RESUMEN

The Saccharomyces cerevisiae RGS protein Sst2p is involved in desensitization to pheromone and acts as a GTPase-activating protein for the Galpha subunit Gpa1p. Other results indicate that Sst2p acts through Mpt5p and that this action occurs downstream of Fus3p and through Cln3p/Cdc28p. Our results indicate that the interaction of Sst2p with Mpt5p requires the N-terminal MPI (Mpt5p-interacting) domain of Sst2p and is independent of the C-terminal RGS domain. Overexpression of the MPI domain results in an Mpt5p-dependent increase in recovery from pheromone arrest. Overexpression of either intact Sst2p or the MPI domain leads to partial suppression of a gpa1 growth defect, and this suppression is dependent on Mpt5p, indicating that MPI function occurs downstream of Gpa1p and through Mpt5p. Combination of an mpt5 mutation with the GPA1(G302S) mutation, which uncouples Gpa1p from Sst2p, results in pheromone supersensitivity similar to the sst2 mutant, and promotion of recovery by overexpression of Sst2p is dependent on both Mpt5p and the Gpa1p interaction. These results indicate that Sst2p is a bifunctional protein and that the MPI domain acts through Mpt5p independently of the RGS domain. RGS family members from other fungi contain N-terminal domains with sequence similarity to the Sst2p MPI domain, suggesting that MPI function may be conserved.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas Fúngicas/química , Subunidades alfa de la Proteína de Unión al GTP , Proteínas RGS/química , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas Activadoras de GTPasa/metabolismo , Genes Fúngicos , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fenotipo , Feromonas/farmacología , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos

RESUMEN

Presented data demonstrate that both HP1c1 and S2 bacteriophages of Haemophilus influenzae can use two related attB sites on the host chromosome. The first indication comes from the analysis of the whole genome sequence of H. influenzae Rd in which a second region of nucleotide sequence identical with the known attB site was detected. The location of both attB sites agrees with results of earlier field-inversion gel electrophoresis (FIGE) analysis of S2 and HP1c1 lysogens of H. influenzae. Functionality of cloned attB sites in the integration process of both phages was confirmed using in vivo integrase-dependent recombination system in Escherichia coli. Presented results seem to extend previously described similarities between S2 and HP1c1 phages.

Asunto(s)

Sitios de Ligazón Microbiológica , Bacteriófagos/genética , Haemophilus influenzae/genética , Haemophilus influenzae/virología , Bacteriófagos/fisiología , Secuencia de Bases , Cromosomas Bacterianos , Escherichia coli/genética , Vectores Genéticos , Integrasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Recombinación Genética , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Integración Viral

RESUMEN

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. In this communication, we present a new approach omitting the conventional PCR amplification step. Bisulfite-converted methylated DNA is directly sequenced. The effectiveness of the new protocol is demonstrated by using it for the detection of 5-methylation of cytosine residues introduced by three different DNA methyltransferases (M.HaeIII, M.HpaII and M.HhaI). A simple experimental system useful to determine the sequence specificity of DNA methyltransferases is also presented.

Asunto(s)

Citosina/análogos & derivados , Metilación de ADN , Técnicas Genéticas , 5-Metilcitosina , Composición de Base , Citosina/análisis , ADN/química , Metilasas de Modificación del ADN/metabolismo , Datos de Secuencia Molecular , Plásmidos/genética , Análisis de Secuencia de ADN , Sulfitos/metabolismo

RESUMEN

Comparison of the nucleotide sequences of the left arms of two Haemophilus influenzae phages, S2 and HP1 is presented. They exhibit a characteristic mosaic pattern of homologous and non-homologous regions. The homology extends over the attP site and int, orf 5 to 9, rep and the 3' part of cI genes. Two major non-homologous regions were detected. One is found between the int and cI genes; the other spans the region of promoters and the cox gene. Variations in the region of the promotors which is involved in the choice between a lysogenic and a lytic pathway and some divergences in the cI coding sequences are probably responsible for the observed immunity differences between the two phages. Distinctions in the distribution of consensus sequences for an integration host factor (IHF) and integrase-binding sites and promoters are described. These data offer an explanation of the relationship between three types of S2/HP1 phages. It allows in turn a final settlement of the nomenclature variation in the literature. The results presented, which are similar to those obtained for other phage groups, suggest that the mosaic structure of phage genomes is a normal outcome of phage divergence.

