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. 1991 May;10(5):1237–1243. doi: 10.1002/j.1460-2075.1991.tb08065.x

An ectopic copy of the Drosophila ftz associated SAR neither reorganizes local chromatin structure nor hinders elution of a chromatin fragment from isolated nuclei.

H Eggert 1, R S Jack 1
PMCID: PMC452778  PMID: 1850697

Abstract

In vitro assays using detergent extracted nuclei allow the operational definition of SARs--specific sequences in the chromosome which are thought to interact with a structural matrix or scaffold. This interaction results in the formation of large stable protein-DNA complexes. We have used P-element transformation to introduce a characterized SAR into the Drosophila genome. The standard assay, which uses detergent extracted nuclei, shows that the ectopic SAR is indeed bound to the scaffold. However, in unextracted nuclei, a chromatin fragment containing the SAR sequence is eluted from the nucleus as readily as a fragment which lacks an SAR. Furthermore, an analysis of the accessibility of the neighbouring chromosomal restriction sites in unextracted nuclei indicates that the introduction of this ectopic SAR does not influence the local structure of chromatin. We conclude that the ectopic SAR site is not attached to a nuclear scaffold or matrix in vivo.

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Selected References

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

  1. Benyajati C., Worcel A. Isolation, characterization, and structure of the folded interphase genome of Drosophila melanogaster. Cell. 1976 Nov;9(3):393–407. doi: 10.1016/0092-8674(76)90084-2. [DOI] [PubMed] [Google Scholar]
  2. Berrios M., Osheroff N., Fisher P. A. In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4142–4146. doi: 10.1073/pnas.82.12.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chao M. V., Gralla J. D., Martinson H. G. lac Operator nucleosomes. 1. Repressor binds specifically to operator within the nucleosome core. Biochemistry. 1980 Jul 8;19(14):3254–3260. doi: 10.1021/bi00555a024. [DOI] [PubMed] [Google Scholar]
  4. Clark D. J., Kimura T. Electrostatic mechanism of chromatin folding. J Mol Biol. 1990 Feb 20;211(4):883–896. doi: 10.1016/0022-2836(90)90081-V. [DOI] [PubMed] [Google Scholar]
  5. Cockerill P. N., Garrard W. T. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell. 1986 Jan 31;44(2):273–282. doi: 10.1016/0092-8674(86)90761-0. [DOI] [PubMed] [Google Scholar]
  6. Dickinson P., Cook P. R., Jackson D. A. Active RNA polymerase I is fixed within the nucleus of HeLa cells. EMBO J. 1990 Jul;9(7):2207–2214. doi: 10.1002/j.1460-2075.1990.tb07390.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Earnshaw W. C., Halligan B., Cooke C. A., Heck M. M., Liu L. F. Topoisomerase II is a structural component of mitotic chromosome scaffolds. J Cell Biol. 1985 May;100(5):1706–1715. doi: 10.1083/jcb.100.5.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Finch J. T., Klug A. Solenoidal model for superstructure in chromatin. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1897–1901. doi: 10.1073/pnas.73.6.1897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gasser S. M., Laemmli U. K. Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell. 1986 Aug 15;46(4):521–530. doi: 10.1016/0092-8674(86)90877-9. [DOI] [PubMed] [Google Scholar]
  10. Gasser S. M., Laemmli U. K. The organisation of chromatin loops: characterization of a scaffold attachment site. EMBO J. 1986 Mar;5(3):511–518. doi: 10.1002/j.1460-2075.1986.tb04240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gasser S. M., Laroche T., Falquet J., Boy de la Tour E., Laemmli U. K. Metaphase chromosome structure. Involvement of topoisomerase II. J Mol Biol. 1986 Apr 20;188(4):613–629. doi: 10.1016/s0022-2836(86)80010-9. [DOI] [PubMed] [Google Scholar]
  12. Harrison S. D., Travers A. A. Identification of the binding sites for potential regulatory proteins in the upstream enhancer element of the Drosophila fushi tarazu gene. Nucleic Acids Res. 1988 Dec 23;16(24):11403–11416. doi: 10.1093/nar/16.24.11403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hiromi Y., Kuroiwa A., Gehring W. J. Control elements of the Drosophila segmentation gene fushi tarazu. Cell. 1985 Dec;43(3 Pt 2):603–613. doi: 10.1016/0092-8674(85)90232-6. [DOI] [PubMed] [Google Scholar]
  14. Izaurralde E., Käs E., Laemmli U. K. Highly preferential nucleation of histone H1 assembly on scaffold-associated regions. J Mol Biol. 1989 Dec 5;210(3):573–585. doi: 10.1016/0022-2836(89)90133-2. [DOI] [PubMed] [Google Scholar]
