Hydrogen peroxide induces G:C to TA and G:C to C:G transversions in the supF gene of Escherichia coli

A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe³⁺/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:C→C:G (28 cases) and G:C→T:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5′-PuGA-3′ was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5′PyGN3′. G:C→T:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:C→C:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.     

Hydrogen peroxide induces G:C to TA and G:C to C:G transversions in the supF gene of Escherichia coli

A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe³⁺/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:CC:G (28 cases) and G:CT:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5-PuGA-3 was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5PyGN3. G:CT:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:CC:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.     

Quinolone-resistant mutations of the gyrA gene of Escherichia coli

Summary DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.     

New Escherichia coli gyrA and gyrB mutations which have a graded effect on DNA supercoiling

Summary We isolated new gyrA and gyrB mutations in Escherichia coli which have a graded effect on DNA supercoiling. The mutants, selected respectively for resistance to nalidixic acid and coumermycin, were sorted by means of a rapid in vivo assay of DNA gyrase activity (Aleixandre and Blanco 1987). Cells carrying a gyrB (Cou^r) mutation usually showed a decrease in DNA supercoiling, which would indicate a reduction in gyrase activity. In contrast, most of the gyrA (Nal^r) mutations had no significant effect on DNA supercoiling. Moreover, they conferred a high level of resistance to nalidixic acid and other quinolones, thus being similar to the gyrA(Nal^r) mutants currently used. We also detected rare gyrA mutants showing a reduction in DNA gyrase activity. These mutants were, in addition, resistant to only low concentrations of quinolones, which allowed us to use the phenotype of partial quinolone resistance as an indicator to score gyrA mutations affecting DNA supercoiling. When gyrB mutations were introduced into the gyrA mutants, these became more sensitive to quinolones and a decrease in supercoiling was observed. Moreover, the topA10 mutation sensitized gyrA(Nal^r) cells to quinolones. We conclude therefore that the GyrA-dependent quinolone resistance is diminished as a consequence of the reduction either in topoisomerase I or gyrase activities.     

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

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 by 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.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.     

Specificity of mutations induced by riboflavin mediated photosensitization in the supF gene of Escherichia coli

Riboflavin-mediated photosensitization has been shown to produce 8-hydroxyguanine (oh⁸Gua) in DNA. We investigated the specificity of mutation of photosensitized supF gene induced in Escherichia coli. The oh⁸Gua repair deficient E. coli mutant mutM and mutY were transformed with plasmid pUB3 carrying the supF gene irradiated with white light in the presence of riboflavin. Under these conditions, riboflavin photosensitization increased the amounts of oh⁸Gua in pUB3 DNA. Three types of a single base substitution occurring at G:C pairs were detected in both wild-type and mutM mutant strains. Almost all base substitutions were transversions to T:A or C:G pairs occurring at a similar extent in both wild-type and mutM strains. Mutations derived from mutY strain transformed with photosensitized DNA were only G:C to T:A transversions. These G:C to T:A transversions observed in the mutY strain were suggested to be the result of mispairing of oh⁸Gua with adenine. Riboflavin-mediated photosensitization may also produce lesions on DNA causing G:C to C:G changes by unknown mechanisms.     

Mutagenic specificity of N-acetoxy-3-aminobenzanthrone, a major metabolically activated form of 3-nitrobenzanthrone, in shuttle vector plasmids propagated in human cells

3-Nitrobenzanthrone (3-NBA) is a potent environmental mutagen and a potential human carcinogen present in diesel exhaust and airborne particulates. N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) has been shown to be a major reactive metabolite of 3-NBA, which mainly produces adducts with guanine and adenine in cellular DNA. Here we analyzed mutations induced by N-Aco-ABA using supF shuttle vector plasmids to elucidate the mutagenic specificity of 3-NBA in human cells. Base sequence analysis of more than 100 plasmids with supF mutations induced in wildtype and DNA repair-deficient XP cells revealed that the major mutation was base substitutions of which the majority (42 and 38%, respectively) were G:C to T:A transversions. The next major mutation was G:C to A:T and A:T to G:C base substitutions in wildtype and XP cells, respectively. The DNA polymerase stop assay using N-Aco-ABA-treated plasmids as a template showed that most stop signals, i.e., adducted sites, appeared at G:C sites. These results suggest that N-Aco-ABA binds preferably to guanine rather than adenine, and adducted adenine is repaired more efficiently by the nucleotide excision repair. Error-prone DNA polymerases could insert adenine at sites opposite to N-Aco-ABA-adducted guanine, which leads to G:C to T:A transversion. These findings could be very important to evaluate the human lung cancer risk of environmental 3-NBA.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Quadruplex DNA formation in a region of the tRNA gene supF associated with hydrogen peroxide mediated mutations

