In Vitro Transcription in Plant Nuclei
Isolated plant nuclei can be used for fundamental studies on the transcription apparatus. Total RNA polymerase activity can be measured using plant nuclei, and by using different α-amanitin concentrations in the enzyme assay, the individual RNA polymerase I, II, and in activities can be measured (1, 2, 3, 4, 5). The assay procedure involves the incubation of nuclei in the presence of the four substrates for RNA synthesis: ATP, GTP, CTP, and UTP. If one of these precursors is supplied as a radio-labeled molecule, transcription can be detected as incorporation of radioactivity into acid-insoluble material. Following incubation, transcription products are precipitated with TCA, collected and washed on glass fiber filter discs, and counted by liquid scintillation counting.
KeywordsMagnesium Glycerol Phenol Chloroform MgCl2
- 1.Guilfoyle, T. J. (1983) DNA-Dependent RNA Polymerases of Plants and Lower Eukaryotes, in Enzymes of Nucleic Add Synthesis and Modification Vol. 2 (Jacob, S. T., ed.) CRC, Florida.Google Scholar
- 4.Guilfoyle, T. J. and Key, J. L. (1977) Purification and Characterisation of Soybean DNA-Dependent RNA Polymerases and the Modulation of Their Activities During Development, in Nucleic Acids and Protein Synthesis in Plants (Bogorad, L. and Weil, J., eds.) Plenum, New York.Google Scholar
- 6.Slater, R. J. (1987) In Vitro Transcription, in Techniques in Molecular Biology Vol. 2 (Walker, J. M., ed.) Croom Helm, London, Sydney, 203–227.Google Scholar
- 9.Marzluff, W. F. and Huang, R. C. C. (1984) Transcription in Isolated Nuclei, in Transcription and Translation: A Practical Approach (Hames, B. D. and Higgins, S. J., eds.) IRL, Oxford, Washington DC, 89–129.Google Scholar