Isolation and Identification of Yeasts from Natural ... - Library Science

CHAPTER 5
Protoplast Fusion
in Saccharomyces cerevisiae
Brendan P. G. Curran
and Virginia C. Bugeja
1. Introduction
Succharomyces cerevisiue can exist as either a haploid cell of either
Mat a or Mat a mating type, a diploid cell or a polyploid cell, but the
transfer of genetic material is normally restricted to conjugation between
haploids of opposite mating type. It is possible, however, to isolate
genetically stable yeast hybrids from strains that are unable to mate by
using protoplast fusion. In this process, the yeast cell walls are enzymati-
tally removed, the resulting protoplasts are fused together using poly-
ethylene glycol (PEG) and calcium ions (Ca2+), before being embedded
in agar under appropriate selective conditions. Colonies of genetically
stable hybrids are normally visible within 5-10 d at 25°C.
The mechanism by which PEG and Ca2+ bring about protoplast
fusion is unknown, but it is thought that the PEG has the dual role of
diminishing the electrostatic field between the lipid membranes (I) and
removing water from the protoplasts. This allows extensive areas of
lipid from different protoplasts to come into contact with one another.
The calcium ions appear to perturb the membrane structure (2), resulting
in the intermingling of lipids from both membranes, and small cyto-
plasmic connections are made. Finally, as the protoplasts are rehy-
drated, they swell and the cytoplasmic connections coalesce, generating
a heterokaryon.
From: Methods m Molecular Biology, Vol 53 Yeast Protocols
Edited by: I Evans Humana Press Inc , Totowa, NJ
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