Isolation and characterisation of a transcribing polynucleosomal chromatin fraction

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Abstract
A method is described for preparation of a fraction of chromatin enriched in transcribing regions from nuclei of mouse GR cells. This fraction, released by mild staphylococcal nuclease digestion of isolated nuclei, contains 2-10% of the DNA as polynucleosomal chromatin together with 50-70% of pulse-labeled RNA and about 90% of all template-engaged RNA polymerase B molecules, titrated with (3H)-.alpha.-amanitin. Hydridization of DNA from this chromatin fraction to total nuclear RNA in excess shows that it is enriched in frequently-transcribed DNA sequences. A modification of the Miller technique, allowing the spreading of the active chromatin fraction for EM, was developed. Examination of the spreads reveals that this chromatin fraction contains 20-100 nucleosome-long polynucleosomal chains bearing lateral RNP [ribonucleoprotein] fibrils interpreted as nascent transcripts. The average length of the DNA fragments in the fraction is greater than that of average transcribed regions, suggesting that the transcribed regions are linked to flanking segments whose chromatin conformation probably contributes to the selective release of transcribing chromatin.