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N Munakata

Publications and source records attributed to N Munakata.

At least 19 recordsLinked to original sources
PubMed1995

Diverse capacities for the adaptive response to DNA alkylation in Bacillus species and strains.

Our previous studies of Bacillus subtilis showed that the genes responsible for the adaptive response to DNA alkylation were organized as a divergent regulon, in contrast to scattered operons in Escherichia coli ada regulon. To study the generality and diversity of gene organization, several species and strains of Bacillus were examined for the responsiveness to DNA alkylation. B. cereus cells exhibited the highest resistance to MNNG treatment. When the cells were grown in the presence of MNNG, 3-methyladenine DNA glycosylase and two species of DNA methyltransferase were induced as in B. subtilis 168 cells. B. licheniformis 749 and B. amyloliquefaciens H cells exhibited a partial response that manifested itself as the induction of one species of DNA methyltransferase. On the other hand, B. thuringiensis var. Tohokuensis, B. megaterium KMT, and B. subtilis W23 cells were totally deficient in this response, and were hypersensitive to alkylating agents. To determine the cause of this deficiency in strain W23, we examined the genomic structure of the corresponding region where three genes (alkA, adaA, and adaB) were located in 168. No homologues for the three genes were detected in W23 DNA by Southern hybridization. Two genes (glmS and ndhF) flanking the adaptive response regulon in 168 were also present in W23. A sequence of about 2750 bp that carried the entire regulon in 168 was replaced with a sequence of about 250 bp that was unique to W23. At the ends of the conserved segments, palindromic sequences corresponding to the transcriptional termination sites of the adaB and glmS genes were observed. The regulon in 168 could be artificially replaced by the W23 sequence, and be regained through DNA-mediated transformation.

Alkylation↗
PubMed1995

Polyethylene glycol-modified avidin: a novel agent for the selective extraction of biotinylated immune-complex in an aqueous two-phase system.

Chicken avidin was chemically modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine (activated PEG2) to form PEG-avidin. The PEG-avidin, in which 78% of the amino groups were modified, retained 49% of the active biotin-binding sites. The modified avidin was partitioned preferentially into the PEG-phase in an aqueous two-phase system (PEG/dextran). Using PEG-avidin, the immune-complex formed between biotinylated anti-mouse IgG and its antigen IgG (mouse) molecules, was successfully transferred into the PEG-phase in an aqueous two-phase system. This finding leads to the effective isolation of a specific antigen among various kinds of antigens by partitioning with a two-phase system using PEG-avidin.

Animals↗
PubMed1994

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis.

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 bp DNA segment from a 5' portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.

Bacillus subtilis↗
PubMed1993

Immunohistochemical examination of tumor-suppressor gene p53 product and pyrimidine dimer in solar keratosis.

In order to find biomarkers to measure the effects of UV irradiation, we examined the accumulation of p53 protein and pyrimidine dimers in 18 solar keratosis specimens. Frozen or AMeX-fixed solar keratosis specimens were immunohistochemically stained by anti-p53 mouse monoclonal antibody, pAb1801 and polyclonal anti-(pyrimidine dimer) antibody. Nuclear accumulation of p53 protein was found in 5/18 (28%) solar keratosis lesions. The percentage of cases showing nuclear p53 protein varied according to the histological type; in the bowenoid type it was 4/7 (57%); in the atrophic type it was 1/7 (14%). Nuclear pyrimidine dimers were not stained in solar keratosis, although the skin of UV-irradiated nude mice was positive. Accumulation of p53 protein is a good marker for early precancerous change caused by UV exposure.

Aged↗
PubMed1993

Biologically effective dose of solar ultraviolet radiation estimated by spore dosimetry in Tokyo since 1980.

