Molecular analysis of mutagenesis in E. coli

In this paper we present a general strategy that is suitable for the analysis of the forward mutation spectrum induced by any physical or chemical mutagen or carcinogen. The assay is based upon the inactivation of the tetracycline resistance gene located on plasmid pBR322. Plasmid DNA is treated in vitro with the mutagen and transformed into the host bacteria of choice. Mutant clones are selected and analysed by sequencing. We present also two techniques that allow the determination of the DNA modification spectrum. The comparison for a given mutagen of the modification spectrum and the induced mutation spectrum permits the identification of hot spot sequences. Using different chemical mutagens (derivatives of the carcinogenic aromatic amide N-2-acetylaminofluorene, cis-platinum, etc.) this assay was found to be able to detect the different classes of mutagenic events: base substitutions, frameshifts, insertions and deletions. The advantages and limitations of this assay are discussed.