EP1165788A2 - Moleküle aus dem immunsystem - Google Patents
Moleküle aus dem immunsystemInfo
- Publication number
- EP1165788A2 EP1165788A2 EP00921733A EP00921733A EP1165788A2 EP 1165788 A2 EP1165788 A2 EP 1165788A2 EP 00921733 A EP00921733 A EP 00921733A EP 00921733 A EP00921733 A EP 00921733A EP 1165788 A2 EP1165788 A2 EP 1165788A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- imol
- polynucleotide
- sequence
- sequences
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- C07K—PEPTIDES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to nucleic acid and amino acid sequences of immune system molecules and to the use of these sequences in the diagnosis, treatment, and prevention of lmmunological disorders, infections, and cell pro ferative disorders including cancer
- the immune system responds to invading microorganisms in two major ways: antibody production and cell mediated responses.
- the cells that recogmze and destroy pathogens include different types of leukocytes: monocytes, lymphocytes, neutrophils, eosinophils, and basophils.
- Neutrophils and monocytes attack invading bacteria, viruses, and other pathogens and destroy them by phagocytosis.
- Monocytes enter tissues and differentiate into macrophages which are extremely phagocytic. Lymphocytes and plasma cells are a part of the immune system which recognizes specific foreign molecules and organisms and inactivates them, as well as signaling other cells to attack the invaders.
- Leukocytes are formed from two stem cell lineages in bone marrow
- the myeloid stem cell line produces granulocytes and monocytes and, the lymphoid stem cell produces lymphocytes.
- Lymphoid cells travel to the thymus, spleen and lymph nodes, where they mature and differentiate into lymphocytes.
- Two classes of lymphocytes are T- and B- lymphocytes, also called T cells and B cells. The maturation of lymphocytes is subject to control by a variety of factors including the mterleukins.
- T cells that react with foreign antigen on the surface of infected or transformed (cancerous) cells.
- T cells There are two major types of T cells-cytotoxic T cells destroy antigen-bea ⁇ ng cells, whereas helper T cells activate other white blood cells via chemical signals such as the mterleukins
- T H 1 activates macrophages to destroy ingested microorganisms
- T H 2 stimulates the production of antibodies by B cells
- Antibodies are immunoglobuhn proteins produced by B-lymphocytes which bind to specific antigens and cause inactivation or promote destruction of the antigen bv other cells
- the prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chams) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules.
- Antibodies are classified based on their H- chain composition.
- the five antibody classes, IgA, IgD, IgE, IgG and IgM are defined by the ⁇ , ⁇ , e, ⁇ , and ⁇ H-chain types.
- L-chains There are two types of L-chains, K and ⁇ , either of which may associate as a pair with any H-chain pair.
- IgG the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure.
- H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region.
- the constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains.
- the amino acid sequence of the constant region is nearly identical among H- or L-chains of a particular class.
- the variable region consists of about 110 amino acids in both H- and L-chains. However, the amino acid sequence of the variable region differs among H- or L-chains of a particular class.
- Within each H- or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigen recognition site.
- Both H-chains and L-chains contain repeated immunoglobulin domains.
- a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site.
- a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region.
- H chains such as ⁇ have been shown to associate with other polypeptides during differentiation of the B-cell.
- the complement system serves as an effector system and is involved in infectious agent recognition. It can function as an independent immune network or in conjunction with other humoral immune responses.
- the complement system is comprised of numerous plasma and membrane proteins that act in a cascade of reaction sequences whereby one component activates the next to generate a rapid and amplified response to infection through either an inflammatory response or increased phagocytosis.
- the first element in this cascade, Cl is composed of subunits Clq, Clr, and Cls.
- Clq-induced cellular responses are thought to be involved in host defense and in protection against autoimmunity since Clq-def ⁇ cient humans are suceptible to infectious disorders and a very high incidence of autoimmune disease (Nicholson-Weller, A. and Vogelstein, L.B. (1999) Curr. Opin. Immunol. 11:42-46).
- the classical pathway requires antibody binding to infectious agent antigens.
- the antibodies serve to define the target and initiate the complement system cascade, culminating in the destruction of the infectious agent.
- the complement can be seen as an effector arm of the humoral immune system.
- the alternative pathway of the complement system does not require the presence of preexisting antibodies for targeting infectious agent destruction. Rather, this pathway, through low levels of an activated component, remains constantly primed and provides surveillance in the non- immune host to enable targeting and destruction of infectious agents. In this case foreign material triggers the cascade, thereby facilitating phagocytosis or lysis (Paul, W. (1993) Fundamental Immunology Raven Press Ltd., New York NY pp.918-919).
- Antigen Recognition A key feature of the immune system is its ability to distinguish foreign molecules, or antigens, from "self molecules. This ability is mediated primarily by secreted and transmembrane proteins expressed by leukocytes (white blood cells) such as lymphocytes, granulocytes, and monocytes.
