Yoshie Shimazu

ULTRAVIOLET REACTIVATION OF A BACTERIOPHAGE CONTAINING A SINGLE-STRANDED DEOXYRIBONUCLEIC ACID AS A GENETIC ELEMENT.

Abstract UV reactivation (UVR) of bacteriophage φR, which contains single-stranded DNA, was studied using Escherichia coli C and E. coli Csyn − as host bacteria. E. coli C can perform host-cell reactivation (HCR) with UV-irradiated phage λ but is incompetent for HCR of φR. However, the UVR extent of φR in E. coli C was comparable with that found with phage λ. An induced enzyme hypothesis is proposed for the UVR property, although the mechanism for repair of UV lesions on the single-stranded DNA molecule during UV reactivation is still unknown. The possibility that the UVR enzyme and HCR enzyme are independent entities is also discussed. The survival of UV or X-irradiated φR is increased when the phage infects host cells pretreated with UV or X-ray.

Reactions of copper(II)-oligopeptide complexes with hydrogen peroxide: Effects of biological reductants

Cu(II) complexes with oligopeptides containing histidyl residue in the second or third position could scarcely activate hydrogen peroxide (H2O2), but they could activate H2O2 to yield hydroxyl radical (.OH) in the presence of biological reductants such as cysteine and ascorbic acid. Further, DNA single strand breakage was also observed during the reactions of Cu(II)-glycylglycylhistidylglycine (GGHG) with H2O2 in the presence of same biological reductants.

Evidence that the geneuvrB is indispensable for a polymerase I deficient strain ofEscherichia coli K-12

SummaryConclusive evidence in presented to show that the geneuvrA is dispensable, but theuvrB is indispensable for anEscherichia coli strain carrying genepolA1. We constructed strains E139 (sup-126 polA1 uvrB59) and E159 (sup-126 polA1 uvrA43) where mutationspolA1, uvrB59 anduvrA43 are amber mutations and mutationsup-126 is an amber suppressor mutation effective at 30°C but not at 42°C. At 42°C, strain E139 is inviable but strain E159 viable whereas both are viable at 30°C. Revertants of E139 viable at 42°C occurred spontaneously at a frequency of about 3×10-4. One of the revertants was shown to be caused by suppressor mutation, designatedspu, rather than back mutation of the geneuvrB59 orpolA1 or amber suppressor mutation. Viabilities of the revertants varied from 10-3 to 1.0 at 42°C compared with those at 30°C. At 42°C, all the revertants with normal viabilities at 42°C were non-filamentous in contrast to the filamentous character of E139. However, strain E159 was viable at 42°C despite its filamentous character. We conclude that the geneuvrB is involved not only in excision repair but also in normal growth in apolA background.

CHO · K1 cell mutants sensitive to active oxygen-generating agents. I. Isolation and genetic studies

Nine mutants isolated from CHO.K1 cells with increased sensitivity to the lethal effect of plumbagin (PG), a powerful superoxide generator, were classified into five groups, A-E, according to their sensitivity to PG and methyl viologen (MV). Two mutants of group B (Pa13 and Pb4) were sensitive to both drugs, and two mutants of group C (Pa14 and Pa15) were moderately sensitive to PG and extremely sensitive to MV. To mitomycin C (MMC) these mutants showed cross-sensitivity; especially Pa13 and Pb4 (group B) were highly sensitive to MMC. Genetic complementation analyses of these four mutants were carried out using MV sensitivity. Sensitivity group B was divided into two complementation group, I and II. Pa14 and Pa15 belonged to the same complementation group III. These four mutants were also classified into three complementation groups for MMC sensitivity. Because Pa13 and Pb4 were also sensitive to cis-diamminedichloroplatinum(II), they may have a defect in the repair of DNA crosslinks induced by these agents. A complementation group IV (Pa2 and Pa8) was also suggested based on the studies of MMC sensitivity.

