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Mutagenesis vol. 11 no. 6 pp. 565-572, 1996
© 1996 UK Environmental Mutagen Society/Oxford University Press

research-article

Regionally-targeted mutagenesis by metabolically-activated steviol: DNA sequence analysis of steviol-induced mutants of guanine phosphoribosyltransferase (gpt) gene of Salmonella typhimurium TM677

M. Matsui, T. Sofuni and T. Nohmi1

Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Health Sciences 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan

Steviol is the aglycone of stevioside, a non–caloric sugar substitute commonly used in Japan. Steviol strongly induces mutations at the guanine phosphoribosyltransferase gene (gpt) of Salmonella typhimurium TM677 when the metabolic activation system (S9 mix) is present. However, it is completely negative in the reverse mutation assays using Escherichia coli WP2 uvrA/pKM101 or S.typhimurium TA strains. In order to characterize the mutations induced by metabolically-activated steviol, the chromosomal gpt alleles of 24 induced (ST clones) and 16 spontaneous mutants (SP clones) of S.typhimurium TM677 were sequenced and the mutation spectra were compared. About 40% of the mutations of ST clones (nine out of 24) were localized in the region between nucleotides 280 and 330 from the starting codon ATG, whereas no mutations of SP clones were found in that region. The mutations identified in the region included transitions (three clones), transversions (four clones), a duplication and a deletion. There were no other marked differences between ST and SP clones: basechange mutations were dominant over frameshifts and deletions (ST clones, 20 versus three; SP clones, 16 versus two) and base change mutations occurred more frequently at G:C pairs rather than at A:T pairs (ST clones, 15 versus five; SP clones, 12 versus four). The possibility that metabolically-activated steviol gives pause to DNA synthesis around nucleotide 280, thereby stimulating the duplication, deletion and untargeted mutagenesis in the defined region of the gpt gene is discussed.

1To whom correspondence should be addressed


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