- Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples.
- the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
- Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occuning environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular finge ⁇ rints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24: 153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly inco ⁇ orated by reference herein).
- a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
- These finge ⁇ rints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, 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 well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in inte ⁇ retation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity.
- the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
- Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels conesponding to the polynucleotides of the present invention may be quantified.
- the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
- proteome refers to the global pattern of protein expression in a particular tissue or cell 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 cell' s proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
- the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by 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 visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
- the optical density of each protein spot is generally proportional to the level of the protein in the sample.
- the optical densities of equivalently positioned protein spots from different samples for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment.
- the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed 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 may be obtained for definitive protein identification.
- a proteomic profile may also be generated using antibodies specific for PMMM to quantify the levels of PMMM 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.
- Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each anay element.
- Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel 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 may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
- the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable 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.
- Microanays may be prepared, used, and analyzed using methods known in the art.
- nucleic acid sequences encoding PMMM may be used to generate hybridization probes useful in mapping the naturally occuning genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
- the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
- BACs bacterial Pl constructions, or single chromosome cDNA libraries.
- BACs bacterial Pl constructions, or single chromosome cDNA libraries.
- BACs bacterial Pl constructions, or single chromosome cDNA libraries.
- RFLP restriction fragment length polymo ⁇ hism
- Fluorescent in situ hybridization may be conelated with other physical and genetic map data.
- 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 (OMIM) World Wide Web site. Conelation between the location of the gene encoding PMMM 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.
- In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian 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 localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
- the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, canier, or affected individuals.
- PMMM, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries 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 solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between PMMM and the agent being tested may be measured.
- Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
- This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with PMMM, or fragments thereof, and washed. Bound PMMM is then detected by methods well known in the art. Purified PMMM can also be coated directly onto plates for use in the aforementioned drug screening techniques.
- non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
- nucleotide sequences which encode PMMM may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are cunently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
- Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ 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 oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
- Stratagene was provided with RNA and constructed the corresponding cDNA libraries.
- cDNA was synthesized and cDNA libraries 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 oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated 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 ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT 1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.
- Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones
- Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
- plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling steps were canied out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
- Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
- Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were canied out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
- the polynucleotide sequences derived from Incyte cDNAs were validated 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 public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens. Rattus norvegicus. Mus musculus, Caenorhabditis elegans.
- HMM hidden Markov model
- 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 full length polynucleotide sequences were translated to derive the conesponding full length polypeptide sequences. Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide.
- Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM.
- Full 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 alignments are generated using default parameters specified by the CLUSTAL algorithm as inco ⁇ orated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
- Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters.
- the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
- Genscan is a general-pu ⁇ ose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Cun. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
- 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 protein modification and maintenance molecules. Potential protein modification and maintenance molecules were also identified by homology to Incyte cDNA sequences that had been annotated as protein modification and maintenance molecules. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases.
- 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 public 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.
- Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example in. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences. V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences
- Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example IE 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 splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
- Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis.
- GenBank primate a GenBank primate
- rodent a rodent
- mammalian a mammalian
- vertebrate eukaryote databases
- eukaryote databases using the BLAST program.
- GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example EV.
- 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.
- GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public 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 PMMM Encoding Polynucleotides The sequences which were used to assemble SEQ ED NO: 17-32 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm.
- Map locations are represented by ranges, or intervals, of human chromosomes.
- the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
- centiMorgan cM
- centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
- the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
- SEQ ID NO:30 was mapped to chromosome 5 within the interval from 174.30 centiMorgans to the q terminus, and to chromosome 10 within the interval from 83.30 to 96.90 centiMorgans. More than one map location is reported for SEQ ID NO:30, indicating that sequences having different map locations were assembled into a single cluster. This situation occurs, for example, when sequences having strong similarity, but not complete identity, are assembled into a single cluster. VII. Analysis of Polynucleotide Expression
- Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs • from a particular cell type or tissue have been bound.
