THE RELATIONSHIP BETWEEN CHROMATIN STRUCTURE AND TRANSCRIPTION

ABSTRACT Chromatin and DNA from Schneider's Drosophila melanogaster cell line 2 and from mouse L-cells were transcribed in vitro with E . coli RNA polymerase. Using mercurated UTP as precursor the newly synthesized RNA could be separated from DNA and endogenous RNA by affinity chromatography on sulfhydryl-Sepharose 6B. Characterization of the Drosophila transcription products with complementary DNA made from polyadenylated nuclear RNA and with fractionated cDNA probe revealed a quantitative fidelity in the in vitro transcript from chromatin which was not evident when DNA was transcribed. However, as shown by hybridization to total nuclear RNA, E. coli RNA polymerase transcribed both DNA strands from chromatin in vitro . Hybridization of L-cell transcription products with purified satellite heavy strand DNA demonstrated that although satellite sequences were undetectable in cellular RNA, a significant amount was present in the in vitro transcripts. Two different transcription systems, and several different preparation procedures for chromatin gave the same result. We therefore conclude that active chromatin is preferentially accessible to E. coli polymerase but that the controls over strand selection and repression of satellite expression are poorly recognized in vitro . Patterns of phosphorylation and acetylation of Drosophila histones were studied after whole cell incubation with 32p-orthophosphate or 3H-acetate. Radioactive phosphate was associated with H1, H3 and H4 and radioactive acetate was associated with H3, H4 and H2B. H3 showed the highest specific activity of both labels. Chromatin fractionation was performed to investigate the distribution of acetate and phosphate groups in histones from template-active and template-inactive regions. The levels of both acetate and phosphate groups were higher in histones from template-active regions.