- Ig immunoglobulin
- MHC major histocompatibility
- MHC proteins are cell surface markers that bind to and present foreign antigens to T cells. MHC molecules are classified as either class I or class II. Class I MHC molecules (MHC I) are expressed on the surface of almost all cells and are involved in the presentation of antigen to cytotoxic T cells. For example, a cell infected with virus will degrade intracellular viral proteins and express the protein fragments bound to MHC I molecules on the cell surface. The MHC I/antigen complex is recognized by cytotoxic T-cells which destroy the infected cell and the virus within. Class II MHC molecules are expressed primarily on specialized antigen-presenting cells of the immune system, such as B-cells and macrophages.
- MHC molecules also play an important role in organ rejection following transplantation. Rejection occurs when the recipient's T-cells respond to foreign MHC molecules on the transplanted organ in the same way as to self MHC molecules bound to foreign antigen.
- Antibodies or immunoglobulins, bind and neutralize antigens in the circulation and other extracellular fluids.
- Antibody classes include IgG, IgA, IgM, IgD, and IgE.
- IgA are found primarily in secretions and play an important role in mucosal immunity. IgA are transcytosed across epithelial cell sheets and secreted along with mucous into the lumenal space. Transcytosis and secretion are mediated by the polymeric Ig receptor, which binds to and transports IgA.
- the polymeric Ig receptor is a transmembrane protein with about two to five Ig domains in its extracellular domain. (Reviewed in Alberts, supra, pp. 1210-1211; Kulseth, M.
- T-cell receptors are both structurally and functionally related to antibodies.
- T-cell receptors are cell surface proteins that bind foreign antigens and mediate diverse aspects of the immune response.
- a typical T-cell receptor is a heterodimer comprised of two disulfide-linked polypeptide chains called ⁇ and ⁇ . Each chain is about 280 amino acids in length and contains one variable region and one constant region. Each variable or constant region folds into an Ig domain. The variable regions from the ⁇ and ⁇ chains come together in the heterodimer to form the antigen recognition site.
- T-cell receptor diversity is generated by somatic rearrangement of gene segments encoding the ⁇ and ⁇ chains.
- T-cell receptors recognize small peptide antigens that are expressed on the surface of antigen-presenting cells and pathogen-infected cells. These peptide antigens are presented on the cell surface in association-with major histocompatibility proteins which provide the proper context for antigen recognition. (Reviewed in Alberts, supra, pp. 1228-1229.)
- Cytokines comprise a family of signaling molecules that modulate the immune system and the inflammatory response. Cytokines are usually secreted by leukocytes, or white blood cells, in response to injury or infection. However, other tissues are capable of secreting cytokines in response to disease or trauma. Cytokines function as growth and differentiation factors that act primarily on cells of the immune system including B- and T-lymphocytes, monocytes, macrophages, granulocytes, and their progenitors, such as the myeloid stem cells and lymphoid stem cells. Like other signaling molecules, cytokines bind to specific plasma membrane receptors and trigger intracellular signal transduction pathways which alter gene expression patterns. There is considerable potential for the use of cytokines in the treatment of inflammation and immune system disorders.
- Cytokine structure and function have been extensively characterized in vitro. Most cytokines are small polypeptides of about 30 kilodaltons or less. Over 50 cytokines have been identified from human and rodent sources. Examples of cytokine subfamilies include the interferons (IFN- ⁇ , - ⁇ , and - ⁇ ), the interleukins (IL-1 through IL-18), the tumor necrosis factors (TNF- ⁇ and - ⁇ ), and the chemokines. Many cytokines have been produced using recombinant DNA techniques, and the activities of individual cytokines have been determined in vitro. These activities include regulation of leukocyte proliferation, differentiation, and motility. (Reviewed in Callard, R. E. and Gearing, A. J.
- Chemokines comprise a cytokine subfamily with over 30 members. (Reviewed in Wells, T. N. C. and Peitsch, M. C. (1997) J. Leukoc. Biol. 61:545-550.) Chemokines were initially identified as chemotactic proteins that recruit monocytes and macrophages to sites of inflammation. Recent evidence indicates that chemokines may also play key roles in hematopoiesis and HIV-l infection. Chemokines are small proteins which range from about 6-15 kilodaltons in molecular weight. Chemokines are further classified as C, CC, CXC, or CX 3 C based on the number and position of critical cysteine residues.
- the CXC chemokines for example, each contain a conserved motif consisting of two cysteines separated by a single residue followed by two additional cysteines which occur downstream at about 23- and 12-residue intervals (ExPASy PROSITE database, documents PS00471 and PDOC00434).
- the presence and spacing of these four cysteine residues are highly conserved, whereas the intervening residues diverge significantly.
- Primary lymphoid organs include the bone marrow, which produces B-cells, and the thymus, which produces T-cells (thymocytes).
