RESUMEN
Short treatment of Escherichia coli cells with antibiotics disturbing synthesis of bacterial cell wall in small concentrations renders the cells capable of absorbing foreign plasmid DNA. A novel express-method for transformation of E. coli cells by plasmid DNA has been developed on the basis of the results obtained. The whole procedure can be performed at room temperature. Depending on cell strain and the plasmid size, the efficiency of transformation can vary from 1.10(4) to 5.10(5) transformants per 1 mkg of DNA. The method suggested improves significantly the every-day work aimed at constructing plasmids.
Asunto(s)
Antibacterianos/farmacología , Pared Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Plásmidos , Transformación Bacteriana , Permeabilidad de la Membrana Celular/efectos de los fármacos , Pared Celular/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
The modern data on Escherichia coli K-12 ilv genes expression are reviewed. The problems of regulation of the ilv genes activity and of their possible role in the process of cell adaptation to changeable environment are discussed.
Asunto(s)
Escherichia coli/genética , Regulación de la Expresión Génica , Genes Bacterianos , Aminoácidos/genética , Medios de Cultivo/metabolismo , AMP Cíclico/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/efectos de los fármacos , Guanosina Tetrafosfato/fisiología , Isoleucina/biosíntesis , Isoleucina/genética , Mutación , Operón , Biosíntesis de Proteínas , Receptores de AMP Cíclico/genética , Transcripción Genética , Valina/biosíntesis , Valina/genéticaRESUMEN
The influence of rifampicin resistance mutations on expression of the ilv operon of Escherichia coli was investigated. Some of these mutations, like those previously described in our works, occur in translation machinery of E. coli and inhibit derepression of the ilv operon. However, another group of mutations stimulated the expression of the operon when mutated cells were transferred from rich to minimal growth medium. The possible mechanisms of the effects of rifampicin resistance mutations on the action of coupled transcription--translation system are discussed.
Asunto(s)
Aminoácidos/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Genes Bacterianos , Mutación , Operón , Farmacorresistencia Microbiana , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Código Genético , Biosíntesis de Proteínas , Rifampin/antagonistas & inhibidores , Transcripción GenéticaRESUMEN
The rate of adaptation of Escherichia coli K-12 NF930 spoT1 cells with elevated intracellular level of ppGpp to various minimal media was studied. It has been found that the rate of adaptation of spoT cells, like that of parent and rel strains, depends mainly on the rate of derepression of the ilv operon. The maximal rate of the ilv operon derepression was observed when an optimal concentration of ppGpp was maintained in cells. Derepression of the ilv operon is sharply delayed when the level of ppGpp is elevated or reduced. Mutations altering the translation system do not change the rate of adaptation of spoT cells. Rifampicin resistance mutations which altered the structure of RNA polymerase change the rate of adaptation of spoT cells to minimal media, especially to those containing serine at high concentrations. The possible role of serine in the regulation of ppGpp degradation system is discussed.
Asunto(s)
Aminoácidos/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Genes Bacterianos , Mutación , Operón , Adaptación Fisiológica , Farmacorresistencia Microbiana , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Biosíntesis de Proteínas , Rifampin/antagonistas & inhibidores , Transcripción GenéticaRESUMEN
Derepression of the ilv operon in rel strains of Escherichia coli is delayed when cells are transferred from rich to minimal medium and is completely blocked when the mixture of amino acids--serine, methionine and glycine is present in the minimal medium. It is shown that alterations in translation machinery caused by streptomycine resistance mutation can also lead to the delay of the ilv operon derepression in rel+ strains or to its complete inhibition in rel strains of E. coli. The possible mechanisms of high sensitivity of the ilv operon to different alterations in E. coli are discussed.
Asunto(s)
Aminoácidos/genética , Escherichia coli/genética , Genes Bacterianos , Mutación , Operón , Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Transcripción Genética , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Regulación de la Expresión Génica , Código Genético , Fenotipo , Proteína Ribosómica S9RESUMEN
It has been shown in our previous study that mutations in genes relA, relC and rpsL result in the delay in Escherichia coli ilv operon derepression; the complete inhibition of derepression of the ilv operon is observed in the double mutants having alterations in rpsL and relA or relC genes. At present, some mutations occurring in the fus gene and altering the structure of the translational elongation G factor have been also found to delay derepression of E. coli ilv operon and complete inhibition in fusr and rel double mutants. Phenotypical ile and val auxotrophy is also detected in the double E. coli mutants with spectinomycin resistance mutation in rpsE gene coding for the structure of ribosomal S5 protein and mutations in relA or relC genes. The suggestion of participation of the ilv operon in regulation of other E. coli amino acid operons expression is discussed.
Asunto(s)
Aminoácidos/genética , Escherichia coli/genética , Genes Bacterianos , Mutación , Operón , Factores de Elongación de Péptidos/genética , Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Regulación de la Expresión Génica , Código Genético , Factor G de Elongación Peptídica , Proteína Ribosómica S9RESUMEN
In quantitative measurements of pyridoxal-5'-phosphate and pyridoxal in enzymes routinely used phenylhydrasine was substituted for 4-nitrophenylhydrasine. This increased the sensitivity of the method by 70%. The modified procedure had another advantage: it allowed measurements of the optic density of resulting 4-nitrophenylhydrasones at 430 nm for acid solutions and at 550 nm for alkaline solutions.