The biologically effective dose of solar UV radiation has been measured in Tokyo since 1980 using Bacillus subtilis spores. To determine the cumulative dose in a half day, several samples of UV-sensitive spores were exposed in successive intervals from the solar-noon time. Because fluence-survival curves were exponential, the number of lethal hits received by the spores was calculated for each interval and termed inactivation dose (ID). The total number of hits obtained in a half day (half-day ID) was correlated with the amount of global insolation by a power-function regression. The regression analyses were performed for the data collected on 35 days from 1980 to 1986 and for the data collected on 53 days from 1989 to 1991. The latter data set yielded significantly larger estimates of half-day ID relative to the insolation than the former. These analyses suggested that the biologically effective dose relative to the insolation increased about 30% at some time in the later part of 1980s at this location. Changes of solar activity, air pollution and stratospheric ozone layer were considered as potentially responsible for this increase, but identification of the causative factors requires further efforts.

Bacillus subtilis↗
PubMed1992

Inactivation action spectra of Bacillus subtilis spores with monochromatic soft X rays (0.1-0.6 nm) of synchrotron radiation.

Five types of Bacillus subtilis spores differing in DNA repair and recombinational capacities were exposed in vacuum to monochromatic soft X rays from synchrotron radiation. The inactivation rate constants were obtained from exposure-survival curves upon irradiations at 12 wavelengths in the range of 0.1000 nm (12.40 keV) to 0.6000 nm (2.066 keV). Spores of two repair-deficient strains, UVS (uvrA ssp) and UVP (uvrA ssp polA), exhibited almost equal sensitivities to those of wild-type UVR+, while those of two recombination-deficient strains, RCE (recE) and RCF (recF), exhibited higher sensitivities in the whole wavelength range. This suggested that the repair of DNA damage produced by soft X rays was dependent on the recombinational capabilities. Inactivation action spectra based on photon fluence showed that the effectiveness of the radiation increased as the wavelengths became longer. Abrupt changes in the effectiveness occurred around the wavelengths corresponding to the absorption edges of K-shell electrons of phosphorus and calcium. In both cases, the sensitivity was the highest at the wavelengths of the resonance absorption peak, the next highest at those of the higher energy, and the lowest at the lower energy. Mass energy absorption coefficients of spores were obtained from the transmission of a flake made of spores. They were used to derive inactivation action spectra based on absorbed doses. In these spectra, basal levels of the sensitivity seemed constant, and enhancements of the sensitivity were observed consistent with the absorption by calcium and phosphorus. Thus calcium and phosphorus atoms were the predominant targets for the absorption events leading to the inactivation of spores in the wavelength range examined.

Bacillus subtilis↗
PubMed1991

Inactivation action spectra of Bacillus subtilis spores in extended ultraviolet wavelengths (50-300 nm) obtained with synchrotron radiation.

Five types of Bacillus subtilis spores (UVR, UVS, UVP, RCE, and RCF) differing in repair and/or recombinational capabilities were exposed to monochromatic radiations at 13 wavelengths from 50 to 300 nm in vacuum. An improved biological irradiation system connected to a synchrotron radiation source was used to produce monochromatic UV radiation in this extended wavelength range with sufficient fluence to inactivate bacterial spores. From the survival curves obtained, the action spectra for the inactivation of the spores were depicted. Recombination-deficient RCE (recE) and RCF (recF) spores were more sensitive than the wild-type UVR spores in the entire range of wavelengths. This was considered to mean that DNA was the major target for the inactivation of the spores. Vacuum-UV radiations of 125-175 nm were effective in killing the spores, and distinct peaks of the sensitivity were seen with all types of the spores. Insensitivities at 190 and 100 nm were common to all five types of spores, indicating that these wavelengths were particularly impenetrant and absorbed by the outer layer materials. The vacuum-UV peaks centering at 150 nm were prominent in the spores defective in recombinational repair, while the far-UV peaks at around 235 and 270 nm were prominent in the UVS (uvrA ssp) and UVP (uvrA ssp polA) spores deficient in removal mechanisms of spore photoproducts. Thus, the profiles of the action spectra were explained by three factors; the penetration depth of each radiation in a spore, the efficiency of producing DNA damage that could cause inactivation, and the repair capacity of each type of spore.

Bacillus subtilis↗
PubMed1991

Molecular analysis of Bacillus subtilis ada mutants deficient in the adaptive response to simple alkylating agents.

