Mutagenesis assays in yeast.
Analyzing mutation spectra is a very powerful method to determine the effects of various types of DNA damage and to understand the workings of various DNA repair pathways. However, compiling sequence-specific mutation spectra is laborious; even with modern sequencing technology, it is rare to obtain spectra with more than several hundred data points. Two assay systems are described for yeast, one for insertion/deletion mutations and one for base substitution mutations, that allow determination of specific mutations without the necessity of DNA sequencing. The assay for insertion/deletion mutations uses a variety of different simple repeats placed in frame with URA3 such that insertions or deletions lead to a selectable Ura(-) phenotype; essentially all such mutations are in the simple repeat sequence. The assay for base substitution mutations uses a series of six strains with different mutations in one essential codon of the CYC1 gene. Because only true reversions lead to a selectable phenotype, the bases mutated in any reversion event are known. The advantage of these assays is that they can quantitatively determine over several orders of magnitude the types of mutations that occur under a given set of conditions, without DNA sequencing.
Crouse
G F
GF
Department of Biology, Emory University, Atlanta, Georgia 30322, USA.
D004249:DNA Damage; D004252:DNA Mutational Analysis / Q000379:methods*; D004260:DNA Repair; D017353:Gene Deletion; D009152:Mutagenicity Tests / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D017354:Point Mutation; D015003:Yeasts / Q000235:genetics*