RESUMEN

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system.

Asunto(s)

Farmacorresistencia Microbiana/genética , Plásmidos/genética , Fagos T/genética , Transducción Genética , Proteínas Virales , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , Desoxirribonucleasa EcoRI/genética , Desoxirribonucleasa EcoRI/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virología , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Marcadores Genéticos , Hidrolasas/genética , Hidrolasas/metabolismo , Factores de Integración del Huésped/biosíntesis , Factores de Integración del Huésped/genética , Mutación , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Fagos T/patogenicidad , Replicación Viral

Asunto(s)

Farmacorresistencia Microbiana/genética , Plásmidos , Fagos T/genética , Transducción Genética , Escherichia coli/genética

RESUMEN

A number of recombinant plasmids, containing EcoRV restriction-modification genes have been constructed. Individual genes of this system were introduced into plasmids of various incompatibility groups. Promoter regions of genes encoding methylase and restrictase have been cloned and studied. With the use of specialized vector pVE8 it was shown that the efficiency of the endonuclease gene promoter is comparable with early lambda phage promoters and produced about 70% of PL efficiency. The efficiency of the methylase gene promoter region was twice less than the efficiency of the restriction endonuclease gene promoter. Plasmid with restriction endonuclease gene promoter located downstream in relation to the additional regulatable phage lambda promoter PL has been obtained. It enabled us to construct strains 30-40 fold overproducing this enzyme under conditions of inactivation of the temperature sensitive phage repressor c1857. This construction directs the production of a high level (10%) of the total cellular soluble proteins) of the EcoRV restriction enzyme. The factors that influenced the level of enzyme synthesis under induction are discussed.

Asunto(s)

Clonación Molecular , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Regulación Enzimológica de la Expresión Génica , Secuencia de Bases , Escherichia coli/enzimología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Recombinación Genética

Asunto(s)

Clonación Molecular , Genes Virales , Fagos T/genética , Secuencia de Bases , ADN/genética , Genes Reguladores , Vectores Genéticos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , Regiones Promotoras Genéticas , Transcripción Genética

Asunto(s)

Secuencia de Bases , ADN Ligasas/genética , Genes Virales , Polinucleótido Ligasas/genética , Homología de Secuencia de Ácido Nucleico , Fagos T/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Especificidad de la Especie , Fagos T/enzimología

RESUMEN

E. coli hsd genes were subcloned from lambda 642 (ral+) into lambda L47.1 vector (ral-after replacement). The influence of bacteriophage lambda ral gene on the expression efficiency of hsdS kappa, hsdM kappa genes was investigated. It was shown, that its presence in vitro enhanced the synthesis of beta-subunit, hsdM gene product, and the increase of modification in vivo was observed. It is proposed that the increase of modification rate of lambda phage fully unmodified DNA is connected with the appearance of E. coli DNA methylase consisting of beta- and gamma-subunits but lacking alpha-subunit.

Asunto(s)

Bacteriófago lambda/genética , Enzimas de Restricción del ADN/biosíntesis , Desoxirribonucleasas de Localización Especificada Tipo I , Genes Virales , Vectores Genéticos , Clonación Molecular , ADN Viral/análisis , Electroforesis en Gel de Agar , Escherichia coli/enzimología , Escherichia coli/genética , Plásmidos

Asunto(s)

Proteínas Bacterianas , ADN Viral/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Fagos T/genética , Mapeo Cromosómico , Enzimas de Restricción del ADN

Asunto(s)

Clonación Molecular/métodos , ADN (Citosina-5-)-Metiltransferasas/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Genes Bacterianos , Metiltransferasas/genética , Plásmidos , Escherichia coli/enzimología , Recombinación Genética

Asunto(s)

Clonación Molecular/métodos , ADN (Citosina-5-)-Metiltransferasas/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Genes Bacterianos , Metiltransferasas/genética , Bacteriófago lambda/genética , Escherichia coli/enzimología , Plásmidos , Biosíntesis de Proteínas , Recombinación Genética , Transcripción Genética , Transfección

Asunto(s)

ADN Ligasas/genética , Genes Virales , Polinucleótido Ligasas/genética , Fagos T/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Conformación de Ácido Nucleico , Fagos T/enzimología

Asunto(s)

Clonación Molecular/efectos de los fármacos , Enzimas de Restricción del ADN/farmacología , Escherichia coli/enzimología , Vectores Genéticos/efectos de los fármacos , Plásmidos/efectos de los fármacos , Fagos T/efectos de los fármacos , Genes Virales/efectos de los fármacos , Hidrólisis , Recombinación Genética/efectos de los fármacos

Asunto(s)

Bacteriófago lambda/genética , Escherichia coli/genética , Genes Bacterianos , Operón , Plásmidos , Recombinación Genética , Mapeo Cromosómico , ADN Bacteriano/genética

Asunto(s)

Bacteriófago lambda/genética , Vectores Genéticos , Operón , Plásmidos , Regulación de la Expresión Génica , Genes Virales , Variación Genética , Supresión Genética

RESUMEN

DNAs of lambda T4 recombinants 596-27 (genes 50-5), 596-30 (genes 50-8), 596-29 (genes 50-12), 591-16 (genes 6-8), 591-1 (genes 9-12), 596-13 (genes 13-16), 596-17 (genes 18-20) and 596-11 (genes 25-29) were mapped with the use of EcoRI, HindIII, SmaI, SalI and BamHI restriction enzymes. T4 dcDNA was digested with HindIII restriction endonuclease and resulting fragments were cloned into HindIII lambda vector 761. The recombinants 761-7, 761-17, 761-19, 761-24, 761-44, 761-50, 761-55 contained the region of genes 25-48 and 761-42, 761-26 and 761-16 contained a single HindIII-fragment with genes 6-12 in both orientations. Data obtained with the DNA of the latter recombinants allowed to show the correctness of the map established earlier which did not contain a full set of overlapping sequences. As a result of the experiments reported, the position of EcoRI and HindIII recognition sites in the region of genes 50-20 and 25-48 was determined and in the region of genes 25-48 BglII and XhoI restriction sites were mapped. The location of a single BamHI restriction site in the region of gene 8 was also established.

Asunto(s)

Replicación del ADN , ADN Viral/genética , Genes Virales , Fagos T/genética , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Recombinante , Escherichia coli/genética , Replicación Viral

Asunto(s)

Clonación Molecular , Enzimas de Restricción del ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Plásmidos , Recombinación Genética

Asunto(s)

Enzimas de Restricción del ADN/genética , Fagos T/enzimología , Mapeo Cromosómico , ADN Viral/genética , Peso Molecular , Hibridación de Ácido Nucleico , Recombinación Genética , Fagos T/genética

Asunto(s)

Colifagos/genética , Genes Reguladores , Genes Virales , Plásmidos , ADN Bacteriano/genética , ADN Recombinante , ADN Viral/genética , Código Genético , Transcripción Genética

Asunto(s)

Colifagos/genética , Enzimas de Restricción del ADN/genética , Recombinación Genética

Asunto(s)

Colifagos/genética , Genes Virales , Recombinación Genética , Mapeo Cromosómico , ADN Viral/análisis , Electroforesis en Gel de Agar , Escherichia coli/genética , Especificidad de la Especie , Transcripción Genética , Proteínas Virales/biosíntesis

Asunto(s)

Escherichia coli/enzimología , Polinucleótido Ligasas/biosíntesis , Colifagos , Inducción Enzimática , Nucleótidos de Guanina