In-vitro transcription in tobacco chromatin.

A method is described to isolate transcriptionally active Nicotiana tabacum L. cv. Xanthi chromatin from suspension-cultured cells. Enzymatic preparation of protoplasts with solubilization of the plasma membrane, Triton X-100 and homogenization resulted in chromatin free from cellular debris. Incroporation of [(3)H]uridine triphosphate into RNA increased for more than 30 min at 30° C. Transcriptional activity was maximally stimulated at 10 mM MgCl2, 200 mM (NH4)2SO4 and 150 mM KCl. The in-vitro synthesized RNA was found to contain 3.8% polyadenylated RNA. The results of digestion studies with ribonuclease, heat and detergent inactivation studies, α-amanitin and actinomycin D inhibitor effects, as well as dependency on ribonucleotide triphosphates, showed that the activity recorded in the tobacco chromatin was bona-fide enzymatic RNA transcription. There also was marked stimulation of transcriptional activity when exogenous DNA was added to the assay mixture.