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. 1988 Jan 1;106(1):1–12. doi: 10.1083/jcb.106.1.1

Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs

PMCID: PMC2114961  PMID: 3339085

Abstract

We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout the chromatin mass. At the periphery this single membrane is surrounded by an outer membrane. In mature nuclei all membranes are at the periphery, the two membranes are linked by pores, and no prepores are seen. Nuclear assembly and replication are inhibited by preincubating the chromatin with the vesicular fraction. However nuclear assembly is accelerated by preincubating the condensed chromatin with the soluble fraction. This also decreases the lag before DNA replication. Initiation of DNA replication is only observed after normal nuclei have fully reassembled, increasing the evidence that replication depends on nuclear structure. The pathway of nuclear assembly and its relationship to DNA replication are discussed.

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Selected References

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

  1. Barry J. M., Merriam R. W. Swelling of hen erythrocyte nuclei in cytoplasm from Xenopus eggs. Exp Cell Res. 1972 Mar;71(1):90–96. doi: 10.1016/0014-4827(72)90267-4. [DOI] [PubMed] [Google Scholar]
  2. Benavente R., Krohne G., Franke W. W. Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis. Cell. 1985 May;41(1):177–190. doi: 10.1016/0092-8674(85)90072-8. [DOI] [PubMed] [Google Scholar]
  3. Blow J. J., Laskey R. A. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986 Nov 21;47(4):577–587. doi: 10.1016/0092-8674(86)90622-7. [DOI] [PubMed] [Google Scholar]
  4. Blow J. J., Watson J. V. Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system. EMBO J. 1987 Jul;6(7):1997–2002. doi: 10.1002/j.1460-2075.1987.tb02463.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burke B., Gerace L. A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell. 1986 Feb 28;44(4):639–652. doi: 10.1016/0092-8674(86)90273-4. [DOI] [PubMed] [Google Scholar]
  6. Comings D. E., Okada T. A. Association of chromatin fibers with the annuli of the nuclear membrane. Exp Cell Res. 1970 Oct;62(2):293–302. doi: 10.1016/0014-4827(70)90557-4. [DOI] [PubMed] [Google Scholar]
  7. Comings D. E., Okada T. A. Nuclear proteins. III. The fibrillar nature of the nuclear matrix. Exp Cell Res. 1976 Dec;103(2):341–360. doi: 10.1016/0014-4827(76)90271-8. [DOI] [PubMed] [Google Scholar]
  8. Forbes D. J., Kirschner M. W., Newport J. W. Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs. Cell. 1983 Aug;34(1):13–23. doi: 10.1016/0092-8674(83)90132-0. [DOI] [PubMed] [Google Scholar]
  9. Gerace L., Blobel G. The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell. 1980 Jan;19(1):277–287. doi: 10.1016/0092-8674(80)90409-2. [DOI] [PubMed] [Google Scholar]
  10. Gerace L., Blum A., Blobel G. Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution. J Cell Biol. 1978 Nov;79(2 Pt 1):546–566. doi: 10.1083/jcb.79.2.546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lohka M. J., Masui Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983 May 13;220(4598):719–721. doi: 10.1126/science.6601299. [DOI] [PubMed] [Google Scholar]
  12. Lohka M. J., Masui Y. Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. J Cell Biol. 1984 Apr;98(4):1222–1230. doi: 10.1083/jcb.98.4.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maul G. G. Nuclear pore complexes. Elimination and reconstruction during mitosis. J Cell Biol. 1977 Aug;74(2):492–500. doi: 10.1083/jcb.74.2.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Newmeyer D. D., Lucocq J. M., Bürglin T. R., De Robertis E. M. Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. EMBO J. 1986 Mar;5(3):501–510. doi: 10.1002/j.1460-2075.1986.tb04239.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Newport J. Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. Cell. 1987 Jan 30;48(2):205–217. doi: 10.1016/0092-8674(87)90424-7. [DOI] [PubMed] [Google Scholar]
  16. Stick R., Hausen P. Changes in the nuclear lamina composition during early development of Xenopus laevis. Cell. 1985 May;41(1):191–200. doi: 10.1016/0092-8674(85)90073-x. [DOI] [PubMed] [Google Scholar]

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