Externally added DNA molecules support initiation of transcription in isolated nuclei from petunia

Nuclei isolated from protoplasts transfected with the pUC8CaMVCAT and pDO432 plasmids were able to support, in run off experiments, the synthesis of specific transcripts as was evident from analysis by dot blot hybridization. Also the addition of the above plasmids to nuclei, prepared from non-transfected protoplasts, supported the synthesis of specific transcripts. Dot blot analysis showed that most of the transcripts obtained were complementary to the relevant gene sequences. alpha-Amanitin, at concentrations which are known to block the activity of RNA polymerase II, significantly inhibited the synthesis of specific transcripts by the isolated nuclei. The transcription activity was found to be predominantly associated with the nuclear fraction while the transcription products (RNA molecules) appeared in the supernatant obtained following sedimentation of the nuclei.