RESUMEN
The catalytic properties of the partially purified deacetoxycephalosporin C (DAOC)-synthetase and DAOC-hydroxylase from an industrial strain of Cephalosporium acremonium were studied. After mechanical breakage of the cells, purification was achieved by fractional (NH4)2SO4 precipitation, gel chromatography on Sephadex G-75, ion exchange chromatography on DEAE-Trisacryl M and two isoelectric focusing steps. The two enzyme activities could not be separated. Indirect evidence was obtained from SDS-polyacrylamide gel electrophoresis of the purest fractions obtained by isoelectric focusing that the two reactions are catalyzed by a single enzyme with a molecular weight of 33,000 +/- 2,000 and a pI of 4.6 +/- 0.1. Both reactions require alpha-ketoglutarate, FeSO4, ascorbate and O2, whereas additional ATP shows only a slight stimulation.
Asunto(s)
Acremonium/metabolismo , Cefalosporinas/biosíntesis , Transferasas Intramoleculares , Isomerasas/aislamiento & purificación , Oxigenasas de Función Mixta/aislamiento & purificación , Proteínas de Unión a las Penicilinas , Adenosina Trifosfato/farmacologíaRESUMEN
The role of the mitochondrial system during sporulation of Saccharomyces cerevisiae was studied. Addition of ethidium bromide (EthBr) to cells growing in acetate medium resulted in the quantitative (>98%) conversion of the culture to the petite genotype in one generation. The cells were respiratory active (derepressed) but contained no mitochondrial deoxyribonucleic acid (mtDNA) as demonstrated by analytical ultracentrifugation in CsCl. When transferred to acetate sporulation medium, the culture sporulated. Ascus production was only slightly below that of the control culture. Synthesis of mtDNA occurred during sporulation in the control but not in the EthBr-treated culture. Mitochondrial protein synthesis was virtually eliminated in the EthBr-treated culture. Therefore, completely derepressed cells can sporulate without a functional mitochondrial genetic system. When partially repressed cells were treated with EthBr, no ascus formation was observed after transfer to sporulation medium. Control cultures underwent respiratory adaptation in sporulation medium and then sporulated. Extensive derepression of the respiratory system is thus required for sporulation, and this adaptation is dependent on a functional mitochondrial system. Our results suggest that once the cells are fully derepressed no mitochondrial genetic information has to be expressed during meiosis and ascus formation.
Asunto(s)
Genes , Mitocondrias/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Sistema Libre de Células , Colorimetría , ADN/biosíntesis , Complejo IV de Transporte de Electrones/metabolismo , Etidio/farmacología , Proteínas Fúngicas/biosíntesis , Isocitratos , Leucina/metabolismo , Mutación , Oxo-Ácido-Liasas/metabolismo , Consumo de Oxígeno , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/crecimiento & desarrollo , Succinato Deshidrogenasa/metabolismo , Tritio , UltracentrifugaciónRESUMEN
A population of petite ascospores (mitochondrial deoxyribonucleic acid [mtDNA]-less), produced by brief ethidium bromide (EthBr) mutagenesis prior to transfer to sporulation medium, was used to examine the role of the mitochondrial genetic system on germination and outgrowth in Saccharomyces cerevisiae. Petite ascospores, which are morphologically indistinguishable by phase-contrast microscopy from wild-type spores, germinate and proceed through outgrowth at a rate and extent only slightly less than that of wild-type spores. Both developmental processes occurred in the absence of mtDNA synthesis and measurable cytochrome oxidase activity. These results indicate that neither respiration nor a functional mitochondrial genome are required for germination and outgrowth. The properties of the petite clones were typical of petites formed during vegetative growth. Individual sporal clones differed markedly from each other in suppressiveness. Petite sporal clones which exhibited a high degree of supressiveness also contained a reduced but detectable amount of mtDNA of altered buoyant density. One clone contained a unique mtDNA with a buoyant density higher than that of wild-type mtDNA.
Asunto(s)
ADN/biosíntesis , Mitocondrias/metabolismo , Mutación , Saccharomyces cerevisiae/crecimiento & desarrollo , Centrifugación por Gradiente de Densidad , ADN/análisis , Complejo IV de Transporte de Electrones/metabolismo , Etidio , Isocitratos , Meiosis , Microscopía de Contraste de Fase , Mutágenos , Oxo-Ácido-Liasas/metabolismo , Consumo de Oxígeno , Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
Mitochondrial membrane proteins synthesized in vivo in the presence of cycloheximide were analyzed on electrofocusing polyacrylamide gels. Five of the six protein bands observed have their isoelectric points below pH 7. The six proteins are not synthesized when both cycloheximide and chloramphenicol are present, and they are absent in a petite mutant lacking mitochondrial deoxyribonucleic acid (DNA), leading to the conclusion that the proteins are synthesized on mitochondrial ribosomes. By labeling cells of Saccharomyces cerevisiae cultivated in a chemostat under different degrees of glucose limitation, the effect of glucose repression on the synthesis of the mitochondrial membrane proteins was determined. Two of the protein bands showed a relatively reduced synthesis under the strongest glucose repression tested. The specific activity of the cytochrome oxidase and the percentage of mitochondrial DNA in the total DNA were found to be influenced at a lower level of glucose repression.