- Bone marrow also contains blood vessels, nerves, fatty tissue, and stromal cells.
- Stromal cells produce a supporting meshwork of collagen fibers and other extracellular matrix components which are important for promoting the growth and differentiation of B-cells and other hematopoietic cells.
- B- and T-cells travel through the lymphatic system and populate secondary lymphoid organs throughout the body such as the lymph nodes, adenoids, tonsils, spleen, and intestinal Peyer's patches.
- Immune System disorders of the immune system include various autoimmune and inflammatory diseases caused by failure of the immune system to discriminate self from non-self molecules. Other immune system disorders are caused by uncontrolled cell proliferation, including leukemias such as multiple myeloma and lymphomas such as Hodgkin's disease. Immunodeficiency, brought on by a variety of diseases and agents including HIV, renders afflicted individuals susceptible to severe and sometimes fatal bacterial and viral infections. (See, for example, Golub, E. S. et al. (1987) Immunology: A Synthesis. Sinauer Associates, Sunderland, MA, pages 481 and 509-530.)
- AIDS Abnormal Immunodeficiency Syndrome
- Leukopenia or agranulocytosis occurs when the bone marrow stops producing white blood cells. This leaves the body unprotected against foreign microorganisms, including those which normally inhabit the skin, mucous membranes, and gastrointestinal tract. Impaired phagocytosis occurs in several diseases, including monocytic leukemia, systemic lupus, and granulomatous disease.
- Leukemias are an excess production of white blood cells, to the point where a major portion of the body's metabolic resources are directed solely at proliferation of white blood cells, leaving other tissues to starve.
- myelogenous leukemias cancerous young myelogenous cells spread from the bone marrow to other organs, especially the spleen, liver, lymph nodes and other highly vascularized regions.
- the extra leukemic cells released are immature, incapable of function, and undifferentiated.
- Leukemias may be caused by exposure to environmental factors such as radiation or toxic chemicals or by genetic aberration.
- Transplant rejection and allergies are examples of situations in which it may be desirable to curtail the immune response.
- Interleukin activity may play a pivotal role in specific immune responses.
- An antagonist to IL-17 has been shown to promote survival in heart tissue grafts (Antonysamy, M.A. et al. (1999) Transplant. Proc. 31:93).
- the invention features purified polypeptides, immune system molecules, referred to collectively as “IMOL” and individually as “IMOL-1,” “IMOL-2,” “IMOL-3,” “IMOL-4,” “IMOL- 5,” “IMOL-6” “IMOL-7,” “IMOL-8,” “IMOL-9,” “IMOL-10,” “IMOL-11,” “IMOL-12,” “IMOL-13,” “IMOL- 14,” and “IMOL- 15.”
- the invention provides an isolated polypeptide comprising a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ED NO: 1-15, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15.
- the invention provides an isolated polypeptide comprising the amino acid sequence of
- the invention further provides an isolated polynucleotide encoding a polypeptide comprising a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15.
- the polynucleotide is selected from the group consisting of SEQ ID NO: 16-30.
- the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15.
- 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 comprising a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15.
- the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
- the invention provides an isolated antibody which specifically binds to a polypeptide comprising a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-15.
- the invention further provides an isolated polynucleotide comprising a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 16-30, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 16-30, c) a polynucleotide sequence complementary to a), or d) a polynucleotide sequence complementary to b).
- 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 comprising a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 16-30, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 16-30, c) a polynucleotide sequence complementary to a), or d) a polynucleotide sequence complementary to b).
- the method comprises a) hybridizing the sample with a probe comprising at least 16 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, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
- the probe comprises at least 30 contiguous nucleotides.
- the probe comprises at least 60 contiguous nucleotides.
- the invention further provides a pharmaceutical composition comprising an effective amount of a polypeptide comprising a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID
- the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional IMOL, comprising administering to a patient in need of such treatment the pharmaceutical composition.
- the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide comprising a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15.
- 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 pharmaceutical 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 IMOL, comprising administering to a patient in need of such treatment the pharmaceutical composition.
- the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide comprising a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-15, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15.
- 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 pharmaceutical 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 IMOL, comprising administering to a patient in need of such treatment the pharmaceutical composition.
- the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 16-30, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
- Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full- length sequences encoding IMOL.
- Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of IMOL.
- Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis; diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA was cloned.
- Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding IMOL were isolated.
- Table 5 shows the tools, programs, and algorithms used to analyze IMOL, along with applicable descriptions, references, and threshold parameters.
- IMOL refers to the amino acid sequences of substantially purified IMOL 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 IMOL.
- Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of IMOL either by directly interacting with IMOL or by acting on components of the biological pathway in which IMOL participates.
- allelic variant is an alternative form of the gene encoding IMOL. 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 IMOL include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as IMOL or a polypeptide with at least one functional characteristic of IMOL. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding IMOL, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding IMOL.
