Mutagenesis, Vol. 15, No. 4, 317-323,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
A comparison of mutation spectra detected by the Escherichia coli Lac+ reversion assay and the Salmonella typhimurium His+ reversion assay
School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 and 1 Institute of Environmental Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187-0011, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Each of the Escherichia coli tester strains in the WP3101P–WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene. Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac+ reversion. We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds. We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A·T
T·A, G·CA·T and G·CT·A mutations, respectively, and we compared the sensitivity and specificity of the two systems. Escherichia coli strain WP3102P was more sensitive than the S.typhimurium strains to G·CA·T transitions induced by N4-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G·CA·T transitions induced by 2-aminopurine and phosmet. Escherichia coli strain WP3104P, which detects G·CT·A transversions, was superior to the S.typhimurium strains in detecting transversions induced by N4-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Escherichia coli WP3105P was also more sensitive than S.typhimurium to A·TT·A transversions induced by N-methyl-N- nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-aminopurine. The present results indicate that the E.coli Lac+ reversion system with tester strains WP3101P–WP3106P is as sensitive as the S.typhimurium His+ reversion system for the detection of specific mutations induced by a variety of direct mutagens. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Analysis of the mutational specificity of a wide variety of mutagens has provided considerable insight into the nature of mutagenesis. Several approaches have been developed for measuring specific base substitutional events. They include reverse mutation analysis with a set of specific tester strains, colony probe hybridization analysis (Miller and Barnes, 1986
; Prival and Cebula, 1992; Koch et al., 1996), genetic suppression pattern analysis with a set of nonsense mutants of bacteriophages (Shinoura et al., 1983; Levin and Ames, 1986) and conventional DNA sequencing analysis. An original set of six Escherichia coli tester strains (CC101–CC106) that use Lac+ reversion to detect each of six possible base pair substitution mutations was developed by Cupples and Miller (1989). We have been employing that system to study the mutational specificity of a large number of mutagens because the procedure is simple and rapid (Watanabe and Ohta, 1993; Watanabe et al., 1994a,b; Watanabe-Akanuma and Ohta, 1994; Watanabe-Akanuma et al., 1997). Several investigations on mutation spectra of chemical mutagens using the Lac+ reversion system have also been reported (Lu et al., 1995; Marwood et al., 1995; Garganta et al., 1999). Since the mutational target of the strains is an F' plasmid-encoded mutant lacZ461 allele, it can be easily transferred into other 
(lac-pro) strains with different genetic backgrounds by means of simple conjugation and appropriate selection. In our recent study, we transferred F' plasmids from E.coli K-12-derived strains CC101–CC106 to E.coli B-derived strain WP2uvrA and designated the new strains WP3101–WP3106 (Ohta et al., 1998). WP2uvrA is widely used in Trp+ reversion assays because of its high sensitivity to many kinds of mutagens and it is recommended for general mutagenicity screening tests as a supplement to the standard Salmonella strains (TA100, TA1535, TA98, TA97 and TA102) (Gatehouse et al., 1994). Therefore, strains WP3101–WP3106 could determine mutational specificity in a Lac+ reversion assay and general mutagenicity in an ordinary Trp+ reversion assay. To further improve the sensitivity of these strains, we constructed pKM101 (mucAB)-carrying strains (WP3101P–WP3106P) and rfa derivatives of them (WP4101P–WP4106P) that are defective in the lipopolysaccharide core of the bacterial outer membrane. With these strains we determined the mutational specificity of different kinds of mutagens, including heterocyclic amines, oxidative compounds, free radical generators and polycyclic aromatic compounds (Ohta et al., 1999).
