[Expression of plasmid R6K in a cell-free coupled transcription-translation system]. / Ekspressiia plazmidy R6K v beskletochnoi sisteme sparennoi transkriptsii--transliatsii.

The modified method for obtaining E. coli extract S 30 for the coupled transcription--translation system is described. The extract treated with immobilized DNAase and micrococcal nuclease had the minimal endogenous protein synthesis and high activity in respect to exogenous DNA matrices. The synthesis of 9-12 proteins with molecular weights ranging from 14,000 to 178,000 daltons was shown to occur in this system in the presence of the DNA of R6K plasmid. The use of the modification of Novick's method allowed to identify active beta-lactamase coded by the amp-gene of R6K plasmid.

[Transforming activity of plasmid R6K DNA on Serratia marcescens strain 20-10. The behavior of the plasmid in the transformants]. / Transformiruiushchaia aktivnost' DNK plazmidy R6K na shtamme Serratia marcescens 20-10. Povedenie plazmidy v transformantakh.

The authors described transformation of S. marcescens, strain 20-10, of the isolated R6K plasmide DNA. As demonstrated by centrifugation in cesium chloride gradient and electrophoresis in agarose, the plasmide was present in the transformants in the form identical to R6K in E. coli K12. Analysis of the transforming activity of R6K plasmide from Serratia and E. coli K12 strains with a complete and defective restriction system showed S. marsescens, strain 20-10, to possess specific system of restriction and modification. In studying beta-lactamase activity and Serratia and E. coli strains ampicillin and streptomycin resistance revealed differences in the phenotypical expression of the plasmide signs in the heterologous and homologous host.

[Characteristics of the deletion mutant of plasmid R6K]. / Kharakteristika deletsionnogo mutanta plazmidy R6K.

Deletion plasmide R6Kdelta with the mol wt of 17.2.10(6) dalton isolated from the E. coli chi 925 (R6K) is described. This plasmide expresses no resistance to streptomycin, is replicated in the E. coli K12 under relaxed control and is resistant to the treatment with the eliminating agents. Analysis of plasmide DNA with the aid of electrophoresis in agarose gel demonstrated that R6K delta has one site attacked by restriction endonucleases Eco. RI and Bam HI. These data were confirmed by the determination of the transforming activity of the corresponding DNA restrictors. It is supposed that the isolated plasmide was identical with plasmide RSF1040. A possibility of using R6K delta as a genetic vector for obtaining recombination DNA molecules in vitro is discussed.

[Entobacterial transformation by R6K plasmid DNA]. / Transformatsiia énterobakterii DNK plazmidy R6K

Preparations of DNA of R6K plasmide obtained by various methods on the basis of the "clarified" lysate: by gel filtration on sepharose 4B, centrifugation in cesium chloride with ethidium bromide gradient, were analysed in the E. coli C600. S. typhimurium AG37, Pr. vulgaris 4636, S. marcescens 20-10 transformation. The frequency of transformation proved to depend on the extent of DNA purification. Factors influencing the E. coli C600 competence and the transformation efficacy (the phase of the culture growth, the concentration of cells in the mixture with DNA, theCaCl2 concentration, the time of the cell incubation at 42 degrees C) were studied. Kinetics of the phenotypical expression of the plasmide signs of ampicillin and streptomycin resistance in transformation was also studied in this work.