Spheroplasting medium, preparation

Approximately 1 p% of wild-type or mutant pCLI plasmid DNA is used for transformation of yeast cells. Spheroplasts of Saccharomyces cerevisiae strain GRF 18023,24 are prepared following the protocol of Burgers and Percival25 with some modification. High copy number transformants are directly selected on minimal medium plates lacking leucine and containing 8% glucose (to repress lysozyme expression) and 1 M sorbitol as an osmotic stabilizer. 

Novobiocin also causes chaining in Streptococcus faecium [23], although this effect is not produced in all cocci [13]. The induction of spheroplasts in Escherichia coli by novobiocin has also been reported [24] and, in fact, the antibiotic has been recommended for preparing spheroplasts in Gramnegative bacteria [25]. In contrast, however, it has been shown by various workers [26-28] that novobiocin does not induce spheroplasts in Serratia marcescens or in various strains of E. coli and [28] that it may even prevent spheroplast induction caused by benzylpenicillin in hypertonic medium (Figure 2.1). 

Yeast strain differences and/or differences in culture medium result in a range of efficiencies of spheroplasting with the enzyme preparations we use. Pretreatment has allowed us to employ the conditions described below with different strain backgrounds grown in different media. Because we have made changes to our previously reported method for preparing yeast nuclei (Aris and Blobel, 1991), which serves as the starting material for the isolation of nucleoli, we present the entire method for nuclei here. 

Due to the presence of a thick cell wall, it is difficult to observe detailed ultrastructural features of ribosome-membrane association in whole yeast cells. To circumvent this problem, we examined preparations of yeast spheroplasts capable to carrying out macromoleculer synthesis. This was done by first removing the cell wall by enzymatic digestion and then placing the spheroplasts in an osmotically stabilized growth medium... 

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