Another set of tester strains that uses the His+ reverse mutation system to detect each of the six possible base pair substitution mutations has been developed in Salmonella typhimurium by Gee et al. (1994). The mutational targets of their strains, designated TA7001–TA7006, are chromosomal hisG or hisC genes. In the present study we have compared the sensitivity of our Lac+ reversion system with the S.typhimurium His+ reversion system. We did this by assaying 21 mutagens for their ability to induce three kinds of base substitutions, G·CA·T, G·CT·A and A·TT·A, in both systems.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bacterial strains
The E.coli tester strains WP3101P–WP3106P [uvrA155, trpE65,
(lac-pro), malB15, lon-11, sulA1/F' (lacI–, lacZ–, proAB+), pKM101 (mucAB+, Apr)] have been described previously (Ohta et al., 1999). Each of the six F' plasmids possesses a unique lacZ mutation at the GAG codon corresponding to Glu461 (Cupples and Miller, 1989) and was originally constructed for E.coli K-12 strains CC101–CC106 [ara, (lac-pro)5/F' (lacI–, lacZ–, proAB+)], which were provided to us by Prof.J.H. Miller (University of California, Los Angeles, CA). The reversion events at the codon required to restore the Lac+ phenotype to each strain are TAGGAG (A·TC·G transversion) for WP3101P, GGGGAG (G·CA·T transition) for WP3102P, CAGGAG (G·CC·G transversion) for WP3103P, GCGGAG (G·CT·A transversion) for WP3104P, GTGGAG (A·TT·A transversion) for WP3105P and AAGGAG (A·TG·C transition) for WP3106P. Since WP3101P–WP3106P were derived from WP2uvrA, all the strains contain the trpE65 (ochre) allele, which is available for a Trp+ reversion assay.
Three out of six tester strains of S.typhimurium in the Ames II kit (Funakoshi, Tokyo) were obtained for the present study from the National Institute of Health Sciences (Tokyo, Japan). They are TA7002 [hisC9138, (uvrB-bio), rfa/pKM101], TA7004 [hisG9133, (uvrB-bio), rfa/pKM101] and TA7005 [hisG9130, (uvrB-bio), rfa/pKM101]. In the hisC9138 mutation, the codon for wild-type Lys217 (AAA) is changed to a codon for Ile217 (ATA). In the hisG9133 and hisG9130 mutations, the codon for Glu169 (GAG) is changed to the codon for Gly169 (GGG) and Ala169 (GCG), respectively. Therefore, in a His+ reversion assay strains TA7002, TA7004 and TA7005 detect A·TT·A transversion, G·CA·T transition and G·CT·A transversion, respectively (Gee et al., 1994). The reversion event in each tester strain of E.coli and S.typhimurium is presented in Table I.
|
Chemicals
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phosmet) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2) and N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2) were obtained from Wako Pure Chemical Industry (Tokyo, Japan). 2-Aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66–27–3) and 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5) were purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). N4-Aminocytidine (CAS no. 57294-74-3) and sodium azide (CAS no. 26628-22-8) were purchased from Funakoshi Co. (Tokyo, Japan) and Nacalai Tesque (Kyoto, Japan), respectively. N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9) and N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0) were from Iwai Kagaku Yakuhin Co. (Tokyo, Japan). 5-Azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7) were obtained from Aldrich Chemical Co. (Madison, WI). 5-Diazouracil (CAS no. 2435-76-9) and t-butyl hydroperoxide (BHP) (CAS no. 75-91-2) were from Sigma Chemical Co. (St Louis, MO). N4-aminocytidine, 5-azacytidine, BHP, CHP, 5-diazouracil, formaldehyde, glyoxal and sodium azide were dissolved in sterile water produced by the Milli-Q Ultra-Pure Water system (Millipore Ltd, Japan). All other mutagens were dissolved in dimethylsulfoxide (Wako Pure Chemical Industry).
Media
For the Lac+ reversion assay in E.coli, MGT medium, MLT agar plates and NB top agar were used. MGT medium consisted of Vogel-Bonner E medium (0.2% citric acid monohydrate, 1% K2HPO4, 0.35% NaNH4HPO4·4H2O and 0.02% MgSO4·7H2O) supplemented with 0.5% glucose and 50 µg/ml L-tryptophan. MLT plates consisted of Vogel-Bonner E medium supplemented with 0.5% lactose, 50 µg/ml tryptophan and 1.5% agar. NB top agar contained 0.5 mg/ml nutrient broth powder (Oxoid no. 2), 0.5% NaCl and 0.6% agar. Ampicillin at a final concentration of 10 µg/ml was added to MGT medium when strains WP3101P–WP3106P were cultured.
