Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1980 May 1;187(2):467–477. doi: 10.1042/bj1870467

Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes.

G J Dimitriadis, J R Tata
PMCID: PMC1161813  PMID: 6156673

Abstract

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.

Full text

PDF
467

Selected References

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

  1. Axel R. Dissection of the eukaryotic chromosome with deoxyribonucleases. Methods Cell Biol. 1978;18:41–54. doi: 10.1016/s0091-679x(08)60131-4. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Berkowitz E. M., Doty P. Chemical and physical properties of fractionated chromatin. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3328–3332. doi: 10.1073/pnas.72.9.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blobel G., Potter V. R. Nuclei from rat liver: isolation method that combines purity with high yield. Science. 1966 Dec 30;154(3757):1662–1665. doi: 10.1126/science.154.3757.1662. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Britten R. J., Graham D. E., Neufeld B. R. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. doi: 10.1016/0076-6879(74)29033-5. [DOI] [PubMed] [Google Scholar]
  8. Chae C. B., Wong T. K., Gadski R. A. Transcription, processing and structure of chromatin in SV40-transformed cell. Biochem Biophys Res Commun. 1978 Aug 29;83(4):1518–1524. doi: 10.1016/0006-291x(78)91393-1. [DOI] [PubMed] [Google Scholar]
  9. Cole R. D., Lawson G. M., Hsiang M. W. H1 histone and the condensation of chromatin and DNA. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 1):253–263. doi: 10.1101/sqb.1978.042.01.027. [DOI] [PubMed] [Google Scholar]
  10. Darnell J. E., Jelinek W. R., Molloy G. R. Biogenesis of mRNA: genetic regulation in mammalian cells. Science. 1973 Sep 28;181(4106):1215–1221. doi: 10.1126/science.181.4106.1215. [DOI] [PubMed] [Google Scholar]
  11. Davie J. R., Candido E. P. Acetylated histone H4 is preferentially associated with template-active chromatin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3574–3577. doi: 10.1073/pnas.75.8.3574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Djondjurov L., Ivanova E., Tsanev R. Two chromatin fractions with different metabolic properties of non-histone proteins and of newly synthesized RNA. Eur J Biochem. 1979 Jun;97(1):133–139. doi: 10.1111/j.1432-1033.1979.tb13094.x. [DOI] [PubMed] [Google Scholar]
  13. Duesberg P. H., Vogt P. K. Gel electrophoresis of avian leukosis and sarcoma viral RNA in formamide: comparison with other viral and cellular RNA species. J Virol. 1973 Sep;12(3):594–599. doi: 10.1128/jvi.12.3.594-599.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Farmer S. R., Henshaw E. C., Berridge M. V., Tata J. R. Translation of Xenopus vitellogenin mRNA during primary and secondary induction. Nature. 1978 Jun 1;273(5661):401–403. doi: 10.1038/273401a0. [DOI] [PubMed] [Google Scholar]
  15. Felsenfeld G. Chromatin. Nature. 1978 Jan 12;271(5641):115–122. doi: 10.1038/271115a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Foe V. E., Wilkinson L. E., Laird C. D. Comparative organization of active transcription units in Oncopeltus fasciatus. Cell. 1976 Sep;9(1):131–146. doi: 10.1016/0092-8674(76)90059-3. [DOI] [PubMed] [Google Scholar]
  18. Franke W. W., Scheer U., Trendelenburg M., Zentgraf H., Spring H. Morphology of transcriptionally active chromatin. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):755–772. doi: 10.1101/sqb.1978.042.01.076. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Garel A., Zolan M., Axel R. Genes transcribed at diverse rates have a similar conformation in chromatin. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4867–4871. doi: 10.1073/pnas.74.11.4867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gordon J. I., Burns A. T., Christmann J. L., Deeley R. G. Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA. J Biol Chem. 1978 Dec 10;253(23):8629–8639. [PubMed] [Google Scholar]
