In vitro transcription in tobacco nicotiana tabacum cultivar xanthi

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

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

1983


ISSN/ISBN: 0032-0935
Accession: 005652752

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Abstract
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 30.degree. 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.