For the His+ reversion assay in S.typhimurium, MG agar plates consisting of Vogel-Bonner E medium supplemented with 0.4% glucose and 1.5% agar were used. The concentration of glucose in the agar plates was reduced from 2% in the original formula (Maron and Ames, 1983) to 0.4%, according to the method of Gee et al. (1994). HB top agar contained 50 µM L-histidine, 50 µM biotin, 0.5% NaCl and 0.6% agar. Nutrient broth (Oxoid no. 2) was used for preculture of S.typhimurium strains.
Lac+ reversion assay
Strains WP3101P–WP3106P were precultured overnight at 37°C with shaking in proline-free MGT medium supplemented with ampicillin to maintain the F' plasmid and pKM101 to an OD600 of 
1.0. The cells were used in the experiments without being washed. The Lac+ reversion assay was performed by the preincubation method: 0.1 ml of bacterial culture and 0.01–0.1 ml of mutagen solution were added to 0.5 ml of 100 mM sodium phosphate buffer (pH 7.4). The mixture was preincubated at 37°C for 20 min with gentle shaking and then 2 ml of molten NB top agar kept at 45°C was added. The contents were mixed thoroughly and overlaid onto MLT agar plates. After 48 h incubation at 37°C, the number of Lac+ revertant colonies was scored. Experiments included a single plate for each dose of mutagen and duplicate plates for the solvent control. We performed all the assays between three and five times to confirm reproducibility and the data presented in the figures are the average values of whole valid experiments. In the Lac+ reversion assay, background growth of Lac– cells on MLT agar plates with NB top agar is almost invisible, so it was difficult to assess the killing effects of the test compounds by means of a reduced background lawn. Instead, we judged toxicity to tester strains by a drastic decrease in the number of revertant colonies in strains WP3102P and WP3104P.
His+ reversion assay
The His+ reversion assay was conducted with S.typhimurium TA7002, TA7004 and TA7005. The bacterial cells were precultured overnight in nutrient broth. Aliquots of 0.1 ml of bacterial culture and 0.01–0.1 ml of a mutagen solution were added to 0.5 ml of sodium phosphate buffer. After preincubation at 37°C for 20 min, treated cells were poured onto MG agar plates with 2 ml of molten HB top agar. His+ revertants were counted following 48 h incubation at 37°C. Experiments included a single plate for each dose of mutagen and duplicate plates for the solvent control. We performed all assays at least three times to confirm reproducibility and the data presented are the average of three or more experiments.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
All mutagens were tested with six strains of E.coli (WP3101P–WP3106P) and three strains of S.typhimurium (TA7002, TA7004 and TA7005). The mutational spectra determined in the E.coli Lac+ reversion system are presented in Table II
. The ranges of spontaneous Lac+ per plate in all experiments were 1–3 in WP3101P, 7–15 in WP3102P, 1–3 in WP3103P, 6–11 in WP3104P, 3–9 in WP3105P and 1–3 in WP3106P. Spontaneous His+ per plate were 4–10 in TA7002, 15–29 in TA7004 and 20–39 in TA7005. In order to compare the sensitivity of tester strains that detect G·CA·T transition (WP3102P and TA7004), G·CT·A transversion (WP3104P and TA7005) and A·TT·A transversion (WP3105P and TA7002), fold increase over the control values for each strain was calculated and these are plotted in Figures 1–5. Since the numbers of spontaneous revertants per plate varied up to 3-fold between experiments, a ratio of E.coli/S.typhimurium >3.5 or less than its reciprocal was considered to reflect a meaningful difference in sensitivity to a mutagen.
|
|
) and S.typhimurium TA7004 (•); G·C
) and S.typhimurium TA7005 (
); A·T
) and S.typhimurium TA7002 (
).
|
|
|
|
Mutation spectra of alkylating agents
In WP3101P–WP3106P all the alkylating agents except MMS induced predominantly G·C
A·T transitions (80–97%), as shown in Table II
. They also induced G·CT·A, A·TT·A and A·TG·C base substitutions weakly. In contrast, MMS efficiently induced G·CT·A and A·TT·A transversions (16 and 22%, respectively) as well as G·CA·T transitions (56%). A·TC·G transversions were weakly induced by MNNG, ENNG, MMS and ENU, while G·CC·G transversions were slightly induced by ENNG, MMS and ENU. As shown in Figure 1, three kinds of base substitutions (G·CA·T, G·CT·A and A·TT·A) were induced with almost equal frequency by MNNG and EMS in the E.coli and Salmonella tester strains. The E.coli Lac+ reversion system was as sensitive as the S.typhimurium His+ reversion system for the detection of G·CA·T transitions by MNU, G·CT·A transversions by MMS and MNU and A·TT·A transversions by MMS. A·TT·A transversion by MNU and G·CA·T transition by MMS were detected more strongly in E.coli strains. Of the base substitutions induced by ENNG and ENU, the E.coli strains were more sensitive than the S.typhimurium strains to induction of G·CA·T and G·CT·A, while Salmonella strains were more sensitive to the induction of A·TT·A (Figure 2).
