Sporulation of products of protoplast fusion without regeneration in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, diploid strains which are respiratory deficient (e.g., rho-) or are homozygous for the mating-type locus (i.e., either a/a or alpha/alpha) are unable to sporulate. In order to induce sporulation in these nonsporulating strains, the technique of protoplast fusion mediated by polyethylene glycol was adopted. In this study, the products of protoplast fusion were induced to sporulate without reversion to normal cells. Protoplasts from a respiratory-deficient diploid strain were mixed with those from a respiratory-competent haploid one carrying mitochondrial drug resistance markers, treated with 30% polyethylene glycol-4000 and 25 mM CaCl2, and incubated in 0.1 M potassium acetate containing 0.8 M sorbitol as an osmotic stabilizer. After two days' incubation, asci with three to eight spores were formed at a frequency of 1 x 10(-3) to 2 x 10(-4). Sporulation was also observed in products of fusion between an a/a diploid and alpha haploid strains and between an alpha/alpha diploid and a haploid strains. The analysis of the genotypes of spores revealed that when fusion products were cultured under conditions for sporulation, karyogamy did not take place, diploid nuclei underwent meiosis, and both diploid and haploid nuclei were able to develop into spores.