PUB - Publikationen an der Universität Bielefeld

Detailed kinetic data suggest that the direct transfer of plasmid DNA (YEp 351, 5.6 kbp, supercoiled, M(r) approximate to 3.5 x 10(6)) by membrane electroporation of yeast cells (Saccharomyces cerevisiae, strain AH 215) is mainly due to electrodiffusive processes. The rate-limiting step for the cell transformation, however, is a bimolecular DNA-binding interaction in the cell interior. Both the adsorption of DNA, directly measured with [P-32]dCTP DNA, and the number of transformants are collinearly enhanced with increasing total concentrations [D-t] and [Ca-t] of DNA and of calcium, respectively. At [Ca-t] = 1 mM, the half-saturation or equilibrium constant is <(K)over bar (D)> = 15 +/- 1 nM al 293 K (20 degrees C). The optimal transformation frequency is TFopt = 4.1 +/- 0.4 x 10(-5) if a single exponential pulse of initial field strength E(0) = 4 kV cm(-1) and decay time constant tau(E) = 45 ms is applied al [D-t] = 2.7 nM and 10(8) cells in 0.1 mi. The dependence of TF on [Ca-t] yields the equilibrium constants K-Ca(0) = 1.8 +/- 0.2 mM (in the absence of DNA) and K-Ca' (at 2.7 nM DNA), comparable with and derived from electrophoresis data. In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event. At E(0) = 4.0 kV cm(-1) and T = 293 K, the flow coefficient of DNA through the porous membrane patches is k(f)(0) = 7.0 +/- 0.7 x 10(3) s(-1) and the electrodiffusion of DNA is approximately 10 times more effective than simple diffusion: D/D-o approximate to 10.3. The mean radius of these pores is r(p) = 0.39 +/- 0.05 nm, and the mean number of pores per cell (of size empty set approximate to 5.5 mu m) is N-p = 2.2 +/- 0.2 x 10(4). The maximal membrane area that is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023% of the total cell surface. The surface penetration is followed either by additional electrodiffusive or by passive (after-field) diffusive translocation of the inserted DNA into the cell interior. For practical purposes of optimal transformation efficiency, 1 mM calcium is necessary for sufficient DNA binding and the relatively long pulse duration of 20-40 ms is required to achieve efficient electrodiffusive transport across the cell wall and into the outer surface of electroporated cell membrane patches.