Mutation spectra of base analogs
N4-aminocytidine preferentially induced G·CA·T transitions (92%) and also induced A·TG·C transitions and G·CT·A transversions (Table II). In E.coli it induced 140 times the control value at 0.1 µg/plate (Figure 3). In S.typhimurium, on the other hand, it did not induce G·CT·A transversions and the induction of G·CA·T transitions was not as great (it induced 19 times the control value at 0.1 µg/plate). N4-aminocytidine did not induce A·TT·A transversions in either E.coli or S.typhimurium. 5-Diazouracil, as reported in our previous work (Ohta et al., 1999), induced base substitutions in all E.coli tester strains, primarily G·CT·A (51%). It induced a lower frequency of G·CT·A transversions in the S.typhimurium strains than in the corresponding E.coli strain (Figure 3). 5-Azacytidine induced predominantly G·CC·G transversions (89%) and also G·CT·A transversions (11%). The prevalent induction of G·CC·G transversions was consistent with previous reports (Cupples and Miller, 1989; Gee et al., 1994; Ohta et al., 1998). In S.typhimurium strains neither G·CA·T transitions nor G·CT·A transversions was detected (Figure 3). 2-Aminopurine induced G·CA·T transitions marginally in the E.coli tester strain and was toxic to E.coli starting at 500 µg/plate. In S.typhimurium, however, 2-aminopurine clearly induced G·CA·T transitions and A·TT·A transversions at 1000 µg/plate, as shown in Figure 3. 6-Mercaptopurine was toxic to E.coli at 10 µg/plate and to S.typhimurium at 500 µg/plate and induced no detectable mutations at lower doses (data not shown). Thus, the E.coli Lac+ reversion system responded well to N4-aminocytidine, 5-diazouracil and 5-azacytidine, but not as well as the S.typhimurium His+ reversion system to 2-aminopurine.
Mutation spectra of oxidative compounds
CHP and BHP induced predominantly G·CA·T (29–36%), G·CT·A (26–28%) and A·TT·A (28%) base substitutions. Glyoxal also induced G·CA·T, G·CT·A and A·TT·A (44, 45 and 11%, respectively), while formaldehyde induced only G·CT·A transversions (Table II). Sensitivity to glyoxal and formaldehyde was almost equal in both sets of tester strains. CHP and BHP appeared to be more toxic to the Salmonella strains. Either negative or marginal results were obtained in S.typhimurium strains (Figure 4). Thus, the E.coli Lac+ reversion system seems to be superior to the S.typhimurium His+ reversion system for some hydroperoxide compounds.
