The effect of micrococcal nuclease and DNAase I on bulk and 5 S RNA syntheses in isolated HeLa cell nuclei

Abstract The effect of micrococcal nuclease and DNAase I on bulk and 5 S RNA syntheses was studied using isolated HeLa cell nuclei. Chromatin DNA isolated from micrococcal nuclease-digested nuclei ran as a series of bands that are multiples of a smallest size on agarose gel. According to the increment of the nuclease used, length of DNA has been shown to converge into mononucleosomal size. To measure transcriptional activity of thus-treated nuclei, EGTA, an effective chelating agent for calcium ion that is an essential component for micrococcal nuclease to exert its activity but not for RNA polymerases, was employed. The level of RNA syntheses stays rather constant up to the digestion with 125 units/ml of the nuclease, but at higher concentrations both bulk and 5 S RNA synthesis decreased. The proportion of 5 S RNA in bulk RNA synthesized by nuclease-treated nuclei remained constant. The size of 5 S RNA sequence as measured by Sephadex G-100 gel filtration in combination with hybridization with cloned Xenopus 5 S DNA coincided with that of authentic in vivo 5 S RNA. On the other hand, chromatin DNA derived from DNAase I-digested nuclei showed rather smear-like pattern. Transcriptional activity was also inhibited as the digestion proceeded. Both bulk and 5 S RNA syntheses appeared to be inhibited to the same extent and the invariable ratio was again observed. The inhibition of transcription was, however, observed already at a very low concentration of DNAase I at which there is no remarkable change in nuclear DNA size from that of untreated control, indicating that active class III genes are also preferentially digested by DNAase I like many class II genes. The majority of 5 S RNA sequences produced by partially DNAase I-digested nuclei was again 5 S in size. These results suggested that micrococcal nuclease and DNAase I inhibit the nuclear transcriptional activity equally effectively, irrespective of size of RNA species to be transcribed, and that long intact structure might be required for the transcription of even small genes.