Structural Investigations on Isolated Chromatin of Higher‐Order Organisation

Abstract

Chromatin of a high structural order was prepared by a method avoiding drastic mechanical stress, as well as strong changes of the ionic strength, and was investigated by small-angle X-ray scattering. The results obtained support the idea that the quaternary structure of completely hydrated chromatin in solution is a solenoid with a diameter of about 32-34 nm. Gel chromatography of this chromatin yields solutions containing mainly quaternary structures with differing portions of tertiary structure. Decrease of the ionic strength results in an increase of loosening and unfolding of quaternary structures. Reconstitution by readjustment of the physiological ionic strength indicates slight differences between reconstituted and original higher-order structures.Chromatin structure appears to be a hierarchical one. The elementary subunit is the nucleosome [I -61 of which the over all shape is well known now. Some details about the arrangement of D N A and histones within the nucleosome are established [7-111 and several models of primary organization of nucleosomes are discussed [8,12 -The next level of organization is the tertiary structure of chromatin which appears to be an arrangement of nucleosomes like 'beads on a string' or tightly packed like a rod with a diameter of about 10-11 nm in solvents of low ionic strength [1,16]. In solvents of higher ionic strength part of the chromatin is organized in a higher-order conformation [ 16 -321 forming a quaternary structure. In electron micrographs this sometimes appears as a 'knobby' fiber of about 25 -30 nm in diameter. However, whether the quaternary structure is organized in the form of 'superbeads' [26,27, 32-34] containing 6-12 nucleosomes or as a solenoid [17,[29][30][31] is still an open question [16].In order to investigate chromatin in solution it has first to be isolated. Even with mild methods avoiding shear and mechanical stress, one can obtain isolated chromatin only as a fragmentary material. A procedure often applied is the lysis of the cell nuclei in 0.2 mM EDTA [38]. In this solvent chromatin appears as a string of nucleosomes, i.e. the tertiary structure. Increasing the ionic strength of the solvent leads to reconstitution of higher-order structures. These quaternary structures appear to be very similar to the original ones in intact cell nuclei; however, it is not known how far they are really identical. By avoiding drastic changes of ionic strength during lysis of the nuclei it is possible to obtain solutions of chromatin having a highly preserved structural order [39,40]. For our structural studies applying small-angle X-ray scattering higher-order chromatin was obtained by a modification of the method of Rees et al. [39,41]. By enzymatic autolysing of the cell nuclei chromatin with a very high sedimentation coefficient can be isolated. Purification of this chromatin by gel chromatography results in solutions containing native quaternary structure and still a certain amount of tertiary structure. This accumulation of quaternary structure material ...

Organization of Transcriptionally Active and Inactive Chromatin

“…1978. Measurements of hydrodynamic properties (BUTLER and THOMAS 1980) and X-ray data (BRUST and HARBERS 1981) have also been interpreted in support of this continuous solenoid model (Fig. 4 a).…”