Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation

Abstract

Murine erythroleukemic cells accumulate cytoplasmic globin mRNA during differentiation induced in tissue culture by dimethyl sulfoxide. Cellular accumulation of globin RNA may reflect transcriptional activation of the globin genes and/or posttranscriptional stabilization of globin RNA during differentiation. To evaluate possible transcriptional controls directly; globin RNA synthesis by isolated erythroleukemic cell nuclei was studied. Conditions were established for optimal nuclear RNA synthesis in vitro in the presence of a mercurinucleotide (Hg-CTP). Mercurated RNA synthesized in vitro was purified free of endogenous RNA by affinity chromatography on sulfhydryl-Sepharose, and analyzed for the presence of newly synthesized globin RNA sequences by molecular hybridization to globin complementary [32P]DNA. The results demonstrate markedly increased synthesis of globin RNA by nuclei isolated from dimethyl sulfoxide-treated cells, even within 5 min of nuclear transcription in vitro. These findings are most consistent with transcriptional activation of the globin genes upon induction of differentiation.