7] Fractionation of Chromatin

Abstract

Publisher Summary This chapter summarizes four simple convenient procedures where one or more obvious criteria for the separation of functionally different fractions have been applied. These include the association of nascent RNA chains, detection of transcribed DNA sequences by molecular hybridization, in vitro template activity, and thermal denaturation behavior. Thymocyte chromatin, fragmented by sonication, was separated by differential centrifugation into two fractions corresponding by biochemical and ultrastructural criteria to eu- and heterochromatin. The sucrose gradient method is modified from earlier methodology by employing a very steep sucrose gradient that facilitates a clean separation of the two fractions. The method was optimized for Drosophila melanogaster cultured cell chromatin in which approximately one-third of the DNA is transcribed, but the general approach appears to be valid for mammalian cells. More recent evidence suggests that although chromatin can be fractionated, the basis of fractionation may be the nonrandom distribution of proteins along the chromatin fiber, rather than the differences in the overall structure of active and inactive regions of chromatin.