A hot spot for H2O2/Fe-mediated mutation has been observed between bases 154 and 170 of the supF gene in the mutation reporter plasmid pZ189 [Moraes et al. (1990) Carcinogenesis 11, 283; Akman et al. (1991) Mutat. Res. (in press)]. To further characterize this hot spot, we synthesized the 33mer d(pAAAGTGATGGTGGTGGGGGAAGGATTCGAACCT) (pZ33), which is complementary to bases 159-191 of the supF gene. pZ33 annealed spontaneously in 10 mM Tris-HCl (pH 8.0)-1 mM EDTA-100 mM NaCl at 50 degrees C into two major forms, one of which migrates more slowly than does d(pT)33 on nondenaturing 12% polyacrylamide gels. We propose that this form is a four-stranded structure stabilized by Hoogsteen-type deoxyguanosine quartets involving all deoxyguanosines of the sequence d-(pGGTGGTGGGGG) because of the following. (1) pZ33 migrates as a single form that comigrates with d(pT)33 on denaturing 20% acrylamide-8 M urea gels. (2) Annealing an equimolar mixture of 5'-32P-labeled pZ33 and the oligodeoxynucleotide d(pTTTTTTTTpZ33TTTTTTTT) (pZ49), as well as 5'-32P-labeled pZ49 and pZ33, caused the formation of four, discreet slowly migrating bands on nondenaturing 12% polyacrylamide gels. Mixing 5'-32P-labeled pZ33 with 5'-32P-labeled pZ49 resulted in five slowly migrating bands. (3) An oligodeoxynucleotide identical with pZ33 except that every deoxyguanosine has been replaced with deoxyinosine did not anneal into a slowly migrating form. (4) Dimethyl sulfate protection studies demonstrated that all deoxyguanosines of the sequence d(pGGTGGTGGGGG) were protected at N-7 in the slowly migrating form but not in single-stranded pZ33. These data suggest that a hot spot for H2O2/Fe-mediated base substitutions is located adjacent to a sequence that can spontaneously adopt a quadruplex structure in which deoxyguanosine quartets are Hoogsteen bonded.     

Glyoxal, a major product of DNA oxidation, induces mutations at G:C sites on a shuttle vector plasmid replicated in mammalian cells.

Glyoxal is a major product of DNA oxidation in which Fenton-type oxygen free radical-forming systems are involved. To determine the mutation spectrum of glyoxal in mammalian cells and to compare the spectrum with those observed in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA gene (supF) in the shuttle vector plasmid pMY189. We treated pMY189 with glyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and mutation frequency increased according to the dose of glyoxal. The majority of glyoxal-induced mutations (48%) were single-base substitutions. Eighty three percent of the single-base substitutions occurred at G:C base pairs. Among them, G:C-->T:A transversions were predominant, followed by G:C-->C:G transversions and G:C-->A:T transitions. A:T-->T:A transversions were also observed. Mutational hotspots within the supF gene were detected. These results suggest that glyoxal may play an important role in mutagenesis induced by oxygen free radicals.     

pZ189质粒DNA体外复制系统的建立

报道了含SV40复制起点的质粒DNA在真核细胞抽提物中进行复制的DNA体外复制系统的建立. 在外源性蛋白质SV40大T抗原(SV40 Tag)的参与下,穿梭质粒pZ189能在猴肾vero细胞胞浆抽提物中,利用其中参与体内DNA复制所需的蛋白质成分,有效地进行体外DNA复制. 从而为研究真核细胞DNA复制系统的结构与功能提供了简单、有效的模型.     

Mutagenic specificity in DNA base sequence by irradiation of health lamp light (UV-B) in Escherichia coli.

A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene, was irradiated with health lamp (HL) light containing UV-B. Plasmid mutations were scored by transforming an indicator strain of Escherichia coli carrying a suppressive blue amber mutation in the beta-galactosidase gene. Plasmid survival was also measured by transforming activity of the indicator strain. The majority of mutations induced by HL light were GC-AT transitions (69%) and the rest were transversions (31%). Some hot-spots in the mutations were observed by sequencing the suppressor gene. Mutagenic specificity in DNA base sequences induced by HL in E. coli agrees well with previous reports about 254-nm or 313-nm light effects on mammalian cells. This agreement may depend on the substitution of the inserted base instead of a G residue at the opposite site of a damaged C residue from conformational change of DNA structure in both bacterial and mammalian cells.     

Mutations induced by glyoxal and methylglyoxal in mammalian cells.

To investigate the mutation spectra of glyoxal and methylglyoxal in mammalian cells, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. The cytotoxicity and the mutation frequency increased according to the doses of glyoxal and methylglyoxal. The majority of glyoxal-induced mutations (65%) were base-pair substitutions, in which G:C-->C:G transversions were predominant. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which G:C-->C:G and G:C-->T:A transversions were predominant.     

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1

Summary The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.     

Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis

The factors maintaining genomic integrity, which have been studied in detail in other species, have yet to be investigated in plants. Recent progress in gene-silencing technology has made it possible to produce transgenic plants with loss-of-function phenotypes for the effective analysis of these factors, even with the high redundancy of genes in plants. Therefore, a mutation-detection system for plants is necessary to estimate the biological function of a target gene for mutation frequencies and spectra. Here, we reported the development of a novel system to analyze mutations in the chromosomal DNA of plants. The supF gene of E. coli was used as a target for the mutation because it was possible to detect all mutational base changes. Based on the plasmid pTN30, which carries supF, we constructed a binary Ti vector for its introduction to Arabidopsis genomes. The system was validated by measuring mutations in both non-treated and mutagen-treated transgenic plants. DNA fragments including pTN30 were rescued from the plants, and introduced into E. coli KS40/pOF105 to isolate the supF mutant clones conferring both nalidixic acid and streptomycin resistance on transformants. We found that the mutation frequency was approximately three times higher with the ethyl methanesulfonate (EMS) treatment than without it and G:C to A:T transitions dominated, which was the most reasonable mutation induced by EMS. These results show that this system allowed for the rapid analysis of mutations in plants, and may be useful for analyzing plant genes related to the functions of genomic stability and monitoring environmental genotoxic substances.