Previously, we isolated and characterized six Bacillus subtilis ada mutants that were hypersensitive to methylnitroso compounds and deficient in the adaptive response to alkylation. Cloning of the DNA complementing the defects revealed the presence of an ada operon consisting of two tandem and partially overlapping genes, adaA and adaB. The two genes encoded proteins with methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase activities, respectively. To locate the six mutations, the ada operon was divided into five overlapping regions of about 350 bp. The fragments of each region were amplified by polymerase chain reaction and analyzed by gel electrophoresis to detect single-strand conformation polymorphism. Nucleotide sequences of the fragments exhibiting mobility shifts were determined. Three of the mutants carried sequence alterations in the adaA gene: the adaA1 and adaA2 mutants had a one-base deletion and insertion, respectively, and the adaA5 mutant had a substitution of two consecutive bases causing changes of two amino acid residues next to the presumptive alkyl-accepting Cys-85 residue. Three mutants carried sequence alterations in the adaB gene: the adaB3 mutant contained a rearrangement, the adaB6 mutant contained a base substitution causing a change of the presumptive alkyl-accepting Cys-141 to Tyr, and the adaB4 mutant contained a base substitution changing Leu-167 to Pro. The adaB mutants produced ada transcripts upon treatment with low doses of alkylating agents, whereas the adaA mutant did not. We conclude that the AdaA protein functions as the transcriptional activator of this operon, while the AdaB protein specializes in repair of alkylated residues in DNA.

Adaptation, Physiological↗
PubMed1990

Bacillus subtilis ada operon encodes two DNA alkyltransferases.

By prophage transformation and subcloning, we have obtained Bacillus subtilis DNA fragments that could complement the hypersensitivity of ada (adaptive response deficient) mutants to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The nucleotide sequence contained two open reading frames that were assigned to the genes adaA and adaB, encoding methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase, respectively. These two genes overlap by 11 bp and comprise a small operon. The 1.6 Kb transcripts derived from the operon were detected in ada+ cells cultured in the presence of MNNG but not in control ada+ cells. From analysis of the syntheses of DNA alkyltransferases in the ada mutant cells harboring the plasmid carrying the complete or partial fragment, we conclude that the adaA gene product functions as a transcriptional activator of the ada operon, while the adaB gene product specializes in repair of mutagenic O6-methylguanine residues. Comparison with Escherichia coli ada operon showed that the two genes correspond to portions of the E. coli ada gene, implicating gene fusion or splitting as the origin of the difference in the organizations of the genes.

Alkyl and Aryl Transferases↗
PubMed1990

Isolation of a Bacillus subtilis mutant defective in constitutive O6-alkylguanine-DNA alkyltransferase.

A mutant of Bacillus subtilis defective in the constitutive activity of O6-alkylguanine-DNA alkyltransferase was isolated from a strain (ada-1) deficient in the adaptive response to DNA alkylation. Cells carrying the mutation dat-1 which was responsible for the defect in constitutive activity exhibited hypersensitivity for lethality and mutagenesis when challenged with methyl-nitroso compounds. The constitutive activity is independent of the adaptive response, and seems to function as a basal defense against environmental alkylating agents.

Alkyl and Aryl Transferases↗
PubMed1989

Bacillus subtilis gene coding for constitutive O6-methylguanine-DNA alkyltransferase.

We have cloned a Bacillus subtilis DNA fragment that could correct the defect in a constitutive O6-methylguanine-DNA alkyltransferase (Dat1). This fragment also corrected the hypersensitivity of the strain TKJ6951(ada-1 dat-1) to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In the fragment, the gene activity resides in a region of about 850 bp which contains an open reading frame capable of coding for a protein of 165 amino acid residues. The amino acid sequence of this protein exhibits striking similarity to those of E. coli O6-methylguanine-DNA alkyltransferases (Ogt and Ada proteins). We conclude that this is a structural gene for the Dat1 protein, which is distinct from inducible DNA alkyltransferases involved in the adaptive response. The dat-1 mutation was shown to be caused by a structural rearrangement affecting the coding region, and the 0.8 kb transcripts of this gene were detected in dat+ cells but not in dat mutant cells.

