Effect of n-alcohols on the electrotransformation and permeability of Saccharomyces cerevisiae

Abstract

The correlation between electrotransformation and electropermeability was studied in yeast cells following the modification of their membranes by treatment with n-alcohols. It was found that the number of transformed cells decreased with increase of chain length of the alcohols used as follows: methanol < ethanol < propanol < butanol. The electropermeability was unaffected by the prepulse n-alcohol treatment. The lack of a unidirectional link between permeability and transformation leads to the assumption that the mechanism of DNA introduction into the cell could not be interpreted solely as a result of the existence of pores.

Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation

“…(5.6 kbp, relative molecular mass Mr = 3.6 X 106) (Hill et al, 1986) compensates leu 2 mutation of S. cerevisiae (Ganeva and Tsoneva, 1993). The isolation and purification of the plasmid DNA in the supercoiled form was performed using the method of Maniatis et al (1982).…”

“…total concentration of Ca concentration of free Ca total concentration of DNA concentration of bound DNA concentration of DNA permeable pores cell radius degree of DNA binding or adsorption to cell surface diffusion coefficient of DNA electrodiffusion coefficient of DNA initial field strength effective field strength elementary charge vacuum permittivity dielectric constant of water dielectric constant of lipids Faraday constant degree of cell transformation conductivity factor mean electrical potential difference across the electroporated membrane patches equilibrium constant of overall DNA binding kp electroporation rate coefficient [s-'] choice in cell biology and medicine. Since the early documentations of direct gene transfer by electroporation producing stable transformants of mammalian culture cells (Neumann et al, 1982;Wong and Neumann, 1982;Falkner et al, 1984;Evans et al, 1984;Potter et al, 1984), intact yeast cells and yeast spheroplasts have also been electrotransformed (Karube et al, 1984;Hashimoto et al, 1985;Meilhoc et al, 1990;Ganeva and Tsoneva, 1993). Among the various electrical and biological parameters characterizing electrotransformations of cells, the electroporative gene transfer is greatly facilitated by the adsorption of DNA to the cell surface (Wolf et al, 1989;Xie and Tsong, 1990a,b, 1992Neumann, 1992).…”