- 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 IMOL.
- Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of IMOL is retained.
- negatively charged amino acids may include aspartic acid and glutamic acid
- positively charged amino acids may include lysine and arginine.
- Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
- Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
- amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence” is recited to refer to an amino acid 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 IMOL.
- Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of IMOL either by directly interacting with IMOL or by acting on components of the biological pathway in which IMOL 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 IMOL 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.
- antisense refers to any composition capable of base-pairing with the "sense" strand of a specific nucleic acid sequence.
- Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
- PNA peptide nucleic acid
- Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
- the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
- biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
- immunologically active refers to the capability of the natural, recombinant, or synthetic IMOL, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
- complementarity refers to the natural binding of polynucleotides by base pairing. For example, the sequence “5" A-G-T 3"' bonds to the complementary sequence “3' T-C-A 5'.”
- Complementarity between two single-stranded molecules may be “partial,” such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules.
- the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acid strands, and in the design and use of peptide nucleic acid (PNA) molecules.
- PNA peptide nucleic acid
- 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 IMOL or fragments of IMOL 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; SDS
- other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
- Consensus sequence refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of one or more Incyte Clones and, in some cases, one or more public domain ESTs, using a computer program for fragment assembly, such as the GEL VIEW fragment assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
- GEL VIEW fragment assembly system GEL VIEW fragment assembly system
- Constant amino acid substitutions are those substitutions that, when made, 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 the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence 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 “fragment” is a unique portion of IMOL or the polynucleotide encoding IMOL which is identical in sequence to but shorter in length than the parent sequence.
- a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
- a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
- a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
- a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence.
- a fragment of SEQ ID NO: 16-30 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: 16-30, for example, as distinct from any other sequence in the same genome.
- a fragment of SEQ ID NO: 16-30 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO: 16-30 from related polynucleotide sequences.
- the precise length of a fragment of SEQ ID NO: 16-30 and the region of SEQ ID NO: 16-30 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
- a fragment of SEQ ID NO:l-15 is encoded by a fragment of SEQ ID NO:16-30.
- a fragment of SEQ ID NO: 1-15 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO: 1-15.
- a fragment of SEQ ID NO: 1-15 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO: 1-15.
- the precise length of a fragment of SEQ ID NO: 1-15 and the region of SEQ ID NO: 1-15 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
- similarity refers to a degree of complementarity. There may be partial similarity or complete similarity. The word “identity” may substitute for the word “similarity.”
- a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
- the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency.
- a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency.
- 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 2.0.9 (May-07-1999) set at default parameters. Such default parameters may be, for example:
- Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
- Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
- Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy 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.
- the phrases "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 hydrophobicity and acidity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
- NCBI BLAST software suite may be used.
- BLAST 2 Sequences Version 2.0.9 (May-07-1999) 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 stable mitotic chromosome segregation and maintenance.
- humanized antibody refers to antibody molecules 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 identity. 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 denatured salmon sperm DNA.
- stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
- wash temperatures are selected to be about 5°C to 20°C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength and pH.
- 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, denatured salmon sperm DNA at about 100-200 ⁇ g/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations.
- 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).
- 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 IMOL 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 IMOL which is useful in any of the antibody production methods disclosed herein or known in the art.
- microarray refers to an arrangement of distinct polynucleotides on a substrate.
- element and “array element” in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
- modulate refers to a change in the activity of IMOL.
- modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of IMOL.
- 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.
- PNA peptide nucleic acid
- operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
- operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
- PNA protein nucleic acid
- PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
- Probe refers to nucleic acid sequences encoding IMOL, 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.
- Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
- PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
- Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
- the Primer3 primer selection program (available to the public from the Whitehead 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.
- 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.
- 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 nucleic acids encoding IMOL, or fragments thereof, or IMOL itself, 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 containing 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 acids or nucleotides by different amino acids 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.
- Transformation describes a process by which exogenous DNA enters and changes 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, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
- the term "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, and 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 95% or at least 98% 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 “polymorphic” variant.
- a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
- the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
- Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
- a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
- Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
- 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 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.
- the invention is based on the discovery of new human immune system molecules (IMOL), the polynucleotides encoding IMOL, and the use of these compositions for the diagnosis, treatment, or prevention of immunological disorders, infections, and cell proliferative disorders including cancer.
- IMOL new human immune system molecules
- the polynucleotides encoding IMOL and the use of these compositions for the diagnosis, treatment, or prevention of immunological disorders, infections, and cell proliferative disorders including cancer.
- Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding IMOL. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each IMOL were identified, and column 4 shows the cDNA libraries from which these clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. The Incyte clones in column 5 were used to assemble the consensus nucleotide sequence of each IMOL and are useful as fragments in hybridization technologies.
- SEQ ID NOs sequence identification numbers
- column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs; column 6 shows homologous sequences as identified by BLAST analysis; and column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied. The methods of column 7 were used to characterize each polypeptide through sequence homology and protein motifs.