Mutation spectra of miscellaneous mutagens
In the E.coli assay 4-NQO induced four types of base substitutions (G·CA·T, G·CC·G, G·CT·A and A·TT·A), most predominantly G·CT·A transversions (82%). MX and AF-2 induced all base substitutions except A·TG·C transitions, with G·CT·A predominating (Table II). Compared with S.typhimurium, E.coli was more sensitive to 4-NQO- and MX-induced G·CT·A transversions and to 4-NQO-induced A·TT·A transversions. On the other hand, there was no obvious difference in responses to AF-2 (Figure 5). Captan induced all possible base substitutions, although G·CC·G transversions were of low frequency, and was an efficient inducer of all three possible base substitutions that can occur at A·T sites (A·TC·G, A·TT·A and A·TG·C). The strong induction of A·TC·G transversions (18%) and A·TG·C transitions (34%) was unique to this mutagen. Escherichia coli and S.typhimurium tester strains responded to captan with similar sensitivities for the mutations tested, as shown in Figure 5. Phosmet, an organophosphorous compound, induced only G·CT·A transversions weakly in E.coli, but clearly induced G·CT·A transversions and G·CA·T transitions in Salmonella. Sodium azide induced only G·CA·T transitions in E.coli. G·CA·T transitions were induced in S.typhimurium at a similar level (Table II and Figure 5).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The six tester strains of the E.coli Lac+ reversion system, WP3101–WP3106, and their pKM101-bearing derivatives, WP3101P–WP3106P, provide a convenient way to measure mutation spectra, as demonstrated in our previous work (Ohta et al., 1998
, 1999). Because the strains were derived from WP2uvrA, they can be used simultaneously in a Trp+ reversion assay. Therefore, the advantage of this system is that mutagens detectable in Trp+ reversion assays with WP2uvrA or WP2uvrA/pKM101 could be analyzed for specificity of mutations in strains WP3101–WP3106 or WP3101P–WP3106P, respectively, in the same genetic background. Similar sets of S.typhimurium tester strains that use His+ reversion have been developed and they have been used to analyze several mutagens (Gee et al., 1994, 1998). It is worth comparing the sensitivity and specificity of the Lac+ reversion system with those of the His+ reversion system in order to elucidate quantitative and qualitative differences between the two systems. For this purpose, we selected three base substitutions (G·CA·T, G·CT·A and A·TT·A), the predominant mutations induced by most of the mutagens investigated in our previous studies (Ohta et al., 1998, 1999). As a result of comparable assays conducted with paired strains (WP3102P/TA7004, WP3104P/TA7005 and WP3105P/TA7002), several interesting characteristics of the test system were found. Due to the differing spontaneous reversion rates in the E.coli and S.typhimurium strains, mutagenic potency was compared with fold increase over the control values rather than the actual number of revertants induced.
The Lac+ reversion system was superior to the His+ reversion system for measuring the mutation spectra of CHP and BHP because the E.coli strains were more resistant to the toxic effects of these compounds. In addition, the Lac+ reversion system was more sensitive than the His+ reversion system for detecting G·CA·T transitions induced by N4-aminocytidine, 5-azacytidine, CHP, BHP, ENNG, MMS and ENU, G·CT·A transversions induced by N4-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-NQO and MX and A·TT·A transversions induced by MNU, CHP and 4-NQO. The better results obtained in the Lac+ reversion system could not have been due to the difference in the number of F'-encoded target lacZ genes compared with chromosomally encoded hisG or hisC genes because there was only a single F' plasmid in the cells.
The His+ reversion system was found to be more sensitive than the Lac+ reversion system in detecting G·CA·T transitions induced by 2-aminopurine and phosmet and A·TT·A transversions induced by 2-aminopurine, ENNG and ENU. 2-Aminopurine showed marginal mutagenic activity in the Lac+ reversion assay with WP3101P–WP3106P due to its strong toxicity to E.coli in the present study. Cupples and Miller (1989), however, reported that 2-aminopurine efficiently induced G·CA·T transitions in the Lac+ reversion system when E.coli CC102 cells were treated overnight in LB medium containing 2-aminopurine at 700 µg/ml. The data suggest that 2-aminopurine mutagenesis is largely affected by treatment conditions. The medium for preculture of bacterial strains employed in this study was minimal MGT medium for E.coli cells in the Lac+ reversion assay and nutrient broth for S.typhimurium in the His+ reversion assay. Since de novo synthesis of nucleotides is required for growth in minimal medium, the inhibitory effect of the base analog 2-aminopurine might be pronounced. Another base analog, 6-mercaptopurine, was also differentially toxic in the two assay systems. 6-Mercaptopurine was reported to be mutagenic to S.typhimurium TA1535 and TA100 at 50–200 µg/plate (Benedict et al., 1977; Seino et al., 1978; Nepomnaschy et al., 1984), but not mutagenic in the Trp+ reversion assay with WP2uvrA/pKM101 (unpublished data). It may be more practical to use nutrient broth for preculture of WP3101P–WP3106P. Without selection pressure on the F' plasmid, F'-free cells would not appear with extremely high frequency and, therefore, would not reduce the sensitivity of the test system.
