Chapter 13 The Nucleoprotein Hybridization Method for Isolating Active and Inactive Genes as Chromatin

Abstract

This chapter describes a method for fractionating chromatin that is based on specific hybridization between soluble chromatin fragments and oligonucleotides. This method, termed “nucleoprotein hybridization,” targets a specific gene on the basis of its DNA sequence. This technique makes it possible to isolate the chromatin of defined DNA sequence irrespective of its functional state. Alternative protocols for chromatin fractionation have employed differential solubility of chromatin fragments after mild nuclease digestion, immunoprecipitation, and mercury affinity columns. These alternative techniques yield a fraction enriched in active or inactive chromatin representing an ensemble of different genes. The chapter schematically represents seven steps of nucleoprotein hybridization. The feasibility of the technique has been proved with the SV40 model system. SV40 could be mixed with an excess of exogenous sea-urchin chromatin and then isolated to 88% purity corresponding to a 115-fold enrichment. The chapter also describes the first purification of active and inactive cellular genes as chromatin using the nucleoprotein hybridization. The Strongylocentrotus purpuratus sea urchin early histone gene repeat (SUEHGR) was chosen as a target for the first application of the technique. © 1991, Elsevier Inc. All rights reserved.