- the columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding IMOL.
- the first column of Table 3 lists the nucleotide SEQ ID NOs.
- Column 2 lists fragments of the nucleotide sequences of column 1. These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID NO: 16-30 and to distinguish between SEQ ED NO: 16-30 and related polynucleotide sequences.
- the polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.
- Column 3 lists tissue categories which express IMOL as a fraction of total tissues expressing IMOL.
- Column 4 lists diseases, disorders, or conditions associated with those tissues expressing IMOL as a fraction of total tissues expressing IMOL.
- Column 5 lists the vectors used to subclone each cDNA library.
- Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding IMOL were isolated.
- Column 1 references the nucleotide SEQ ID NOs
- column 2 shows the cDNA libraries from which these clones were isolated
- column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
- SEQ ED NO:26 maps to chromosome 16 within the interval from 19.70 to 33.30 centiMorgans. This interval also contains genes and ESTs associated with B cell maturation and MHC Class II transactivation. SEQ ED NO:29 maps to chromosome 11 within the interval from 104.80 to 123.50 centiMorgans. This interval also contains genes and ESTs associated with human lymphoma.
- the invention also encompasses IMOL variants.
- a preferred IMOL 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 IMOL amino acid sequence, and which contains at least one functional or structural characteristic of IMOL.
- the invention also encompasses polynucleotides which encode IMOL.
- the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO: 16-30, which encodes IMOL.
- the polynucleotide sequences of SEQ ID NO: 16-30 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences 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 IMOL.
- a variant polynucleotide sequence will have at least about 80%, or alternatively at least about 90%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding IMOL.
- a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 16-30 which has at least about 80%, or alternatively at least about 90%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 16-30.
- any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of IMOL. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding IMOL, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring IMOL, and all such variations are to be considered as being specifically disclosed.
- nucleotide sequences which encode IMOL and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring IMOL under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding IMOL 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 occurring sequence.
- the invention also encompasses production of DNA sequences which encode IMOL and IMOL 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 EMOL 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: 16-30 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 (Perkin- Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), 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 (Perkin-Elmer).
- Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
- the resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
- the nucleic acid sequences encoding IMOL may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
- PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
- one method which may be employed restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
- Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
- the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
- a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
- capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
- multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
- Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
- 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.
- OLIGO 4.06 Primer Analysis software National Biosciences, Plymouth MN
- 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, Perkin-Elmer), 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.
- appropriate software e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer
- polynucleotide sequences or fragments thereof which encode IMOL may be cloned in recombinant DNA molecules that direct expression of IMOL, 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 IMOL.
- nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter IMOL-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides 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 Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of IMOL, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
- MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et
- DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
- genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. 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 IMOL 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.
- IMOL itself or a fragment thereof may be synthesized using chemical methods.
- peptide synthesis can be performed using various solid-phase techniques.
- Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of IMOL, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
- the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
- the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties. WH Freeman, New York NY.)
- the nucleotide sequences encoding IMOL 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 IMOL. Such elements may vary in their strength and specificity.
- Specific initiation signals may also be used to achieve more efficient translation of sequences encoding IMOL. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
- exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
- Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
- a variety of expression vector/host systems may be utilized to contain and express sequences encoding IMOL. 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.
- 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 IMOL.
- routine cloning, subcloning, and propagation of polynucleotide sequences encoding IMOL can be achieved using a multifunctional E. coli vector such as PBLUESCRU (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding IMOL into the vector's multiple cloning site disrupts the lacL gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
- these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
- vectors which direct high level expression of IMOL may be used.
- vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
- Yeast expression systems may be used for production of IMOL.
- 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 IMOL. Transcription of sequences encoding IMOL may be driven 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.
- a number of viral-based expression systems may be utilized.
- sequences encoding IMOL 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 IMOL 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 purposes. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.)
- sequences encoding IMOL 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 purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
- Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
- selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, 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. 55:121-131.)
- marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
- sequence encoding IMOL is inserted within a marker gene sequence, transformed cells containing sequences encoding IMOL can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding IMOL 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 IMOL and that express IMOL 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 IMOL 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
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding IMOL include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- the sequences encoding IMOL, 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 IMOL 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 IMOL may be designed to contain signal sequences which direct secretion of IMOL 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.
- 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.
- CHO, HeLa, MDCK, HEK293, and WI38 Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities
- ATCC American Type Culture Collection
- HEK293, and WI38 Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities
- ATCC American Type Culture Collection
- HEK293, and WI38 natural, modified, or recombinant nucleic acid sequences encoding IMOL may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
- a chimeric IMOL protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of IMOL 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 IMOL encoding sequence and the heterologous protein sequence, so that IMOL 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 IMOL 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.
