Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Feb 1;124(3):235–248. doi: 10.1083/jcb.124.3.235

Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate

PMCID: PMC2119932  PMID: 8294509

Abstract

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha- amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205- 217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

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

  1. BERNFELD P. Enzymes of starch degradation and synthesis. Adv Enzymol Relat Subj Biochem. 1951;12:379–428. doi: 10.1002/9780470122570.ch7. [DOI] [PubMed] [Google Scholar]
  2. Berrios M., Avilion A. A. Nuclear formation in a Drosophila cell-free system. Exp Cell Res. 1990 Nov;191(1):64–70. doi: 10.1016/0014-4827(90)90036-a. [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., Sleeman A. M. Replication of purified DNA in Xenopus egg extract is dependent on nuclear assembly. J Cell Sci. 1990 Mar;95(Pt 3):383–391. doi: 10.1242/jcs.95.3.383. [DOI] [PubMed] [Google Scholar]
  5. Boman A. L., Delannoy M. R., Wilson K. L. GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. J Cell Biol. 1992 Jan;116(2):281–294. doi: 10.1083/jcb.116.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Booher R., Beach D. Involvement of a type 1 protein phosphatase encoded by bws1+ in fission yeast mitotic control. Cell. 1989 Jun 16;57(6):1009–1016. doi: 10.1016/0092-8674(89)90339-5. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  9. Coimbra A., Leblond C. P. Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-H3. J Cell Biol. 1966 Jul;30(1):151–175. doi: 10.1083/jcb.30.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cox L. S., Laskey R. A. DNA replication occurs at discrete sites in pseudonuclei assembled from purified DNA in vitro. Cell. 1991 Jul 26;66(2):271–275. doi: 10.1016/0092-8674(91)90617-8. [DOI] [PubMed] [Google Scholar]
  11. Dabauvalle M. C., Loos K., Scheer U. Identification of a soluble precursor complex essential for nuclear pore assembly in vitro. Chromosoma. 1990 Dec;100(1):56–66. doi: 10.1007/BF00337603. [DOI] [PubMed] [Google Scholar]
  12. Dasso M., Nishitani H., Kornbluth S., Nishimoto T., Newport J. W. RCC1, a regulator of mitosis, is essential for DNA replication. Mol Cell Biol. 1992 Aug;12(8):3337–3345. doi: 10.1128/mcb.12.8.3337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dilworth S. M., Black S. J., Laskey R. A. Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts. Cell. 1987 Dec 24;51(6):1009–1018. doi: 10.1016/0092-8674(87)90587-3. [DOI] [PubMed] [Google Scholar]
  14. Doonan J. H., Morris N. R. The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell. 1989 Jun 16;57(6):987–996. doi: 10.1016/0092-8674(89)90337-1. [DOI] [PubMed] [Google Scholar]
  15. Dworkin M. B., Dworkin-Rastl E. Carbon metabolism in early amphibian embryos. Trends Biochem Sci. 1991 Jun;16(6):229–234. doi: 10.1016/0968-0004(91)90091-9. [DOI] [PubMed] [Google Scholar]
  16. Dworkin M. B., Dworkin-Rastl E. Metabolic regulation during early frog development: glycogenic flux in Xenopus oocytes, eggs, and embryos. Dev Biol. 1989 Apr;132(2):512–523. doi: 10.1016/0012-1606(89)90246-7. [DOI] [PubMed] [Google Scholar]
  17. Eyal-Giladi H., Feinstein N., Friedlander M., Raveh D. Glycogen metabolism and the nuclear envelope-annulate lamella system in the early chick embryo. J Cell Sci. 1985 Feb;73:399–407. doi: 10.1242/jcs.73.1.399. [DOI] [PubMed] [Google Scholar]
  18. Fernandez A., Brautigan D. L., Lamb N. J. Protein phosphatase type 1 in mammalian cell mitosis: chromosomal localization and involvement in mitotic exit. J Cell Biol. 1992 Mar;116(6):1421–1430. doi: 10.1083/jcb.116.6.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ferrans V. J., Maron B. J., Buja L. M., Ali N., Roberts W. C. Intranuclear glycogen deposits in human cardiac muscle cells: ultrastructure and cytochemistry. J Mol Cell Cardiol. 1975 Jun;7(6):373–386. doi: 10.1016/0022-2828(75)90044-9. [DOI] [PubMed] [Google Scholar]
  20. Finlay D. R., Forbes D. J. Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. Cell. 1990 Jan 12;60(1):17–29. doi: 10.1016/0092-8674(90)90712-n. [DOI] [PubMed] [Google Scholar]
  21. Flaks B. Formation of membrane-glycogen arrays in rat hepatoma cells. J Cell Biol. 1968 Feb;36(2):410–414. doi: 10.1083/jcb.36.2.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Granzow C., Kopun M., Zimmermann H. P. Role of nuclear glycogen synthase and cytoplasmic UDP glucose pyrophosphorylase in the biosynthesis of nuclear glycogen in HD33 Ehrlich-Lettré ascites tumor cells. J Cell Biol. 1981 Jun;89(3):475–484. doi: 10.1083/jcb.89.3.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harland R. M., Laskey R. A. Regulated replication of DNA microinjected into eggs of Xenopus laevis. Cell. 1980 Oct;21(3):761–771. doi: 10.1016/0092-8674(80)90439-0. [DOI] [PubMed] [Google Scholar]
  26. Hart G. W., Haltiwanger R. S., Holt G. D., Kelly W. G. Glycosylation in the nucleus and cytoplasm. Annu Rev Biochem. 1989;58:841–874. doi: 10.1146/annurev.bi.58.070189.004205. [DOI] [PubMed] [Google Scholar]
  27. Hartl P., Gottesfeld J., Forbes D. J. Mitotic repression of transcription in vitro. J Cell Biol. 1993 Feb;120(3):613–624. doi: 10.1083/jcb.120.3.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hirano T., Mitchison T. J. Cell cycle control of higher-order chromatin assembly around naked DNA in vitro. J Cell Biol. 1991 Dec;115(6):1479–1489. doi: 10.1083/jcb.115.6.1479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hubbard M. J., Cohen P. On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem Sci. 1993 May;18(5):172–177. doi: 10.1016/0968-0004(93)90109-z. [DOI] [PubMed] [Google Scholar]
  30. Hubbard M. J., Cohen P. The glycogen-binding subunit of protein phosphatase-1G from rabbit skeletal muscle. Further characterisation of its structure and glycogen-binding properties. Eur J Biochem. 1989 Mar 15;180(2):457–465. doi: 10.1111/j.1432-1033.1989.tb14668.x. [DOI] [PubMed] [Google Scholar]
  31. Hubbard M. J., Dent P., Smythe C., Cohen P. Targetting of protein phosphatase 1 to the sarcoplasmic reticulum of rabbit skeletal muscle by a protein that is very similar or identical to the G subunit that directs the enzyme to glycogen. Eur J Biochem. 1990 Apr 30;189(2):243–249. doi: 10.1111/j.1432-1033.1990.tb15483.x. [DOI] [PubMed] [Google Scholar]
  32. Karasaki S. Cytoplasmic and nuclear glycogen synthesis in Novikoff ascites hepatoma cells. J Ultrastruct Res. 1971 Apr;35(1):181–196. doi: 10.1016/s0022-5320(71)80150-8. [DOI] [PubMed] [Google Scholar]
  33. Kessel R. G., Beams H. W. Freeze fracture and scanning electron microscope studies on the nuclear envelope and perinuclear cytomembranes (parabasal apparatus) in the protozoan, Lophomonas blattarum. J Submicrosc Cytol Pathol. 1990 Jul;22(3):367–378. [PubMed] [Google Scholar]
  34. Kessel R. G. The annulate lamellae--from obscurity to spotlight. Electron Microsc Rev. 1989;2(2):257–348. doi: 10.1016/0892-0354(89)90003-8. [DOI] [PubMed] [Google Scholar]
  35. Kopun M., Granzow C., Krisman C. R. Comparative study of nuclear and cytoplasmic glycogen isolated from mutant HD33 ascites cells. J Cell Biochem. 1989 Feb;39(2):185–195. doi: 10.1002/jcb.240390210. [DOI] [PubMed] [Google Scholar]
  36. Laskey R. A., Leno G. H. Assembly of the cell nucleus. Trends Genet. 1990 Dec;6(12):406–410. doi: 10.1016/0168-9525(90)90301-l. [DOI] [PubMed] [Google Scholar]
  37. Laskey R. A., Mills A. D., Morris N. R. Assembly of SV40 chromatin in a cell-free system from Xenopus eggs. Cell. 1977 Feb;10(2):237–243. doi: 10.1016/0092-8674(77)90217-3. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Minton A. P. The effect of volume occupancy upon the thermodynamic activity of proteins: some biochemical consequences. Mol Cell Biochem. 1983;55(2):119–140. doi: 10.1007/BF00673707. [DOI] [PubMed] [Google Scholar]
  41. Mori M., Dempo K., Abe M., Onoe T. Electron microscopic study of intranuclear glycogen. J Electron Microsc (Tokyo) 1970;19(2):163–169. [PubMed] [Google Scholar]
  42. Newmeyer D. D., Finlay D. R., Forbes D. J. In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2091–2102. doi: 10.1083/jcb.103.6.2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Newmeyer D. D., Forbes D. J. An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore. J Cell Biol. 1990 Mar;110(3):547–557. doi: 10.1083/jcb.110.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Newmeyer D. D., Forbes D. J. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. doi: 10.1016/0092-8674(88)90402-3. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Newport J. W., Wilson K. L., Dunphy W. G. A lamin-independent pathway for nuclear envelope assembly. J Cell Biol. 1990 Dec;111(6 Pt 1):2247–2259. doi: 10.1083/jcb.111.6.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Newport J., Dunphy W. Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components. J Cell Biol. 1992 Jan;116(2):295–306. doi: 10.1083/jcb.116.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. Newport J., Spann T., Kanki J., Forbes D. The role of mitotic factors in regulating the timing of the midblastula transition in Xenopus. Cold Spring Harb Symp Quant Biol. 1985;50:651–656. doi: 10.1101/sqb.1985.050.01.079. [DOI] [PubMed] [Google Scholar]
  51. Pfaller R., Smythe C., Newport J. W. Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro. Cell. 1991 Apr 19;65(2):209–217. doi: 10.1016/0092-8674(91)90155-r. [DOI] [PubMed] [Google Scholar]
  52. Pflugrath J. W., Wiegand G., Huber R., Vértesy L. Crystal structure determination, refinement and the molecular model of the alpha-amylase inhibitor Hoe-467A. J Mol Biol. 1986 May 20;189(2):383–386. doi: 10.1016/0022-2836(86)90520-6. [DOI] [PubMed] [Google Scholar]
  53. Philpott A., Leno G. H. Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts. Cell. 1992 May 29;69(5):759–767. doi: 10.1016/0092-8674(92)90288-n. [DOI] [PubMed] [Google Scholar]
  54. Roberts S. B., Segil N., Heintz N. Differential phosphorylation of the transcription factor Oct1 during the cell cycle. Science. 1991 Aug 30;253(5023):1022–1026. doi: 10.1126/science.1887216. [DOI] [PubMed] [Google Scholar]
  55. Sheehan M. A., Mills A. D., Sleeman A. M., Laskey R. A., Blow J. J. Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. J Cell Biol. 1988 Jan;106(1):1–12. doi: 10.1083/jcb.106.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Simpson R. T., Thoma F., Brubaker J. M. Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure. Cell. 1985 Oct;42(3):799–808. doi: 10.1016/0092-8674(85)90276-4. [DOI] [PubMed] [Google Scholar]
  57. Smythe C., Cohen P. The discovery of glycogenin and the priming mechanism for glycogen biogenesis. Eur J Biochem. 1991 Sep 15;200(3):625–631. doi: 10.1111/j.1432-1033.1991.tb16225.x. [DOI] [PubMed] [Google Scholar]
  58. Smythe C., Newport J. W. Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2. Cell. 1992 Feb 21;68(4):787–797. doi: 10.1016/0092-8674(92)90153-4. [DOI] [PubMed] [Google Scholar]
  59. Smythe C., Newport J. W. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods Cell Biol. 1991;35:449–468. doi: 10.1016/s0091-679x(08)60583-x. [DOI] [PubMed] [Google Scholar]
  60. Solomon M. J., Glotzer M., Lee T. H., Philippe M., Kirschner M. W. Cyclin activation of p34cdc2. Cell. 1990 Nov 30;63(5):1013–1024. doi: 10.1016/0092-8674(90)90504-8. [DOI] [PubMed] [Google Scholar]
  61. Stone E. M., Yamano H., Kinoshita N., Yanagida M. Mitotic regulation of protein phosphatases by the fission yeast sds22 protein. Curr Biol. 1993 Jan;3(1):13–26. doi: 10.1016/0960-9822(93)90140-j. [DOI] [PubMed] [Google Scholar]
  62. Ulitzur N., Gruenbaum Y. Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos. FEBS Lett. 1989 Dec 18;259(1):113–116. doi: 10.1016/0014-5793(89)81507-8. [DOI] [PubMed] [Google Scholar]
  63. Vigers G. P., Lohka M. J. A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs. J Cell Biol. 1991 Feb;112(4):545–556. doi: 10.1083/jcb.112.4.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Vigers G. P., Lohka M. J. Regulation of nuclear envelope precursor functions during cell division. J Cell Sci. 1992 Jun;102(Pt 2):273–284. doi: 10.1242/jcs.102.2.273. [DOI] [PubMed] [Google Scholar]
  65. Vértesy L., Oeding V., Bender R., Zepf K., Nesemann G. Tendamistat (HOE 467), a tight-binding alpha-amylase inhibitor from Streptomyces tendae 4158. Isolation, biochemical properties. Eur J Biochem. 1984 Jun 15;141(3):505–512. doi: 10.1111/j.1432-1033.1984.tb08221.x. [DOI] [PubMed] [Google Scholar]
  66. Walker D. H., DePaoli-Roach A. A., Maller J. L. Multiple roles for protein phosphatase 1 in regulating the Xenopus early embryonic cell cycle. Mol Biol Cell. 1992 Jun;3(6):687–698. doi: 10.1091/mbc.3.6.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wilson K. L., Newport J. A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. J Cell Biol. 1988 Jul;107(1):57–68. doi: 10.1083/jcb.107.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wolffe A. P., Brown D. D. Differential 5S RNA gene expression in vitro. Cell. 1987 Dec 4;51(5):733–740. doi: 10.1016/0092-8674(87)90096-1. [DOI] [PubMed] [Google Scholar]
  69. Zimmerman S. B., Harrison B. Macromolecular crowding increases binding of DNA polymerase to DNA: an adaptive effect. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1871–1875. doi: 10.1073/pnas.84.7.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES