An economic method for a rapid estimation of the number of copies of plasmid R6K delta 1 in E. coli cells using lysis of the cells directly on the agarose gel and electrophoretic separation of radioactively labelled plasmid and chromosomal DNA's is described. The method, particularly useful for screening purposes, permits detection of both the CCC and OC forms of plasmids of molar mass 2-150 Mg/mol and it can be applied to other bacterial systems.

• MeSH
• bakteriologické techniky MeSH
• DNA bakterií analýza   MeSH
• Escherichia coli analýza   genetika   MeSH
• kruhová DNA analýza   MeSH
• R-plasmidy * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• kruhová DNA MeSH

Intracellular location of plasmid NRI (M = 58 Mg/mol, stringent control of replication 1-2 copies per Escherichia coli chromosomal equivalent) was compared with that of plasmid R6D delta 1 (M = 21 Mg/mol, relaxed control of replication, 10-15 copies per E. coli chromosomal equivalent), both in E. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA's between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lysates were fractionated by sedimentation in a double-linear gradient of caesium chloride and sucrose. Also the differences in relative numbers of NRI DNA and R6K delta 1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NRI DNA molecules with E. coli minicell membrane was found to be a much more frequent event than such association of R6K delta 1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NRI and R6K delta 1.

• MeSH
• buněčná membrána analýza   MeSH
• centrifugace - gradient hustoty MeSH
• DNA bakterií izolace a purifikace   MeSH
• Escherichia coli analýza   cytologie   genetika   MeSH
• kruhová DNA izolace a purifikace   MeSH
• R-plasmidy * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• kruhová DNA MeSH

Minicells of Escherichia coli P678-54 containing plasmid RIdrd19 were submitted to careful controlled lysis. By sedimentation of the resulting lyzate in a sucrose gradient, the material absorbing at 260 nm was separated into three distinct bands. Among the most rapidly sedimenting particles, double-stranded topological circles of DNA attached to patches of membrane were visualized by electron microscopy, while single-stranded molecules (probably RNA) with associated proteins were detected in the medium band. Covalently closed and open circles of the RIdrd19 DNA were found at the top of the gradient. Their contour lengths correspond to the size of the DNA sedimenting together with the membrane in the first peak. This finding implies a direct intracellular interaction between RIdrd19 DNA and membrane in E. coli minicelle.

• MeSH
• bakteriolýza MeSH
• buněčná membrána analýza   ultrastruktura   MeSH
• centrifugace - gradient hustoty MeSH
• DNA bakterií analýza   MeSH
• elektronová mikroskopie MeSH
• Escherichia coli analýza   ultrastruktura   MeSH
• kruhová DNA analýza   MeSH
• R-plasmidy * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• kruhová DNA MeSH

Some properties of streptomycin-resistant mutants of Escherichia coli were analyzed. In a R+ culture, the phenotype under study may be significantly selected at a frequency of 10(-5) on media with higher streptomycin level. The lrs mutation is present in the cells prior to the action of streptomycin and remains in the cells even after curing of the R1 plasmid. The mapping of the lrs gene by conjugation with a concomitant transfer of chromosome and the R1 plasmid in different Hfr strains of E. coli failed to establish the localization of this gene in the tested chromosome regions. The presence of a cryptic plasmid was detected in cells with the lrs mutation after curing of the R1 plasmid. This plasmid codes neither fertility functions nor chloramphenicol-acetyltransferase, streptomycin-adenyltransferase, or ampicillin-beta-lactamase.

• MeSH
• antibiotická rezistence MeSH
• Escherichia coli účinky léků   enzymologie   genetika   MeSH
• mapování chromozomů * MeSH
• mutace MeSH
• R-plasmidy * MeSH
• streptomycin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• streptomycin MeSH