Chapter 15 Methods for Selecting Auxotrophic and Temperature-Sensitive Mutants in Yeasts

Publisher Summary This chapter describes the methods for selecting auxotrophic and temperature-sensitive mutants in yeasts. Mutant selection is defined as those processes that enrich a culture for desired mutants by decreasing the number of viable nonmutant cells. Considerable pressure toward increasing the percentage of specific mutants can be exerted at each of the four basic steps in mutant isolation: (1) choice of parental strain, (2) mutagenesis, (3) enrichment of the culture for auxotrophic or temperature-sensitive ( ts ) mutants, and (4) identification of the desired mutants in the remaining cell population. Auxotrophic and ts mutations are usually recessive and therefore should be induced in a haploid strain. It is advisable to use a standard strain carrying at least one easily identified marker mutation. The chapter describes general enrichment procedures applicable to the selection of a wide variety of auxotrophic and ts mutants in yeasts. Although used primarily for Saccharomyces cerevisiae , these techniques appear to be usable with most species of yeast. Generalized procedures for selecting auxotrophs with nystatin and for selecting ts mutants by tritium suicide are given. These are followed by specific protocols used for S. cerevisiae.