A comparison of procedures for fractionating hepatic chromatin

Abstract

Rat hepatic chromatin was fractionated into three components, a slow sedimenting fraction, a fast sedimenting fraction, and a fraction designated pelleted chromatin using either glycerol gradient centrifugation, or selective MgCl 2 precipitation. An enhanced amount of nascent RNA was found to be associated with both the slow sedimenting chromatin and the MgCl 2 soluble chromatin. The nascent RNA was shown to be physically complexed with the chromatin DNA on the glycerol gradient. The slow sedimenting chromatin also had a significantly greater in vitro template activity when compared to the fast sedimenting and the pelleted chromatin. A direct comparison of the MgCl 2 and glycerol gradient procedures demonstrated an overlap of 60–85% in the corresponding chromatin fractions. The pelleted chromatin was shown to be partially composed of chromatin that was not converted to a less dense conformation by extensvie sonication. Furthermore, studies indicated that less of the DNA isolated from the pelleted chromatin was available for transcription by E. coli RNA polymerase when compared to the slow and fast sedimenting chromatin. We conclude that the pelleted chromatin is not an artifact of the fractionation procedure, but may contain DNA base sequences not found in the other two chromatin fractions. These methods should be useful in reproducibly obtaining chromatin fractions demonstrating many of the biochemical characteristics of euchromatin and heterochromatin.