Of great interest was the higher sensitivity of S.typhimurium strain TA7002 compared with E.coli strain WP3105P to A·TT·A transversions induced by two ethylating agents, ENNG and ENU. Several explanations come to mind. (i) O6-methylguanine-DNA methyltransferase (O6-MGT) plays an important role in the repair of premutagenic DNA alkylation, such as O6-alkylguanine and O4-alkylthymine. Both E.coli and S.typhimurium have two enzymes with O6-MGT activity that are encoded by the ada and ogt genes (Potter et al., 1987; Lindahl et al., 1988; Hakura et al., 1991; Yamada, et al., 1995). The Ada protein (O6-MGT-I), however, may not play a major role in protecting S.typhimurium against the mutagenic effects of methylating agents because ada mutants show almost the same sensitivity to MNNG as the wild-type strain (Yamada et al., 1993). In contrast, ogt mutants are hypersensitive compared with the parent strain to the mutagenicity of MNNG and particularly ENNG in S.typhimurium (Yamada et al., 1995). The Ogt protein (O6-MGT-II) is more active than the Ada protein (O6-MGT-I) in the repair of ethylated DNA in E.coli (Wilkinson et al., 1989) and S.typhimurium (Yamada et al., 1995). Possible differences in O6-MGT-II activity between E.coli and S.typhimurium may reflect on their sensitivity to A·TT·A transversions. (ii) Influence of the DNA sequence context is another plausible explanation. For example, Koch et al. (1994) demonstrated that the majority of base substitutions induced by MNNG and sodium azide at the CCC target site in the hisG46 allele of S.typhimurium were G·CA·T transitions. Base substitutions occurred preferentially at the codon first position with MNNG, resulting in TCC, and at the second position with sodium azide, resulting in CTC. The codon reversion sites are the same in the E.coli and S.typhimurium strains that detect G·CA·T transitions (GGGGAG) and in those that detect G·CT·A transversions (GCGGAG). They are different, however, in the strains that detect A·TT·A transversions (GTGGAG in lacZ; ATAAAA in hisC). The 5'- and 3'-neighboring bases are guanines in E.coli WP3105P and adenines in S.typhimurium TA7002. An effect of sequence context on sensitivity to ethylating agents cannot be excluded. (iii) The His+ reversion of hisC9138 in TA7002 is reported to be a specific change from Ile (ATA) to Lys (AAA), based on a study of ~100 His+ revertants induced by several mutagens (not including ENNG and ENU) that were analyzed by DNA sequencing (Gee et al., 1994). However, there still remains a slight possibility that amino acid replacement of Ile (ATA) by Thr (ACA), Arg (AGA), Val (GTA) or Leu (CTA) could partially restore the hisC-encoded enzyme activity so that the strain would exhibit a His+ phenotype. If this is the case, base substitutions other than A·TT·A transversions could contribute to His+ reversion in TA7002. In the Lac+ reversion system, the presence of any amino acid other than Glu at position 461 yields a Lac– phenotype (Cupples and Miller, 1988). Such a study has not yet been reported on hisC9138.
As mentioned above and also discussed previously with regard to the effect of sequence context (Ohta et al., 1998), the reversion frequency induced by mutagens often varies with the sequences adjacent to the mutational target site. It is important to comprehend that the information obtained by monitoring reversions at a specific site is not always consistent with the mutational specificity obtained by DNA sequencing analysis. Recent progress in mutation analysis using transgenic mice has revealed the mutational spectra induced by many mutagens in mammalian cells. However, information on mutation spectra in bacterial cells is not always enough for these mutagens. The convenient Lac+ reversion system comprising tester strains WP3101–WP3106 and WP3101P–WP3106P would help us to detect specific mutations induced by a variety of mutagens. Since plasmid pKM101 generally enhanced G·CT·A and A·TT·A transversions strongly over other base substitutions (Watanabe et al., 1994b), pKM101-free strains WP3101–WP3106 may be suitable to determine the nature of the mutagenesis induced by mutagens. On the other hand, a large number of mutagens are known to exhibit mutagenicity only in pKM101-carrying tester strains, such as S.typhimurium TA100, TA102 and TA92, in mutagenicity tests. Strains WP3101P–WP3106P would be useful to investigate the mutational profiles of such mutagens. Further investigations of mutational specificities in bacterial cells as well as mammalian cells will provide important information about the mechanisms of mutagenesis and DNA repair.
|
Acknowledgments |
|---|
We thank Prof. Jeffrey H.Miller for providing F' plasmids. We also thank Hiromi Ishihara and Yoko Kita for their technical assistance and Dr Miriam Bloom for her critical reading of the manuscript.
|
Notes |
|---|
2 To whom correspondence should be addressed. Tel: +81 426 76 7093; Fax: +81 426 76 7081; Email: ohta{at}ls.toyaku.ac.jp
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Benedict,W.F., Baker,M.S., Haroun,L., Choi,E. and Ames,B.N. (1977) Mutagenicity of cancer chemotherapeutic agents in the Salmonella/microsome test. Cancer Res., 37, 2209–2213.