- Fragments of IMOL may be produced not only by recombinant means, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra, pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431 A peptide synthesizer (Perkin-Elmer). Various fragments of IMOL may be synthesized separately and then combined to produce the full length molecule. THERAPEUTICS
- IMOL Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of IMOL and immune system molecules.
- the expression of IMOL is closely associated with immunological disorders, infections, and cell proliferative disorders including cancer.
- SEQ ID NO:26 maps to a chromosomal interval which also contains genes and ESTs associated with B cell maturation and MHC Class II transactivation.
- SEQ ID NO:29 maps to a chromosomal interval which also contains genes and ESTs associated with human lymphoma. Therefore, IMOL appears to play a role in immunological disorders, infections, and cell proliferative disorders including cancer. In the treatment of disorders associated with increased IMOL expression or activity, it is desirable to decrease the expression or activity of IMOL.
- IMOL 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 IMOL.
- disorders include, but are not limited to, an immunological disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
- a vector capable of expressing IMOL 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 IMOL including, but not limited to, those described above.
- a pharmaceutical composition comprising a substantially purified IMOL in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of IMOL including, but not limited to, those provided above.
- an agonist which modulates the activity of IMOL may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of IMOL including, but not limited to, those listed above.
- an antagonist of IMOL may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of IMOL.
- disorders include, but are not limited to, those immunological disorders, infections, and cell proliferative disorders including cancer, listed above.
- an antibody which specifically binds IMOL 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 IMOL.
- a vector expressing the complement of the polynucleotide encoding IMOL may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of IMOL 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 IMOL may be produced using methods which are generally known in the art. In particular, purified IMOL may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind IMOL.
- Antibodies to IMOL 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) are generally preferred for therapeutic use.
- various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with IMOL 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
- Corynebacterium parvum are especially preferable.
- the oligopeptides, peptides, or fragments used to induce antibodies to IMOL 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 and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of IMOL 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 IMOL 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.
- the hybridoma technique the human B-cell hybridoma technique
- EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, 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 IMOL-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 IMOL 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 IMOL and its specific antibody.
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering IMOL epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
- K association constant
- the K determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple IMOL epitopes, represents the average affinity, or avidity, of the antibodies for IMOL.
- the K determined for a preparation of monoclonal antibodies, which are monospecific for a particular IMOL epitope, represents a true measure of affinity.
- High-affinity antibody preparations with j ranging from about 10 9 to 10 12 L ⁇ nole are preferred for use in immunoassays in which the IMOL-antibody complex must withstand rigorous manipulations.
- Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of IMOL, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington, DC; Liddell, J.E. and Cryer, A. (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 IMOL-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 IMOL, or any fragment or complement thereof may be used for therapeutic purposes.
- the complement of the polynucleotide encoding IMOL may be used in situations in which it would be desirable to block the transcription of the mRNA.
- cells may be transformed with sequences complementary to polynucleotides encoding IMOL.
- complementary molecules or fragments may be used to modulate IMOL activity, or to achieve regulation of gene function.
- sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding IMOL.
- Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding IMOL. (See, e.g., Sambrook, supra: Ausubel, 1995, supra.)
- Genes encoding IMOL can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding IMOL. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
- modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions of the gene encoding IMOL.
- Oligonucleotides derived from the transcription initiation site e.g., between about positions -10 and +10 from the start site, may be employed.
- 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.
- 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 IMOL.
- RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the 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. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding IMOL. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into 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.
- any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
- An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
- Such pharmaceutical compositions may consist of IMOL, antibodies to IMOL, and mimetics, agonists, antagonists, or inhibitors of IMOL.
- the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
- the compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
- 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, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
- compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
- Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired.
- Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
- disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
- Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
- Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
- Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
- the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
- compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
- Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
- suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
- Non-lipid polycationic amino polymers may also be used for delivery.
- the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
- suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
- penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
- compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
- the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
- the preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1 % to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
- compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
- labeling would include amount, frequency, and method of administration.
- compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
- the determination of an effective dose is well within the capability of those skilled in the art.
- 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, 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 IMOL or fragments thereof, antibodies of IMOL, and agonists, antagonists or inhibitors of IMOL, 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.
- Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
- the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
- the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with 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.
- 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 pharmaceutical 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
- antibodies which specifically bind IMOL may be used for the diagnosis of disorders characterized by expression of IMOL, or in assays to monitor patients being treated with IMOL or agonists, antagonists, or inhibitors of IMOL.
- Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for IMOL include methods which utilize the antibody and a label to detect IMOL in human body fluids or in extracts of cells or tissues.
- the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
- a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
- a variety of protocols for measuring IMOL including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of IMOL expression.
- Normal or standard values for IMOL expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibody to IMOL under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of IMOL 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 IMOL may be used for diagnostic purposes.
- 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 IMOL may be correlated with disease.
