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The isolation of multiple forms of dna dependent rna polymerases from nuclei of quiescent wheat embryos by affinity chromatography


, : The isolation of multiple forms of dna dependent rna polymerases from nuclei of quiescent wheat embryos by affinity chromatography. Zeitschrift fuer Pflanzenphysiologie 97(1): 43-58

A method is described which facilitated the rapid mass isolation of nuclei from wheat embryos (wheat germ). Routinely 5 .times. 107 nuclei/g wheat embryos were obtained. The nuclei showed a DNA:RNA:protein ratio of 1:0.3:3.5. DNA-dependent RNA polymerases were solubilized from isolated nuclei by sonication in TGED buffer including 0.5 M (NH4)2SO4. The isolation of all classes of RNA polymerases was achieved by affinity chromatography on heparin-Sepharose 4B containing 400 .mu.g covalently bound heparin/ml wet gel. Using heparin-sepharose and DEAE-cellulose chromatography it could be shown that nuclei of quiescent wheat embryos contained all 3 classes of RNA polymerases (the tripartite transcriptive system). RNA polymerase II which was further purified showed the typical subunit pattern as revealed by 10-15% acrylamide sodium dodecyl sulfate gel electrophoresis. The combination of heparin-Sepharose affinity chromatography and conventional chromatography on DEAE-cellulose and DEAE-Sephadex as a general procedure for the simultaneous isolation of all 3 classes of eukaryotic RNA polymerases is discussed.

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