In vitro transcription in tobacco nicotiana tabacum cultivar xanthi

Calza, R.E.; Lurquin, P.F.

Planta (Heidelberg) 159(2): 172-177


ISSN/ISBN: 0032-0935
Accession: 005652752

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A method is described to isolate transcriptionally active N. tabacum L. cv. Xanthi chromatin from suspension-cultured cells. Enzymatic preparations of protoplasts with solubilization of the plasma membrane, Triton X-100 and homogenization resulted in chromatin free from cellular debris. Incorporation of [3H]uridine triphosphate into RNA increased for more than 30 min at C. Transcriptional activity was maximally stimulated at 10 mM MgCl2, 200 mM (NH4)2SO4 and 150 mM KCl. The in-vitro synthesized RNA contained 3.8% polyadenylated RNA. The results of digestion studies with ribonuclease, heat and detergent inactivation studies, .alpha.-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.