RESUMO
The COP9 signalosome (originally described as the COP9 complex) is an essential multi-subunit repressor of light-regulated development in plants [1] [2]. It has also been identified in mammals, though its role remains obscure [3] [4] [5]. This complex is similar to the regulatory lid of the proteasome and eIF3 [5] [9] [10] [11] [12] and several of its subunits are known to be involved in kinase signaling pathways [4] [6] [7] [8]. No proteins homologous to COP9 signalosome components were identified in the Saccharomyces cerevisiae genome, suggesting that the COP9 signalosome is specific for multi-cellular differentiation [13]. In order to reveal the developmental function of the COP9 signalosome in animals, we have isolated Drosophila melanogaster genes encoding eight subunits of the COP9 signalosome, and have shown by co-immunoprecipitation and gel-filtration analysis that these proteins are components of the Drosophila COP9 signalosome. Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain. Disruption of one of the subunits by either a P-element insertion or deletion of the gene caused lethality at the late larval or pupal stages. This lethality is probably a result of numerous pleiotropic effects. Our results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Proteínas de Insetos/metabolismo , Proteínas , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Complexo do Signalossomo COP9 , Drosophila melanogaster/genética , Genes de Insetos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Complexos Multiproteicos , Peptídeo Hidrolases , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais , Especificidade da Espécie , Técnicas do Sistema de Duplo-HíbridoRESUMO
Ty elements belong to the family of LTR-containing retrotransposons. Ty RNA is reverse transcribed by Ty-encoded proteins. The cDNA then transposes to new locations in the genome by a process that involves the integrase protein encoded by the element. We have previously shown that the Ty cDNA molecule can participate in recombination events with genomic Tys. In this study we have analyzed the role of the integrase protein in cDNA-mediated Ty recombination. We found that this process involves the integrase protein in a temperature-dependent manner. In addition we have investigated whether double-stranded DNA is the only molecule that can participate in cDNA-mediated Ty recombination. We have shown that mutations in the polypurine tract that abolish plus-strand synthesis do not prevent cDNA-mediated recombination, implying that other types of intermediates from the reverse-transcription process (e.g. single-stranded DNA or a hybrid RNA-cDNA molecule) can participate in cDNA-mediated Ty recombination.
Assuntos
DNA Complementar/genética , Integrases/fisiologia , Recombinação Genética/genética , Retroelementos/genética , DNA Complementar/biossíntese , DNA Fúngico/biossíntese , DNA Fúngico/genética , Integrases/genética , Saccharomyces cerevisiae/genéticaRESUMO
In the yeast Saccharomyces cerevisiae ectopic recombination has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. Little is known about the mechanism(s) that prevent recombination between repeated sequences. We have previously shown that nonreciprocal recombination (gene conversion) of a genetically marked Ty can be induced either by the presence of high levels of Ty cDNA or by transcription of the marked Ty from a GAL1 promoter. These two kinds of induction act in a synergistic manner. To further characterize these two kinds of Ty recombination, we have investigated the role played by the RAD52 and RAD1 genes. We have found that the RAD52 and RAD1 gene products are essential to carry out transcription-induced Ty conversion whereas cDNA-mediated conversion can take place in their absence.
Assuntos
Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteínas Fúngicas/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Enzimas Reparadoras do DNA , DNA Complementar , Regulação Fúngica da Expressão Gênica , Proteína Rad52 de Recombinação e Reparo de DNA , Proteínas de Saccharomyces cerevisiaeRESUMO
Families of repeated sequences are present in the genomes of all eukaryotes. Little is known about the mechanism(s) that prevents recombination between repeated sequences. In the yeast Saccharomyces cerevisiae, recombination between homologous sequences placed at nonhomologous locations in the genome (ectopic recombination) has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. We have previously shown that a high level of Ty cDNA in the cell causes an increase in the rate of nonreciprocal recombination (gene conversion) of a marked Ty element. In the present study, we show that it is also possible to elevate the rate of recombination of a marked Ty by increasing its transcription. This induction is different from, and acts synergistically to, the one seen upon increased levels of donor Ty cDNA. We show that the induction by transcription does not require the products of the RAD50, RAD51, and RAD57 genes. In contrast, cDNA-mediated recombination is dependent on the product of the RAD51 gene but not on products of the genes RAD50 or RAD57.