- a membrane on which RNAs • from a particular cell type or tissue have been bound See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.
- Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LEFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations.
- the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
- the basis of the search is the product score, which is defined as:
- the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
- the product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
- the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment 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 pair with the highest BLAST score is used to calculate the product score.
- the product score represents a balance between fractional overlap and quality in a BLAST alignment.
- 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 PMMM 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 IH). Each cDNA sequence is derived from a cDNA library constructed from a human tissue.
- Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
- the number of libraries in each category is counted and divided by the total number of libraries across all categories.
- each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding PMMM.
- cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of PMMM Encoding Polynucleotides
- Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide 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 hai ⁇ in structures and primer-primer dimerizations was avoided.
- Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
- 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 well of an opaque fluorimeter plate (Coming Costar, Acton MA), allowing the DNA to bind to the reagent.
- the plate was scanned in a Fluoroskan ⁇ (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
- a 5 l to 10 ⁇ l aliquot 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, transfened to 384-well plates, digested with CviJI cholera virus endonuc lease (Molecular Biology Research, Madison Wl), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
- CviJI cholera virus endonuc lease Molecular Biology Research, Madison Wl
- sonicated or sheared prior to religation 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 fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media.
- the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
- SNPs Single nucleotide polymo ⁇ hisms
- Preliminary filters removed the majority of basecall enors by requiring a minimum Phred quality score of 15, and removed sequence alignment enors and enors resulting from improper trimming of vector sequences, chimeras, and splice variants.
- An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP.
- Clone enor filters used statistically generated algorithms to identify enors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
- Clustering enor filters used statistically generated algorithms to identify enors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences.
- a final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.
- Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations.
- the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
- the African population comprised 194 individuals (97 male, 97 female), all African Americans.
- the Hispanic population comprised 324 individuals (162 male, 162 female), all 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. Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
- Hybridization probes derived from SEQ ID NO: 17-32 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides 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 oligonucleotides are substantially purified using a
- SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl ⁇ , Eco RI, Pst I, Xba I, or Pvu B (DuPont NEN). The DNA from each digest is fractionated on a 0.7% agarose gel and transfened to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH).
- Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared. XL Microarrays
- the linkage or synthesis of anay elements upon a microanay can be achieved utilizing photolithography, 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 solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, 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, UV, chemical, or mechanical bonding procedures.
- a typical anay may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
- Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microanay. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
- the anay elements are hybridized with polynucleotides in a biological sample.
- the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
- a fluorescence scanner is used to detect hybridization at each anay element. Altematively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
- RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
- Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-(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. (CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS. Microarray Preparation
- Sequences of the present invention are used to generate anay elements.
- Each anay element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
- PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
- Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified anay elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
- Purified anay elements are immobilized on polymer-coated glass slides.
- Glass microscope slides (Coming) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
- Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Co ⁇ oration (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
- Anay elements are applied to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, inco ⁇ orated herein by reference.
- 1 ⁇ l of the anay element DNA is loaded into the open capillary printing element by a high-speed robotic apparatus.
- the apparatus then deposits about 5 nl of anay element sample per slide.
- Microarrays are UV -crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distilled 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 followed by washes in 0.2% SDS and distilled water as before. Hybridization
- Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
- the sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microanay surface and covered with an 1.8 cm 2 coverslip.
- the anays are transfened to a wate ⁇ roof chamber having a cavity just slightly larger than a microscope slide.
- the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a comer of the chamber.
- 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
- 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 light is focused on the anay using a 20X microscope objective (Nikon, Inc., Melville NY).
- the slide containing the anay is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
- the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
- a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) conesponding to the two fluorophores. Appropriate filters positioned between the anay and the photomultiplier tubes are used to filter the signals.
- the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
- Each array is typically scanned twice, one scan per 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 typically calibrated 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, allowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1: 100,000.
- the calibration 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 photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
- the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
- the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first 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 conesponding to the average intensity of the signal.
