Release of transcriptionally active chromatin from liver nuclei by micrococcus nuclease

Abstract

Chromatin-bound and free RNA polymerase activity was measured in nuclei before and after treatment with micrococcus nuclease. Isolated rat liver nuclei were incubated with the enzyme at concentrations of 0.2, 1.0, or 20 units/mg nuclear DNA. Chromatin was fractionated by differential centrifugation into a low-speed pellet, a high-speed pellet, and a supernatant fraction. In the low-speed pellet increasing concentrations of nuclease resulted in decreasing absorbance at 260 nm. A diminution in total chromatin-bound RNA polymerase I and II activities was also observed. The high-speed pellet showed a 2-fold increase in absorbance, total RNA polymerase I activity rose 4-fold, and RNA polymerase II activity 40-fold. An enrichment of chromatin-bound enzymes was also found in the high-speed supernatant fraction. Free poly[d(A-T)] dependent RNA polymerases were distributed in all 3 chromatin subfractions but the majority was found in the high-speed supernatant fraction. The results showed that transcriptionally active chromatin was released from total chromatin by treatment with micrococcus nuclease. The transcriptionally active regions were verified by their bound RNA polymerase activity.