Purification of Soybean DNA-Dependent RNA Polymerase I on a Column of Plasmid pHFK 206 Covalently Attached to Agarose

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Abstract
The plasmid pHFK 206 consisted of plasmid pBR 322 and an 1.3 × 10-6 D insert, a cloned seg­ment of a soybean rRNA repeating unit with a preferential binding region for soybean RNA polymerase I, was coupled to cyanogen bromide activated agarose (Sepharose 4B) and used for affinity chromatography of RNA polymerases. It could be shown by elution profiles and sodium dodecylsulphate/polyacrylamide slab gels that highly purified RNA polymerase from Escherichia coli MRE 600 bound to pHFK 206- Sepharose as holoenzyme with an apparently full complement of σ-subunit. With initially purified soybean RNA polymerase I from chromatin of 2,4-D treated hypocotyls it was demonstrated that pHFK 206-Sepharose could be used as an affinity chromatography method for purification of soybean RNA polymerase I in high degrees and economy of time.

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