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.

Electrical breakdown, electropermeabilization and electrofusion

“…The field conditions were similar to those described by both Halfmann et al (178)(179)(180) and Schnettler et al (90). This demonstrates that the application of an alternating field and high-intensity field pulses in the appropriate manner does not lead to any severe side effects within the cells and the membrane, but rather that more consideration needs to be given to the composition of the fusion media and to the system parameters (chambers, temperature, etc.…”

“…Halfmann et al (178) were the first to report that yeast protoplasts can be fused with high yields by means of the standard electrofusion technique (Fig. 34).…”

“…The progress in electrofusion of yeast protoplasts was mainly attributable to the use of new fusion chambers and new fusion media. Instead of the simple two electrode wire chamber used by Halfmann et al (178)(179)(180). Schnettler and Zimmermann (90,181)used the helical chamber (Fig.…”

Saccharomyces cerevisiae nuclear fusion requires prior activation by alpha factor.

“…After treatment with alpha factor, as many as 10% of the regenerated spheroplasts had fused nuclei. The requirement for activation by alpha factor explains the observation of previous workers that untreated spheroplasts can be made to fuse at high frequency, whereas nuclear fusion occurs at low frequency (12,28,29). In the absence of alpha factor, spheroplasts fused to form unstable heterokaryons.…”

“…Moreover, when spheroplasts fuse and karyogamy fails, the products of the fusion reaction are unstable heterokaryons, just as they are in karl-i crosses. The spheroplast fusion system is a good way to transfer mitochondria when cells are not treated with mating hormone (12), and it is an efficient way to produce a/a diploids when mating hormone is used. The spheroplast fusion system should therefore provide an efficient method for mating sterile mutants (11) that have defects in the pathway of cell fusion but still respond to mating hormones.…”