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J Nesvera

Publications and source records attributed to J Nesvera.

29 records · Page 2Linked to original sources
PubMed1985

New replication mutant pNH602 and its relationship to plasmid pAS3, another deletion derivative of plasmid R6K.

A stable deletion derivative pNH602 was obtained when the recently described higher-copy-number point mutant pNH601 of plasmid R6K was introduced to a minicells-producing strain of Escherichia coli. The size of plasmid pNH602 is 18.8 Mg/mol as determined by electron microscopy. The 7.2 Mg/mol fragment of R6K genome missing in pNH602 carries the Smr-determinant and the region finO and, according to the results of restriction analysis, it includes one EcoRI site. With its radioisotopically determined 33 copies of pNH602 per E. coli K-12 chromosome (npc), representing a 23% increase of the point mutant pNH601 and 150% enhancement of R6K npc, plasmid pNH602 differs from another closely related R6K deletion derivative pAS3 of the same size which exhibits only 20 npc. Both pNH602 and pAS3 plasmids are conjugative.

Chromosome Deletion↗
PubMed1983

Isolation and characterization of a higher-copy-number mutant of plasmid R6K.

A stable copy-number mutant (pNH601) of plasmid R6K was isolated by selection for increased resistance to ampicillin determined by this plasmid. The size of the mutant plasmid was found to be unchanged (26 Mg/mol) but it is present in 27 copies of pNH601 per E. coli K-12 chromosome which represents a two-fold increase of R6K copy number value. The following genetic properties of pNH601 are reported and compared with those of R6K: conjugative transfer, fertility inhibition of plasmids belonging to other incompatibility groups, incompatibility with plasmid R485 under both non-selective and selective conditions and the integrative suppression of the dnaA ts mutation. The mutant plasmid pNH601 was found to be different from the original R6K in most of these properties.

Ampicillin↗
PubMed1982

A rapid and economic method for estimation of the number of plasmid copies in Escherichia coli cells.

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.

Bacteriological Techniques↗
PubMed1982

The recA-dependent spontaneous degradation of proximal F merogenotes in Escherichia coli.

Proximal F' elements of KLF-1 type are relatively stable in Escherichia coli recA recipients. In such merodiploids the transferability of F'-DNA and the plasmid determined fertility functions are expressed. When introduced into the wild type recA+ cells the F'-DNA is degraded and several classes of DNA molecules of molar mass about 66 Mg/mol and lower exist in the cell in 1-2 copies per bacterial chromosome. As was detected by complementation analysis, the chromosomal genes determining the host specificity for DNA (hsd) originally located on the F' element seem to be salvaged during the process of DNA degradation probably by recombination with the bacterial chromosome.

Bacterial Proteins↗
PubMed1981

Intracellular location of NR1-plasmid DNA in Escherichia coli minicells.

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.

Cell Membrane↗
PubMed1980

Visualization of membrane-associated R-plasmid DNA in fraction of Escherichia coli minicell lyzate.

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.

Bacteriolysis↗
PubMed1978

Extranuclear mutant of Schizophyllum commune.

A mutant of the hymenomycete Schizophyllum commune was isolated which, owing to an extranuclear mutation, did not utilize acetate as the sole carbon source for growth. The growth of the mutant on glucose minimal medium was completely inhibited by sodium azide but was resistant to the effect of 2,4-dinitrophenol or oligomycin. Its endogenous respiration was cyanide-sensitive and was stimulated by 2,4-dinitrophenol to a considerably smaller degree than that of the wild-type strain. The experimental results obtained with this mutant suggest a defect in aerobic phosphorylation.

Acetates↗
PubMed1978

Application of R-plasmid DNA's from Escherichia coli minicells in genetic transformation.

Components of minicell lyzates of Escherichia coli P678.54 (R1 drd 19) and escherichia coli P678.54 (R6K) were visualized in an electron microscope and used for the transformation of Escherichia coli JC7623. The frequency of the resulting transformants (of the order of 10(-6)%) was not appreciably influenced by the manner of lyzate preparation. The presence of covalently closed circular DNA was detected in two different transformants using radioisotopes, thus demonstrating an autonomous existence of R1 drd 19 or R6K plasmids in tested transformants. This finding corresponds with the results of their genetic analysis.

Ampicillin↗
PubMed1977

Transforming activity of plasmid and chromosomal DNA in Escherichia coli.

An auxotrophic strain of Escherichia coli with the recB recC sbcB genotype was transformed by chromosomal DNA of the prototrophic strain and by plasmid DNA carrying genes for antibiotic resistance (R1 drd19). The donor plasmid DNA obtained by cell lysis in the presence of Triton X-100 and subsequent centrifugation in a caesium chloride-ethidium bromide gradient was shown to have a circular molecule and to retain its completeness after penetration into the recipient. Experiments with mixtures or plasmid and chromosomal DNA indicate a competition between these two DNA types during the transformation reaction in the given system.

Chromosomes, Bacterial↗
PubMed1977

Cyanide-insensitive respiration in Schizophyllum commune.

Two mutants of the hymenomycete Schizophyllum commune, unable to use acetate as the sole carbon source for growth, were isolated. Growth of the mutants on a glucose minimal medium was only slightly inhibited by sodium azide. Genetic analysis revealed mutations in different chromosomal genes in the respective mutants. Both these mutants exhibited a high cyanide-insensitive endogenous respiration. The inhibition of the respiration by 8-hydroxyquinoline showed the mutants to respire predominantly by an alternative respiratory pathway observed in many fungal species, but not in the hymenomycetes so far. An enhanced cyanide-insensitive respiration was also found in a wild-type strain of Schizophyllum commune grown in the presence of sodium azide.

Acetates↗