RESUMO
1. The antimycin binding sites were found to be independent of cytochrome(s) b synthesis in pseudo-wild-type revertants of cytochrome b mutants. These revertants, whose primary mutation is located in the introns of the cob-box gene of mitochondrial DNA, have modified contents of cytochromes b-562 and b-565 and fully functional respiratory chain. 2. Missense mutations in three genetic loci allocated to the exons of the cytochrome b gene, abolish the strong affinity binding of antimycin. 3. It is proposed that the antimycin-binding component is not cytochrome b itself, but interacts with it in such a way that an alteration of the cytochrome b structure affects the antimycin-binding site.
Assuntos
Antimicina A/metabolismo , Grupo dos Citocromos b/metabolismo , Mitocôndrias/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Quinona Redutases/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Ligação Competitiva , Grupo dos Citocromos b/genética , Complexo III da Cadeia de Transporte de Elétrons , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
In order to study the functional role of the spectral species of cytochrome b-565 observed in mitochondria, genetically manipulated strains of Saccharomyces cerevisiae have been used. Strains have been found which are devoid of cytochrome b-565 under certain conditions and which nevertheless are able to grow on a respirable substrate. Two different methods have been used to determine the cytochrome b-565 content: anaerobic titrations and antimycin-A-induced reduction of cytochrome b-565. Both yield the same results.
Assuntos
Grupo dos Citocromos b , Citocromos/metabolismo , DNA Mitocondrial/metabolismo , Proteínas de Escherichia coli , Saccharomyces cerevisiae/metabolismo , Aerobiose , Anaerobiose , Antimicina A , Genótipo , Mutação , Oxirredução , Especificidade da Espécie , Espectrofotometria , TemperaturaAssuntos
Citocromos/genética , Genes , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Grupo dos Citocromos b , Mitocôndrias , Mutação , Conformação de Ácido Nucleico , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
To uncover the functional circuitry both within the mitochondrial genome and between the mitochondrial and the nuclear genome, we have developed a general method for selecting and characterizing genetically suppressor mutations that restore the respiratory capacity of mit- mitochondrial mutants. Several hundreds of pseudo-wild type revertants due to a second unlinked mutation which suppresses a target mit- mutation were isolated. The suppressor mutations were found located either in the nuclear (abbreviated NAM for 'nuclear accommodation of mitochondria') or in the mitochondrial genome (abbreviated MIM for 'mitochondrial-mitochondrial interaction'). The specificity of action of various suppressors upon some 250 different mit- mutations located in several genes was tested. According to this specificity of action, suppressors were subdivided into two major classes: allele specific or gene specific suppressors. Because the cob-box mitochondrial gene has a mosaic organization, we were able to find a novel third class of extragenic suppressors specific for mit- mutations within the introns of this gene. Four examples of suppressors showing various specificities of action illustrate our approach. (1) a nuclear gene controlling specific alleles of different mitochondrial genes; (2) a nuclear gene controlling selectively one intron of a split mitochondrial gene; (3) a mitochondrial gene controlling specific alleles of different mitochondrial genes; (4) a region in one complex mitochondrial gene which controls selectively one intron of another split mitochondrial gene. Different mechanisms of suppression are discussed stressing the alleviation of splicing deficiencies of intron mutations.
Assuntos
DNA Fúngico/genética , DNA Mitocondrial/genética , Saccharomyces cerevisiae/genética , Supressão Genética , Células Clonais/metabolismo , Cruzamentos Genéticos , Metanossulfonato de Etila/farmacologia , Mutagênicos , Mutação/efeitos dos fármacos , FenótipoRESUMO
To determine the tryptophan content in proteins,an analytical ultraviolet fluroescence method is proposed based on making uniform the environment of aromatic chromophores in 6-7 M guanidine hydrochloride. The fluorescence intensity scale is calibrated using standard solutions of free tryptophan. A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins. This method is particularly suited to proteins carrying groups absorbing in the 290-370 nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2-mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods. It is less time-consuming than techniques requiring hydrolysis or chemical reactions.