  15. Izaurralde E., Mirkovitch J., Laemmli U. K. Interaction of DNA with nuclear scaffolds in vitro. J Mol Biol. 1988 Mar 5;200(1):111–125. doi: 10.1016/0022-2836(88)90337-3. [DOI] [PubMed] [Google Scholar]
  16. Jack R. S., Eggert H. Restriction enzymes have limited access to DNA sequences in Drosophila chromosomes. EMBO J. 1990 Aug;9(8):2603–2609. doi: 10.1002/j.1460-2075.1990.tb07442.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jackson D. A., Cook P. R. A general method for preparing chromatin containing intact DNA. EMBO J. 1985 Apr;4(4):913–918. doi: 10.1002/j.1460-2075.1985.tb03718.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jackson D. A., Cook P. R. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. doi: 10.1002/j.1460-2075.1985.tb03719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jackson D. A., Dickinson P., Cook P. R. Attachment of DNA to the nucleoskeleton of HeLa cells examined using physiological conditions. Nucleic Acids Res. 1990 Aug 11;18(15):4385–4393. doi: 10.1093/nar/18.15.4385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jackson D. A., Dickinson P., Cook P. R. The size of chromatin loops in HeLa cells. EMBO J. 1990 Feb;9(2):567–571. doi: 10.1002/j.1460-2075.1990.tb08144.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Keith T. P., Riley M. A., Kreitman M., Lewontin R. C., Curtis D., Chambers G. Sequence of the structural gene for xanthine dehydrogenase (rosy locus) in Drosophila melanogaster. Genetics. 1987 May;116(1):67–73. doi: 10.1093/genetics/116.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Keppel F. Transcribed human ribosomal RNA genes are attached to the nuclear matrix. J Mol Biol. 1986 Jan 5;187(1):15–21. doi: 10.1016/0022-2836(86)90402-x. [DOI] [PubMed] [Google Scholar]
  23. Mirkovitch J., Gasser S. M., Laemmli U. K. Scaffold attachment of DNA loops in metaphase chromosomes. J Mol Biol. 1988 Mar 5;200(1):101–109. doi: 10.1016/0022-2836(88)90336-1. [DOI] [PubMed] [Google Scholar]
  24. Mirkovitch J., Mirault M. E., Laemmli U. K. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984 Nov;39(1):223–232. doi: 10.1016/0092-8674(84)90208-3. [DOI] [PubMed] [Google Scholar]
  25. Mirkovitch J., Spierer P., Laemmli U. K. Genes and loops in 320,000 base-pairs of the Drosophila melanogaster chromosome. J Mol Biol. 1986 Jul 20;190(2):255–258. doi: 10.1016/0022-2836(86)90296-2. [DOI] [PubMed] [Google Scholar]
  26. Piña B., Brüggemeier U., Beato M. Nucleosome positioning modulates accessibility of regulatory proteins to the mouse mammary tumor virus promoter. Cell. 1990 Mar 9;60(5):719–731. doi: 10.1016/0092-8674(90)90087-u. [DOI] [PubMed] [Google Scholar]
  27. Richmond T. J., Finch J. T., Rushton B., Rhodes D., Klug A. Structure of the nucleosome core particle at 7 A resolution. Nature. 1984 Oct 11;311(5986):532–537. doi: 10.1038/311532a0. [DOI] [PubMed] [Google Scholar]
  28. Rubin G. M., Spradling A. C. Genetic transformation of Drosophila with transposable element vectors. Science. 1982 Oct 22;218(4570):348–353. doi: 10.1126/science.6289436. [DOI] [PubMed] [Google Scholar]
  29. Samal B., Worcel A., Louis C., Schedl P. Chromatin structure of the histone genes of D. melanogaster. Cell. 1981 Feb;23(2):401–409. doi: 10.1016/0092-8674(81)90135-5. [DOI] [PubMed] [Google Scholar]
  30. Sander M., Hsieh T. S. Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site. Nucleic Acids Res. 1985 Feb 25;13(4):1057–1072. doi: 10.1093/nar/13.4.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thoma F., Koller T., Klug A. Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin. J Cell Biol. 1979 Nov;83(2 Pt 1):403–427. doi: 10.1083/jcb.83.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Travers A. A. DNA conformation and protein binding. Annu Rev Biochem. 1989;58:427–452. doi: 10.1146/annurev.bi.58.070189.002235. [DOI] [PubMed] [Google Scholar]
  33. Widom J. Physicochemical studies of the folding of the 100 A nucleosome filament into the 300 A filament. Cation dependence. J Mol Biol. 1986 Aug 5;190(3):411–424. doi: 10.1016/0022-2836(86)90012-4. [DOI] [PubMed] [Google Scholar]
  34. Worcel A., Gargiulo G., Jessee B., Udvardy A., Louis C., Schedl P. Chromatin fine structure of the histone gene complex of Drosophila melanogaster. Nucleic Acids Res. 1983 Jan 25;11(2):421–439. doi: 10.1093/nar/11.2.421. [DOI] [PMC free article] [PubMed] [Google Scholar]

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