Isolation and characterization of rDNA-containing chromatin from nucleoli

Abstract

Fractions corresponding to ‘euchromatin’ and ‘heterochromatin’ were isolated from Novikoff hepatoma nucleoli by a modification of the DNase II method of Gottesfeld et al. [18, 19]. Nucleolar chromatin [36] was digested with DNase II, and ‘euchromatin’ was obtained as a soluble fraction following differential precipitation of ‘heterochromatin’ with Mg 2+. In this ‘euchromatin’ fraction, the rDNA was enriched approx. 8.5-fold over the unfractionated nucleolar chromatin, as determined by DNA-RNA hybridization. On 8–80% glycerol gradients, the ‘euchromatin’ separated into three discrete subfractions as with euchromatin obtained from the bulk chromatin [18, 19]. Two subfractions which sedimented at 14S and 20S, respectively, were consistently found after DNase II digestion (to 15 min) which contained all histone fractions. All euchromatin subfractions were enriched in rDNA and had a high protein/DNA ratio, a high RNA-DNA ratio, and a high template activity with exogenous RNA polymerase I. Fraction B with a sedimentation of 14S was most enriched in the parameters related to rRNA synthesis. The Mg 2+-precipitable fractions reported to be ‘heterochromatin’ [18, 19] not only contained substantial amounts of ribosomal genes, but also had higher template activities than unsheared chromatin. These results imply (1) nucleolar ‘euchromatin’ has a structural heterogeneity similar to that of the euchromatin containing active single copy sequences; (2) at least a portion of rDNA in ‘euchromatin’ is protected by proteins, including histones, which does not preclude active transcription of rDNA; (3) none of the currently used criteria for ‘euchromatin’ alone defines the chromatin containing rDNA; and (4) an appreciable enrichment of rDNA occurs in isolated euchromatin but not all the rDNA was separated into ‘euchromatin’ by the DNase II method.