J. Štokrová

Transfection ofBrevibacterium flavum with bacteriophage BFB10 DNA

The virulent bacteriophageBFB10 is infective toBrevibacterium flavum Its DNA (about 48 kilobase pairs) was used for optimization of the DNA transfer intoB. flavum cells treated with lysozyme. Efficiencies of transfection up to 60 transfectants per ng DNA were obtained The described procedure can also be used for transformation ofB. flavum with plasmid DNA.

Plurimodal heterogeneity of base composition of calf thymus DNA

Abstract Calf thymus DNA was fractionated by three cycles of chromatography on methylated serum albumin columns. The separate fractions, as well as the unfractionated DNA, were characterized by the first derivatives of absorption-melting curves, continuous thermal chromatograms on hydroxyapatite columns and isopycnic sedimentation in gradients of CsCl. All these methods indicate a plurimodal distribution of base composition of calf thymus DNA. Fractions with the highest G+C content, rich in satellite Components I, II and III (1.713, 1.720–1.721 and 1.707 g/cm3, respectively) give polyphasic denaturation curves, contrasting with more simple isopycnic-sedimentation profiles; therefore, these molecules seem to contain large cooperatively-melting segments of widely different base composition. Similarly the denaturation profiles of fractions with the lowest average G+C content indicate the presence of independently-melting segments with G+C contents varying between 24 and 40 %.

The dsRNA ofTrichomonas vaginalis is associated with virus-like particles and does not correlate with metornidazole resistance

Twelve metronidazole-resistant and twelve metronidazole-susceptible strains ofTrichomonas vaginalis were tested for the presence of dsRNA. Three resistant and five susceptible strains were found to contain dsRNA which indicated that metronidazole resistance does not correlate with the absence of dsRNA. Electron microscopy showed the homogenates of all dsRNA -positive strains to contain virus-like particles 32 –38 nm in diameter, while no such particles were found in the dsRNA-negative strains. A mutual relationship between the dsRNA and virus-like particles seems to exist.

Temperate and virulent forms of phage theta attacking Bacillus licheniformis.

SummaryClear-plaque phage ϑc, attacking bacitracin-producing strains of B. licheniformis, yields spontaneous temperate mutants at high frequency; the temperate mutants fall into several classes phenotypically different in plaque morphology and properties of lysogenised bacteria. The most common phenotype ϑ3 has DNA restriction fragment patterns identical with those of the parent ϑc; some less common temperate forms, i.e. ϑ1 and ϑ2, produce different restriction fragment patterns, sugesting that a part of the original ϑc DNA has been reorganized or replaced by some foreign genetic material. The changed fragment pattern remains stable upon subsequent passaging of the phage or of the lysogenic bacteria. Neither class of temperate phage mutants gives clearplaque revertants at measurable frequency. Lysogenisation of bacteria with any class of temperate phage confers immunity to all temperate forms and to ϑc; virulent mutants ϑvir, which plate with 100% efficiency on lysogens for ϑ1 and ϑ2 but not for ϑ3, occur in stocks of ϑc at a frequency of 10−7. The mutation from ϑc to ϑvir is not accompanied by any change of the restriction fragment patterns of DNA.

Multiple populations of double-stranded RNA in two virus-harbouring strains of Trichomonas vaginalis

The existence of six dsRNA segments ofTrichomonas vaginalis virus was confirmed and the molar mass and relative abundance of these segments were determined by agarose gel electrophoresis with reovirus dsRNA serving as a standard. TheM’s were 3.5, 3.4, 3.2, 2.5, 1.4 and 0.34 Mg/mol for the two strains studied, the relative abundances, however, were 1.0, 1.4, 3.0, 0.3, 2.7, 4.2 and 1.0, 0.6, 1.7, 0.5, 3.4 1.0 for these strains, respectively. Cell homogenate fractionation showed that all dsRNA segments were associated with viral particles. The data appeared to support the hypothesis of a relationship between viruses of the protozoanT. vaginalis and of the yeastSaccharomyces cerevisiae.

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 ofEscherichia 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 regionfinO and, according to the results of restriction analysis, it includes one EcoRI site. With its radioisotopically determined 33 copies of pNH602 perE. 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.

Transforming activity of plasmid and chromosomal DNA inEscherichia coli

An auxotrophic strain ofEscherichia coli with therecB recC sbcB genotype was transformed by chromosomal DNA of the prototrophic strain and by plasmid DNA carrying genes for antibiotic resistance (R1drd 19). 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 circulaf 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.

Application of R-plasmid DNA’s fromEscherichia coli minicells in genetic transformation

Components of minicell lyzatesof Escherichia coli P678-54 (Rldrd19) andEscherichia col P678-54 (R6K) were visualized in an electron microscope and used for the transformation ofEscherichia 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 Rldrd19 or R6K plasmids in tested transformants. This finding corresponds with the results of their genetic analysis.

Correlation of physical maps and some genetic functions in the genomes of the κ-ϑ phage family of Bacillus licheniformis

SummaryNatural recombinant genomes between several, phenotypically distinct forms of phages κ and ϑ were isolated and characterized by DNA restriction fragment mapping and electron microscopic heteroduplex analysis. The phenotypes of the recombinants were correlated with the physical maps of the genomes, and several genetic functions were therfore defined and mapped. All genes necessary for the assembly of infectious virus particles map in a contiguous tract of DNA comprising about 20 kb, or nearly one third of the genome length. No DNA homology occurs within these domains of the two genomes, so that homologous recombination does not take place here and phenotypic mixing of the phages is eo ipso excluded. Other regions of heterology contain regulatory genes responsible for the lytic or temperate character of the phages, and for exclusion of phage κ by ϑ.

MULTICOPY PLASMID R6Kδ1 IN ESCHERICHIA COLI MINICELLS-INTERACTION OF ITS DNAMOLECULES WITH THE CELL MEMBRANE

ABSTRACT Intracellular location of DNA of plasmid R6Kδ1 in chromosome-free Escherichia coli minicells was studied. Mini-cells harboring this plasmid were carefully lysed and the mixture of released subcellular components was fractionated by sedimentation. In a double cesium chloride - sucrose gradient material absorbing at 260 nm separated into three bands. Electron microscopy revealed R6Kδ1 DNA in two of them. The greater part of plasmid DNA was found on the top of the gradient as free molecules, a lower amount was associated with cellular membrane fragments which had sedimented most rapidly. Majority of R6Kδ1 DNA in minicells is therefore probably located in the cytoplasm. Stability of all membrane-R6Kδ1 DNA complexes does not seem to be equal, as the released plasmid molecules were observed in the membrane fraction. The percentage of R6Kδ1 DNA stably associated with cellular membrane is lower than that of R1 drd19 DNA (Hochmannova and others, 1979) and is comparable with the data for ColE1 DNA (Sparks and Helinski, 1979). Cosedimentation of R6Kδ1 DNA with membrane-attached chromosome during the fractionation of a lyzate of normal plasmid-harboring E.coli cells indicates the possible nonintegrative association of R6Kδ1 DNA with the membrane-complexed folded chromosome.