RESUMO
In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.
Assuntos
Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Proteínas de Ligação ao GTP/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas ras , ras-GRF1 , Sequência de Bases , Northern Blotting , Clonagem Molecular , Análise Mutacional de DNA , DNA Fúngico/genética , Regulação Fúngica da Expressão Gênica , Genes Supressores , Dados de Sequência Molecular , Fenótipo , RNA Fúngico/genética , RNA Mensageiro/genética , Mapeamento por Restrição , Proteínas rap de Ligação ao GTPRESUMO
In Saccharomyces cerevisiae, the product of the CDC25 gene controls the RAS-mediated production of adenosine 3',5'-monophosphate (cAMP). In vivo the carboxyl-terminal third of the CDC25 gene product is sufficient for the activation of adenylate cyclase. The 3'-terminal part of SCD25, a gene of S. cerevisiae structurally related to CDC25, can suppress the requirement for CDC25. Partially purified preparations of the carboxy-terminal domain of the SCD25 gene product enhanced the exchange rate of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) of pure RAS2 protein by stimulating the release of GDP. This protein fragment had a similar effect on the human c-H-ras-encoded p21 protein. Thus, the SCD25 carboxyl-terminal domain can enhance the regeneration of the active form of RAS proteins.
Assuntos
Proteínas de Ciclo Celular , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Nucleotídeos de Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas de Saccharomyces cerevisiae , Proteínas ras , ras-GRF1 , Escherichia coli/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Cinética , Plasmídeos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , TransfecçãoRESUMO
In Saccharomyces cerevisiae, the product of the CDC25 gene is required for progression in the cell division cycle. It is necessary for cAMP production. It has been suggested that the CDC25 gene product acts through Ras proteins. We report the cloning of a DNA fragment from a new gene able to suppress the thermosensitive phenotype of the cdc25-5 mutation. It is unable to suppress the defect of a mutant of the adenylate cyclase gene or of the ras1, ras2ts double mutant. This DNA fragment prevents the drop in cAMP level in cdc25-5 mutant cells shifted to restrictive temperature. The complementing part of this fragment contains a truncated open reading frame (ORF) corresponding to the 3' end of a gene we named SCD25. The 584-amino acid sequence deduced from this ORF shares 45% identity with the 592-aa C-terminal part of the CDC25 ORF which is sufficient for complementation of cdc25 mutations. Some of the common sequences between these two genes are also partially homologous with the amino acid sequence of LTE1, another gene of S. cerevisiae. The capacity of the SCD25 fragment to suppress a cdc25 mutation and its homology to the C-terminal part of the CDC25 led us to propose that the CDC25 and the SCD25 C-terminal fragments each encode a protein domain which is capable in itself to support a similar biochemical function.
