In-vitro transcription in tobacco chromatin

✍ Scribed by Roger E. Calza; Paul F. Lurquin

• Book ID: 104753369
• Publisher: Springer-Verlag
• Year: 1983
• Tongue: English
• Weight: 592 KB
• Volume: 159
• Category: Article
• ISSN: 0032-0935
• DOI: 10.1007/bf00392989

✦ Synopsis

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 rain at 30 ~ C. Transcriptional activity was maximally stimulated at 10 mM MgC12, 200 mM (NHr 4 and 150 mM KC1. 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, ~-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.