Cupples,C.G. and Miller,J.H. (1988) Effect of amino acid substitutions at the active site in Escherichia coli ß-galactosidase. Genetics, 120, 637–644.
Cupples,C.G. and Miller,J.H. (1989) A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc. Natl Acad. Sci. USA, 86, 5345–5349.
Garganta,F., Krause,G. and Scherer,G. (1999) Base-substitution profiles of externally activated polycyclic aromatic hydrocarbons and aromatic amines determined in a lacZ reversion assay. Environ. Mol. Mutagen., 33, 75–85.[Web of Science][Medline]
Gatehouse,D., Haworth,S., Cebula,T.,Gocke,E., Kier,L., Matsushima,T., Melcion,C., Nohmi,T., Ohta,T., Venitt,S. and Zeiger,E. (1994) Recommendations for the performance of bacterial mutation assays. Mutat. Res., 312, 217–233.[Web of Science][Medline]
Gee,P., Maron,D.M. and Ames,B.N. (1994) Detection and classification of mutagens: a set of base-specific Salmonella tester strains. Proc. Natl Acad. Sci. USA, 91, 11606–11610.
Gee,P., Sommers,C.H., Melick,A.S., Gidrol,X.M., Todd,M.D., Burris,R.B., Nelson,M.E., Klemm,R.C. and Zeiger,E. (1998) Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens: the results of a validation study. Mutat. Res., 412, 115–130.[Web of Science][Medline]
Hakura,A., Morimoto,K., Sofuni,T. and Nohmi,T. (1991) Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase. J. Bacteriol., 173, 3663–3672.
Koch,W.H., Henrikson,E.N., Kupchella,E. and Cebula,T.A. (1994) Salmonella typhimurium strain TA100 differentiates several classes of carcinogens and mutagens by base substitution specificity. Carcinogenesis, 15, 79–88.
Koch,W.H., Henrikson,E.N. and Cebula,T.A. (1996) Molecular analysis of Salmonella hisG428 ochre revertants for rapid characterization of mutational specificity. Mutagenesis, 11, 341–348.
Levin,D.E. and Ames,B.N. (1986) Classifying mutagens as to their specificity in causing the six possible transitions and transversions. Environ. Mutagen., 8, 9–28.[Web of Science][Medline]
Lindahl,T., Sedgwick,B., Sekiguchi,M. and Nakabeppu,Y. (1988) Regulation and expression of the adaptive response to alkylating agents. Annu. Rev. Biochem., 57, 133–157.[Web of Science][Medline]
Lu,C., Pfeil,R.M. and Rice,C.P. (1995) Determination of mutational spectrum of the pesticide, captan, with an improved set of Escherichia coli lacZ mutants. Mutat. Res., 343, 219–227.[Web of Science][Medline]
Maron,D.M. and Ames,B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutat. Res., 113, 173–215.[Web of Science][Medline]
Marwood,T.M., Meyer,D. and Josephy,P.D. (1995) Escherichia coli lacZ strains engineered for detection of frameshift mutations induced by aromatic amines and nitroaromatic compounds, Carcinogenesis, 16, 2037–2043.
Miller,J.K. and Barnes,W.M. (1986) Colony probing as an alternative to standard sequencing as a means of direct analysis of chromosomal DNA to determine the spectrum of single-base changes in regions of known sequence. Proc. Natl Acad. Sci. USA, 83, 1026–1030.
