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Mutational specificity of 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea in Escherichia coli : Comparison of in vivo with in vitro exposure of the supF gene
Author(s) -
LuqueRomero Francisco L.,
Ian Raffaella,
Inga Alberto,
Fronza Gilberto,
Pueyo Carmen
Publication year - 1997
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/(sici)1098-2280(1997)30:1<65::aid-em9>3.0.co;2-b
Subject(s) - mutagenesis , biology , in vivo , mutation frequency , microbiology and biotechnology , mutant , dna , genetics , mutation , shuttle vector , in vitro , gene , point mutation , recombinant dna , vector (molecular biology)
Forward mutations induced by 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea (CCNU) in the supF gene of Escherichio coli were recovered from bacteria deficient in nucleotide excision repair and in DNA‐alkyltransferase activity. Bacteria were exposed to 0.4 mM CCNU (in vivo supF mutagenesis), increasing the overall mutation frequency 15.7‐fold above the spontaneous value. A total of 73 independent supF − mutants were sequenced. The resulting mutation spectrum was compared with those obtained in bacteria and mammalian cells following the classical shuttle‐vector approach (in vitro supF mutagenesis). In vivo CCNU mutagenesis in E. coli yielded a large number of deletions (20/73), in agreement with mammalian data but distinct from in vitro bacterial spectra, which are almost exclusively composed of G:C → A:T transitions. A substantial proportion (6/18) of CCNU‐induced deletions (> 3 bp) involved repeated DNA sequences, suggesting a contribution of a slippage‐misalignment process in the generation of this mutation class. Substitutions occurred primarily at G:C base pairs (44/53) and were predominantly G:C → A:T transitions (39/53). This mutational change was attributed to the mispair potential of the O 6 ‐chloroethylguanine lesion with thymine. Most G:C → A:T transitions (34/39) were located at three 5′‐GG‐3′ hotspot sites (positions 123, 160, and 168). The distribution of hotspot sites for G:C → A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF ‐bearing plasmids and revealed significant differences in the DNA strand distribution of this mutational event. Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O 6 ‐chloroethylguanine formation, reducing its incidence in the transcribed DNA strand. Environ. Mal. Mutagen. 30:65–71, 1997. © 1997 Wiley‐Liss, Inc.