Asunto(s)

Bacteriófagos/genética , Haemophilus influenzae/virología , Secuencia de Aminoácidos , Secuencia de Bases , Genoma Viral , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/genética

RESUMEN

The cos region of Haemophilus influenzae phage HP1/S2 type B has been cloned and its nucleotide (nt) sequence determined. The nt sequence of the cohesive ends (cos) and whole cos site of type-B phage have been compared to corresponding sequences of the HP1c1 phage. The results of a search for symmetry elements and IHF-binding sites in this region are presented.

Asunto(s)

Bacteriófagos/genética , Genoma Viral , Haemophilus influenzae/virología , Secuencia de Bases , ADN Viral , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos

RESUMEN

Site specific restriction endonuclease R. BcrAI has been purified from Bacillus cremoris. The enzyme recognize the sequence 5' CTCTTC 3'.

Asunto(s)

Bacillus/enzimología , Enzimas de Restricción del ADN/aislamiento & purificación , Enzimas de Restricción del ADN/metabolismo , Plásmidos/metabolismo , Especificidad por Sustrato

RESUMEN

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.

Asunto(s)

Cromosomas Bacterianos , ADN Bacteriano/genética , Haemophilus influenzae/genética , Plásmidos/genética , Transformación Genética , Resistencia a la Ampicilina/genética , Secuencia de Bases , Desoxirribonucleasa I/metabolismo , Estimulación Eléctrica , Marcadores Genéticos , Datos de Secuencia Molecular , Mutación/genética

RESUMEN

The resolution of high molecular weight DNA fragments by field-inversion gel electrophoresis (FIGE) demonstrate the presence of two phage (S2 and HP1c1) integration sites (attB) in the Haemophilus influenzae Rd chromosome. In a population of wild-type cells either prophage site appears to be occupied in a single cell by one to at least three, tandemly repeated, amplified phage DNA molecules. The attL of the second bacterial attachment site present in the host SmaI fragment 7 and the leftmost part of phage S2 type B DNA of its genome organization (Piekarowicz et. al., 1986) have been sequenced. A comparison of the two bacterial att sites demonstrated that their homology is limited to the core region. A comparison of the DNA sequences of phage S2 type B and HP1c1 type C revealed a 530-bp insertion in the HP1c1 type C (not present in S2 type B) in addition to DNA variants due mostly to single-base mismatches. We postulate that phage S2 and HP1c1 genome variants (A, B, and C) evolved from a single phage origin and might stem from passage history arisen through accumulation of mutations.

Asunto(s)

Bacteriófagos/genética , Mapeo Cromosómico , Haemophilus influenzae/genética , Lisogenia , Secuencia de Bases , ADN Viral/química , Electroforesis , Datos de Secuencia Molecular

RESUMEN

Physical maps constructed by localization of the cleavage sites of several restriction endonucleases have shown that the chromosome of Haemophilus influenzae bacteriophage S2 and HP1c1 can possess different types of molecular organization (Piekarowicz, Brzezinski, Smorawinska, Kauc, Skowronek, Lenarczyk & Golembiewska, 1986). We have compared the physical maps of the HP1c1 and S2 phage DNAs of type B and the results led us to conclude that there are no differences between these two phages of the same type of molecular organization of the genomes, i.e. S2 and HP1c1 is the same phage. We also suggest that some of the fine differences between these two phages noted in some laboratories were induced only by different type of genome of the same phage.

Asunto(s)

Bacteriófagos/genética , ADN Viral/análisis , Genes Virales , Clonación Molecular , Enzimas de Restricción del ADN , Electroforesis en Gel de Poliacrilamida , Haemophilus influenzae , Plásmidos

RESUMEN

Physical maps constructed by the localization of the cleavage site of several restriction endonucleases have shown that the genomes of the Haemophilus bacteriophages S2 and HP1c1 exist in variant forms which differ in the molecular organization of the genomes. At least three regions of different organization of the bacteriophage chromosomes have been identified. The different types of molecular organization can be detected both in the DNA isolated from the mature phage particles and after integration of the phage DNA into the bacterial chromosome.

Asunto(s)

Bacteriófagos/genética , Genes Bacterianos , Genes Virales , Haemophilus influenzae/genética , Secuencia de Bases , Cromosomas Bacterianos/fisiología , Enzimas de Restricción del ADN , Variación Genética , Hibridación de Ácido Nucleico