Takesi Kato

Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light

SummaryMutants of E. coli defective in susceptibility to UV-induction of mutations were isolated by direct screening for their UV nonmutable phenotype (Umu−). Screening of about 30,000 mutagenized clones of a uvrB− derivative of AB1157 yielded six Umu− strains. The mutants can be classified into three groups by the location of the mutations, umuA, umuB and umuC. Mutations umuA and umuB are, respectively, mapped close to lexA and recA genes and mutations at both loci partially reduce UV mutagenesis. The locus of umuC is between hemA and purB and the mutations at this new locus result in a moderate increase of UV sensitivity. The mutation diminishes UV mutagenesis and UV reactivation of phage λ without affecting the inducibility of phophage λ nor the inhibition of cell division following UV irradiation. Related properties of an isogenic strain of a recF− mutant are compared with those of umuC−.

Let dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions

We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.

ACTION SPECTRA FOR PHOTOREACTIVATION OF KILLING AND MUTATION TO PROTOTROPHY IN U.V.-SENSITIVE STRAINS OF ESCHERICHIA COLI POSSESSING AND LACKING PHOTOREACTIVATING ENZYME*

Abstract— Ultraviolet‐sensitive strains of E. coli, Hs30 (uvr‐ phr‐ arg‐), lacking the ability to repair ultraviolet damage in the dark (uvr‐), lacking photoreactivating‐enzyme activity (phr‐), and lacking the ability to synthesize arginine (arg‐), and Hs30–R (uvr‐ phr+ arg‐), having the same auxotrophic marker and the same high u.v.‐sensitivity, were derived, respectively, from resistant strain H/r30 (uvr+ phr‐ urg‐) and its revertant H/r30–R (uvr+ phr+ urg‐). Hs30 and Hs30–R have about 25 and 35 times higher sensitivity than H/r30 and H/r30–R for u.v.‐induced killing and mutation to prototrophy, respectively. Efficiency of photoreactivation (PR) of mutation in strain Hs30–R is about ten times higher than the PR efficiency in the parent strain H/r30–R at wavelengths between 3341 and 4358 Å but not that much higher at 3132 Å. On the other hand, Hs30 shows no PR at any of these wavelengths, although the parent strain H/r30 does show PR of mutation around 3341 Å., These results support the. previously described model that PR in H/r30 is an indirect effect of PR light which enhances dark repair. It is concluded that Hs30 and Hs30–R lack dark‐repair capacity for u.v.‐induced mutational damage and that the same type of photoreactivable DNA lesions as those responsible for U.V. killing, i.e. pyrimidine dimers, are the primary cause of u.v.‐induced mutation in the uvr‐strains. PR spectra for killing in Hs30–R and mutation in H/r30–R and H/r30 were obtained and compared, and the quantitative conclusions reached are discussed in connection with the hypothesis that the same kind of lesion (pyrimidine dimer) is the primary cause of mutation in the uvr+ strains as well.

Spectrum of spontaneous mutations in a cDNA of the human hprt gene integrated in chromosomal DNA.

SummaryAltered sequences were determined of 52 independent spontaneous mutations occuring in a cDNA of the human hypoxanthine phosphoribosyltransferase (hprt) gene, which was integrated into chromosomal DNA of the mouse cell as a part of the retroviral shuttle vector. Spontaneous mutations comprised a variety of events: base substitutions, frameshifts, deletions, duplications, and complex mutational events, and were distributed randomly over the coding region of the gene. Frameshifts were the most frequent mutational event (38%), and base substitutions were the next most frequent (25%), followed by deletions (19%). Frameshift and deletion mutations commonly occurred preferentially at sites flanked by short direct repeats. Short inverted repeats were frequently found to be associated with duplication and complex mutational events. Analysis of the sequence alterations in the mutant genes suggests that misalignment mutagenesis represents an important molecular mechanism for the generation of spontaneous mutations in eukaryotic cells.

Analysis of ultraviolet light-induced suppressor mutations in the strain of Escherichia coli K-12 AB1157: An implication for molecular mechanisms of UV mutagenesis

SummaryGenetic analysis of histidine independent (His4) revertants induced by ultraviolet light in the his-4 E. coli strain AB1157 was carried out: 83% carried ochre (UAA) suppressor mutations and 17% carried back mutations to his+ or (intragenic?) suppressors not detectably separable from his-4. Using the specialized transducing λpsu 2int− phage, which carries supE-supB, it was determined that 87% of the ochre suppressors mapped in the supE-supB region. We were able to deduce that 56% of these affected tRNA1Glnby a CAA→TAA change in the tRNA gene while 31% affected tRNA2Glnby TAG→TAA change. Although we were unable to deduce the base substitution of the remaining 13%, the results indicated that most of the suppressor mutations are caused by a G:C to A:T transition.These results suggest that the high incidence of supE-supB region suppressor mutation in E. coli by UV would be a reflection of the general feature of UV mutagenesis; i.e. preferential induction of G:C to A:T transition in repairing nonparing DNA lesions.

