A collection of 196 spontaneous mutations in the SUP4-o gene of the yeast Saccharomyces cerevisiae was analyzed by DNA sequencing. The classes of mutation identified included all possible types of base-pair substitution, deletions of various lengths, complex alterations involving multiple changes, and insertions of transposable elements. Our findings demonstrate that at least several different mechanisms are responsible for spontaneous mutagenesis in S. cerevisiae.Although spontaneous mutagenesis has been well studied in the yeast Saccharomyces cerevisiae, relatively little is known about the molecular nature of spontaneous mutational events or the factors that influence such events in this organism (for reviews, see references 6, 10, and 18). To improve our understanding of mutational mechanisms in S. cerevisiae, we recently developed a system in which mutations occurring in the tyrosine suppressor tRNA gene SUP4-o can be rapidly analyzed to determine the DNA sequence changes responsible (12). Here we describe our use of the SUP4-o system to analyze spontaneous mutations in S. cerevisiae.In this system, SUP4-o is carried on the centromere plasmid YCpMP2. Autonomous yeast centromere-containing plasmids mimic the behavior of yeast chromosomes. They are maintained predominantly as single copies in haploid cells (a feature essential for mutant selection), exhibit typical chromatin organization, and replicate once per cell cycle in S phase (2). Forward mutations in the SUP4-o gene are detected by their elimination of suppressor activity (13). The haploid yeast strain MKP-o (12) carries ochresuppressible markers which, in the absence of suppression, confer resistance to the arginine analog canavanine (cani-100), cause red pigmentation (ade2-1), or result in lysine auxotrophy (lys2-1). Cells harboring YCpMP2 are canavanine sensitive and form white, lysine-independent colonies.Loss of suppressor activity leads to the formation of canavanine-resistant, red or pink colonies unable to grow when replicated to lysine omission medium. Following mutant characterization in vivo, yeast DNA is isolated and transformed into Escherichia coli JF1754 (12) to retrieve the shuttle vector. Then the mutant SUP4-o genes are sequenced directly on linearized double-stranded YCpMP2 plasmid DNA by the dideoxynucleotide chain termination technique (17) by the procedure of Korneluk et al. (8).
We have characterized mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v. irradiation. Mutants were selected following treatment with 60 J/m2 u.v. light which reduced cell survival to 10% and increased the SUP4-o mutation frequency 100-fold above background. DNA sequence analysis of 120 mutants revealed that u.v. induced all types of base substitutions, although transitions, in particular G:C----A:T events predominated. In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered. The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting that they were targeted by u.v. photolesions. A substantial fraction of the mutations were detected at hotspots for u.v. mutagenesis. The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and [6-4]-photoproducts could form. Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where [6-4]-photoproducts would be expected to occur rarely. The possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v. lesions in yeast are considered.
A collection of 196 spontaneous mutations in the SUP4-o gene of the yeast Saccharomyces cerevisiae was analyzed by DNA sequencing. The classes of mutation identified included all possible types of base-pair substitution, deletions of various lengths, complex alterations involving multiple changes, and insertions of transposable elements. Our findings demonstrate that at least several different mechanisms are responsible for spontaneous mutagenesis in S. cerevisiae.
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