  22. Gottesfeld J. M., Bagi G., Berg B., Bonner J. Sequence composition of the template-active fraction of rat liver chromatin. Biochemistry. 1976 Jun 1;15(11):2472–2483. doi: 10.1021/bi00656a034. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Gottesfeld J. M. Methods for fractionation of chromatin into transcriptionally active and inactive segments. Methods Cell Biol. 1977;16:421–436. doi: 10.1016/s0091-679x(08)60117-x. [DOI] [PubMed] [Google Scholar]
  25. Gottesfeld J. M., Murphy R. F., Bonner J. Structure of transcriptionally active chromatin. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4404–4408. doi: 10.1073/pnas.72.11.4404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Hendrick D., Tolstoshev P., Randlett D. Enrichment for the globin coding region in a chromatin fraction from chick reticulocytes by endonuclease digestion. Gene. 1977;2(3-4):147–158. doi: 10.1016/0378-1119(77)90014-2. [DOI] [PubMed] [Google Scholar]
  28. Hentschel C. C., Kay R. M., Williams J. G. Analysis of Xenopus laevis globins during development and erythroid cell maturation and the construction of recombinant plasmids containing sequences derived from adult globin mRNA. Dev Biol. 1979 Oct;72(2):350–363. doi: 10.1016/0012-1606(79)90124-6. [DOI] [PubMed] [Google Scholar]
  29. Hentschel C. C., Tata J. R. Template-engaged and free RNA polymerases during Xenopus erythroid cell maturation. Dev Biol. 1978 Aug;65(2):496–507. doi: 10.1016/0012-1606(78)90044-1. [DOI] [PubMed] [Google Scholar]
  30. Igo-Kemenes T., Greil W., Zachau H. G. Prepartation of soluble chromatin and specific chromatin fractions with restriction nucleases. Nucleic Acids Res. 1977 Oct;4(10):3387–3400. doi: 10.1093/nar/4.10.3387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kornberg R. D. Structure of chromatin. Annu Rev Biochem. 1977;46:931–954. doi: 10.1146/annurev.bi.46.070177.004435. [DOI] [PubMed] [Google Scholar]
  32. Levy B., Dixon G. H. Partial purification of transcriptionally active nucleosomes from trout testis cells. Nucleic Acids Res. 1978 Nov;5(11):4155–4163. doi: 10.1093/nar/5.11.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lutter L. C. Deoxyribonuclease I produces staggered cuts in the DNA of chromatin. J Mol Biol. 1977 Nov 25;117(1):53–69. doi: 10.1016/0022-2836(77)90022-5. [DOI] [PubMed] [Google Scholar]
  34. Lutter L. C. Kinetic analysis of deoxyribonuclease I cleavages in the nucleosome core: evidence for a DNA superhelix. J Mol Biol. 1978 Sep 15;124(2):391–420. doi: 10.1016/0022-2836(78)90306-6. [DOI] [PubMed] [Google Scholar]
  35. Marushige K. Activation of chromatin by acetylation of histone side chains. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3937–3941. doi: 10.1073/pnas.73.11.3937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mathis D. J., Oudet P., Wasylyk B., Chambon P. Effect of histone acetylation on structure and in vitro transcription of chromatin. Nucleic Acids Res. 1978 Oct;5(10):3523–3547. doi: 10.1093/nar/5.10.3523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Matthews H. R., Bradbury E. M. The role of H1 histone phosphorylation in the cell cycle. Turbidity studies of H1-DNA interaction. Exp Cell Res. 1978 Feb;111(2):343–351. doi: 10.1016/0014-4827(78)90179-9. [DOI] [PubMed] [Google Scholar]
  38. McKnight S. L., Miller O. L., Jr Ultrastructural patterns of RNA synthesis during early embryogenesis of Drosophila melanogaster. Cell. 1976 Jun;8(2):305–319. doi: 10.1016/0092-8674(76)90014-3. [DOI] [PubMed] [Google Scholar]
  39. Miller D. M., Turner P., Nienhuis A. W., Axelrod D. E., Gopalakrishnan T. V. Active conformation of the globin genes in uninduced and induced mouse erythroleukemia cells. Cell. 1978 Jul;14(3):511–521. doi: 10.1016/0092-8674(78)90237-4. [DOI] [PubMed] [Google Scholar]
  40. Neumann J. R., Housman D., Ingram V. M. Nuclear protein synthesis and phosphorylation in Friend erythroleukemia cells stimulated with DMSO. Exp Cell Res. 1978 Feb;111(2):277–284. doi: 10.1016/0014-4827(78)90171-4. [DOI] [PubMed] [Google Scholar]
  41. Noll M., Kornberg R. D. Action of micrococcal nuclease on chromatin and the location of histone H1. J Mol Biol. 1977 Jan 25;109(3):393–404. doi: 10.1016/s0022-2836(77)80019-3. [DOI] [PubMed] [Google Scholar]
