RESUMO
Recombination-deficient strains have been proven useful for the understanding of the genetic control of homologous recombination. As the genetic screens used to isolate recombination-deficient (rec(-)) yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with low or elevated rates of recombination. Using this system we isolated a collection of rec(-) mutants. We report the characterization of the REC41 gene identified in this way. REC41 is required for normal levels of interplasmid recombination and gamma-ray induced mitotic interchromosomal recombination. The rec41-1 mutant failed to grow at 37 degrees C. Microscopic analysis of plated cells showed that 45-50% of them did not form visible colonies at permissive temperature. Haploid cells of the rec41 mutant show the same gamma-ray sensitivity as wild type ones. However, the diploid rec41 mutant shows gamma-ray sensitivity which is comparable with heterozygous REC41/rec41-1 diploid cells. This fact indicates semidominance of the rec41-1 mutation. Diploid strains homozygous for the rec41 rad52 mutations had the same gamma-ray sensitivity as single rad52 diploids and exhibited dramatically decreased growth rate. The expression of the HO gene does not lead to inviability of rec41 cells. The rec41 mutation has an effect on meiosis, likely meiotic recombination, even in the heterozygous state. We cloned the REC41 gene. Sequence analysis revealed that the REC41 gene is encoded by ORF YDR245w. Earlier, this ORF was attributed to MNN10, BED1, SLC2, CAX5 genes. Two multicopy plasmids with suppressers of the rec41-1 mutation (pm21 and pm32) were isolated. The deletion analysis showed that only DNA fragments with the CDC43 and HAC1 genes can partially complement the rec41-1 mutation.
Assuntos
Recombinação Genética , Saccharomyces cerevisiae/química , Divisão Celular/efeitos da radiação , Análise Mutacional de DNA , Relação Dose-Resposta à Radiação , Raios gama , Deleção de Genes , Haploidia , Heterozigoto , Homozigoto , Meiose , Mitose , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Tolerância a Radiação/genética , Saccharomyces cerevisiae/genética , Supressão Genética , TemperaturaRESUMO
An alpha-L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving alpha1 --> 6-, alpha1 -->3-, alpha1 --> 4-, and alpha1 --> 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The alpha-L-fucosidase hydrolyzes p-nitrophenyl alpha-L-fucopyranoside with V(max) of 12.0 +/- 0.1 microM/min/mg and K(m) = 0.20 +/- 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 +/- 10 kDa consisting of four identical subunits with a molecular mass of 61.0 +/- 0.5 kDa. The N-terminal sequence showed homology to some alpha-L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl alpha-L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the alpha-L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl alpha-L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.
Assuntos
Thermus/enzimologia , alfa-L-Fucosidase/metabolismo , Sequência de Aminoácidos , Cromatografia/métodos , Sequência Conservada , Eletroforese em Gel de Poliacrilamida , Fucose/biossíntese , Fucose/química , Glicosilação , Cinética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Ultrafiltração , alfa-L-Fucosidase/isolamento & purificaçãoRESUMO
The RAD58 (XRS4) gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that RAD58 also encodes an essential meiotic function. The spore inviability of rad58 strains is not rescued by a spo13 mutation. The rad50 mutation suppresses spore inviability of a spo13 rad58 strain suggesting that RAD58 acts after RAD50 in meiotic recombination. The rad58-4 mutation does not prevent mitotic recombination events. Haploid rad58 cells fail to carry out G2-repair of gamma-induced lesions, whereas rad58/rad58 diploids are able to perform some diploid-specific repair of these lesions.
Assuntos
Epistasia Genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Dano ao DNA , Reparo do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Fúngico/efeitos da radiação , Proteínas Fúngicas/fisiologia , Raios gama , Teste de Complementação Genética , Meiose , Plasmídeos/genética , Ploidias , Recombinação Genética , Reprodução , Saccharomyces cerevisiae/metabolismoRESUMO
The cdc28-srm mutation in Saccharomyces cerevisiae decreases spontaneous and induced mitochondrial rho- mutability and the mitotic stability of native chromosomes and recombinant circular minichromosomes. The effects of cdc28-srm on the genetic stability of cells support the hypothesis that links cell cycle regulation in yeast to changes in chromatin organization dependent on the start gene CDC28 (Hayles and Nurse, 1986).