Abstract
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 [3H]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.
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Abbreviations
- SCV:
-
sedimented cell volume
- TCA:
-
trichloroacetic acid
- UTP:
-
uridine-5′-triphosphate
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Calza, R.E., Lurquin, P.F. In-vitro transcription in tobacco chromatin. Planta 159, 172–177 (1983). https://doi.org/10.1007/BF00392989
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DOI: https://doi.org/10.1007/BF00392989