- the diagnostic assay may be used to determine absence, presence, and excess expression of IMOL, and to monitor regulation of IMOL levels during therapeutic intervention.
- hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding IMOL or closely related molecules may be used to identify nucleic acid sequences which encode IMOL.
- 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 IMOL, 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 IMOL encoding sequences.
- the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ED NO: 16-30 or from genomic sequences including promoters, enhancers, and introns of the IMOL gene.
- Means for producing specific hybridization probes for DNAs encoding IMOL include the cloning of polynucleotide sequences encoding IMOL or IMOL derivatives into vectors for the production of mRNA probes.
- Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
- Hybridization probes may be labeled by a variety of reporter groups, for example, by 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 IMOL may be used for the diagnosis of disorders associated with expression of IMOL.
- disorders include, but are not limited to, an immunological disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, anky losing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gas
- the polynucleotide sequences encoding IMOL 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 microarrays utilizing fluids or tissues from patients to detect altered IMOL expression. Such qualitative or quantitative methods are well known in the art.
- the nucleotide sequences encoding IMOL may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
- the nucleotide sequences encoding IMOL 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 IMOL 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 IMOL, 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 IMOL 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 IMOL, or a fragment of a polynucleotide complementary to the polynucleotide encoding IMOL, 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.
- Methods which may also be used to quantify the expression of IMOL include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
- radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating 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 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 targets in a microarray.
- the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
- Microarrays may be prepared, used, and analyzed using methods known in the art.
- methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150- 2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.
- nucleic acid sequences encoding IMOL may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
- the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
- HACs human artificial chromosomes
- YACs yeast artificial chromosomes
- BACs bacterial artificial chromosomes
- PI constructions or single chromosome cDNA libraries.
- Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques 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. Correlation between the location of the gene encoding IMOL on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
- the nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
- 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 number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
- any sequences mapping to that area may represent associated or regulatory genes for further investigation.
- the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
- IMOL in another embodiment, IMOL, 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 IMOL 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 IMOL, or fragments thereof, and washed. Bound IMOL is then detected by methods well known in the art. Purified IMOL 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 IMOL may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
- RNA was purchased from Clontech or isolated from tissues described in Table 4. 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.
- 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), PSPORTl plasmid (Life Technologies), pcDNA2.1 plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Pharmaceuticals, Palo Alto CA).
- 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.
- 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 carried 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.
- cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) 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 (Perkin-Elmer).
- Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) 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 VI.
- Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters.
- the first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences).
- Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
- Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
- the polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis.
- sequences 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, and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS.
- GenBank primate rodent, mammalian, vertebrate, and eukaryote databases
- BLOCKS PRINTS
- DOMO DOMO
- PRODOM PRODOM
- PFAM PFAM
- HMM Hidden Markov Model
- 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 nucleotide databases such as GenBank or LIFESEQ (Incyte Pharmaceuticals). 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: % sequence identity x % maximum BLAST score
- the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
- the results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding IMOL occurred.
- Analysis involved the categorization of cDNA libraries by organ/tissue and disease.
- the organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic.
- the disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories. Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
- the cDNA sequences which were used to assemble SEQ ID NO: 16-30 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ID NO: 16-30 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped.
- SHGC Stanford Human Genome Center
- WIGR Whitehead Institute for Genome Research
- a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
- the genetic map locations of SEQ ID NO:26 and SEQ ID NO:29 are described in the Invention as a range, or interval, of a particular human chromosome.
- the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm.
- centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers.
- the full length nucleic acid sequences of SEQ ID NO: 16-30 were 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, to initiate 3' extension of the known fragment.
- the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
- Selected human cDNA 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 (Corning Costar, Acton MA), allowing the DNA to bind to the reagent.
- the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
- a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
- the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
- CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
- 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, 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.
- nucleotide sequences of SEQ ID NO: 16-30 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library.
- 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 ⁇ (DuPont NEN).
- the DNA from each digest is fractionated on a 0.7% agarose gel and transferred 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.
- Microarrays A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements. After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
- Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
- Fluorescent probes are prepared and used for hybridization to the elements on the substrate.
- the substrate is analyzed by procedures described above.
- oligonucleotides Sequences complementary to the IMOL-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring IMOL. 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 IMOL. 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 IMOL-encoding transcript.
- IMOL Expression and purification of IMOL 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 IMOL upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
- IMOL 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 IMOL 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.
- IMOL 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 IMOL obtained by these methods can be used directly in the following activity assay.
- XI Demonstration of IMOL Activity An assay for IMOL activity measures the proliferation of leukocytes in response to IMOL.
- the amount of tritiated thymidine incorporated into newly synthesized DNA is used to estimate proliferative activity.
- Varying amounts of IMOL are added to cultured leukocytes, such as granulocytes, monocytes, or lymphocytes, in the presence of [ 3 H]thymidine, a radioactive DNA precursor.
