Selection of spontaneous mutants by inositol starvation in yeast

A new method for the routine isolation of mutations of spontaneous origin in the yeast, Saccharomyces cerevisiae, is reported. The technique is based on the observation that inositol auxotrophs die when deprived of inositol. However, if macromolecular synthesis is inhibited, most of the cells survive. Appropriate manipulation of inositol requiring mutants can therefore result in the selective survival of cells possessing mutations which affect macromolecular synthesis. Since reversion to inositol prototrophy can be a major source of interference in efficient selection, a haploid double mutant strain has been constructed which reverts to inositol prototrophy with a frequency estimated to be several orders of magnitude lower than the expected frequency of single, spontaneous mutational events. Using this strain, enrichment in excess of 10,000 fold has been obtained for various classes of auxotrophic mutants. Spontaneous temperature sensitive mutants have also been obtained.