Purification of Plant Ribosomal DNA: Use of Buoyant Density Centrifugation in Cesium Chloride-Actinomycin D Gradients and DNA-RNA Hybridization

The general features of isopycnic centrifugation of DNA and the separation of main-band from satellite DNAs in solutions of cesium chloride are discussed in the preceeding chapter. This chapter describes a modification of the procedure that enhances the separation of specific DNA sequences from main-band DNA. The method is based on the fact that actinomycin D binds preferentially to DNA molecules with a high-GC content. This lowers their density relative to main-band and other satellite DNAs so that they can be resolved separately. Cesium chloride-actinomycin D gradients have been used to isolate sea urchin histone genes (1 ) and plant ribosomal DNA (2 ), and to separate genes coding for plant 25S and 18S from 5S RNA genes (3 ) and as a prelude to the cloning of ribosomal DNA (4 ).