Bacillus subtilis↗
PubMed1989

Genotoxic action of sunlight upon Bacillus subtilis spores: monitoring studies at Tokyo, Japan.

Samples of Bacillus subtilis spores dried on membrane filter were exposed to natural sunlight from solar-noon time at Tokyo. The survival and mutation induction of wild-type (UVR) and repair-deficient (UVS) spores were determined on 66 occasions since 1979. Two of the values were considered to be useful in monitoring solar UV intensity; the inverse of the time (in minutes) of exposure to kill 63% of the UVS spores ("sporocidal index") and the induced mutation frequency at 60 minutes of exposure of the UVR spores ("mutagenic index"). Both values were varied greatly due to time of a year, weather and other conditions. Estimates of year-round changes under clear skies were obtained by connecting the maximum values attained in these years. In these curves, there are more than 7-fold differences in the genotoxicity between winter and summer months, with major increases observed in early spring and decreases through autumn. Using a series of UV cut-off filters, the wavelengths most effective for the sporocidal actions were estimated to be in the range of 308-325 nm, shorter wavelengths being effective when the genotoxicity was higher. Sunburn meter of Robertson-Berger type seems to respond to slightly longer wavelength components of the solar spectrum. However, a reasonable correlation was obtained between the reading of the meter and the sporocidal index.

Bacillus subtilis↗
PubMed1987

Multiple species of Bacillus subtilis DNA alkyltransferase involved in the adaptive response to simple alkylating agents.

Three molecular species of methyl-accepting proteins exist in Bacillus subtilis cells, which collect methyl groups from methylated DNA. A 20-kilodalton (kDa) protein was constitutively present in the cells of the ada+ (proficient in adaptive response) strain as well as in those of six ada (deficient in adaptive response) mutant strains and was assigned to the O6-methylguanine:DNA methyltransferase. Another species of O6-methylguanine:DNA methyltransferase, which had a molecular size of 22 kDa, emerged after adaptive treatment of the ada+ but not any of the ada mutant cells. A 27-kDa methyl-accepting protein, which preferred methylated poly(dT) to methylated calf thymus DNA as a substrate, was assigned to the methylphosphotriester:DNA methyltransferase. It was produced, after adaptive treatment, in the cells of ada+, ada-3, ada-4, and ada-6 strains but not in the cells of ada-1, ada-2, or ada-5 strains. These results support and extend our proposition that ada mutants can be classified into two groups; one (the ada-4 group) is defective only in the inducible synthesis of O6-methylguanine:DNA methyltransferase (22-kDa protein), and the other (the ada-1 group) is deficient in the adaptive response in toto. The finding that inducible and constitutive methyltransferases reside in different molecular species of methyl-accepting proteins is intriguing compared with the regulatory mechanisms of the adaptive response to simple alkylating agents in other organisms.

Bacillus subtilis↗
PubMed1986

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents.

Six mutant strains of Bacillus subtilis hypersensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada+) cells contained similar constitutive levels of O6-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada+ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada+, augmented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N'-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.

Alkylating Agents↗
PubMed1985

Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents.

Three mutant strains exhibiting hyper-sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine, but not to methyl methanesulfonate, were selected by a replica method from mutagenized spores of Bacillus subtilis. All three were totally deficient in the adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine with regard to both lethality and mutagenesis. The activity to destroy O6-methylguanine residues in the methylated DNA was not elevated in the mutant cells by the pretreatment with sublethal concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. This deficiency corresponded to the persistence of O6-methylguanine residues in the DNA of both control and pretreated mutant cells challenged with the drug. The lethal and mutagenic sensitivity of the mutant strains were observed only for methyl- or ethyl-nitroso compounds that are thought to be active as inducers and are also active in O-alkylation. Except for the insensitivity to methyl methanesulfonate, the phenotypes of these mutants look very similar to those of ada mutants isolated previously in Escherichia coli.

Alkylating Agents↗