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. 1990 Dec 1;111(6):2839–2850. doi: 10.1083/jcb.111.6.2839

Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II

PMCID: PMC2116389  PMID: 2176652

Abstract

We report the development of a new method for producing mitotic extracts from tissue culture cells. These extracts reproducibly promote the condensation of chromatin in vitro when incubated with purified interphase nuclei. This condensation reaction is not species specific, since nuclei from chicken, human, and hamster cell lines all undergo chromatin condensation upon incubation with the extract. We have used this extract to investigate the role of DNA topoisomerase II (topo II) in the chromosome condensation process. Chromatin condensation does not require the presence of soluble topo II in the mitotic extract. However, the extent of formation of discrete chromosome-like structures correlates with the level of endogenous topo II present in the interphase nuclei. Our results further suggest that chromatin condensation in this extract may involve two processes: chromatin compaction and resolution into discrete chromosomes.

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Selected References

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  1. Adolphs K. W., Cheng S. M., Paulson J. R., Laemmli U. K. Isolation of a protein scaffold from mitotic HeLa cell chromosomes. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4937–4941. doi: 10.1073/pnas.74.11.4937. [DOI] [PMC free article] [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. Cooke C. A., Heck M. M., Earnshaw W. C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J Cell Biol. 1987 Nov;105(5):2053–2067. doi: 10.1083/jcb.105.5.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davies P. F., Ross R. Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor. J Cell Biol. 1978 Dec;79(3):663–671. doi: 10.1083/jcb.79.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DiNardo S., Voelkel K., Sternglanz R. DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc Natl Acad Sci U S A. 1984 May;81(9):2616–2620. doi: 10.1073/pnas.81.9.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Duguet M., Lavenot C., Harper F., Mirambeau G., De Recondo A. M. DNA topoisomerases from rat liver: physiological variations. Nucleic Acids Res. 1983 Feb 25;11(4):1059–1075. doi: 10.1093/nar/11.4.1059. [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. Earnshaw W. C., Halligan N., Cooke C., Rothfield N. The kinetochore is part of the metaphase chromosome scaffold. J Cell Biol. 1984 Jan;98(1):352–357. doi: 10.1083/jcb.98.1.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Earnshaw W. C., Heck M. M. Localization of topoisomerase II in mitotic chromosomes. J Cell Biol. 1985 May;100(5):1716–1725. doi: 10.1083/jcb.100.5.1716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Earnshaw W. C., Laemmli U. K. Architecture of metaphase chromosomes and chromosome scaffolds. J Cell Biol. 1983 Jan;96(1):84–93. doi: 10.1083/jcb.96.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Earnshaw W. C., Rothfield N. Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma. 1985;91(3-4):313–321. doi: 10.1007/BF00328227. [DOI] [PubMed] [Google Scholar]
  12. Gasser S. M., Amati B. B., Cardenas M. E., Hofmann J. F. Studies on scaffold attachment sites and their relation to genome function. Int Rev Cytol. 1989;119:57–96. doi: 10.1016/s0074-7696(08)60649-x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Gurdon J. B. Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. J Embryol Exp Morphol. 1968 Nov;20(3):401–414. [PubMed] [Google Scholar]
  15. Heck M. M., Earnshaw W. C. Topoisomerase II: A specific marker for cell proliferation. J Cell Biol. 1986 Dec;103(6 Pt 2):2569–2581. doi: 10.1083/jcb.103.6.2569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heck M. M., Hittelman W. N., Earnshaw W. C. Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1086–1090. doi: 10.1073/pnas.85.4.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hiraoka Y., Minden J. S., Swedlow J. R., Sedat J. W., Agard D. A. Focal points for chromosome condensation and decondensation revealed by three-dimensional in vivo time-lapse microscopy. Nature. 1989 Nov 16;342(6247):293–296. doi: 10.1038/342293a0. [DOI] [PubMed] [Google Scholar]
  18. Hoffmann A., Heck M. M., Bordwell B. J., Rothfield N. F., Earnshaw W. C. Human autoantibody to topoisomerase II. Exp Cell Res. 1989 Feb;180(2):409–418. doi: 10.1016/0014-4827(89)90067-0. [DOI] [PubMed] [Google Scholar]