- IMOL for this assay can be obtained by recombinant means or from biochemical preparations.
- Incorporation of [ 3 H]thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is directly proportional to the amount of newly synthesized DNA.
- a linear dose-response curve over at least a hundred-fold IMOL concentration range is indicative of IMOL activity.
- One unit of activity per milliliter is conventionally defined as the concentration of IMOL producing a 50% response level, where 100% represents maximal incorporation of [ 3 H]thymidine into acid-precipitable DNA.
- an assay for IMOL activity measures the expression of IMOL on the cell surface.
- cDNA encoding IMOL is transfected into a non-leukocytic cell line.
- Cell surface proteins are labeled with biotin as described (de la Fuente, M. A. et al. (1997) Blood 90:2398-2405).
- Immunoprecipitations are performed using IMOL-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of IMOL expressed on the cell surface.
- IMOL activity is exemplified by that of immunoglobulins, which recognize and precipitate antigens from serum. The quantitative precipitin reaction measures this activity.
- IMOL is isotopically labeled using methods known in the art. Various serum concentrations are added to constant amounts of labeled IMOL.
- IMOL-antigen complexes precipitate out of solution and are collected by centrifugation. The amount of precipitable IMOL-antigen complex is proportional to the amount of radioisotope detected in the precipitate. The amount of precipitable IMOL-antigen complex is plotted against the serum concentration.
- the amount of precipitable IMOL-antigen complex is a measure of IMOL activity which is characterized by sensitivity to both limiting and excess quantities of antigen.
- IMOL function is assessed by expressing the sequences encoding IMOL 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 plasmid (Life Technologies) and pCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial 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.
- Expression of a 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.
- FCM Flow cytometry
- FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry. Oxford, New York NY.
- IMOL The influence of IMOL on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding IMOL 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 IMOL and other genes of interest can be analyzed by northern analysis or microarray techniques.
- PAGE polyacrylamide gel electrophoresis
- the IMOL amino acid sequence is analyzed using LASERGENE software
- oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St.
- Naturally occurring or recombinant IMOL is substantially purified by immunoaffinity chromatography using antibodies specific for IMOL.
- An immunoaffinity column is constructed by covalently coupling anti-IMOL antibody to an activated chromatographic resin, such as
- Media containing IMOL are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of IMOL (e.g., high ionic strength buffers in the presence of detergent).
- the column is eluted under conditions that disrupt antibody/IMOL 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 IMOL is collected.
- IMOL or biologically active fragments thereof, are labeled with 1 5 I Bolton-Hunter reagent.
- Bolton-Hunter reagent See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
- Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled IMOL, washed, and any wells with labeled IMOL complex are assayed. Data obtained using different concentrations of IMOL are used to calculate values for the number, affinity, and association of IMOL with the candidate molecules.
- molecules interacting with IMOL 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).
- ABI FACTURA A program that removes vector sequences and masks Perkin-Elmer Applied Biosystems, ambiguous bases in nucleic acid sequences. Foster City, CA.
- ABI/PARACEL FDF A Fast Data Finder useful in comparing and annotating Perkin-Elmer Applied Biosystems, Mismatch ⁇ 50% amino acid or nucleic acid sequences. Foster City, CA; Paracel Inc., Pasadena, CA.
- ABI AuloAssembler A program that assembles nucleic acid sequences. Perkin-Elmer Applied Biosystems, Foster City, CA.
- BLAST includes five functions: blastp, blastn, Nucleic Acids Res. 25: 3389-3402. Full Length sequences: Probability blastx, tblastn, and tblastx. value-- l.OE-10 or less
- fastx score 100 or greater
- Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome sequencer traces with high sensitivity and probability. Res. 8: 175-185; Ewing. B. and P. Green ( 1998) Genome Res. 8: 186- 194.
- Motifs A program that searches amino acid sequences for patterns Bairoch et al. supra; Wisconsin that matched those defined in Prosite. Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI.
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US6521233B1 (en) | 1999-04-20 | 2003-02-18 | Zymogenetics, Inc. | Adipocyte complement related protein homolog zacrp3 |
US20020081625A1 (en) * | 1999-10-14 | 2002-06-27 | John Ford | Member of the immunoglobulin superfamily and uses thereof |
AR030554A1 (es) * | 2000-03-16 | 2003-08-27 | Amgen Inc | Moleculas similares a receptores il-17 y usos de las mismas |
US7094566B2 (en) | 2000-03-16 | 2006-08-22 | Amgen Inc., | IL-17 receptor like molecules and uses thereof |
AU2001263236A1 (en) * | 2000-05-17 | 2001-11-26 | Immunex Corporation | Human myd88 adapter-like protein and functional fragments thereof |
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US6692748B2 (en) | 2000-12-07 | 2004-02-17 | Zymogenetics, Inc. | Adipocyte complement related protein zacrp3x2 and nucleic acids encoding zacrp3x2 |
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