RNA Is an Integral Component of Chromatin that Contributes to Its Structural Organization
Figure 3
Treatment with RNase A alters the sedimentation behavior of purified chicken liver chromatin.
A) Chicken liver chromatin, prepared by micrococcal nuclease digestion of purified nuclei, was subjected to sedimentation through a linear 5%–30% sucrose gradient after treatment with RNase A (bottom panel) or not (top panel). After centrifugation and fractionation, samples were subjected to total nucleic acids extraction and analyzed by electrophoresis in 1% agarose-TBE gels. Fraction numbers are indicated. Lanes M correspond to molecular weight markers. Quantitation of the results is shown on the right where the average molecular weight (M) of the chromatin fragments contained in each fraction, expressed as bp of DNA, is presented as a function of the fraction number: (°) untreated chromatin, (•) chromatin treated with RNase A. B) Histone content of chromatin fractions 11 and 12 of the gradients shown in A) was analyzed by SDS-PAGE: untreated chromatin (lanes 4 and 5), chromatin treated with RNase A (lanes 6 and 7) and, as controls, H1 from calf thymus (lane 1), RNase A (lane 2) and hydroxylapatite-purified core histones from chicken liver (lane 3). The gel was stained with silver. Quantitation of the results is shown on the right where the H1/H2A ratio of fractions 11 and 12 is presented before (white columns) and after (black columns) treatment with RNase A. C) The sedimentation behavior of chicken liver chromatin was determined before (left panel) and after treatment RNase A either in the presence of anti-RNase (Ambion) (central panel) or in the absence of any added inhibitor (right panel). Fraction numbers are indicated. Lanes M, correspond to molecular weight markers. Quantitation of the results is shown on the right: (°) untreated chromatin, (▴) chromatin treated with RNase A in the presence of anti-RNase, (•) chromatin treated with RNase A in the absence of any added inhibitor.