  19. Holm C., Goto T., Wang J. C., Botstein D. DNA topoisomerase II is required at the time of mitosis in yeast. Cell. 1985 Jun;41(2):553–563. doi: 10.1016/s0092-8674(85)80028-3. [DOI] [PubMed] [Google Scholar]
  20. Högner D., Lepper K., Seibold G., Jost E. Lamin disassembly kinetics: a cell-free system with extracts from mitotic HeLa cells. Exp Cell Res. 1988 Jun;176(2):281–296. doi: 10.1016/0014-4827(88)90331-x. [DOI] [PubMed] [Google Scholar]
  21. Langlois A. J., Lapis K., Ishizaki R., Beard J. W., Bolognesi D. P. Isolation of a transplantable cell line induced by the MC29 avian leukosis virus. Cancer Res. 1974 Jun;34(6):1457–1464. [PubMed] [Google Scholar]
  22. Lehner C. F., Kurer V., Eppenberger H. M., Nigg E. A. The nuclear lamin protein family in higher vertebrates. Identification of quantitatively minor lamin proteins by monoclonal antibodies. J Biol Chem. 1986 Oct 5;261(28):13293–13301. [PubMed] [Google Scholar]
  23. Lewis C. D., Laemmli U. K. Higher order metaphase chromosome structure: evidence for metalloprotein interactions. Cell. 1982 May;29(1):171–181. doi: 10.1016/0092-8674(82)90101-5. [DOI] [PubMed] [Google Scholar]
  24. Lohka M. J., Maller J. L. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. doi: 10.1083/jcb.101.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Luke M., Bogenhagen D. F. Quantitation of type II topoisomerase in oocytes and eggs of Xenopus laevis. Dev Biol. 1989 Dec;136(2):459–468. doi: 10.1016/0012-1606(89)90271-6. [DOI] [PubMed] [Google Scholar]
  26. Miake-Lye R., Kirschner M. W. Induction of early mitotic events in a cell-free system. Cell. 1985 May;41(1):165–175. doi: 10.1016/0092-8674(85)90071-6. [DOI] [PubMed] [Google Scholar]
  27. Nakagawa J., Kitten G. T., Nigg E. A. A somatic cell-derived system for studying both early and late mitotic events in vitro. J Cell Sci. 1989 Nov;94(Pt 3):449–462. doi: 10.1242/jcs.94.3.449. [DOI] [PubMed] [Google Scholar]
  28. Newport J., Spann T. Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell. 1987 Jan 30;48(2):219–230. doi: 10.1016/0092-8674(87)90425-9. [DOI] [PubMed] [Google Scholar]
  29. Shero J. H., Bordwell B., Rothfield N. F., Earnshaw W. C. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science. 1986 Feb 14;231(4739):737–740. doi: 10.1126/science.3003910. [DOI] [PubMed] [Google Scholar]
  30. Sullivan D. M., Glisson B. S., Hodges P. K., Smallwood-Kentro S., Ross W. E. Proliferation dependence of topoisomerase II mediated drug action. Biochemistry. 1986 Apr 22;25(8):2248–2256. doi: 10.1021/bi00356a060. [DOI] [PubMed] [Google Scholar]
  31. Sundin O., Varshavsky A. Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication. Cell. 1981 Sep;25(3):659–669. doi: 10.1016/0092-8674(81)90173-2. [DOI] [PubMed] [Google Scholar]
  32. Suprynowicz F. A., Gerace L. A fractionated cell-free system for analysis of prophase nuclear disassembly. J Cell Biol. 1986 Dec;103(6 Pt 1):2073–2081. doi: 10.1083/jcb.103.6.2073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Taudou G., Mirambeau G., Lavenot C., der Garabedian A., Vermeersch J., Duguet M. DNA topoisomerase activities in concanavalin A-stimulated lymphocytes. FEBS Lett. 1984 Oct 29;176(2):431–435. doi: 10.1016/0014-5793(84)81212-0. [DOI] [PubMed] [Google Scholar]
  34. Tobey R. A., Anderson E. C., Petersen D. F. Properties of mitotic cells prepared by mechanically shaking monolayer cultures of Chinese hamster cells. J Cell Physiol. 1967 Aug;70(1):63–68. doi: 10.1002/jcp.1040700109. [DOI] [PubMed] [Google Scholar]
  35. Tsao Y. P., Wu H. Y., Liu L. F. Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies. Cell. 1989 Jan 13;56(1):111–118. doi: 10.1016/0092-8674(89)90989-6. [DOI] [PubMed] [Google Scholar]
  36. Uemura T., Ohkura H., Adachi Y., Morino K., Shiozaki K., Yanagida M. DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe. Cell. 1987 Sep 11;50(6):917–925. doi: 10.1016/0092-8674(87)90518-6. [DOI] [PubMed] [Google Scholar]
  37. Uemura T., Tanagida M. Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis. EMBO J. 1986 May;5(5):1003–1010. doi: 10.1002/j.1460-2075.1986.tb04315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  39. Wu H. Y., Shyy S. H., Wang J. C., Liu L. F. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. doi: 10.1016/0092-8674(88)90163-8. [DOI] [PubMed] [Google Scholar]

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