Ultrastructural and biochemical characteristics of reconstructed chromatin and synthetic nucleohistones

Abstract Calf thymus DNA was complexed with whole calf histones or individual histone fractions F1, F2a1, F2a2, F2b and F3. The composition of the precipitated nucleohistones was determined by the histone preparation rather than by the histone/DNA ratio used. Moreover, electron microscopic examination of the mixtures showed mainly aggregates of nucleohistone against a background of free DNA at histone/DNA ratios of less than 1. Hence, the complexing of histones to DNA occurs in a co-operative manner. Even in conditions of histone excess, 45–60% of the DNA-phosphate groups were free to react with toluidine blue. In these preparations no free DNA was seen with the electron microscope and the template activity of the complexes was very low. Hence, the occurrence of free DNA phosphate groups is more likely to be due to steric interference with further histone binding than to the presence of free DNA. Chromatin was also reconstituted from DNA, whole histones and non-histone proteins. This material had a template activity similar to that of native chromatin and had 47% free DNA-phosphate groups, like native chromatin. Moreover, the addition of extra histones to native chromatin did not significantly reduce either the template activity or the fraction of free DNA-phosphate groups. These findings substantiate the hypothesis that non-histone proteins facilitate transcription from nucleo-proteins by opposing the effect of histones. The electron microscopic appearance of ‘reconstituted chromatin’ was indistinguishable from that of native chromatin. The appearance of a complex formed with DNA and whole histones was very similar but not identical. That formed between DNA and F3 histone contained some regions resembling the structure of native chromatin but also different structures. The remaining histones formed complexes with DNA which were clearly different. The complexes formed with F1 histone had some unique features which distinguished them from other complexes.