Transfer of mitochondrial function into a cytoplasmic respiratory-deficient mutant of Saccharomyces yeast by electro-fusion

Abstract

Electric field-induced fusion was induced between Saccharomyces cerevisiae protoplasts from the ρ (-) heterozygous diploid strain 2114 and the respiratory-competent diploid strain 3441, carrying chromosomal markers. Close membrane contact between the cells of the two different strains (ratio 1:1) was achieved by dielectrophoresis in a weak inhomogeneous alternating field (about 1 kV/cm, 2 MHz). Due to dielectrophoresis pearl chains of two or more cells of the two strains are formed between the electrodes. Cell fusion was induced by application of two single square field pulses sufficiently high to induce reversible electrical breakdown in the membrane contact zone between cells within a pearl chain (about 7 to 8 kV/cm field strength and 40 Ms duration). The two subsequent pulses were applied at an interval of about 10 s.Hybrids could be isolated on selection medium in a high yield (compared with conventional fusion techniques). The hybrids were diploid, respiratory-competent and produced prototrophic spores. Thus, the fused hybrids contained only the chromosomal markers of strain 2114 and the cytoplasmic marker for respiratory competence from strain 3441; electro-fusion thus resulted mainly in plasmogamy.