RESUMO
Six mutations blocking the function of a seven intron form of the mitochondrial gene encoding subunit I of cytochrome c oxidase (COXI) and mapping upstream of exon 3 were isolated and characterized. A cis-dominant mutant of the group IIA intron 1 defines a helical portion of the C1 substructure of domain 1 as essential for splicing. A trans-recessive mutant confirms that the intron 1 reading frame encodes a maturase function. A cis-dominant mutant in exon 2 was found to have no effect on the splicing of intron 1 or 2. A trans-recessive mutant, located in the group IIA intron 2, demonstrates for the first time that intron 2 encodes a maturase. A genetic dissection of the five missense mutations present in the intron 2 reading frame of that strain demonstrates that the maturase defect results from one or both of the missense mutations in a newly-recognized conserved sequence called domain X.
Assuntos
DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Endorribonucleases/metabolismo , Íntrons , Nucleotidiltransferases/metabolismo , Splicing de RNA/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA Fúngico , Genes Fúngicos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , RNA Fúngico , Mapeamento por RestriçãoRESUMO
We have found that intron 5 alpha of the COXI gene (al5 alpha) of yeast mtDNA is a mobile group I intron in crosses between strains having or lacking the intron. We have demonstrated the following hallmarks of that process: 1) co-conversion of flanking optional intron markers; 2) mutations that truncate the intron open reading frame block intron mobility; and 3) the intron open reading frame encodes an endonuclease activity that is required for intron movement. The endonuclease activity, termed I-Sce IV, cleaves the COXI allele lacking al5 alpha near the site of intron insertion, making a four-base staggered cut with 3' OH overhangs. Three cloned DNAs derived from different forms of the COXI gene, which differ in primary sequence at up to seven nucleotides around the cleavage site, are all good substrates for in vitro I-Sce IV cleavage activity. Two of the strains from which these substrates were derived were tested in crosses and are comparably efficient as al5 alpha recipients. When compared with omega mobility occurring simultaneously in one cross, al5 alpha is less efficient as a mobile element.
Assuntos
Elementos de DNA Transponíveis/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Íntrons/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Cruzamentos Genéticos , DNA Fúngico/genética , Endonucleases/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Mutação/genética , Plasmídeos/genéticaRESUMO
From a group of 436 Drosophila melanogaster cDNA clones, we selected 39 that are expressed exclusively or predominantly in the adult visual system. By sequence analysis, 20 of the clones appear to represent previously unreported distinct cDNAs. The corresponding transcripts are detected in the retina and optic lobes. The genes are scattered throughout the genome, some near mutations known to affect eye function. One of these clones has been identified, by sequence analysis, as the structural gene (Arr) for a Drosophila homolog of human arrestin. Vertebrate arrestin interacts with rhodopsin in phototransduction and has been associated with an autoimmune form of uveitis in primates. The presence of an arrestin homolog in Drosophila suggests that both the vertebrate and invertebrate phototransduction cascades are regulated in a similar manner.
Assuntos
Antígenos/genética , DNA/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Genes , Proteínas de Membrana/genética , Inibidores de Fosfodiesterase/metabolismo , Sequência de Aminoácidos , Animais , Arrestina , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA/isolamento & purificação , Drosophila melanogaster/embriologia , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Pupa , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
We have investigated the in vitro self-splicing of a class II mitochondrial intron. A model pre-mRNA containing intron 5 gamma of the oxi 3 gene of yeast mitochondrial DNA undergoes an efficient intramolecular rearrangement reaction in vitro. This reaction proceeds under conditions distinct from those optimal for self-splicing of class I introns, such as the Tetrahymena nuclear rRNA intron. Intron 5 gamma is excised as a nonlinear RNA indistinguishable from the in vivo excised intron product by gel electrophoresis and primer extension analysis. Studies of the in vitro excised intron product strongly indicate that it is a branched RNA with a circular component joined by a linkage other than a 3'-5' phosphodiester. Two other products, the spliced exons and the broken form of the lariat, were also characterized. These results show that the class II intron products are similar to those of nuclear pre-mRNA splicing.
