Maurice S. Fox

Induction kinetics of mutagenic DNA repair activity in E. coli following ultraviolet irradiation

SummaryUltraviolet mutagenesis of phage λ is produced by host functions which are inducible by ultraviolet irradiation of the host cell. Induction kinetics and the half life of the inducible mutagenic DNA repair (SOS-repair) in E. coli have been determined using phage λ assays. At 37°C, both mutagenic and repair activities are maximal approximately 30 min following irradiation and decay with a half life of approximately 30 min. The presence of 100 μg/ml chloramphenicol during the first 40 min after irradiation completely abolishes induction of repair and mutagenesis. The ultraviolet induction pattern of SOS repair very much resembles that of λ prophage in lysogenic induction (Monk and Kinross, 1975).

Generalized transduction by phage P22 in Salmonella typhimurium. I. Molecular origin of transducing DNA.

Abstract P22, a temperate phage which grows on Salmonella typhimurium , is capable of carrying out generalized transduction in this host. The generalized transducing particles are the same size as P22 phage particles but instead of phage DNA they contain bacterial DNA which was synthesized before phage infection. Further, DNA molecules isolated from the transducing particles have the same molecular weight as P22 DNA, 27 × 10 6 daltons. Examination of the formation of transducing particles under conditions in which the S. typhimurium recombination system ( rec ) and/or the P22 recombination system ( erf ) are defective indicates that neither of these general recombination systems is necessary for the formation of transducing particles.

Physical and genetic hybrids formed in bacterial transformation.

Abstract Pneumococcus was transformed with DNA labeled with the heavy isotopes 2H and 15N. DNA was extracted from the recipient cells immediately after transformation and also after various amounts of growth following termination of the exposure to the heavy DNA. Re-extracted DNA was broken to desired ranges of molecular weights by shear, and then fractionated in cesium chloride density-gradients. We examined the distributions of transforming activity in these gradients for two markers, one carried by the heavy DNA and the other by the light recipient cells. Both markers were present at hybrid density immediately after transformation. The recipient marker largely disappeared from hybrid material during the first DNA replication after transformation. The donor marker began to appear at the light density during the second replication. We concluded from these observations that the physical hybrid observed was a genetic hybrid between donor and recipient for the two markers examined, and that either strand of the duplex could participate in the breakage and rejoining mechanism which effects transformation. From the ratios of donor to recipient activities observed at the hybrid density, we further concluded that either strand of the duplex can transform with equal probability. From experiments in which we varied the molecular weight of the DNA applied to the cesium chloride gradients, we concluded that DNA can be integrated into the recipient genome in a continuum of lengths ranging from an undetermined lower limit below 1 × 106 daltons to more than 5 × 106 daltons, in single-stranded molecular weight. From our experiments using markers for streptomycin-resistance and erythromycin-resistance, we estimated a weight-average length of integration near 2 × 106 daltons.

Transformation of yeast with linearized plasmid DNA: Formation of inverted dimers and recombinant plasmid products

The molecular products of DNA double strand break repair were investigated after transformation of yeast (Saccharomyces cerevisiae) with linearized plasmid DNA. DNA of an autonomous yeast plasmid cleaved to generate free ends lacking homology with the yeast genome, when used in transformation along with sonicated non-homologous carrier DNA, gave rise to transformants with high frequency. Most of these transformants were found to harbor a head-to-head (inverted) dimer of the linearized plasmid. This outcome of transformation contrasts with that observed when the carrier DNA is not present. Transformants occur at a much reduced frequency and harbor either the parent plasmid or a plasmid with deletion at the site of the cleavage. When the linearized plasmid is introduced along with sonicated carrier DNA and a homologous DNA restriction fragment that spans the site of plasmid cleavage, homologous recombination restores the plasmid to its original circular form. Inverted dimer plasmids are not detected. This relationship between homologous recombination and a novel DNA transaction that yields rearrangement could be important to the cell, as the latter could lead to a loss of gene function and lethality.

Effects of disintegration of incorporated 3H and 32P on the physical and biological properties of DNA

