Other

University of Cincinnati

Incyte Pharmaceuticals, Inc.

Medical College of Georgia Research Institute, Inc.

Jay A Tischfield

Peter J Stambrook

Jeffrey J Seilhamer

Mary Jeanne Kreek

Karl Steven LaForge

Lei Yu

Novel mammalian phospholipase (PLA ) nucleotide sequences and low molecular weight (about 14 KD) amino acid sequences encoded thereby are disclosed. More particularly, a cloned human HPLA cDNA expressing HPLA -10 and its cloned rat RPLA cDNA counterpart, expressing RPLA -10, which are characterized as PLA Type IV, are disclosed. A second type of PLA cDNA, characterized as PLA Type III and exemplified by a rat PLA cDNA encoding RPLA -8 and a partial human PLA nucleotide sequence encoding HPLA -8, is disclosed. Expression of the cDNAs encode the two new types of PLA enzymes which have phospholipase activity. The novel PLA s do not include disulfide bridges between cysteine amino acids 11 and 77 or elapid loops. However, the novel PLA s may include amino acid COOH-terminal extensions which can vary in length. Seventeen of the eighteen absolutely conserved amino acids in all active 14 KD PLA s are believed to be conserved in RPLA -8 and HPLA -8, whereas all eighteen are believed to be conserved in RPLA -10 and HPLA -10. Because the encoded sequences of RPLA -8 and HPLA -8 include only 16 cysteine amino acids, they have been designated as Type III. RPLA -10 and HPLA -10 are designated as Type IV since their encoded sequences include only 12 cysteine amino acids.

Mammalian phospholipase A2 nucleotide sequences, low molecular weight amino acid sequences encoded thereby, antisense sequences and nucleotide sequences having internal ribosome binding sites

Provided herein are variant alleles of a gene encoding a mu opioid receptor, along with cloning vectors for replicating such variant alleles, expressing vectors for expressing the variant alleles to produce variant mu opioid receptors, and antibodies to such variant receptors. Also disclosed are binding characteristics of such variant receptors regarding binding to opioid ligands, and the using of such binding characteristics to diagnose a subjects susceptibility to pain, susceptibility to an addictive disease, selecting an appropriate pain reliever along with a therapeutically effective amount of the reliever to administer to a subject suffering from pain. In addition, diagnostic methods for diagnosing a disease or disorder such as infertility, constipation, diarrhea, decreased immune response relative to a standard, and decreased ability to withstand stress relative to a standard, along with commercial kits for diagnosing such diseases or disorders.

Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon

Provided herein are variant alleles of a gene encoding a mu opioid receptor, along with cloning vectors for replicating such variant alleles, expressing vectors for expressing the variant alleles to produce variant mu opioid receptors, and antibodies to such variant receptors. Also disclosed are binding characteristics of such variant receptors regarding binding to opioid ligands, and the using of such binding characteristics to diagnose a subjects susceptibility to pain, susceptibility to an addictive disease, selecting an appropriate pain reliever along with a therapeutically effective amount of the reliever to administer to a subject suffering from pain. In addition, diagnostic methods for diagnosing a disease or disorder such as infertility, constipation, diarrhea, decreased immune response relative to a standard, and decreased ability to withstand stress relative to a standard, along with commercial kits for diagnosing such diseases or disorders. Furthermore, the invention is also directed to identification of targeted prevention methods, early therapeutic intervention, and improved treatment of opioid addiction, infertility, constipation, diarrhea, impaired immune responsiveness, and stress.

Alleles of the human mu opioid receptor and diagnostic methods based thereon

Novel transgenic nonhuman animals for detecting and characterizing mutations in vivo are disclosed. When detecting reverse mutations, the transgenic nonhuman animal now afford the unique advantage of detecting and characterizing mutations in vivo without having to sacrifice the animals as required heretofore. Moreover, since the transgenic nonhuman animals do not need to be sacrificed, they provide the unique opportunity to correlate the incidence and location of tumors (carcinogenesis) with the incidence and location of mutagenesis. Also disclosed are novel constructs, cell lines and chimeric animals for producing the novel transgenic animals. Novel methods for detecting and characterizing the mutations in vivo and producing animals for use in accordance with the methods of the instant invention are disclosed.

