A stepwise physico-chemical dissection of nucleoli isolated from Chinese hamster ovary cells has allowed the isolation of actively transcribed regions of chromatin, highly enriched in ribosomal genes. Nucleolar chromatin could be separated into its various topographical components. This separation should improve our knowledge of the structure/function relationships in chromatin as the fine structure of the nucleolus is very sensitive to changes in ribosomal RNA synthesis.
The spatial distribution of ribosomal genes among the various chromatin areas of the nucleolus was assayed by hybridization with rRNA. In exponentially growing Chinese hamster ovary cells ribosomal genes are predominantly located in the intranucleolar stretches of nucleolus-associated chromatin. However, ribosomal sequences represent but a very minor part (less than 1 %) of intranucleolar DNA. In an attempt to improve the analysis of the actively transcribed chromatin areas, intranucleolar chromatin was submitted to additional fractionation steps involving, among others, isopycnic separation in metrizamide gradients. In these latter conditions a small subset of intranucleolar chromatin banded with preribosomal ribonucleoprotein. In this subfraction, about one-third of the DNA sequences corresponded to preribosomal RNA matrix units. The yield of recovery of this intranucleolar chromatin subfraction being closely related to the level of transcriptional activity of ribosomal genes, it appears likely that this separating procedure depends on the presence of ribosomal RNA transcription complexes and could, therefore, represent a general procedure for the isolation of actively transcribed areas from chromatin.