RESUMO
Using antibodies directed against the TYB1 protein of the transpositionally competent retrotransposon Ty1-H3, we have identified three mature proteins of 23, 60, and 90 kDa and processing intermediates of 140 and 160 kDa that are derived from the 190-kDa TYA1-TYB1 polyprotein. Mature proteins and variable amounts of the precursors cofractionate with Ty viruslike particles. The map locations and precursor-product relationships of mature TYB1 polypeptides suggest that p23 is Ty1 protease, p90 is integrase, and p60 contains reverse transcriptase and RNase H. Immunoprecipitation and immunoblot analyses of Ty1 proteins show that p190 is cleaved to form p160. The p160 intermediate is cleaved to form p23 and p140, and p140 is cleaved to form p90 and p60. Processing of TYB1 proteins is dependent on Ty1 protease. Immunoblot analysis of TYB proteins from different Ty1 isolates reveal that correct processing of TYB1 proteins is a characteristic of functional Ty1 elements, whereas aberrant processing is a common defect found in transposition-incompetent elements.
Assuntos
Elementos de DNA Transponíveis , Genes Fúngicos , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/metabolismo , Técnicas Imunológicas , Integrases , Dados de Sequência Molecular , Peso Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Ribonuclease HRESUMO
Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element is transcribed into RNA, and this RNA is reverse transcribed into a DNA copy capable of insertion into many different chromosomal locations. Maturation of proteins and reverse transcription take place within noninfectious intracellular viruslike particles. We have studied the element Ty, which is found dispersed in the genome of the yeast Saccharomyces cerevisiae. The frequency of Ty element transposition is normally quite low but can be greatly increased by expressing an element from a strong promoter. We have used the ability to control the level of Ty transposition to investigate the functions of Ty proteins, the regulation of Ty transposition, and the exploitation of Ty elements as insertional mutagens in yeast. The information gained from these experiments should be applicable to the study of retrotransposons found in multicellular organisms.
Assuntos
Elementos de DNA Transponíveis , Genes Fúngicos , Saccharomyces cerevisiae/genética , Clonagem Molecular , DNA Fúngico/genética , Regulação Fúngica da Expressão Gênica , Plasmídeos , RNA Fúngico/genética , Recombinação Genética , Transcrição GênicaRESUMO
We used several mutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae. Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition. The Ty protease is required for processing of both TyA and TyB proteins. Mutations in the protease resulted in the synthesis of morphologically and functionally aberrant Ty viruslike particles. The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro. Ty RNA was present in the mutant particles, but at very low levels. Transposition of a genetically tagged element ceased when the protease domain was mutated, demonstrating that Ty protease is essential for transposition. One of these mutations also defined a segment of TyB encoding an active reverse transcriptase. These results indicate that the Ty protease, like its retroviral counterpart, plays an important role in particle assembly, replication, and transposition of these elements.
Assuntos
Elementos de DNA Transponíveis , Endopeptidases/genética , Genes Fúngicos , Genes , Mutação , Saccharomyces cerevisiae/genética , Endopeptidases/metabolismo , Genótipo , Microscopia Eletrônica , Plasmídeos , DNA Polimerase Dirigida por RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/ultraestruturaRESUMO
Monoclonal antibodies directed against the feline leukemia virus (FeLV) envelope proteins, gp70 and p15E, were identified by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six of these monoclonal antibodies were specific for the gp70; two for the p15E. Enzyme-linked immunosorbent assay binding assays against FeLV subtypes A, B, and C showed that most of the monoclonal antibodies bound to more than one subtype but have a greater affinity for subtype B. One monoclonal bound exclusively to the FL74 isolate. These studies also indicate that antigenic variability exists between FeLV isolates previously classified as being the same subtype. One antibody was found to bind the gp70s of all FeLV isolates tested and to be directed against a viral neutralizing site. A p15E-specific monoclonal antibody, in addition to binding all the FeLV subtypes, also bound to Moloney and Rauscher murine leukemia viruses, suggesting a group determinant is involved. No binding was seen to human T-cell leukemia virus, bovine leukemia virus, equine infectious anemia virus, or RD114V proteins, however.
