[8] Isolation of Template Active and Inactive Regions of Chromatin

This chapter discusses the isolation of template active and inactive regions of chromatin. To minimize the cross-contamination of active and inactive regions, chromatin should be sheared to less than the size of an average unit of transcription. Most investigators have used mechanical methods to shear chromatin. Nucleolytic cleavage of chromatin DNA is gentle and does not lead to detectable levels of protein rearrangement during shearing or fractionation. Sheared chromatin has been separated into active and inactive regions by differential centrifugation and by chromatography on hydroxyapatite, agarose, and anion-exchange resin. A simple and rapid fractionation technique has been developed that involves selective precipitation of the inactive region with standard saline-citrate. Sheared chromatin can be fractionated into template active and inactive portions on the basis of predicted differences in the physical properties of the two regions. If separation without gross cross contamination is to be achieved, the size of the fragments is critical. Mild digestion with DNase II has the advantage over other reported techniques in that it yields substantially smaller fragments. Fractionation of chromatin with MgC1₂ provides a rapid and reliable means of separating active and inactive regions.