Nepomnaschy,I., Sommer,S.E. and Nagel,R. (1984) Effect of the rat liver S9 fraction on the mutagenicity of azathioprine in the Salmonella/mammalian microsome assay. Experientia, 40, 370–371.[Web of Science][Medline]
Ohta,T., Watanabe-Akanuma,M., Tokishita,S., Shiga,Y. and Yamagata,H. (1998) Development of new tester strains derived from E.coli WP2uvrA for the determination of mutational specificity. Mutat. Res., 413, 219–225.[Web of Science][Medline]
Ohta,T., Watanabe-Akanuma,M., Tokishita,S. and Yamagata,H. (1999) Mutation spectra of chemical mutagens determined by Lac+ reversion assay with Escherichia coli WP3101P–WP3106P tester strains. Mutat. Res., 440, 59–74.[Web of Science][Medline]
Potter,P.M., Wilkinson,M.C., Fitton,J., Carr,F.J.S., Brennand,J., Cooper,D.P. and Margison,G.P. (1987) Characterization and nucleotide sequence of ogt, the O6-alkylguanine-DNA alkyltransferase gene of E.coli. Nucleic Acids Res., 15, 9177–9193.
Prival,M.J. and Cebula,T.A. (1992) Sequence analysis of mutations arising during prolonged starvation of Salmonella typhimurium. Genetics, 132, 303–310.[Abstract]
Seino,Y., Nagao,M., Yahagi,T., Hoshi,A., Kawachi,T. and Sugimura,T. (1978) Mutagenicity of several classes of antitumor agents to Salmonella typhimurium TA98, TA100 and TA92. Cancer Res., 38, 2148–2156.
Shinoura,Y., Kato,T. and Glickman,B.W. (1983) A rapid and simple method for the determination of base substitution and frameshift specificity of mutagens. Mutat. Res., 111, 43–49.
Watanabe,M. and Ohta,T. (1993) Analysis of mutational specificity induced by heterocyclic amines in the lacZ gene of Escherichia coli. Carcinogenesis, 14, 1149–1153.
Watanabe,M., Nohmi,T. and Ohta,T. (1994a) Effects of the umuDC, mucAB and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: I. Frameshift mutagenesis. Mutat. Res., 314, 27–37.[Web of Science][Medline]
Watanabe,M., Nohmi,T. and Ohta,T. (1994b) Effects of the umuDC, mucAB and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: II. Base-substitutional mutagenesis. Mutat. Res., 314, 39–49.[Web of Science][Medline]
Watanabe-Akanuma,M. and Ohta,T. (1994) Effects of DNA repair deficiency on the mutational specificity in the lacZ gene of Escherichia coli. Mutat. Res., 311, 295–304.[Web of Science][Medline]
Watanabe-Akanuma,M., Woodgate,R. and Ohta,T. (1997) Enhanced generation of A·TT·A transversions in a recA730 lexA(Def) mutant of Escherichia coli. Mutat. Res., 373, 61–66.[Web of Science][Medline]
Wilkinson,M.C., Potter,P.M., Cawkwell,L., Georgiadis,P., Patel,D., Swann,P.F. and Margison,G.P. (1989) Purification of the E.coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides. Nucleic Acids Res., 17, 8475–8484.
Yamada,M., Hakura,A., Sofuni,T. and Nohmi,T. (1993) New method for gene disruption in Salmonella typhimurium: construction and characterization of an ada-deletion derivative of Salmonella typhimurium TA1535. J. Bacteriol., 175, 5539–5547.
Yamada,M., Sedgwick,B., Sofuni,T. and Nohmi,T. (1995) Construction and characterization of mutants of Salmonella typhimurium deficient in DNA repair of O6-methylguanine. J. Bacteriol., 177, 1511–1519.
Received on October 4, 1999; accepted on March 31, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Watanabe-Akanuma, Y. Inaba, and T. Ohta Mutagenicity of UV-irradiated maltol in Salmonella typhimurium Mutagenesis, January 1, 2007; 22(1): 43 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. R. Hoffmann, D. J. Crowley, and P. J. Theophiles Comparative potencies of induction of point mutations and genetic duplications by the methylating agents methylazoxymethanol and dimethyl sulfate in bacteria Mutagenesis, September 1, 2002; 17(5): 439 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ohta, S.-i. Tokishita, R. Tsunoi, S. Ohmae, and H. Yamagata Characterization of Trp+reversions in Escherichia coli strain WP2uvrA Mutagenesis, July 1, 2002; 17(4): 313 - 316. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||