  42. Ord M. G., Stocken L. A. Initiation of transcription in normal and phosphorylated rat liver nucleosomes. Biochem J. 1978 Nov 15;176(2):615–618. doi: 10.1042/bj1760615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Oudet P., Germond J. E., Bellard M., Spadafora C., Chambon P. Nucleosome structure. Philos Trans R Soc Lond B Biol Sci. 1978 May 11;283(997):241–258. doi: 10.1098/rstb.1978.0021. [DOI] [PubMed] [Google Scholar]
  44. Oudet P., Gross-Bellard M., Chambon P. Electron microscopic and biochemical evidence that chromatin structure is a repeating unit. Cell. 1975 Apr;4(4):281–300. doi: 10.1016/0092-8674(75)90149-x. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Rill R. L., Shaw B. R., Van Holde K. E. Isolation and characterization of chromatin subunits. Methods Cell Biol. 1978;18:69–103. doi: 10.1016/s0091-679x(08)60134-x. [DOI] [PubMed] [Google Scholar]
  47. Scheer U. Changes of nucleosome frequency in nucleolar and non-nucleolar chromatin as a function of transcription: an electron microscopic study. Cell. 1978 Mar;13(3):535–549. doi: 10.1016/0092-8674(78)90327-6. [DOI] [PubMed] [Google Scholar]
  48. Sealy L., Chalkley R. DNA associated with hyperacetylated histone is preferentially digested by DNase I. Nucleic Acids Res. 1978 Jun;5(6):1863–1876. doi: 10.1093/nar/5.6.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Simpson R. T. Structure of chromatin containing extensively acetylated H3 and H4. Cell. 1978 Apr;13(4):691–699. doi: 10.1016/0092-8674(78)90219-2. [DOI] [PubMed] [Google Scholar]
  50. Smith D. F., Searle P. F., Williams J. G. Characterisation of bacterial clones containing DNA sequences derived from Xenopus laevis vitellogenin mRNA. Nucleic Acids Res. 1979 Feb;6(2):487–506. doi: 10.1093/nar/6.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sollner-Webb B., Melchior W., Jr, Felsenfeld G. DNAase I, DNAase II and staphylococcal nuclease cut at different, yet symmetrically located, sites in the nucleosome core. Cell. 1978 Jul;14(3):611–627. doi: 10.1016/0092-8674(78)90246-5. [DOI] [PubMed] [Google Scholar]
  52. Tata J. R., Baker B. Differential subnuclear distribution of polyadenylate-rich ribonuclei acid during induction of egg-yolk protein synthesis in male Xenopus liver by oestradiol-17 beta. Biochem J. 1975 Sep;150(3):345–355. doi: 10.1042/bj1500345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tata J. R., Baker B. Enzymatic fractionation of nuclei: polynucleosomes and RNA polymerase II as endogenous transcriptional complexes. J Mol Biol. 1978 Jan 25;118(3):249–272. doi: 10.1016/0022-2836(78)90227-9. [DOI] [PubMed] [Google Scholar]
  54. Tata J. R., Baker B. Sub-nuclear fractionation. I. Procedure and characterization of fractions. Exp Cell Res. 1974 Jan;83(1):111–124. doi: 10.1016/0014-4827(74)90694-6. [DOI] [PubMed] [Google Scholar]
  55. Tata J. R., Baker B. Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro. Exp Cell Res. 1974 Jan;83(1):125–138. doi: 10.1016/0014-4827(74)90695-8. [DOI] [PubMed] [Google Scholar]
  56. Thomas N., Maclean N. The erythroid cells of anaemic Xenopus laevis. I. Studies on cellular morphology and protein and nucleic acid synthesis during differentiation. J Cell Sci. 1975 Dec;19(3):509–520. doi: 10.1242/jcs.19.3.509. [DOI] [PubMed] [Google Scholar]
  57. Vidali G., Boffa L. C., Bradbury E. M., Allfrey V. G. Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H3 and H4 and increased DNase I sensitivity of the associated DNA sequences. Proc Natl Acad Sci U S A. 1978 May;75(5):2239–2243. doi: 10.1073/pnas.75.5.2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. Weisbrod S., Weintraub H. Isolation of a subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin. Proc Natl Acad Sci U S A. 1979 Feb;76(2):630–634. doi: 10.1073/pnas.76.2.630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wu C., Bingham P. M., Livak K. J., Holmgren R., Elgin S. C. The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence. Cell. 1979 Apr;16(4):797–806. doi: 10.1016/0092-8674(79)90095-3. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES