In vitro mutagenesis of the yeastSUP4-o gene to identify all substitutions that can be detected in vivo with theSUP4-o system

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Abstract
SUP4-o, a suppressor tRNA gene, is the target in a system for characterizing mutational specificity in the yeast Saccharomyces cerevisiae. To date, screening for loss of suppression has detected 172 of the 267 base-pair substitutions possible in the exons and intron of the SUP4-o gene. Although many of the remaining 95 changes might not be detected by this screen, some might occur spontaneously, or be induced, only at very low frequencies. For the purpose of analyzing mutational specificity, it would be valuable to determine which of these substitutions can be detected with the genetic screen employed in this system and which cannot. Thus we used in vitro mutagenesis to generate the 95 substitutions not yet detected in SUP4-o in vivo. Assessment of the phenotypes conferred by these 95 directed mutations revealed that 50 would completely escape detection and only 45 would pass through the first stage of the screen. Of these 45 substitutions, 2 are detectable but have not yet been found among more than 5,000 characterized SUP4-o mutations that arose in vivo. In addition, 4 others should be detected by slightly relaxing the current criteria for selection of SUP4-o mutants. The results indicate that with these modifications the system can detect 174/225 substitutions possible in the SUP4-o exons and 4/42 in the intron.