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Isolation and characterisation of a transcribing polynucleosomal chromatin fraction
Author(s) -
Wurtz T.,
Fakan S.
Publication year - 1984
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/j.1768-322x.1984.tb00206.x
Subject(s) - chromatin , biology , dna , rna , nucleosome , nuclease , microbiology and biotechnology , rna polymerase , cell nucleus , polymerase , fraction (chemistry) , biophysics , genetics , gene , nucleus , chemistry , chromatography
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 to 10% of the DNA as polynucleosomal chromatin together with 50‐70% of pulse‐labelled RNA and about 90% of all template‐engaged RNA polymerase B molecules, titrated with (3H)‐alpha‐amanitin. Hybridisation 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 electron microscopy, has been developed. Examination of the spreads reveals that this chromatin fraction contains 20‐100 nucleosome‐long polynucleosomal chains bearing lateral RNP 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.