Genetic Analysis of ultraviolet-sensitive mutants of Escherichia coli K12; URT−43 (urt-1) and KMBL99dar3, carrying temperature-sensitive mutations at the locus uvrA

Abstract URT- 43 ( urt-1 ) is a thermosensitive ultraviolet-sensitive mutant of Escherichia coli K12. A second mutant KMBL 99 dar3 also has properties almost identical with those of URT-43. To perform complementation analysis with respect to ultraviolet sensitivity, merodiploid strains carrying urt-1 or dar3 on the host chromosome and uvrA6 on an F′ episome were constructed. Neither the mutation urt-1 nor dar3 complemented the uvrA6 mutation. Transductional crosses with P1 phage were carried out among mutants uvr6, urt-1 and dar3 : the pairwise combination all gave rise to only 0.1 ∼ 0.6% ultraviolet-resistant transductants among those selected for the nearby malB marker. It is thus concluded that both urt-1 and dar3 mutations are located within the uvrA cistron.

A mutant of Escherichia coli K12 exhibiting varying ultraviolet sensitivities depending on the temperature II. Cross-sensitivity, recovery in liquid and genetic analysis

Abstract The properties of a mutant of E. coli K12, URT-43, were investigated. Genetic analysis was also carried out. Cross-sensitivity was checked for 4-nitroquinoline-1-oxide and mitomycin C. In parallel with ultraviolet irradiation, URT-43 was more sensitive to both agents than its wild-type strain. However there was an apparent large temperature effect of the action of 4-nitroquinoline-1-oxide, whereas with mitomycin C the effect was only very small. Reflecting the temperature-dependent ultraviolet sensitivity of URT-43 on aga plates, the irradiated URT-43 recovered well in a liquid medium at 30° but not at 42°. The recovery was shown to require all the substances essential for its normal growth, suggesting that the recovery is accompanied by growth. Irradiated URT-43 was still restorable after photoreactivation to the maximal extent. This was true for the bacteria irradiated at dry-ice temperature in which less photoreactivable injuries would be produced than in the bacteria irradiated at romm temperature. These results, as well as the observation that the extent of recovery in a liquid medium of the bacteria irradiated at low tempwerature was much larger than that of the same bacteria after photoillumination, led the authors to assume that the recovery is due to the repair or to a certain tolerance mechanism for injuries which are not limited to the cyclobutane pyrimidine dimers. the mechanism of this recovery has not yet been elucidated. The mutation of URT-43 was mapped by mating and transduction experiments. The efficiency of mating with an Hfr strain was not greatly affected by low and high temperatures, suggesting that the whole mechanism of recombination is not involved in the temperature-dependent recovery in question. The mutated locus was shown to be closely linked to the mal B gene by transduction experiments with P1 phage. The linkage to met A was much weaker, suggesting that the mutation is located in the uvr A gene. Two typical phenotypes of URT-43, namely those with negative host-cell reactivation and temperature-dependent ultraviolet sensitivity, could not be separated in these experiments.

A mutant of Escherichia coli K-12, URT-34, with a temperature-sensitive defect at the incision step of the excision repair mechanism

URT-43, which has a defect in excision repair, exhibits a temperature-dependent ultraviolet survival. It was shown that URT-43 requires protein synthesis but not DNA synthesis for recovery, by examining recovery in a growth medium containing chloramphenicol or nalidixic acid. The recovery of irradiated bacteriophage lambda in URT-43 took place in a medium containing nalidixic acid at 30 degrees, but not at 41 degrees, and chloramphenicol prevented this recovery. These results seem to imply that the product of the mutated gene in URT-43 is labile. URT-43 was confirmed to have a temperature-sensitive mutation at the incision step of the excision repair mechanism by examining the nick formation of parental DNA in alkaline sucrose gradients. The release of pyrimidine dimers was reinvestigated directly by one- and two-dimensional paper-chromatography and indirectly by examining the distribution of DNA molecules synthesized after irradiation. Dimers were excised into the acid-soluble fraction when growing bacteria were incubated, but were not excised when in amino acid starved bacteria. These results suggest that URT-43 is a mutant slowly excising pyrimidine dimers because the product of a mutated gene concerned with the incision step of the excision repair mechanism is unstable.