Reconstitution of chromatin at physiological ionic strength and regulation of transcription of the reconstituted chromatin.

Rat liver and simian virus 40 (SV40) chromatin were reconstituted in vitro under physiological conditions of ionic strength and temperature. The nucleosome assembly under the conditions was mediated in the presence of chromatin extracts, rich in nicking-closing activity, from rat liver or cultured CV-1 cells. The frequency of nucleosome assembly on DNA was dependent on both the incubation time and the weight ratio of histone to DNA. The regulatory effects of host cellular histones on the transcription of SV40 DNA were investigated by using reconstituted SV40 chromatin containing or lacking histone H1. The cellular histones composing the chromatin were prepared from permissive CV-1 cells. Transcription of chromatin was analyzed in vitro using Escherichia coli DNA-dependent RNA polymerase. The rate of incorporation of ribonucleoside triphosphates into RNA synthesized on SV40 chromatin containing Hl as the template was 5 to 10% of the rate for RNA synthesized on supercoiled SV40 DNA. The rate of incorporation for SV40 chromatin lacking Hl was approximately 40 to 50% of that for SV40 DNA. RNA products transcribed from both these chromatin and SV40 DNA were fairly homogeneous 16 to 28S species with several identical peaks.