Abstract Pneumococcal DNA was labeled with high specific activities of phosphorus-32 or tritiated pyrimidines and stored at −30 °C in a casein hydrolysate medium. The kinetics of inactivation of genetic markers on the labeled DNA was examined using a transformation assay. In addition, the kinetics of DNA strand scission were examined as a function of isotope disintegration. Strand scission was determined by sedimenting labeled DNA in neutral and alkaline sucrose density-gradients containing sedimentation reference markers. Sedimentation coefficients were converted to molecular weight using Studiers (1965) relationship. The efficiency of strand scission per disintegration was then calculated. Disintegration of 32 P causes single-strand breaks with unit efficiency and double-strand breaks with an efficiency of 5%. Tritium disintegration produces single-strand breaks with an efficiency of 30% and double-strand breaks with an efficiency less than 1%. The biological activity of pneumococcal transforming DNA labeled either with 32 P or tritium declines as a simple exponential function of the number of disintegrations of the incorporated isotope. Heterozygous DNA hybrids containing a radioisotope in only one strand were prepared by annealing radioactive DNA with DNA labeled with the heavy isotopes 2 H and 15 N. The hybrid molecules were fractionated on cesium chloride density-gradients and the inactivation kinetics of genetic markers in the hybrid DNA were examined. The rates of inactivation of markers on the labeled strand were nearly equal to the rates observed with homozygous DNA labeled in both strands. From this result we conclude that single strands of transforming DNA constitute the predominant “sensitive element” inactivated by disintegration of the radioisotopes. Genetic markers on the unlabeled strand of a tritium containing hybrid are four to five times less sensitive than are markers on the labeled strand. Homozygous DNA duplexes containing 32 P in only one strand appear to have multicomponent inactivation kinetics. These observations lend further support to single-strand integration models and lead us to postulate that a single-strand break constitutes an inactivating event.

Genetic consequences of transfection with heteroduplex bacteriophage λ DNA

SummaryThe role of rectification of heteroduplex heterozygotes in the formation of recombinant genotypes involving closely liked markers has been examined. Heteroduplex molecules of bacteriophage λ DNA, heterozygous at several alleles, have been constructed and the genetic composition of phage present in infective centers derived by transfection with such molecules has been determined. Allele loss and concomitant recombinant formation is frequent, and appears to reflect marker specificity as well as specificities imposed by whether or not the transfection recipient is permissive or nonpermissive for DNA duplication of the transfecting genome. The observations support the proposal that many, perhaps most, of the events involving separation of closely linked markers occur by rectification of non-recombinant heterozygotes.

Integration of donor DNA in bacterial conjugation

Abstract Conjugation between 13 C 15 N- and 3 H-labelled hybrid donors and 13 C 15 N-labelled hybrid recipients of Escherichia coli gives rise to recombinant radioactive DNA of density greater than labelled hybrid. The donor radioactivity is present, in these molecules, in discrete heavy segments covalently attached to the light strand. When light radioactive Hfr cells are mated to heavy F − cells in light medium, the donor label appears, in DNA extracted from the F − cells, in labelled hybrid molecules. The radioactivity in these molecules is exclusively in the light strand. The insertion of donor material is thus restricted to a single newly formed strand of the recipient DNA and double-strand integrations do not occur. A temperature-sensitive recipient containing the dna B mutation ts 43 accumulates single-stranded Hfr DNA if mating is carried out at the nonpermissive temperature. The formation of a complementary strand in the recipient does not, therefore, appear to be necessary for continued transfer of Hfr DNA.

Electron microscope visualization of the products of Bacillus subtilis transformation

Abstract The reduced alkalinity required to denature DNA in which bromouracil has replaced thymine has permitted the visualization of the molecular products of transformation of Bacillus subtilis with bromouracil-substituted DNA. The appearance of these molecules suggests that several distinct segments of a single bound DNA molecule can be integrated into the genome of a recipient bacterium.

Generalized transduction by bacteriophage P22 in Salmonella typhimurium: II. Mechanism of integration of transducing DNA

Abstract Generalized transduction by bacteriophage P22 occurs by the transfer of DNA from one bacterium to another by means of a phage-like particle (i.e. a transducing particle) which contains bacterial DNA formed before infection (preceding paper). Transduction of a 32 P-labeled, light recipient with purified transducing particles whose DNA is labeled with 3 H, 2 H, 15 N, 13 C results in the physical association of 12 to 15% of the transducing DNA with the DNA of the recipient bacteria. The remainder of the transducing DNA persists as unreplicated phage-size fragments during further bacterial growth. Physical and genetic analysis of the DNA integrated as large fragments reveals that recombination in generalized transduction occurs by a displacement mechanism, often resulting in the integration of double strand fragments of transducing DNA into the DNA of the recipient bacteria. The molecular weight of most of the large integrated fragments is greater than 2 to 4 × 10 6 daltons but substantially less than 27 × 10 6 daltons, the molecular weight of the transducing DNA injected into the bacteria. The integrated large double strand fragments of transducing DNA can be detected in DNA isolated soon after transduction, but not in DNA isolated late after transduction, when the transductants have been permitted to replicate.

Apparent changes in mouse liver dna content due to interference by non-dna diphenylamine-reacting cytoplasmic material.

Abstract A significant decrease occurs in the amount of diphenylamine-reacting material extracted with a nucleic acid fraction of liver if rats or mice are deprived of food for several days. However, this decrease, which is rapidly regained on refeeding the animals, does not represent loss of liver DNA, for no corresponding decrease was found if DNA was assayed by phosphate content or ultraviolet absorbance. The interfering material, which is predominantly cytoplasmic, yields a colored product in the diphenylamine reaction, resembling that given by deoxyribose but having an absorbance maximum at about 660 mμ rather than at 600 mμ. Also, the rate of color development with the interfering substance is slower than that observed with deoxyribose.