Distribution of messenger RNA-coding sequences in fractionated chromatin

Abstract

Chromatin from Friend erythroleukemia cells has been fractionated according to the DNAase II/ Mg ++ solubility method of Gottesfeld et al. (1974). Chromatin was separated into nuclease-sensitive and -resistant fractions after mild DNAase II digestion; the enzyme-solubilized material was further fractionated into Mg ++-soluble and -insoluble fractions. The Mg ++-soluble fraction of Friend cell chromatin represents 15–18% of total chromosomal DNA. The distribution of transcribed and nontranscribed DNA sequences in these chromatin fractions has been determined. When grown under standard conditions of cell culture, Friend cells contain only low levels of globin mRNA (about 130 copies per cell cytoplasm). Upon stimulation with 1.5% dimethylsulfoxide, these cells produce 10-fold higher amounts of cytoplasmic globin message. While no enrichment for globin nucleotide sequences is found in the Mg ++-soluble fraction of nonstimulated cells, globin sequences are significantly more abundant in the Mg ++-soluble fraction of dimethylsulfoxide-treated cells. DNA sequences complementary to poly(A)-containing cytoplasmic RNA are also preferentially localized in the Mg ++-soluble fraction of Friend cell chromatin. Upon DMSO induction, transcription of 45S rRNA precursor is inhibited ( Sherton and Kabat, 1976). We find that DNA sequences coding for rRNA are 4–5 fold more abundant in the Mg ++-soluble fraction of uninduced cells than in the insoluble chromatin from these cells; no such difference, however, is found between the chromatin fractions obtained from DMSO-induced cells. Nontranscribed highly repetitive DNA sequences are concentrated in the inactive chromatin fraction. These data support the hypothesis that the Mg ++-soluble fraction of isolated chromatin corresponds to the portion of the genome which is transcriptionally active in vivo.