Effects of chloramphenicol and caffeine on postreplication repair in uvrA−umuC−and uvrA−recF−strains of Escherichia coli K-12

SummaryPostreplication repair and its inhibition by chloramphenicol and caffeine, as seen in alkaline sucrose gradients, were compared between a UV nonmutable strain uvrA−umuC−and normally mutable strains uvrA−recF−and uvrA−umu+rec+of Escherichia coli K-12. The uvrA−umuC−strain performed postreplication repair as efficiently as the parental strain, while the repair in uvrA−recF−strain was dependent on UV dose. Both chloramphenicol and caffeine inhibited postreplication repair to an equal extent of about 25%, and 10%, respectively, in all three uvrA−strains of umuC36, recF−and umu+rec+. These observations suggest that postreplication repair is largely not responsible for UV mutagenesis.

umuC-Mediated misrepair mutagenesis in Escherichia coli: extent and specificity of SOS mutagenesis

Abstract The role of the error-prone misrepair pathway in mutagenesis was examined for a series of mutagens in umuC + and umuC36 strains of Escherichia coli . Mutagenesis by ENU, MNU, MNNG and EMS was independent of the umuC + gene function, while mutagenesis by MMS, 4NQO, γ-rays and UV was largely umuC + -dependent. Residual mutagenesis following UV-treatment of a umuC − strain showed the same mutational specificity seen in the umuC + strain. In contrast, the umuC mutation altered specificity substantially in an excision-repair-defective strain that showed a UV-spectrum strikingly different from that seen in an excision-repair-proficient strain. Only one of nine trpE frameshift mutations examined was reverted by UV-light and its reversion was umuC -dependent. In comparison, the dependence of frameshift mutagenesis following ICR 191 treatment was site-specific, suggesting at least two mechanisms of frameshift mutagenesis, one dependent upon misrepair, the other not. The results, together with those of previous reports (Kato and Nakano, 1981; Shinoura et al., 1983), suggest that the umuC + gene exerts its mutator activity via misrepair of DNA lesions provoking the induction of all types of mutational events, though following UV-irradiation mainly transition events are recovered.

Two types of X-ray-sensitive mutants of Escherichia coli B: Their phenotypic character compared with UV-sensitive mutants

Abstract From radiation-resistant strains (of B/r type) of Escherichia coli, B 18 mutants were selected as UV-sensitive (uvr−) mutants without any increased sensitivity to UV-induced filament formation. 11 mutants are of UV-hcr− type: they are highly sensitive to UV killing in various degrees but only slightly sensitive to X-ray killing; and they lack the ability to reactivate UV-irradiated T3 phage but have only slightly reduced host cell reactivation ability for X-irradiated T1 phage. Compared with these UV-hcr− mutants, 5 mutants are highly sensitive to X-ray killing (exr−), and they retain host cell reactivation ability for Uv-irradiated t3 phage (UV-hcr+) One of them retains the normal host cell reactivation ability for X-irradiated t1 phage (X-hcr+), but 2 of them have reduced ability (X-hcr−). Only 2 of all the selected mutants have intermediate UV-hcr− character. UV-hcr− mutants are also highly sensitive to UV-induction of mutation, but 1 of the exr− mutants has the same resistance to UV-induction of mutation as its parent (uvr+) strain in spite of the uvr− character for killing. It is suggested that resistance mechanisms for X-ray damage may be largely independent of the well-known excision-resynthesis repair mechanism for damage to pyramidine dimers and other DNAs.

Mutational specificity of the umuC mediated mutagenesis in Eschericha coli

A current concept of UV mutagenesis in E. coli is one of the inducible functions triggered by the arrest of DNA synthesis following exposure to various mutagens [18, 29]. We have previously identified the gene umuC which specifically controls the inducible mutagenic function [9]. The function, however, does not seem essential for the cell, since many bacterial genera have been known to lack this function [27]. Besides, a gene (mue) or genetic elements which confer the umuC genelike function have been found on several bacterial plasmids [18]. This may imply a transposable element associated origin of those genetic materials.

A rapid and simple method for the determination of base substitution and frameshift specificity of mutagens

Abstract A rapid method for the determination of mutagenic specificity has been developed which makes use of the ochre mutation (TAA) in the his-4 gene of Escherichia coli. Reversion to His+ may occur by suppressor mutation (Type I) or by mutation within the his-4 gene (Type II). The Type I mutations may be further subdivided with respect to the type of suppressor mutation by their ability to suppress nonsense mutants of bacteriophage T4, thus allowing the identification of the responsible base substitution (Kato et al., 1980). The system has the ability to identify mutagens which produce A:T → G:C transitions since only Type II mutants can arise through this base substitution; and in fact, the system confirms the A:T → G:C specificity of the mutagen, N4-hydroxycytidine (Janion and Glickman, 1980) since only Type II mutants were induced by treatment with this base analogue. When this system was further tested with several additional mutagens, the results indicate that ethyl methanesulphonate, methyl nitrosourea and ethyl nitrosourea produce primarily Type I revertants which were primarily G:C → A:T transitions. UV-light, γ-rays, 4NQO and methyl methanesulphonate produced all types of base substitutions. The tester strain was further improved by introducing a series of sequenced trp− frameshift mutations, thus allowing the simultaneous monitoring of frameshift and base-substitution mutations.