Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Sep;15(9):4873–4883. doi: 10.1128/mcb.15.9.4873

RNA polymerase III transcription in synthetic nuclei assembled in vitro from defined DNA templates.

K S Ullman 1, D J Forbes 1
PMCID: PMC230733  PMID: 7651406

Abstract

Although much is known of the basic control of transcription, little is understood of the way in which the structural organization of the nucleus affects transcription. Synthetic nuclei, assembled de novo in extracts of Xenopus eggs, would be predicted to have a large potential for approaching the role of nuclear structure in RNA biogenesis. Synthetic nuclei provide a system in which the genetic content of the nuclei, as well as the structural and enzymatic proteins within the nuclei, can be manipulated. In this study, we have begun to examine transcription in such nuclei by using the most simple of templates, RNA polymerase III (pol III)-transcribed genes. DNA encoding tRNA or 5S genes was added to an assembly extract, and nuclei were formed entirely from the pol III templates. Conditions which allowed nuclear assembly and pol III transcription to take place efficiently and simultaneously in the assembly extract were found. To examine whether pol III transcription could initiate within synthetic nuclei, or instead was inhibited in nuclei and initiated only on rare unincorporated templates, we identified transcriptional inhibitors that were excluded from nuclei. We found that these inhibitors, heparin and dextran sulfate, blocked pol III transcription in the absence of assembly but did not do so following nuclear assembly. At the concentrations used, the inhibitors had no deleterious effect on nuclear structure itself or on nuclear import. We conclude that pol III transcription is active in synthetic nuclei, and this conclusion is further strengthened by the finding that pol III transcripts could be coisolated with synthetic nuclei. The rapid and direct transcriptional analysis possible with pol III templates, coupled with the simple experimental criteria developed in this study for distinguishing between nuclear and non-nuclear transcription, should now allow a molecular analysis of the effect of nuclear structure on transcriptional and posttranscriptional control.

Full Text

The Full Text of this article is available as a PDF (395.1 KB).

Selected References

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

  1. Adachi Y., Laemmli U. K. Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts. EMBO J. 1994 Sep 1;13(17):4153–4164. doi: 10.1002/j.1460-2075.1994.tb06733.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adeniyi-Jones S., Romeo P. H., Zasloff M. Generation of long read-through transcripts in vivo and in vitro by deletion of 3' termination and processing sequences in the human tRNAimet gene. Nucleic Acids Res. 1984 Jan 25;12(2):1101–1115. doi: 10.1093/nar/12.2.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Adolph K. W., Cheng S. M., Laemmli U. K. Role of nonhistone proteins in metaphase chromosome structure. Cell. 1977 Nov;12(3):805–816. doi: 10.1016/0092-8674(77)90279-3. [DOI] [PubMed] [Google Scholar]
  4. Almouzni G., Méchali M., Wolffe A. P. Competition between transcription complex assembly and chromatin assembly on replicating DNA. EMBO J. 1990 Feb;9(2):573–582. doi: 10.1002/j.1460-2075.1990.tb08145.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Almouzni G., Wolffe A. P. Nuclear assembly, structure, and function: the use of Xenopus in vitro systems. Exp Cell Res. 1993 Mar;205(1):1–15. doi: 10.1006/excr.1993.1051. [DOI] [PubMed] [Google Scholar]
  6. Almouzni G., Wolffe A. P. Replication-coupled chromatin assembly is required for the repression of basal transcription in vivo. Genes Dev. 1993 Oct;7(10):2033–2047. doi: 10.1101/gad.7.10.2033. [DOI] [PubMed] [Google Scholar]
  7. Amberg D. C., Goldstein A. L., Cole C. N. Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. Genes Dev. 1992 Jul;6(7):1173–1189. doi: 10.1101/gad.6.7.1173. [DOI] [PubMed] [Google Scholar]
  8. Barton M. C., Emerson B. M. Regulated expression of the beta-globin gene locus in synthetic nuclei. Genes Dev. 1994 Oct 15;8(20):2453–2465. doi: 10.1101/gad.8.20.2453. [DOI] [PubMed] [Google Scholar]
  9. Barton M. C., Madani N., Emerson B. M. The erythroid protein cGATA-1 functions with a stage-specific factor to activate transcription of chromatin-assembled beta-globin genes. Genes Dev. 1993 Sep;7(9):1796–1809. doi: 10.1101/gad.7.9.1796. [DOI] [PubMed] [Google Scholar]
  10. Bauer D. W., Murphy C., Wu Z., Wu C. H., Gall J. G. In vitro assembly of coiled bodies in Xenopus egg extract. Mol Biol Cell. 1994 Jun;5(6):633–644. doi: 10.1091/mbc.5.6.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bell P., Dabauvalle M. C., Scheer U. In vitro assembly of prenucleolar bodies in Xenopus egg extract. J Cell Biol. 1992 Sep;118(6):1297–1304. doi: 10.1083/jcb.118.6.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Carter K. C., Taneja K. L., Lawrence J. B. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. doi: 10.1083/jcb.115.5.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Dargemont C., Schmidt-Zachmann M. S., Kühn L. C. Direct interaction of nucleoporin p62 with mRNA during its export from the nucleus. J Cell Sci. 1995 Jan;108(Pt 1):257–263. doi: 10.1242/jcs.108.1.257. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Doye V., Wepf R., Hurt E. C. A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution. EMBO J. 1994 Dec 15;13(24):6062–6075. doi: 10.1002/j.1460-2075.1994.tb06953.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dworetzky S. I., Feldherr C. M. Translocation of RNA-coated gold particles through the nuclear pores of oocytes. J Cell Biol. 1988 Mar;106(3):575–584. doi: 10.1083/jcb.106.3.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Elliott D. J., Stutz F., Lescure A., Rosbash M. mRNA nuclear export. Curr Opin Genet Dev. 1994 Apr;4(2):305–309. doi: 10.1016/s0959-437x(05)80058-9. [DOI] [PubMed] [Google Scholar]
  21. Fabre E., Boelens W. C., Wimmer C., Mattaj I. W., Hurt E. C. Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif. Cell. 1994 Jul 29;78(2):275–289. doi: 10.1016/0092-8674(94)90297-6. [DOI] [PubMed] [Google Scholar]
  22. Fang F., Newport J. W. Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell. 1991 Aug 23;66(4):731–742. doi: 10.1016/0092-8674(91)90117-h. [DOI] [PubMed] [Google Scholar]
  23. Featherstone C., Darby M. K., Gerace L. A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo. J Cell Biol. 1988 Oct;107(4):1289–1297. doi: 10.1083/jcb.107.4.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Feldherr C. M., Akin D. Role of nuclear trafficking in regulating cellular activity. Int Rev Cytol. 1994;151:183–228. doi: 10.1016/s0074-7696(08)62633-9. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Finlay D. R., Meier E., Bradley P., Horecka J., Forbes D. J. A complex of nuclear pore proteins required for pore function. J Cell Biol. 1991 Jul;114(1):169–183. doi: 10.1083/jcb.114.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Forbes D. J. Structure and function of the nuclear pore complex. Annu Rev Cell Biol. 1992;8:495–527. doi: 10.1146/annurev.cb.08.110192.002431. [DOI] [PubMed] [Google Scholar]
  29. Gerace L. Molecular trafficking across the nuclear pore complex. Curr Opin Cell Biol. 1992 Aug;4(4):637–645. doi: 10.1016/0955-0674(92)90083-o. [DOI] [PubMed] [Google Scholar]
  30. Gottesfeld J. M., Wolf V. J., Dang T., Forbes D. J., Hartl P. Mitotic repression of RNA polymerase III transcription in vitro mediated by phosphorylation of a TFIIIB component. Science. 1994 Jan 7;263(5143):81–84. doi: 10.1126/science.8272869. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Hartl P., Olson E., Dang T., Forbes D. J. Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate. J Cell Biol. 1994 Feb;124(3):235–248. doi: 10.1083/jcb.124.3.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hassan A. B., Errington R. J., White N. S., Jackson D. A., Cook P. R. Replication and transcription sites are colocalized in human cells. J Cell Sci. 1994 Feb;107(Pt 2):425–434. doi: 10.1242/jcs.107.2.425. [DOI] [PubMed] [Google Scholar]
  34. Hirano T., Mitchison T. J. Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts. J Cell Biol. 1993 Feb;120(3):601–612. doi: 10.1083/jcb.120.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hozák P., Cook P. R. Replication factories. Trends Cell Biol. 1994 Feb;4(2):48–52. doi: 10.1016/0962-8924(94)90009-4. [DOI] [PubMed] [Google Scholar]
  36. Hozák P., Jackson D. A., Cook P. R. Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle. J Cell Sci. 1994 Aug;107(Pt 8):2191–2202. doi: 10.1242/jcs.107.8.2191. [DOI] [PubMed] [Google Scholar]
  37. Huang S., Spector D. L. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev. 1991 Dec;5(12A):2288–2302. doi: 10.1101/gad.5.12a.2288. [DOI] [PubMed] [Google Scholar]
  38. Jackson D. A., Hassan A. B., Errington R. J., Cook P. R. Visualization of focal sites of transcription within human nuclei. EMBO J. 1993 Mar;12(3):1059–1065. doi: 10.1002/j.1460-2075.1993.tb05747.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Jackson D. A. The organization of replication centres in higher eukaryotes. Bioessays. 1990 Feb;12(2):87–89. doi: 10.1002/bies.950120207. [DOI] [PubMed] [Google Scholar]
  40. Jarmolowski A., Boelens W. C., Izaurralde E., Mattaj I. W. Nuclear export of different classes of RNA is mediated by specific factors. J Cell Biol. 1994 Mar;124(5):627–635. doi: 10.1083/jcb.124.5.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kadowaki T., Chen S., Hitomi M., Jacobs E., Kumagai C., Liang S., Schneiter R., Singleton D., Wisniewska J., Tartakoff A. M. Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants. J Cell Biol. 1994 Aug;126(3):649–659. doi: 10.1083/jcb.126.3.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kamakaka R. T., Bulger M., Kadonaga J. T. Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly. Genes Dev. 1993 Sep;7(9):1779–1795. doi: 10.1101/gad.7.9.1779. [DOI] [PubMed] [Google Scholar]
  43. Kassavetis G. A., Bartholomew B., Blanco J. A., Johnson T. E., Geiduschek E. P. Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7308–7312. doi: 10.1073/pnas.88.16.7308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Kassavetis G. A., Riggs D. L., Negri R., Nguyen L. H., Geiduschek E. P. Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes. Mol Cell Biol. 1989 Jun;9(6):2551–2566. doi: 10.1128/mcb.9.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Kornbluth S., Sebastian B., Hunter T., Newport J. Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14. Mol Biol Cell. 1994 Mar;5(3):273–282. doi: 10.1091/mbc.5.3.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Krug R. M. The regulation of export of mRNA from nucleus to cytoplasm. Curr Opin Cell Biol. 1993 Dec;5(6):944–949. doi: 10.1016/0955-0674(93)90074-z. [DOI] [PubMed] [Google Scholar]
  47. Lang I., Scholz M., Peters R. Molecular mobility and nucleocytoplasmic flux in hepatoma cells. J Cell Biol. 1986 Apr;102(4):1183–1190. doi: 10.1083/jcb.102.4.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Laybourn P. J., Kadonaga J. T. Threshold phenomena and long-distance activation of transcription by RNA polymerase II. Science. 1992 Sep 18;257(5077):1682–1685. doi: 10.1126/science.1388287. [DOI] [PubMed] [Google Scholar]
  50. Lazarides E., Lindberg U. Actin is the naturally occurring inhibitor of deoxyribonuclease I. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4742–4746. doi: 10.1073/pnas.71.12.4742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Leno G. H., Laskey R. A. The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts. J Cell Biol. 1991 Feb;112(4):557–566. doi: 10.1083/jcb.112.4.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Lewin B. Chromatin and gene expression: constant questions, but changing answers. Cell. 1994 Nov 4;79(3):397–406. doi: 10.1016/0092-8674(94)90249-6. [DOI] [PubMed] [Google Scholar]
  53. Logan K., Ackerman S. Effects of antibiotics on RNA polymerase III transcription. DNA. 1988 Sep;7(7):483–491. doi: 10.1089/dna.1.1988.7.483. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. 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]
  57. 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]
  58. Mills A. D., Blow J. J., White J. G., Amos W. B., Wilcock D., Laskey R. A. Replication occurs at discrete foci spaced throughout nuclei replicating in vitro. J Cell Sci. 1989 Nov;94(Pt 3):471–477. doi: 10.1242/jcs.94.3.471. [DOI] [PubMed] [Google Scholar]
  59. Méchali M., Harland R. M. DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro. Cell. 1982 Aug;30(1):93–101. doi: 10.1016/0092-8674(82)90015-0. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. 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]
  62. 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]
  63. Newmeyer D. D., Wilson K. L. Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol. 1991;36:607–634. doi: 10.1016/s0091-679x(08)60299-x. [DOI] [PubMed] [Google Scholar]
  64. Newport J. W., Forbes D. J. The nucleus: structure, function, and dynamics. Annu Rev Biochem. 1987;56:535–565. doi: 10.1146/annurev.bi.56.070187.002535. [DOI] [PubMed] [Google Scholar]
  65. 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]
  66. 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]
  67. 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]
  68. 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]
  69. 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]
  70. Pazin M. J., Kamakaka R. T., Kadonaga J. T. ATP-dependent nucleosome reconfiguration and transcriptional activation from preassembled chromatin templates. Science. 1994 Dec 23;266(5193):2007–2011. doi: 10.1126/science.7801129. [DOI] [PubMed] [Google Scholar]
  71. Peterson R. C., Doering J. L., Brown D. D. Characterization of two xenopus somatic 5S DNAs and one minor oocyte-specific 5S DNA. Cell. 1980 May;20(1):131–141. doi: 10.1016/0092-8674(80)90241-x. [DOI] [PubMed] [Google Scholar]
  72. Powers M. A., Macaulay C., Masiarz F. R., Forbes D. J. Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication. J Cell Biol. 1995 Mar;128(5):721–736. doi: 10.1083/jcb.128.5.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Schlenstedt G., Saavedra C., Loeb J. D., Cole C. N., Silver P. A. The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):225–229. doi: 10.1073/pnas.92.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. 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]
  75. Smith D. R., Jackson I. J., Brown D. D. Domains of the positive transcription factor specific for the Xenopus 5S RNA gene. Cell. 1984 Jun;37(2):645–652. doi: 10.1016/0092-8674(84)90396-9. [DOI] [PubMed] [Google Scholar]
  76. 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]
  77. Spector D. L. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. 1993;9:265–315. doi: 10.1146/annurev.cb.09.110193.001405. [DOI] [PubMed] [Google Scholar]
  78. Sullivan K. M., Busa W. B., Wilson K. L. Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function. Cell. 1993 Jul 2;73(7):1411–1422. doi: 10.1016/0092-8674(93)90366-x. [DOI] [PubMed] [Google Scholar]
  79. 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]
  80. Wallrath L. L., Lu Q., Granok H., Elgin S. C. Architectural variations of inducible eukaryotic promoters: preset and remodeling chromatin structures. Bioessays. 1994 Mar;16(3):165–170. doi: 10.1002/bies.950160306. [DOI] [PubMed] [Google Scholar]
  81. Wansink D. G., Schul W., van der Kraan I., van Steensel B., van Driel R., de Jong L. Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J Cell Biol. 1993 Jul;122(2):283–293. doi: 10.1083/jcb.122.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Wente S. R., Blobel G. A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic. J Cell Biol. 1993 Oct;123(2):275–284. doi: 10.1083/jcb.123.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. 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]
  84. Wolffe A. P. Replication timing and Xenopus 5S RNA gene transcription in vitro. Dev Biol. 1993 May;157(1):224–231. doi: 10.1006/dbio.1993.1126. [DOI] [PubMed] [Google Scholar]
  85. Wolffe A. P. Transcriptional activation of Xenopus class III genes in chromatin isolated from sperm and somatic nuclei. Nucleic Acids Res. 1989 Jan 25;17(2):767–780. doi: 10.1093/nar/17.2.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Workman J. L., Buchman A. R. Multiple functions of nucleosomes and regulatory factors in transcription. Trends Biochem Sci. 1993 Mar;18(3):90–95. doi: 10.1016/0968-0004(93)90160-o. [DOI] [PubMed] [Google Scholar]
  87. Xing Y., Johnson C. V., Dobner P. R., Lawrence J. B. Higher level organization of individual gene transcription and RNA splicing. Science. 1993 Feb 26;259(5099):1326–1330. doi: 10.1126/science.8446901. [DOI] [PubMed] [Google Scholar]
  88. Yan H., Newport J. An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor. J Cell Biol. 1995 Apr;129(1):1–15. doi: 10.1083/jcb.129.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES