RESUMO
The pUB110 and pE194 plasmid cointegrates have been isolated and examined in rec+ and recE4 strains of Bacillus subtilis. Cointegrates were shown to be formed by recombination at the specific site present on both parental plasmids as a short region of homology designated RSA. The RSA consists of 63 nucleotides in pE194 and 49 in pUB110; the length of its fully conserved core segment is 10 nucleotides. All cointegrates examined were formed by single crossover event taking place within the core segment, and as a result they have identical nucleotide sequences of recombination junctions. No conversion of mismatched base pairs to nucleotide sequences originally belonging to one of the parental plasmids was found. Though the action of RecE gene did not affect the frequency of cointegrate formation, it was reduced in rec149 host by one order of magnitude. Cointegrates retained their stability during transformation.
Assuntos
Bacillus subtilis/genética , Genes Bacterianos , Plasmídeos , Recombinação Genética , Sequência de Bases , Enzimas de Restrição do DNA , DNA Bacteriano/genética , Conformação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
A portion of purA16 chromosomal locus of Bacillus subtilis was cloned into Rec+ cells of this microorganism with pBD12 plasmid (carrying chloramphenicol and kanamycin resistance determinants) serving as a vector. The hybrid plasmids were stably maintained in cells grown on media supplemented with antibiotics and were lost from cells in the absence of drugs. The cloned fragment could incorporate into the chromosome some with a frequency of 10(-2) per cell per generation. A clone carrying the hybrid plasmid inserted into the chromosome was detected.
Assuntos
Bacillus subtilis/genética , Mapeamento Cromossômico , Clonagem Molecular/métodos , Bacillus subtilis/efeitos dos fármacos , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/ultraestrutura , Clonagem Molecular/efeitos dos fármacos , DNA Bacteriano/genética , Vetores Genéticos/efeitos dos fármacos , Plasmídeos/efeitos dos fármacos , Transformação Bacteriana/efeitos dos fármacosAssuntos
Bacillus subtilis/genética , Cromossomos Bacterianos/efeitos dos fármacos , DNA Bacteriano/genética , DNA Viral/genética , Genes Bacterianos/efeitos dos fármacos , Plasmídeos/efeitos dos fármacos , Transformação Bacteriana/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Bacteriófagos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos/efeitos dos fármacos , FenótipoRESUMO
Following the restriction and ligation of pBD12 plasmid and phi 105 phage DNA and subsequent transformation, the insertion of pBD12 DNA into the phi 105 prophage region of the Bacillus subtilis chromosome was achieved. The plasmid markers were linked with the region of prophage immunity and with the markers of the adjacent region of the B. subtilis chromosome. Their behaviour in transformation of cells and protoplasts of rec+ recipients and recE4 mutants was typical of chromosomal markers. The phenotypic expression (resistance to chloramphenicol and kanamycin) of plasmid genes inserted into the chromosome was preserved; however, the resistance levels were reduced.
Assuntos
Bacillus subtilis/genética , DNA Recombinante , Bacteriófagos/genética , Cloranfenicol/farmacologia , DNA Bacteriano/genética , DNA Viral/genética , Resistência Microbiana a Medicamentos , Ligação Genética , Plasmídeos , Transformação GenéticaRESUMO
The effect of different factors which inhibit transformation with chromosomal DNA on plasmid transformation of competent cells and protoplasts was examined. The treatment with EDTA, dinitrophenol, CdCl2 and different temperature shifts affects significantly less plasmid transformation of protoplasts than plasmid and chromosomal transformation of competent cells. These data point out the different mechanisms of DNA uptake by competent cells and protoplasts.
Assuntos
Bacillus subtilis/genética , Plasmídeos , Protoplastos/ultraestrutura , Transformação Bacteriana , Bacillus subtilis/efeitos dos fármacos , Cádmio/farmacologia , Cloreto de Cádmio , DNA Bacteriano/genética , Dinitrofenóis/farmacologia , Ácido Edético/farmacologia , Plasmídeos/efeitos dos fármacos , Protoplastos/efeitos dos fármacos , Temperatura , Transformação Bacteriana/efeitos dos fármacosRESUMO
Marker rescue in plasmid transformation of competent cells of different rec mutants of B. subtilis was studied. In most cases the value of marker rescue decreased proportionally to reduction of plasmid transformation efficiency (although there were certain exceptions). Marker rescue was not observed either in plasmid transformation of protoplasts or in plasmid transduction of intact cells.
Assuntos
Bacillus subtilis/genética , Plasmídeos , Recombinação Genética , Transdução Genética , Transformação Genética , MutaçãoAssuntos
Bacillus subtilis/genética , Cromossomos Bacterianos/ultraestrutura , DNA Bacteriano/genética , DNA Viral/genética , Genes Bacterianos , Genes Virais , Hibridização de Ácido Nucleico , Recombinação Genética , Bacteriófagos/genética , Engenharia Genética/métodos , Marcadores Genéticos , Plasmídeos , Transformação BacterianaRESUMO
Transformation of Bacillus subtilis cells carrying pUB110 plasmid by DNA of homologous plasmid pBD12 results in the significant increase in the number of plasmid transformants. This phenomenon named "augmentation" was not observed when instead of intact cells, regenerating protoplasts were used, or if pBD12 DNA was introduced into the cells via transduction.
Assuntos
Bacillus subtilis/genética , Marcadores Genéticos , Plasmídeos , Transdução Genética , Transformação Bacteriana , DNA Bacteriano/genética , Lisossomos/ultraestrutura , Protoplastos/ultraestruturaRESUMO
A class of revertants of Bacillus subtilis mutant rec H, which completely restored the ability to transformation but without restoring the activity of ATP-dependent deoxyribonuclease, is isolated and studied. Reversions are located in the same chromosome region as the original mutation. The detection of such revertants points out the existence of more than one recombination pathway for Bac. subtilis transformation.
