Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

Abstract

PHYSIOLOGIA PLANTARUM 90: 445-450. 1994 cv.,,vr,>A/© Wra;,,(o»«,J P'iiiii'd iit Ih'iuiHii'k -nil I'l^'his rt'scrrfd O. 1994. Analy.sis of DNA polymerase acti\itv in Petunia protoplasts treated with clastogenic agents. -Physiol. Plant. 90: 445-450.Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the rate of direct gene transfer lo protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DN.^ polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. Wben comparing total DN.A polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DN.A polymerase activity negatively, but dideoxy thymidine trtphosphate-sensitive activity was not influenced. We conclude thai the DNA strand-breaks induced by low doses of X-rays. UV-Iight or bleomycin do not increase the total or the repair-DN.A polymerase activity and. therefore, that the increase in the transfomiation rates after DNA strand-breaking is not preceded by enhanced DN.A polymerase activity.