RESUMO
Many of the cis-dominant mutations that lead to respiratory deficiency by preventing maturation of specific yeast mitochondrial transcripts are found to affect the ribozyme core of group I and group II introns. We have searched for suppressors of mutations in the ribozyme-encoding sections of a group II intron, the first intron in the COX1 gene of Saccharomyces cerevisiae, which was independently subjected to in vitro site-directed mutagenesis. Three of the original mutants bore multiple mutations, which act synergistically, since for most individual mutations, suppressors could be obtained that ensured at least partial recovery of respiratory competence and splicing. Out of a total of ten suppressor mutations that were identified, three were second-site substitutions that restored postulated base-pairings in the ribozyme core. Remarkably, and as is observed for group I introns, at least half of the cis-dominant mutations in the first two group II introns of the COX1 gene affect sites that have been shown to participate in RNA tertiary interactions. We propose that this bias reflects cooperativity in the formation of ribozyme tertiary but not secondary structure, on the one hand, and the need for synergistic effects in order to generate a respiratory-deficient phenotype in the laboratory on the other. Finally, a novel in vivo splicing product of mutant cells is attributed to bimolecular splicing at high concentrations of defective transcripts.
Assuntos
Íntrons/genética , Splicing de RNA/genética , RNA Mensageiro/genética , Supressão Genética , Sequência de Bases , Complexo IV da Cadeia de Transporte de Elétrons/genética , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Fúngico/química , RNA Fúngico/genética , RNA Mensageiro/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNARESUMO
Sequence analysis was used to define the repeat unit that constitutes the mitochondrial genome of a petite (rho-) mutant of the yeast Saccharomyces cerevisiae. This mutant has retained and amplified in tandem a 2,547 bp segment encompassing the second exon of the oxi3 gene excised from wild-type mtDNA between two direct repeats of 11 nucleotides. The identity of the mtDNA segment retained in this petite has recently been questioned (van der Veen et al., 1988). The results presented here confirm the identity of this mtDNA segment to be that determined previously by restriction mapping (Carignani et al., 1983).
Assuntos
DNA Fúngico , DNA Mitocondrial , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mutação , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , DNA Fúngico/genética , DNA Mitocondrial/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
In the mitochondrial genome of Saccharomyces cerevisiae, introns aI1 and aI2 of the gene encoding the COX1 subunit are the only group II introns with open reading frames (ORFs); these can be translated into two homologous proteins, the maturase aI1 and aI2. These proteins are structurally related to viral reverse transcriptases and have been shown genetically to be involved in pre-mRNA splicing and in the deletion of introns from mitochondrial DNA. To identify these mitochondrial proteins and study their properties more directly, we raised antibodies against a part of the intron aI2 ORF translation product. For this purpose, we constructed series of fusion genes, by joining parts of the genes for protein A or lacZ to different portions of the intron aI2. These were expressed in Escherichia coli as hybrid polypeptides, which were used for the production and identification of specific antibodies against the yeast mitochondrial protein. The antibodies recognized the 57 kDa protein (maturase aI2) that accumulates in two yeast mutants deficient in the splicing of aI2. This protein corresponds to the translation product of the 3' part of intron aI2 and accumulates unaltered in the two cis-acting mutants.
Assuntos
DNA Mitocondrial/genética , Proteínas Fúngicas/análise , Íntrons , DNA Polimerase Dirigida por RNA/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Anticorpos Antifúngicos , Sequência de Bases , Western Blotting , Clonagem Molecular , Genes Fúngicos , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas Recombinantes de Fusão/análise , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologiaRESUMO
We have studied the splicing pathway leading to the synthesis of cytochrome oxidase subunit I (COX I) mRNA, by analysing the transcription pattern of several oxi3- splicing deficient mutants located in the first four introns of the gene. The four introns contain long open reading frames (ORFs) in phase with the upstream exons. All the mutations block the excision of the mutated intervening sequence (IVS) from the pre-mRNA, and accumulate characteristic novel polypeptides of sizes which could correspond to the translation products of the intron's ORF. Most of the mutations do not affect the splicing of the following intervening sequences; only in the case of mutations in the aI1 intron is a polar effect observed on the splicing of the second intron, aI2. Our results indicate that the splicing of these two intervening sequences which both belong to the class II of introns described by Michel et al. (1982), is controlled by the activity of the maturases encoded by their respective ORFs and that the translation of the aI2 maturase depends on the previous excision of aI1 IVS. (Moreover, the aI1 maturase, which accumulates in some mutants, can efficiently splice aI2 IVS when the translation of the latter's proper maturase cannot occur).
