Yeast spheroplasting

Yeast spheroplasts are more fragile than cells with an intact cell wall. To avoid a reduction in transformation efficiency, the yeast should be handled with care once the Zymolyase solution is added. For example, yeast spheroplasts should not be vortexed, and pipetting should be carried out with wide-bore pipette tips. 

It is normal for it to take 2-3 min to completely resuspend the yeast spheroplasts. Yeast spheroplasts are more difficult to resuspend than yeast cells with intact cell walls. 

Optional At this point, yeast spheroplasts can be resuspended in sorbitol/DMSO solution and stored at -80°C for later use if a seven to ten times reduction in transformation efficiency is acceptable. If this route is chosen, resuspend the yeast... 

Optional The yeast transformation procedure can be carried out from this point using previously frozen yeast spheroplasts (see Note 10). If this route is chosen, thaw spheroplasts on ice, harvest them at 1,500 for 8 min in a microfuge, and then resuspend them in 200 pi STC. 

Large molecules are transformed into yeast by incubation of yeast spheroplasts with the DNA in the presence of polyethylene glycol (5). The transforming DNA must be isolated carefully to avoid breakage (this can be done in agarose), and the spheroplasts must be handled gently to avoid lysis. 

BurgersPM,PercivalKJ(1987)Transformation of yeast spheroplasts without cell fusion. Anal Biochem 163 391-397... 

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... 

Fig. 10. Electron micrographs of mitochondrial profiles in growing yeast spheroplasts. Cytoplasmic ribosomal particles can be seen attached to the outer membrane of the mitochondria (M), the outer membrane of the nucleus (N), and to the membrane of the endoplasmic reticulum (ER), but not to the plasma membrane (PM) or the vacuolar membrane (VM). (A) 55,000 X (B) 48,000 x (C) 48,500x (D) 66,000 x (E) 38,000 x. (From Kellems et al )...

The removal of 30-35 % PolyPs from yeast cells during the lysis of cell walls by the snail enzyme was observed in Saccharomyces carlbergensis (Vagabov et al., 1973) - these were alkali-soluble fractions (Table 5.3). A comparative investigation of the amounts of various PolyP fractions was carried out in E. magnusii spheroplasts (Table 5.4.) and some sub-cellular fractions (Table 5.5). All of these data confirm the idea of PolyP localization in different compartments of cells of the lower eukaryotes. 

Table 8.2 PolyP content (mg of Pi per g of dry cell biomass) in the cells, spheroplasts and vacuoles of S. cerevisiae. The yeast was grown for 4 h in a medium with 9 mM P (+P), then for 7 h in Pi-free medium (—P) and finally for 2 h in a medium with 9 mM P (+P, phosphate overplus) (Trilisenko et al., 2002).

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. 

Yeast Genetics. The yeast strain used in these studies was YGXD8 (MAT a /ew2-3 leu2-112). Yeast cells were transformed by the spheroplast method of Hinnen et al. (21). The transformed cells were maintained in YNB medium (0.7% yeast nitrogen base) supplemented with 5% glucose. For induction of polyphenolic protein synthesis, the cells were cultured in YP medium (1% yeast extract, 1% bacto-peptone) supplemented with 4% glucose and 2% galactose. 

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. 

Verwoert, I.I.G.S., Ykema, A., Valkenburg, J.A.C., Verbree, E.C., Nijkamp, H.J.J. and Smit, H. (1989) Modification of the fatty acid composition in lipids of the oleaginous yeast Apiotrichum curvatum by intraspecific spheroplast formation. Appl Microbiol Biotechnol. 32, 327-333. 

This is a late-log-phase culture. VL6-48 spheroplasts efficiently even at this late growth stage. Other yeast strains may not spheroplast efficiently unless harvested at early mid-log phase (about 10 cells/ml). 

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