Yeast transformation without the spheroplasting process.

Abstract

A plasmid, YRp7, carrying the yeast TRP1 gene transformed a trpl strain JH of Saccharomyces cerevisiae without spheroplasting. The transformation conditions were that intact cells were incubated with the plasmid DNA in chilled CaCl2 solution and then the incubation temperature was raised. The transformation frequency changed markedly with the CaCl2 concentration of the solution and the temperature rise after incubation at 0°. The optimal CaCl2 concentration and temperature for strain JH were 200 him and 37°, respectively. Polyethylene glycol and the age of cells showed little effect on the

Genetic Transformation of Yeast

“…Cells were apparently transformed with plasmid vectors after treatment with Triton ® X-100; unfortunately, no experimental details or data were given, and we have been unable to find further information on this work. Two years later, Iimura et al (62) showed that intact yeast cells could be induced to take up the plasmid YRp7 by treatment with a 200 mM CaCl 2 solution. Transformants were recovered by plating onto directly selective medium.…”

Transformation in fungi.

“…A second and more widely adopted way to avoid making protoplasts is to use high concentrations of alkali metal ions to induce permeability to DNA in intact cells. limura et al (59) used 0.2 M calcium chloride to make yeast cells transformable with cloned DNA and obtained somewhat better transformation frequencies than when they used protoplasts. Ito et al (61) tried a variety of cations and found that 0.1 M lithium, supplied as the acetate salt, gave the best results.…”

“…The use of protoplasts for transformation was extended to the filamentous members of the class Ascomycetes N. crassa and Aspergil/lus nidlulans by Case et al in 1979 (26) and Tilburn et al in 1983 (134), respectively, and to several other species over the next few years ( Table 1). The original protocols have been varied and improved in detail (see below), but have not been fundamentally changed, except for the adoption by some groups (31,37) of the use of high concentrations of lithium ions as a means of rendering cell walls permeable to DNA without forming protoplasts: a procedure devised for S. (cere,isiae (59,61).…”

Genetic Transformation Systems in Fungi, Volume 1

“…Several methods were developed as alternatives to protoplasts such as the use of mutant strains with more permeable membranes or cell walls , the use of high concentrations of alkali metal ions to induce permeability to DNA in intact cells (Iimura et al 1983 ) and encapsulation of DNA in liposomes and fusion with protoplasts (Radford et al 1981 ). All of these methods were very ineffi cient (low transformant yields), non-reproducible, and in some cases the mutants were unstable.…”