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Nucleic Acids Research, 1980, Vol. 8, No. 15 3393-3411
© 1980

MOLECULAR BIOLOGY

The extraction by micrococcal nuclease of ghucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated

Jean André+, André Raynaud and Henri Rochefort*

Unité 148 I.N.S.E.R.M., 60, Rue de Navacelles, 34100 Montpellier, France

* Address for reprint requests

Received May 27, 1980.

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2°C with micrococcal nuclease. Three chromatin fractions were successively obtained : an isotonic extract (ne1), a hypotonic extract (ne2) and the residual pellet (P). The 3H Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear H Dex and the MMTV DNA sequences were extracted in a concentration dependent manner while only 10–15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per µg DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20–30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.

+ Present address : Unité 34 I.N.S.E.R.M.-Höpital Debrousse-29, Chetnin Soeur-Bouvier-69332 Lyon France.


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