- the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
- oligonucleotide sequences complementary to the PMMM-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occuning PMMM. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of PMMM. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the PMMM-encoding transcript.
- PMMM Expression and purification of PMMM 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 trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
- Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
- Antibiotic resistant bacteria express PMMM upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
- PMMM in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
- AcMNPV Autographica californica nuclear polyhedrosis virus
- the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding PMMM by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
- Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
- PMMM is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell 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 PMMM obtained by these methods can be used directly in the assays shown in Examples XVH, XVIH, and XIX, where applicable. XIV. Functional Assays PMMM function is assessed by expressing the sequences encoding PMMM at physiologically elevated levels in mammalian cell culture systems.
- cDNA is subcloned into a mammalian 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 cell line, for example, an endothehal or hematopoietic cell line, using either liposome formulations or electroporation.
- 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
- marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable 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 an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death.
- the influence of PMMM on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding PMMM and either CD64 or CD64-GFP.
- CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
- Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
- mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding PMMM and other genes of interest can be analyzed by northern analysis or microanay techniques.
- the PMMM amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
- LASERGENE software DNASTAR
- Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
- oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
- ABI 431 A peptide synthesizer Applied Biosystems
- KLH Sigma- Aldrich, St. Louis MO
- MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
- Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
- Resulting antisera are tested for antipeptide and anti-PMMM activity by, for example, binding the peptide or PMMM to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
- Naturally occuning or recombinant PMMM is substantially purified by immunoaffinity chromatography using antibodies specific for PMMM.
- An immunoaffinity column is constructed by covalently coupling anti-PMMM antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
- Media containing PMMM are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PMMM (e.g., high ionic strength buffers in the presence of detergent).
- the column is eluted under conditions that disrupt antibody/PMMM 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 PMMM is collected.
- PMMM or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
- Bolton-Hunter reagent See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
- Candidate molecules previously anayed in the wells of a multi-well plate are incubated with the labeled PMMM, washed, and any wells with labeled PMMM complex are assayed. Data obtained using different concentrations of PMMM are used to calculate values for the number, affinity, and association of PMMM with the candidate molecules.
- molecules interacting with PMMM are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
- PMMM may also be used in the PATHCALLING process (CuraGen Co ⁇ ., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
- Protease activity is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantified by spectrophotometric (or fluorometric) abso ⁇ tion of the released chromophore (Beynon, R.J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York NY, pp.25-55).
- Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases, or metalloproteases), aminopeptidase (leucine aminopeptidase), or carboxypeptidase (carboxypeptidases A and B, procollagen C-proteinase).
- Commonly used chromogens are 2-naphthylamine, 4-nitroaniline, and furylacrylic acid.
- Assays are performed at ambient temperature and contain an aliquot of the enzyme and the appropriate substrate in a suitable buffer. Reactions are canied out in an optical cuvette, and the increase/decrease in absorbance of the chromogen released during hydrolysis of the peptide substrate is measured. The change in absorbance is proportional to the enzyme activity in the assay.
- An alternate assay for ubiquitin hydrolase activity measures the hydrolysis of a ubiquitin precursor.
- the assay is performed at ambient temperature and contains an aliquot of PMMM and the appropriate substrate in a suitable buffer. Chemically synthesized human ubiquitin-valine may be used as substrate. Cleavage of the C-terminal valine residue from the substrate is monitored by capillary electrophoresis (Franklin, K. et al. (1997) Anal. Biochem. 247:305-309).
- an assay for protease activity takes advantage of fluorescence resonance energy transfer (FRET) that occurs when one donor and one acceptor fluorophore with an appropriate spectral overlap are in close proximity.
- FRET fluorescence resonance energy transfer
- a flexible peptide linker containing a cleavage site specific for PMMM is fused between a red-shifted variant (RSGFP4) and a blue variant (BFP5) of Green Fluorescent Protein.
- RGFP4 red-shifted variant
- BFP5 blue variant
- This fusion protein has spectral properties that suggest energy transfer is occuning from BFP5 to RSGFP4.
- Phage display libraries can be used to identify optimal substrate sequences for PMMM.
- a random hexamer followed by a linker and a known antibody epitope is cloned as an N-terminal extension of gene HI in a filamentous phage library.
- Gene HI codes for a coat protein, and the epitope will be displayed on the surface of each phage particle.
- the library is incubated with PMMM under proteolytic conditions so that the epitope will be removed if the hexamer codes for a PMMM cleavage site.
- An antibody that recognizes the epitope is added along with immobilized protein A. Uncleaved phage, which still bear the epitope, are removed by centrifugation.
- Phage in the supernatant are then amplified and undergo several more rounds of screening. Individual phage clones are then isolated and sequenced. Reaction kinetics for these peptide substrates can be studied using an assay in Example XVB, and an optimal cleavage sequence can be derived (Ke, S.H. et al. (1997) J. Biol. Chem. 272: 16603-16609).
- this method can be expanded to screen a cDNA expression library displayed on the surface of phage particles (T7SELECT 10-3 Phage display vector, Novagen, Madison Wl) or yeast cells (pYDl yeast display vector kit, Invitrogen, Carlsbad CA).
- phage particles T7SELECT 10-3 Phage display vector, Novagen, Madison Wl
- yeast cells pYDl yeast display vector kit, Invitrogen, Carlsbad CA.
- entire cDNAs are fused between Gene DI and the appropriate epitope.
- Compounds to be tested are arrayed in the wells of a multi-well plate in varying concentrations along with an appropriate buffer and substrate, as described in the assays in Example XVB.
- PMMM activity is measured for each well and the ability of each compound to inhibit PMMM activity can be determined, as well as the dose-response kinetics. This assay could also be used to identify molecules which enhance PMMM activity.
- phage display libraries can be used to screen for peptide PMMM inhibitors.
- Candidates are found among peptides which bind tightly to a protease.
- multi-well plate wells are coated with PMMM and incubated with a random peptide phage display library or a cyclic peptide library (Koivunen, E. et al. (1999) Nat. Biotechnol. 17:768-774). Unbound phage are washed away and selected phage amplified and rescreened for several more rounds. Candidates are tested for
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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US10/467,042 US20040077048A1 (en) | 2002-01-30 | 2002-01-30 | Protein modification and maintenance molecules |
EP02723076A EP1356028A2 (fr) | 2001-01-31 | 2002-01-30 | Molecules de modification et de maintenance de proteines |
AU2002253883A AU2002253883A1 (en) | 2001-01-31 | 2002-01-30 | Protein modification and maintenance molecules |
CA002434953A CA2434953A1 (fr) | 2001-01-31 | 2002-01-30 | Molecules de modification et de maintenance proteiques |
JP2002561510A JP2005503108A (ja) | 2001-01-31 | 2002-01-30 | タンパク質修飾およびメンテナンス分子 |
US11/046,868 US20050142600A1 (en) | 2001-01-31 | 2005-02-01 | Protein modification and maintenance molecules |
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US26570501P | 2001-01-31 | 2001-01-31 | |
US60/265,705 | 2001-01-31 | ||
US26676201P | 2001-02-05 | 2001-02-05 | |
US60/266,762 | 2001-02-05 | ||
US26958101P | 2001-02-16 | 2001-02-16 | |
US60/269,581 | 2001-02-16 | ||
US27119801P | 2001-02-23 | 2001-02-23 | |
US60/271,198 | 2001-02-23 | ||
US27281301P | 2001-03-01 | 2001-03-01 | |
US60/272,813 | 2001-03-01 | ||
US27558601P | 2001-03-13 | 2001-03-13 | |
US60/275,586 | 2001-03-13 | ||
US27850501P | 2001-03-23 | 2001-03-23 | |
US60/278,505 | 2001-03-23 | ||
US28053901P | 2001-03-30 | 2001-03-30 | |
US60/280,539 | 2001-03-30 |
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JP (1) | JP2005503108A (fr) |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002050277A2 (fr) * | 2000-12-21 | 2002-06-27 | Curagen Corporation | Proteines et acides nucleiques codant pour celles-ci |
WO2003091280A1 (fr) * | 2002-04-26 | 2003-11-06 | Kirin Beer Kabushiki Kaisha | Polypeptide a activite entretenant la proliferation ou la survie de cellules souches hematopoietiques ou de progeniteurs hematopoietiques, et adn codant ce polypeptide |
NL1022252C2 (nl) * | 2000-02-18 | 2003-11-18 | Hoffmann La Roche | Nieuwe metallo-proteasen met trombospondine domeinen en daarvoor coderende nucle´nezuur samenstellingen. |
EP1427747A2 (fr) * | 2001-08-30 | 2004-06-16 | Nuvelo, Inc. | Procedes et materiaux relatifs aux polynucleotides et polypeptides de type facteur de croissance de cellules souches |
WO2005040418A2 (fr) * | 2003-10-10 | 2005-05-06 | Deutsches Krebsforschungszentrum | Compositions pour le diagnostic et le traitement de maladies associees a l'expression aberrante des futrins (r-spondins) et/ou wnt |
WO2006053955A2 (fr) * | 2004-11-19 | 2006-05-26 | Oy Jurilab Ltd | Procede et kit de detection d'un risque d'hypertension arterielle essentielle |
US7125701B2 (en) | 2001-07-27 | 2006-10-24 | Wyeth | Aggrecanase molecules |
US7320880B2 (en) | 2001-06-11 | 2008-01-22 | Nuvelo, Inc. | Polypeptide having an activity to support proliferation or survival of hematopoietic stem cell and hematopoietic progenitor cell, and DNA coding for the same |
US8926970B2 (en) | 2006-10-20 | 2015-01-06 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Rspondin antibodies as inhibiting factors of angiogenesis and vaculogenesis |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001098503A2 (fr) * | 2000-06-21 | 2001-12-27 | Bayer Aktiengesellschaft | Regulation d'une enzyme de type serine protease 1 d'eosinophile humain |
WO2002000860A2 (fr) * | 2000-06-26 | 2002-01-03 | Sugen, Inc. | Nouvelles proteases |
-
2002
- 2002-01-30 CA CA002434953A patent/CA2434953A1/fr not_active Abandoned
- 2002-01-30 EP EP02723076A patent/EP1356028A2/fr not_active Withdrawn
- 2002-01-30 WO PCT/US2002/002813 patent/WO2002060942A2/fr not_active Application Discontinuation
- 2002-01-30 JP JP2002561510A patent/JP2005503108A/ja active Pending
- 2002-01-30 AU AU2002253883A patent/AU2002253883A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001098503A2 (fr) * | 2000-06-21 | 2001-12-27 | Bayer Aktiengesellschaft | Regulation d'une enzyme de type serine protease 1 d'eosinophile humain |
WO2002000860A2 (fr) * | 2000-06-26 | 2002-01-03 | Sugen, Inc. | Nouvelles proteases |
Non-Patent Citations (4)
Title |
---|
DATABASE EMBL [Online] 1 September 1998 (1998-09-01) RICKE,D.O. ET AL.: "Homo sapiens chromosome 16, cosmid clone 407D8 (LANL), complete sequence" retrieved from EBI Database accession no. AC005570 XP002225576 * |
DATABASE EMBL [Online] 4 August 1998 (1998-08-04) RICKE D.O. ET AL.: "Homo sapiens chromosome 16, cosmid clone 352F10 (LANL), complete sequence." retrieved from EBI Database accession no. AC005361 XP002225575 * |