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Genes Fúngicos , Genes , Splicing de RNA , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Íntrons , Substâncias Macromoleculares , Mutação , Saccharomyces cerevisiae/enzimologiaRESUMO
We report the sequence of a 6.3 kb segment of DNA mapping near the end of the right arm of chromosome III of Saccharomyces cerevisiae. The sequence reveals a major open reading frame coding for a putative protein of 1047 amino acids with a striking similarity to the bacterial proteins involved in recognition of mismatched DNA base pairs. This is particularly interesting as the existence of a yeast mismatch repair system similar to that of bacteria has been postulated for some years, but a yeast protein homologous to the bacterial mismatch binding protein had not been identified. The results of a comparison of the putative yeast mismatch binding protein with the bacterial mismatch binding proteins and with two cognate mammalian sequences, support the idea that a similar mismatch repair system may be present also in mammalian cells. The possibility that all of these proteins may have evolved from a common ancestral gene is also discussed.
Assuntos
Cromossomos Fúngicos/química , Reparo do DNA/genética , DNA Fúngico/química , Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Fúngicas/química , Humanos , Camundongos , Dados de Sequência Molecular , Proteína 3 Homóloga a MutS , Fases de Leitura Aberta , Mapeamento por RestriçãoRESUMO
A Synechocystis 6803 mutant carrying a chimaeric photosystem II (PSII), in which the Zea mays PsbH subunit (7.7 kDa calculated molecular mass) replaces the cyanobacterial copy (7.0 kDa), was constructed. With the exception of the N-terminal 12 amino acid extension, which has a phosphorylatable threonine, the eukaryotic polypeptide is 78% homologous to its bacterial counterpart. Biochemical characterization of this mutant shows that it expresses the engineered gene correctly and is competent for photoautotrophic growth. Fluorescence analysis and oxygen evolution measurements in the presence of exogenous acceptors indicate that the observed phenotype results from a chimaeric PSII rather than from the absence of function associated with PsbH, suggesting that the heterologous protein is assembled into a functional PSII. Inhibition of oxygen evolution by herbicides belonging to different classes shows that the sensitivity of the mutant PSII is changed only towards phenolic compounds. This result indicates slight conformational modification of the QB/herbicide binding pocket of the D1 polypeptide caused by the bulky PsbH protein in the mutant, and also suggests close structural interaction of the D1 and PsbH subunits in the topological arrangement of PSII.
Assuntos
Cianobactérias/genética , Fosfoproteínas/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Sequência de Aminoácidos , Cianobactérias/efeitos dos fármacos , Cianobactérias/metabolismo , Expressão Gênica/efeitos dos fármacos , Herbicidas/farmacologia , Dados de Sequência Molecular , Mutagênese , Fosfoproteínas/genética , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Zea mays/metabolismoRESUMO
The CP29 subunit of Photosystem II is reversibly phosphorylated in Zea mays upon exposure to high light in the cold (Bergantino et al., J Biol Chem 270 (1995) 8474-8481). This phenomenon was previously proposed to be restricted to C4 plants. We present the complete sequence of the CP29 protein, deduced from a maize Lhcb4 cDNA clone, and its comparison with the previously known Lhcb4 sequences of two C3 plants: Hordeum vulgare and Arabidopsis thaliana. Despite the relatively low degree of homology in their amino-terminal region, i.e. the part of the molecule which is phosphorylated in maize, the three polypeptides conserve consensus sequences for the site of phosphorylation. We proved by immunoblotting and 33P-labelling that the same post-translational modification occurs in barley. Being thus common to C3 and C4 plant species, the phosphorylation of this minor antenna complex of Photosystem II appears now as a widespread phenomenon, possibly part of the phosphorylation cascade which signals the redox status of the plastoquinone to the nuclear transcription apparatus. Arabidopsis plants do not show phosphorylation of CP29 in the same conditions, but other low-molecular-weight phosphoproteins, whose role need to be elucidated, become evident.
Assuntos
Complexos de Proteínas Captadores de Luz , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Sequência de Bases , Hordeum/metabolismo , Dados de Sequência Molecular , Fosforilação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Plantas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Zea mays/metabolismoRESUMO
We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa). We have called this gene KIN82. The amino acid sequence of KIN82 is most similar to the cyclic nucleotide-dependent protein kinase subfamily and the protein kinase C subfamily. Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditions. Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy-terminal catalytic domain of KIN82.
Assuntos
Genes Fúngicos/genética , Proteínas Quinases/genética , Saccharomyces cerevisiae/genética , Serina/metabolismo , Treonina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Fúngicos , Clonagem Molecular , Dados de Sequência Molecular , Mutagênese , Hibridização de Ácido Nucleico , Proteínas Quinases/classificação , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The resistance of maize plants to cold stress has been associated with the appearance of a new chlorophyll a/b binding protein in the thylakoid membrane following chilling treatment in the light. The cold-induced protein has been isolated, characterized by amino acid sequencing, and pulse labeled with radioactive precursors, showing that it is the product of post-translational modification by phosphorylation of the minor chlorophyll a/b protein CP29 rather than the product of a cold-regulated gene or an unprocessed CP29 precursor. We show here that the CP29 kinase activity displays unique characteristics differing from previously described thylakoid kinases and is regulated by the redox state of a quinonic site. Finally, we show that maize plants unable to perform phosphorylation have enhanced sensitivity to cold-induced photoinhibition.
Assuntos
Complexos de Proteínas Captadores de Luz , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Processamento de Proteína Pós-Traducional , Zea mays/metabolismo , Sequência de Aminoácidos , Temperatura Baixa , Hidrólise , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Ácidos Fosfóricos , Fosforilação , Alinhamento de Sequência , Espectrometria de FluorescênciaRESUMO
We have localized ten oxi3- mutations in the first, al1, intron of the coxl gene. All are splicing deficient, being unable to excise the intron. Complementation experiments disclose several domains in the intron al1: the 5'-proximal and 3'-proximal domains harbor cis-dominant mutations, while trans-recessive ones are located in the intron's open reading frame. Comprehensive analyses of allele-specific polypeptides accumulating in mutants show that they result from the translation of the intron's ORF. We conclude that a specific mRNA maturase involved in splicing of oxidase mRNA is encoded by the intron al1 in a manner similar to the cytochrome b mRNA maturase.
Assuntos
DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Endorribonucleases , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Genes , Substâncias Macromoleculares , Peso Molecular , Nucleotidiltransferases/genética , Biossíntese de Proteínas , Transcrição GênicaRESUMO
The SH2 domain of c-Fgr (class 1A) has been expressed in E. coli as GST fusion protein and tested for its ability to prevent the dephosphorylation of a variety of phosphotyrosyl (poly)peptides by three distinct protein tyrosine phosphatases (TC-PTPase, YOP, and Low Mr PTPase). Dephosphorylation of HS1 protein and of a derived phosphopeptide, HS1 (388-402), exhibiting the motif selected by class 1A SH2 domains is inhibited in a dose dependent manner with full inhibition promoted by a 2- to 3-molar excess of GST/SH2 domain irrespective of either the nature or the amount of phosphatase used. The IC50 values for inhibition of these and other phosphotyrosyl substrates roughly correlates with their expected affinity for class 1A SH2 domain. Inhibition is partially reversed by the addition of D-myo-inositol 1,4,5-triphosphate, which competes for the binding to the SH2 domains. Our data on one side show that additional mechanism(s) besides mere competition must assist PTPases to dissociate SH2-PTyr complexes and on the other suggest a role for SH2 domains in protecting phosphotyrosyl residues from premature dephosphorylation.