RESUMO
Evidence is reported for the existence of a parity doublet of Delta resonances with total angular momentum J=3/2 from photoproduction of the ppi;{0}eta final state. The two parity partners Delta(1920)P33 and Delta(1940)D33 make significant contributions to the reaction. Cascades of resonances into Delta(1232)eta, N(1535)pi, and Na0(980) are clearly observed.
RESUMO
The second intron (bi2) of the cyt b gene from related Saccharomyces species has an extraordinarily conserved sequence and can have different functions in wild-type cells. The protein encoded by the S. cerevisiae intron functions as a maturase to promote intron splicing, while the homologous S. capensis intron encodes a bifunctional protein that acts both as a maturase and as a homing endonuclease (I-ScaI) promoting intron mobility. The protein encoded by intron bi2 belongs to a large gene family characterized by the presence of two conserved LAGLIDADG motifs (P1 and P2). In this study, we analysed a set of splicing-deficient mutants of the S. cerevisiae intron bi2 that carry non-directed mutations affecting the maturase activity, and a set of directed missense mutations introduced into the bifunctional protein encoded by the S. capensis intron. Analysis of these mutations has allowed identification of the residues in the conserved P1 and P2 motifs which are crucial for splicing and homing activities. Moreover, several mutations which are located in the C-terminal part of the protein have been found to affect both functions.
Assuntos
Grupo dos Citocromos b/genética , Mitocôndrias/genética , Mutação , Splicing de RNA , Leveduras/genética , Motivos de Aminoácidos , Sequência Conservada , Endorribonucleases/genética , Endorribonucleases/metabolismo , Íntrons , Mutação de Sentido IncorretoRESUMO
The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I- Sca I endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3'-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site.
Assuntos
DNA Mitocondrial/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Íntrons/genética , Sequência de Aminoácidos , Sequência de Bases , Grupo dos Citocromos b/genética , Escherichia coli , Genes Fúngicos , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Análise de Sequência de DNARESUMO
Two homologous group I introns, the second intron of the cyt b gene, from related Saccharomyces species differ in their mobility. The S.capensis intron is mobile and encodes the I-ScaI endonuclease promoting intron homing, whilst the homologous S.cerevisiae intron is not mobile, but functions as an RNA maturase promoting splicing. These two intron-encoded proteins differ by only four amino acid substitutions. Taking advantage of the remarkable similarity of the two intron open reading frames and using biolistic transformation of mitochondria, we show that the replacement of only two non-adjacent residues in the S.cerevisiae maturase carboxy-terminal sequence is sufficient to induce a homing-endonuclease activity without losing the splicing function. Also, we demonstrate that these two activities reside in the S.capensis bi2-encoded protein which functions in both splicing and intron mobility in the wild-type cells. These results provide new insight into our understanding of the activity and the evolution of group I intron-encoded proteins.
Assuntos
Grupo dos Citocromos b/genética , Desoxirribonuclease I/metabolismo , Endorribonucleases/metabolismo , Íntrons , Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , DNA/metabolismo , DNA Fúngico , Desoxirribonuclease I/química , Desoxirribonuclease I/genética , Endorribonucleases/química , Endorribonucleases/genética , Dados de Sequência Molecular , Mutagênese , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Saccharomyces/genética , Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
The product of the nuclear gene SUV3 is implicated in a variety of post-transcriptional processes in yeast mitochondria. We have analysed the effect of SUV3 gene-disruption on the expression of intron-containing alleles of the mitochondrial cytb and cox1 genes. We have constructed several strains with mitochondrial genomes containing different combinations of cytb and cox1 introns, and associated these genomes with the disruption of SUV3. The resulting strains were tested for their respiratory competence and spectral cytochrome content. All the strains containing only two or three introns showed normal expression of cytb and cox1, whereas the strains containing more introns were unable to express the appropriate gene. The analysis of mitochondrial RNAs by Northern hybridisation showed that the loss of respiratory competence in the strains containing more introns is due to the decrease of mRNA level with no over-accumulation of high-molecular-weight precursors. However, the transcription of the genes was not affected. These results led us to the notion that SUV3 is required for the stability of intron-containing cytb and cox1 transcripts in a cumulative way, not dependent on any particular intron.
Assuntos
Grupo dos Citocromos b/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Íntrons , Mitocôndrias/metabolismo , RNA Nucleotidiltransferases/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Carbono/metabolismo , Núcleo Celular , RNA Helicases DEAD-box , DNA Fúngico/genética , DNA Mitocondrial/genética , Proteínas Fúngicas/fisiologia , RNA Helicases , RNA Nucleotidiltransferases/fisiologia , RNA Mitocondrial , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
We have studied gene conversion initiated by the ai3 intron of the Saccharomyces cerevisiae mitochondrial (mt) COXI gene and its homologous intron (S.cap.ai1) from Saccharomyces capensis. The approach used involved the measurement of intron transmission amongst the progeny of crosses between constructed recipient and donor strains. We found that the S. cerevisiae ai3 intron is extremely active as a donor in gene conversion, whereas its homologous S. capensis intron is not. We have established the sequence of S.cap.ai1 and compared its open reading frame (ORF) with that of I-SceIII encoded by the homologous S. cerevisiae intron. The two protein-coding intron sequences are almost identical, except that the S. capensis ORF contains an in-frame stop codon. This finding provides a strong indication that the 3' part of the S. cerevisiae intron ORF encoding I-SceIII (which should not be translated in the S. capensis intron) must be critical for function of mtDNA endonucleases to mediate intron mobility.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Genes Fúngicos , Íntrons , Saccharomyces cerevisiae/genética , Saccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Mitocondrial/genética , Genótipo , Substâncias Macromoleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Recombinação Genética , Mapeamento por Restrição , Saccharomyces/enzimologia , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We have undertaken a comprehensive study of the gene conversion of all the mitochondrial introns of Saccharomyces capensis. The approach used involved the measurements of intron transmission amongst the progeny of crosses between a recipient strain (Saccharomyces cerevisiae intronless mitochondria) and various donor strains (Saccharomyces capensis, with various combinations of mitochondrial introns). We have shown that the S. capensis second intron (bi2 of cytochrome b gene) is extremely active as a donor in gene conversion whereas its homologous S. cerevisiae intron is not. Determination of sequence of the S. capensis intron demonstrates that it differs from that of the homologous S. cerevisiae intron (bi2) by a very small number of nucleotide substitutions.
