CONDITIONS FOR GENE-SPECIFIC TRANSCRIPTION IN ISOLATED-NUCLEI FROM TOMATO CELL-CULTURES

An improved method for the isolation of nuclei in a high yield from cultured tomato cells (Lycopersicon peruvianum) is described. The nuclei are active in RNA synthesis by run-on transcription. The transcripts were polydisperse in size without discrete peaks of RNA after polyacrylamide gel electrophoresis. Hybridization of the in vitro labelled RNA to DNA probes showed, that rRNA and mRNA genes were transcribed. Optimal conditions for alpha-amanitin sensitive (polymerase II) and insensitive (polymerase I) transcriptional activities were determined. Only the alpha-amantin sensitive activity was stimulated by increasing concentrations of monovalent (ammonium sulfate) and divalent (magnesium) cations. Dot blot hybridization of the in vitro labelled transcripts to clones containing sequences for rRNA and two heat shock proteins has shown that the optimal specific transcription of rRNA but also of the mRNAs occurred at lower salt concentrations. Under conditions of maximal RNA polymerase II activity (high salt concentrations) no increase in transcription of specific mRNA genes could be observed.