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

Abstract

Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the rale of direct gene transfer to 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 DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane‐permeabilizing compounds. When comparing total DNA 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 DNA polymerase activity negatively. but dideoxythymidine triphosphate‐sensitive activity was not influenced. We conclude that the DNA strand‐breaks induced by low doses of X‐rays. UV‐light or bleomycin do not increase the total or the repair‐DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand‐breaking is not preceded by enhanced DNA polymerase activity.

Studies of the mechanism of transgene integration into plant protoplasts: improvement of the transformation rate

“…It must be regarded as unlikely that irradiation eases the access of DNA into the nucleus, otherwise we would expect increased transient expression after that treatment . Measurement of the activity of DNA polymerases in intact protoplasts (Benediktsson et al ., 1994) showed that adding external template DNA increased' the activity of these nuclear enzymes . This effect of added DNA was further increased in the presence of PEG .…”