Composition of native and reconstituted chromatin particles: direct mass determination by scanning transmission electron microscopy

Abstract

Chromatin particles reconstituted from 145-base-pair lengths of DNA and either the arginine-rich histones H3 and H4 only or all four nucleosomal core histones have been compared with native nucleosomes in terms of their ultrastructure and mass distribution, as determined by scanning transmission electron microscopy (STEM). The mass of the nucleosome derived from STEM analysis was very close to that calculated for its DNA and histone components. The reconstituted particles showed a broader mass distribution, but it was clear that the majority contained at least eight histone molecules. This was to be expected for structures reconstituted from all four core histones, but in the case of H3H4-DNA complexes clearly showed that an octamer rather than tetramer of these histones was required to fold nucleosomal DNA into a stable compact particle. The significance of the H3H4 octamer complex with respect to nucleosomal structure is discussed, and the evidence that nucleosomal DNA can accept even greater numbers of histones is considered.