Mutations in a shuttle vector exposed to activated mitomycin C.
ABSTRACT The cytotoxicity of the potent antibiotic and antitumor agent mitomycin C (MMC) is due to its irreversible binding to DNA. Alkylating species generated by bioreductive activation of MMC are known to cause monoadducts and cross-links in DNA by specifically binding to guanine residues. To gain insight into how these lesions lead to base- and sequence-specific mutations, shuttle vector pSP189 was treated with MMC chemically reduced by treatment with sodium borohydride, replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence. The MMC-induced mutations were predominantly base substitutions. Eighty-four percent of the base substitutions were transversions, with G:C-->T:A the major transversion. Single base deletions were the other major mutational event, and 77% of these were G:C deletions. Base positions 115, 123, and 163 were mutational hot spots based on the frequency of independent mutations. Identification of a single MMC adduct (presumed to be a modified G on the basis of its Rf value) and clustering of MMC-induced mutations at three GC-rich areas (nt 100-123, 152-163, and 168-176) suggested that the mutational spectrum we found was due to binding of MMC to guanine on either strand of the plasmid DNA.
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ABSTRACT: The last decade has witnessed a remarkable increase in the number of mutations identified both in human disease-related genes and mutation reporter genes including those in mammalian cells and transgenic animals. This has led to the curation of a number of computerised databases, which make mutation data freely available for analysis. A primary interest of both the clinical researcher and the genetic toxicologist is determination of location and types of mutation within a gene of interest. Collections of mutation data observed for a disease-related gene or, for a gene exposed to a particular chemical, permits discovery of regions of sequence along the gene prone to mutagenesis and may provide clues to the origin of a mutation. The principal tool for visualising the distribution pattern of mutant data along a gene is the mutation spectrum: the distribution and frequency of mutations along a nucleotide sequence. In genetic toxicology, the current wealth of mutation data available allows us to construct many mutation spectra of interest to investigate the mutagenic mechanisms and mutational sites for one or a group of mutagens. Using the multivariate statistical methods principal components analysis (PCA) and cluster analysis (CA) we have tested the ability of these methods to establish the underlying patterns within and between 60 UV-induced, mitomycin C-induced and spontaneous mutations in the supF gene. The spectra were derived from human, monkey and mouse cells including both repair efficient and repair deficient cell lines. We demonstrate and support the successful application of multivariate statistical methods for exploring large sets of mutation spectra to reveal underlying patterns, groupings and similarities. The methods clearly demonstrate how different patterns of spontaneous and UV-induced supF mutation spectra can result from variation in plasmid, culture medium, species origin of cell line and whether mutations arose in vivo or in vitro.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 08/2002; 518(2):163-80. · 2.85 Impact Factor