INOUE MASAHIRO ET AL: "Structural analysis of esp-1 gene (PRSS 21)" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 266, no. 2, 20 December 1999 (1999-12-20), pages 564-568, XP002208048 ISSN: 0006-291X * |
RAWLINGS N D ET AL: "FAMILIES OF SERINE PEPTIDASES" METHODS IN ENZYMOLOGY, ACADEMIC PRESS INC, SAN DIEGO, CA, US, vol. 244, 1994, pages 19-61, XP008008505 ISSN: 0076-6879 cited in the application * |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NL1022252C2 (nl) * | 2000-02-18 | 2003-11-18 | Hoffmann La Roche | Nieuwe metallo-proteasen met trombospondine domeinen en daarvoor coderende nucle´nezuur samenstellingen. |
WO2002050277A3 (fr) * | 2000-12-21 | 2004-02-26 | Curagen Corp | Proteines et acides nucleiques codant pour celles-ci |
WO2002050277A2 (fr) * | 2000-12-21 | 2002-06-27 | Curagen Corporation | Proteines et acides nucleiques codant pour celles-ci |
US7320880B2 (en) | 2001-06-11 | 2008-01-22 | Nuvelo, Inc. | Polypeptide having an activity to support proliferation or survival of hematopoietic stem cell and hematopoietic progenitor cell, and DNA coding for the same |
US7125701B2 (en) | 2001-07-27 | 2006-10-24 | Wyeth | Aggrecanase molecules |
EP1427747A2 (fr) * | 2001-08-30 | 2004-06-16 | Nuvelo, Inc. | Procedes et materiaux relatifs aux polynucleotides et polypeptides de type facteur de croissance de cellules souches |
WO2003091280A1 (fr) * | 2002-04-26 | 2003-11-06 | Kirin Beer Kabushiki Kaisha | Polypeptide a activite entretenant la proliferation ou la survie de cellules souches hematopoietiques ou de progeniteurs hematopoietiques, et adn codant ce polypeptide |
US7439332B2 (en) | 2002-04-26 | 2008-10-21 | Kirin Pharma Kabushiki Kaisha | Polypeptide having an activity to support proliferation or survival of hematopoietic stem or progenitor cells |
WO2005040418A2 (fr) * | 2003-10-10 | 2005-05-06 | Deutsches Krebsforschungszentrum | Compositions pour le diagnostic et le traitement de maladies associees a l'expression aberrante des futrins (r-spondins) et/ou wnt |
WO2005040418A3 (fr) * | 2003-10-10 | 2005-11-10 | Deutsches Krebsforsch | Compositions pour le diagnostic et le traitement de maladies associees a l'expression aberrante des futrins (r-spondins) et/ou wnt |
WO2006053955A3 (fr) * | 2004-11-19 | 2006-08-31 | Jurilab Ltd Oy | Procede et kit de detection d'un risque d'hypertension arterielle essentielle |
WO2006053955A2 (fr) * | 2004-11-19 | 2006-05-26 | Oy Jurilab Ltd | Procede et kit de detection d'un risque d'hypertension arterielle essentielle |
US8926970B2 (en) | 2006-10-20 | 2015-01-06 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Rspondin antibodies as inhibiting factors of angiogenesis and vaculogenesis |
US9226963B2 (en) | 2006-10-20 | 2016-01-05 | Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts | Antagonist anti-Rspondin3 antibodies |
US10273276B2 (en) | 2006-10-20 | 2019-04-30 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Rspondins as modulators of angiogenesis and vasculogenesis |
US10538563B2 (en) | 2006-10-20 | 2020-01-21 | Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts | Rspondins as modulators of angiogenesis and vasculogenesis |
Also Published As
Publication number | Publication date |
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WO2002060942A3 (fr) | 2003-07-24 |
JP2005503108A (ja) | 2005-02-03 |
EP1356028A2 (fr) | 2003-10-29 |
CA2434953A1 (fr) | 2002-08-08 |
AU2002253883A1 (en) | 2002-08-12 |
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