RESUMO
The cya-5 nuclear mutant of Neurosopora crassa was previously shown to be deficient in cytochrome aa3, cytochrome c oxidase activity, and the immunologically detectable COXI protein. We have now demonstrated that the mitochondria of this mutant contain mRNA for the COXI protein and that COXI cannot be detected during pulse-chase labeling experiments of mitochondrial translation products. Cloning and analysis of the cya-5 gene reveal a long open reading frame capable of encoding a 1136 amino-acid protein. Sequence analysis suggests that the potential CYA-5 protein contains a mitochondrial targeting sequence at its amino-terminus. The long open reading frame also contains a 200 amino-acid region with homology to the PET309 protein, which is required for the production or stability of intron-containing coxI mRNAs, as well as the translation of mature coxI mRNAs, in the yeast Saccharomyces cerevisiae. These data suggest that the CYA-5 protein of N. crassa is required in a post-transcriptional step for COXI expression, most probably for the efficient translation of coxI mRNA.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Membrana , Neurospora crassa/genética , Neurospora crassa/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Fases de Leitura Aberta , Fatores de Iniciação de Peptídeos , Plantas Tóxicas , Biossíntese de Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , NicotianaAssuntos
Artiodáctilos/genética , Repetições de Microssatélites , Rena/genética , Animais , Sequência de Bases , Bison/genética , DNA/sangue , DNA/isolamento & purificação , Primers do DNA , Cervos/genética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Ovinos/genéticaRESUMO
The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa3 and c. Using a sib-selection procedure we have isolated the cyt-12+ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12+ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa3.
Assuntos
Grupo dos Citocromos c/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Genes Fúngicos , Mutação , Neurospora crassa/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Grupo dos Citocromos c/metabolismo , DNA Fúngico/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria.