The reconstitution of the histone octamer

Bibliography: pages 110-126.This thesis describes methodology for the reconstitution of the chicken erythrocyte octamer from acid-denatured histones or the natural H3-H4 tetramer and H2A-H2B dimers. Oligomeric properties of reconstituted octamers were elucidated during column chromatographic and chemical cross-linking studies. The conformational identity of the natural and reconstituted octamers was demonstrated by the ability of all preparations to crystallise as helical octamer tubes. The application of the reconstitution methodology in addressing fundamental problems of chromatin research, was demonstrated during subsequent studies, namely (i) The reconstitution of hybrid histone octamers containing a structural variant of a specific histone. These studies were undertaken to study the effect on histone-histone interactions in hybrid octamers of which erythrocyte H2B was substituted for by sea urchin sperm H2B(l) or erythrocyte H3 and H4 were substituted for by dethiolated H3 and sea urchin sperm H4 respectively. (ii) The reconstitution of an octamer suitable for the sitespecific derivatisation of a specific histone, or covalently labelled with aurothiomalate in a specific histone complex. These studies were concluded to represent general labelling strategies which may be of use in crystallographic or physico-chemical studies of nucleosome structure