- Email: [email protected]
Cloning of Salmonella typhimurium genes for nitroreductase and acetyltransferase: construction of Salmonella highly sensitive to mutagenic nitroarenes
393 sponding synthetic specimen. AAF N-sulfate showed a potent direct-acting mutagenicity toward TA98 and little mutagenicity toward TA100.
62 Yajima, N., S. Hochi, T. Kishi and G. Kawanishi, Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., Ishibashi, Shimotsugagun, Tochigi 329-05 (Japan)
61 Watanabe, M., T. Nohmi and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan)
DNA single-strand breaks induced by 3 cross-linking agents
Cloning of Salmonella typhimurium genes for nitroreductase and acetyltransferase: construction of Salmonella highly sensitive to mutagenic nitroarenes The nitroreductase (NRase) and acetyltransferase (ATase) genes of S. typhimurium TA1538 were cloned into pBR322, and the resuiting plasmids, p Y G l l l , pYG121, and pYG122, were introduced into TA1538 derivatives, which resulted in higher production of these enzymes. The activities, NRase for TA1538NR ( p Y G l l l ) and ATase for TA1538/1,8-DNP with pYG121 or pYG122, were 50 times higher than in TA1538 (pBR322). In the reverse mutation assay, TA1538NR ( p Y G l l l ) exhibited increased sensitivity to 2-nitrofluorene (2-NF) and 1-nitropyrene, while TA1538/1,8-DNP (pYG121 or pYG122) was highly sensitive to both 2-NF and 1,8-dinitropyrene (see Biochem. Biophys. Res. Commun., 147 (1987) 974 for details). The cloned genes were further characterized through subcloning. The constructed plasmids with the NRase gene (pYG216 and pYG217) showed increased expression of NRase (50-fold) in TA98, TA100 and TA1538NR. TA98 with pYG216 or pYG217 was more sensitive to 2-NF than TA1538NR with pYG216, pYG217 or p Y G l l l , while the sensitivity of TA100 with pYG216 or pYG217 was only moderately increased. TA1538NR with pYG213 derived from pYG122 produced 500 times more ATase than strain TA1538 (pBR322), which also resulted in an increment of the sensitivity to 2-NF. These strains will provide an advanced tool for the detection of mutagenic potential among environmental nitroarenes.
DNA single-strand (s-s) breaks induced by 3 cross-linking agents, SN-07, mitomycin C (MMC) and 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) were studied on mouse leukemia L1210 cells in culture by the alkaline elution technique. DNA of L1210 was prelabeled for 24 h with [14C]or [3H]dThd and chased for 24 h. 14C-Labeled cells were treated with a chemical for 1 h and post-incubated for 24 h. Cross-linking and s-s breaks were examined with and without irradiation of 300-R X-rays, respectively, just before the alkaline elution. 3H-Labeled ceils were irradiated with 150 R for use as an internal standard. The results indicated that SN-07 induced s-s breaks, cross-linking induced by MMC was repaired during the post-incubation, but that by ACNU was unrepairable after 24-h post-incubation. Novobiocin, an inhibitor of topoisomerase, enhanced s-s breaks in the cells that had been treated with SN-07 and induced s-s breaks in the cells that had been treated with MMC, but did not affect the ACNU-induced cross-linking.
63 Yamashita, K., A. Umemoto, S. Grivas, S. Kato, S. Sato and T. Sugimura, Biochemistry Division, National Cancer Center Research Institute, Chuoku, Tokyo 104 (Japan) Reaction of N-hydroxy derivatives of heterocyclic amines with DNA In vitro reactivity toward DNA of chemically synthesized N-hydroxy derivatives of 3 mutagenic and carcinogenic heterocyclic amines present in cooked foods and amino acid pyrolysates, 2amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MelQx), 2-amino-6-methyldipyrido[1,2-a: 3',2'-
62 Yajima, N., S. Hochi, T. Kishi and G. Kawanishi, Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., Ishibashi, Shimotsugagun, Tochigi 329-05 (Japan)
61 Watanabe, M., T. Nohmi and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan)
DNA single-strand breaks induced by 3 cross-linking agents
Cloning of Salmonella typhimurium genes for nitroreductase and acetyltransferase: construction of Salmonella highly sensitive to mutagenic nitroarenes The nitroreductase (NRase) and acetyltransferase (ATase) genes of S. typhimurium TA1538 were cloned into pBR322, and the resuiting plasmids, p Y G l l l , pYG121, and pYG122, were introduced into TA1538 derivatives, which resulted in higher production of these enzymes. The activities, NRase for TA1538NR ( p Y G l l l ) and ATase for TA1538/1,8-DNP with pYG121 or pYG122, were 50 times higher than in TA1538 (pBR322). In the reverse mutation assay, TA1538NR ( p Y G l l l ) exhibited increased sensitivity to 2-nitrofluorene (2-NF) and 1-nitropyrene, while TA1538/1,8-DNP (pYG121 or pYG122) was highly sensitive to both 2-NF and 1,8-dinitropyrene (see Biochem. Biophys. Res. Commun., 147 (1987) 974 for details). The cloned genes were further characterized through subcloning. The constructed plasmids with the NRase gene (pYG216 and pYG217) showed increased expression of NRase (50-fold) in TA98, TA100 and TA1538NR. TA98 with pYG216 or pYG217 was more sensitive to 2-NF than TA1538NR with pYG216, pYG217 or p Y G l l l , while the sensitivity of TA100 with pYG216 or pYG217 was only moderately increased. TA1538NR with pYG213 derived from pYG122 produced 500 times more ATase than strain TA1538 (pBR322), which also resulted in an increment of the sensitivity to 2-NF. These strains will provide an advanced tool for the detection of mutagenic potential among environmental nitroarenes.
DNA single-strand (s-s) breaks induced by 3 cross-linking agents, SN-07, mitomycin C (MMC) and 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) were studied on mouse leukemia L1210 cells in culture by the alkaline elution technique. DNA of L1210 was prelabeled for 24 h with [14C]or [3H]dThd and chased for 24 h. 14C-Labeled cells were treated with a chemical for 1 h and post-incubated for 24 h. Cross-linking and s-s breaks were examined with and without irradiation of 300-R X-rays, respectively, just before the alkaline elution. 3H-Labeled ceils were irradiated with 150 R for use as an internal standard. The results indicated that SN-07 induced s-s breaks, cross-linking induced by MMC was repaired during the post-incubation, but that by ACNU was unrepairable after 24-h post-incubation. Novobiocin, an inhibitor of topoisomerase, enhanced s-s breaks in the cells that had been treated with SN-07 and induced s-s breaks in the cells that had been treated with MMC, but did not affect the ACNU-induced cross-linking.
63 Yamashita, K., A. Umemoto, S. Grivas, S. Kato, S. Sato and T. Sugimura, Biochemistry Division, National Cancer Center Research Institute, Chuoku, Tokyo 104 (Japan) Reaction of N-hydroxy derivatives of heterocyclic amines with DNA In vitro reactivity toward DNA of chemically synthesized N-hydroxy derivatives of 3 mutagenic and carcinogenic heterocyclic amines present in cooked foods and amino acid pyrolysates, 2amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MelQx), 2-amino-6-methyldipyrido[1,2-a: 3',2'-