Random-spore analysis in Saccharomyces.

Abstract

An efficient method for separating ascospores and diploid cells in a mixed suspension has been developed. The sporulating culture is treated with snail enzyme to digest the ascus wall; the mixed suspension is treated with ultrasound which parts haploid ascospores and selectively kills diploid cells. The remaining portion of diploid cells can be further separated from the ascospores by a liquid paraffin phase. The ascospores being lipophilic tend to move into the paraffin phase and can, therefore, be separated from the hydrophilic diploid cells. Purified random-spore preparations have been obtained containing about 97% spores from an original population containing about 62% spores. The reliability and efficiency of the new method were evaluated by comparing the genetic linkage map of five genes obtained using this method with the map obtained by the method of tetrad analysis of the same five genes by Hawthorne and Mortimer (1963, 1966). The results of this work show that the random-spore suspension obtained by the new technique known as the ultrasonic-paraffin technique, can be used for genetic analysis of Saccharomyces cerevisiae. The technique as such introduced no factors that affected the viability of spores, at least, not those genotypes that segregated from the diploid strains used in these experiments.