Live mouse mutagenesis systems for testing mutagenic agents in vivo

Novel transgenic nonhuman animals, such as transgenic mice, for detecting and characterizing mutations in vivo are disclosed. When detecting reverse mutations, such as mutations of the APRT gene, the transgenic nonhuman animal now afford the unique advantage of detecting and characterizing mutations in vivo without having to sacrifice the animals as required heretofore. Moreover, since the transgenic nonhuman animals do not need to be sacrificed, they provide the unique opportunity to correlate the incidence and location of tumors (carcinogenesis) with the incidence and location of mutagenesis. Also disclosed are novel constructs, cell lines and chimeric animals for producing the novel transgenic animals. Novel methods for detecting and characterizing the mutations in vivo and producing animals for use in accordance with the methods of the instant invention are disclosed.

Novel mammalian phospholipase (PLA.sub.2) nucleotide sequences and low molecular weight (about 14KD) amino acid sequences encoded thereby are disclosed. More particularly, a cloned human HPLA.sub.2 cDNA expressing HPLA.sub.2 -10 and its cloned rat RPLA.sub.2 cDNA counterpart, expressing RPLA.sub.2 -10, which are characterized as PLA.sub.2 Type IV, are disclosed. A second type of PLA.sub.2 cDNA, characterized as PLA.sub.2 Type III and exemplified by a rat PLA.sub.2 cDNA encoding RPLA.sub.2 -8 and a partial human PLA.sub.2 nucleotide sequence encoding HPLA.sub.2 -8, is disclosed. Expression of the cDNAs encode the two new types of PLA.sub.2 enzymes which have phospholipase activity. The novel PLA.sub.2 s do not include disulfide bridges between cysteine amino acids 11 and 77 or elapid loops. However, the novel PLA.sub.2 s may include amino acid COOH-terminal extensions which can vary in length. Seventeen of the eighteen absolutely conserved amino acids in all active 14KD PLA.sub.2 s are believed to be conserved in RPLA.sub.2 -8 and HPLA.sub.2 -8, whereas all eighteen are believed to be conserved in RPLA.sub.2 -10 and HPLA.sub.2 -10. Because the encoded sequences of RPLA.sub.2 -8 and HPLA.sub.2 -8 include only 16 cysteine amino acids, they have been designated as Type III. RPLA.sub.2 -10 and HPLA.sub.2 -10 are designated as Type IV since their encoded sequences include only 12 cysteine amino acids.

Mammalian phospholipase A.sub.2 nucleotide sequences low molecular

A new and improved cell assay has been developed for mutagens and potential carcinogens to precisely identify the predominant type of mutation(s) each such compound induces in the DNA of cells. The test utilizes, for example, a selectable genetic marker such as adenine phosphoribosyltransferase (APRT) which is now extensively characterized on the DNA, protein and cellular phenotype levels. Specific mutations such as transitions, transversions, point insertions or point deletions are engineered at specific known sites in a mouse APRT gene deduced from the determined gene sequence, such that the gene cannot be properly expressed. These mutant genes are then introduced into non-reverting APRT deficient mammalian cells. These hybrid constructs represent the basic test medium for detection of mutagenic activity. The tester cells are treated with mutagens known to preferentially induce specific DNA mutations in mammalian cells. Reversion within the appropriate tester cell culture detected by growth in selection medium or other detection systems, will confirm or refute the mode of action of these mutagens. As an additional approach for the identification of mutagens that produce frameshifts, the amino acid sequences of major frameshift peptides have been determined from the nucleotide sequence of the mouse APRT gene. These frameshift peptides are synthesized and individually used to elicit antisera. Mutant colonies, arising as a consequence of frameshift mutation, are identified in situ by virtue of their binding one of the specific antisera which are coupled to a color-development assay. Additionally, methods for identifying mutagens which produce mutations at the APRT locus, and which exert their effect by inducing DNA rearrangements, transpositions or excision are employed. The tester lines and reagents enable the exact nature of mutation that any mutagen produces in cells to be rapidly established. Further, portions of the nucleotide sequence of a mouse APRT DNA strand as well as its encoded amino acid sequence are disclosed.

Growing community of inventors

Carmel, IN, United States of America

Jay A Tischfield