Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1980 Aug 11;8(15):3393–3411. doi: 10.1093/nar/8.15.3393

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

J André, A Raynaud, H Rochefort
PMCID: PMC324159  PMID: 6255415

Abstract

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 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The 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 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and 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 3H 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 microgram 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.

Full text

PDF
3393

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ALLFREY V. G., LITTAU V. C., MIRSKY A. E. METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS. J Cell Biol. 1964 May;21:213–231. doi: 10.1083/jcb.21.2.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alberga A., Tran A., Baulieu E. E. Distribution of estradiol receptor and vitellogenin gene in chick liver chromatin fractions. Nucleic Acids Res. 1979 Dec 11;7(7):2031–2044. doi: 10.1093/nar/7.7.2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andre J., Raynaud A., Rochefort H. Characterization of the estradiol receptor extracted from nuclei by micrococcal nuclease. Biochemistry. 1978 Aug 22;17(17):3619–3626. doi: 10.1021/bi00610a031. [DOI] [PubMed] [Google Scholar]
  4. Andre J., Rochefort H. Estrogen receptor: loss of DNA binding ability following trypsin or Ca2+ treatment. FEBS Lett. 1973 Jun 1;32(2):330–334. doi: 10.1016/0014-5793(73)80866-x. [DOI] [PubMed] [Google Scholar]
  5. André J., Pfeiffer A., Rochefort H. Inhibition of estrogen-receptor-DNA interaction by intercalating drugs. Biochemistry. 1976 Jul 13;15(14):2964–2969. doi: 10.1021/bi00659a005. [DOI] [PubMed] [Google Scholar]
  6. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bellard M., Gannon F., Chambon P. Nucleosome structure III: the structure and transcriptional activity of the chromatin containing the ovalbumin and globin genes in chick oviduct nuclei. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):779–791. doi: 10.1101/sqb.1978.042.01.078. [DOI] [PubMed] [Google Scholar]
  8. Bloom K. S., Anderson J. N. Conformation of ovalbumin and globin genes in chromatin during differential gene expression. J Biol Chem. 1979 Oct 25;254(20):10532–10539. [PubMed] [Google Scholar]
  9. Bloom K. S., Anderson J. N. Fractionation of hen oviduct chromatin into transcriptionally active and inactive regions after selective micrococcal nuclease digestion. Cell. 1978 Sep;15(1):141–150. doi: 10.1016/0092-8674(78)90090-9. [DOI] [PubMed] [Google Scholar]
  10. Dudley J. P., Rosen J. M., Butel J. S. Differential expression of poly(A)-adjacent sequences of mammary tumor virus RNA in murine mammary cells. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5797–5801. doi: 10.1073/pnas.75.12.5797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Erdos T., Best-Belpomme M., Bessada R. A rapid assay for binding estradiol to uterine receptor(s). Anal Biochem. 1970 Oct;37(2):244–252. doi: 10.1016/0003-2697(70)90044-8. [DOI] [PubMed] [Google Scholar]
  12. Eriksson H., Upchurch S., Hardin J. W., Peck E. J., Jr, Clark J. H. Heterogeneity of estrogen receptors in the cytosol and nuclear fractions of the rat uterus. Biochem Biophys Res Commun. 1978 Mar 15;81(1):1–7. doi: 10.1016/0006-291x(78)91622-4. [DOI] [PubMed] [Google Scholar]
  13. Flint S. J., Weintraub H. M. An altered subunit configuration associated with the actively transcribed DNA of integrated adenovirus genes. Cell. 1977 Nov;12(3):783–794. doi: 10.1016/0092-8674(77)90277-x. [DOI] [PubMed] [Google Scholar]
  14. Garel A., Axel R. Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3966–3970. doi: 10.1073/pnas.73.11.3966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gottesfeld J. M., Butler P. J. Structure of transcriptionally-active chromatin subunits. Nucleic Acids Res. 1977 Sep;4(9):3155–3173. doi: 10.1093/nar/4.9.3155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  17. Hemminki K., Vauhkonen M. Distribution of estrogen receptors in hen oviduct chromatin fractions in the course of DNAase II digestion. Biochim Biophys Acta. 1977 Jan 3;474(1):109–116. doi: 10.1016/0005-2787(77)90218-0. [DOI] [PubMed] [Google Scholar]
  18. Houts G. E., Miyagi M., Ellis C., Beard D., Beard J. W. Reverse transcriptase from avian myeloblastosis virus. J Virol. 1979 Feb;29(2):517–522. doi: 10.1128/jvi.29.2.517-522.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Howard D. K., Schlom J. Isolation of host-range variants of mouse mammary tumor viruses that efficiently infect cells in vitro. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5718–5722. doi: 10.1073/pnas.75.11.5718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jensen E. V., DeSombre E. R. Mechanism of action of the female sex hormones. Annu Rev Biochem. 1972;41:203–230. doi: 10.1146/annurev.bi.41.070172.001223. [DOI] [PubMed] [Google Scholar]
  21. Lebeau M. C., Massol N., Baulieu E. E. Extraction, partial purification and characterization of 'the insoluble estrogen receptor' from chick liver nuclei. FEBS Lett. 1974 Jul 1;43(1):107–111. doi: 10.1016/0014-5793(74)81117-8. [DOI] [PubMed] [Google Scholar]
  22. Levy B., Baxter J. D. Distribution of thyroid and glucocorticoid hormone receptors in transcriptionally active and inactive chromatin. Biochem Biophys Res Commun. 1976 Feb 23;68(4):1045–1051. doi: 10.1016/0006-291x(76)90301-6. [DOI] [PubMed] [Google Scholar]
  23. Massol N., Lebeau M. C., Baulieu E. E. Estrogen receptor in hen oviduct chromatin, digested by micrococcal nuclease. Nucleic Acids Res. 1978 Mar;5(3):723–738. doi: 10.1093/nar/5.3.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Noll M. Subunit structure of chromatin. Nature. 1974 Sep 20;251(5472):249–251. doi: 10.1038/251249a0. [DOI] [PubMed] [Google Scholar]
  25. Panet A., Cedar H. Selective degradation of integrated murine leukemia proviral DNA by deoxyribonucleases. Cell. 1977 Aug;11(4):933–940. doi: 10.1016/0092-8674(77)90304-x. [DOI] [PubMed] [Google Scholar]
  26. Parks W. P., Ransom J. C., Young H. A., Scolnick E. M. Mammary tumor virus induction by glucocorticoids. Characterization of specific transcriptional regulation. J Biol Chem. 1975 May 10;250(9):3330–3336. [PubMed] [Google Scholar]
  27. Rennie P. S. Binding of androgen receptor to prostatic chromatin requires intact linker DNA. J Biol Chem. 1979 May 25;254(10):3947–3952. [PubMed] [Google Scholar]
  28. Rochefort H., Baulieu E. E. Oestradiol "neo nuclear receptor" in the rat uterus: sedimentation constant and conditions of formation. Biochimie. 1972;54(10):1303–1317. doi: 10.1016/s0300-9084(72)80071-3. [DOI] [PubMed] [Google Scholar]
  29. Rosenberg B. H. The periodic structure of chromatin: implications for DNA function. Biochem Biophys Res Commun. 1976 Oct 18;72(4):1384–1391. doi: 10.1016/s0006-291x(76)80167-2. [DOI] [PubMed] [Google Scholar]
  30. Senior M. B., Frankel F. R. Evidence for two kinds of chromatin binding sites for the estradiol-receptor complex. Cell. 1978 Aug;14(4):857–863. doi: 10.1016/0092-8674(78)90341-0. [DOI] [PubMed] [Google Scholar]
  31. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]
  32. Wrange O., Gustafsson J. A. Separation of the hormone- and DNA-binding sites of the hepatic glucocorticoid receptor by means of proteolysis. J Biol Chem. 1978 Feb 10;253(3):856–865. [PubMed] [Google Scholar]
  33. Young H. A., Shih T. Y., Scolnick E. M., Parks W. P. Steroid induction of mouse mammary tumor virus: effect upon synthesis and degradation of viral RNA. J Virol. 1977 Jan;21(1):139–146. doi: 10.1128/jvi.21.1.139-146.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zalta J., Zalta J. P., Simard R. Isolation of nucleoli. A method that combines high yield, structural integrity, and biochemical preservation. J Cell Biol. 1971 Nov;51(21):563–568. doi: 10.1083/jcb.51.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES