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. 1991 Jan;11(1):299–308. doi: 10.1128/mcb.11.1.299

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

N J Marini 1, R M Benbow 1
PMCID: PMC359620  PMID: 1986227

Abstract

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

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Selected References

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

  1. Andres A. C., Muellener D. B., Ryffel G. U. Persistence, methylation and expression of vitellogenin gene derivatives after injection into fertilized eggs of Xenopus laevis. Nucleic Acids Res. 1984 Mar 12;12(5):2283–2302. doi: 10.1093/nar/12.5.2283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bayne M. L., Alexander R. F., Benbow R. M. DNA binding protein from ovaries of the frog, Xenopus laevis which promotes concatenation of linear DNA. J Mol Biol. 1984 Jan 5;172(1):87–108. doi: 10.1016/0022-2836(84)90416-9. [DOI] [PubMed] [Google Scholar]
  3. Bendig M. M. Persistence and expression of histone genes injected into Xenopus eggs in early development. Nature. 1981 Jul 2;292(5818):65–67. doi: 10.1038/292065a0. [DOI] [PubMed] [Google Scholar]
  4. Bendig M. M., Williams J. G. Replication and expression of Xenopus laevis globin genes injected into fertilized Xenopus eggs. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6197–6201. doi: 10.1073/pnas.80.20.6197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benedetti P., Baldi M. I., Mattoccia E., Tocchini-Valentini G. P. Purification and characterization of Xenopus laevis topoisomerase II. EMBO J. 1983;2(8):1303–1308. doi: 10.1002/j.1460-2075.1983.tb01585.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blow J. J., Laskey R. A. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. doi: 10.1038/332546a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Buongiorno-Nardelli M., Micheli G., Carri M. T., Marilley M. A relationship between replicon size and supercoiled loop domains in the eukaryotic genome. Nature. 1982 Jul 1;298(5869):100–102. doi: 10.1038/298100a0. [DOI] [PubMed] [Google Scholar]
  10. Chambers J. C., Watanabe S., Taylor J. H. Dissection of a replication origin of Xenopus DNA. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5572–5576. doi: 10.1073/pnas.79.18.5572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dingwall C., Laskey R. A. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. doi: 10.1146/annurev.cb.02.110186.002055. [DOI] [PubMed] [Google Scholar]
  12. Endean D. J., Smithies O. Replication of plasmid DNA in fertilized Xenopus eggs is sensitive to both the topology and size of the injected template. Chromosoma. 1989 Jan;97(4):307–314. doi: 10.1007/BF00371971. [DOI] [PubMed] [Google Scholar]
  13. Etkin L. D., Pearman B. Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs. Development. 1987 Jan;99(1):15–23. doi: 10.1242/dev.99.1.15. [DOI] [PubMed] [Google Scholar]
  14. Etkin L. d., Pearman B., Roberts M., Bektesh S. L. Replication, integration and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis. Differentiation. 1984;26(3):194–202. doi: 10.1111/j.1432-0436.1984.tb01395.x. [DOI] [PubMed] [Google Scholar]
  15. Flytzanis C. N., McMahon A. P., Hough-Evans B. R., Katula K. S., Britten R. J., Davidson E. H. Persistence and integration of cloned DNA in postembryonic sea urchins. Dev Biol. 1985 Apr;108(2):431–442. doi: 10.1016/0012-1606(85)90046-6. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Gargiulo G., Wasserman W., Worcel A. Properties of the chromatin assembled on DNA injected into Xenopus oocytes and eggs. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):549–556. doi: 10.1101/sqb.1983.047.01.064. [DOI] [PubMed] [Google Scholar]
  18. Glikin G. C., Ruberti I., Worcel A. Chromatin assembly in Xenopus oocytes: in vitro studies. Cell. 1984 May;37(1):33–41. doi: 10.1016/0092-8674(84)90298-8. [DOI] [PubMed] [Google Scholar]
  19. Graham C. F. The regulation of DNA synthesis and mitosis in multinucleate frog eggs. J Cell Sci. 1966 Sep;1(3):363–374. doi: 10.1242/jcs.1.3.363. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Hines P. J., Benbow R. M. Initiation of replication at specific origins in DNA molecules microinjected into unfertilized eggs of the frog Xenopus laevis. Cell. 1982 Sep;30(2):459–468. doi: 10.1016/0092-8674(82)90243-4. [DOI] [PubMed] [Google Scholar]
  22. Hutchison C. J., Cox R., Ford C. C. The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro. Development. 1988 Jul;103(3):553–566. doi: 10.1242/dev.103.3.553. [DOI] [PubMed] [Google Scholar]
  23. Hutchison C., Kill I. Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs. J Cell Sci. 1989 Aug;93(Pt 4):605–613. doi: 10.1242/jcs.93.4.605. [DOI] [PubMed] [Google Scholar]
  24. Jong A. Y., Scott J. F. DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli. Nucleic Acids Res. 1985 Apr 25;13(8):2943–2958. doi: 10.1093/nar/13.8.2943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kaiserman H. B., Ingebritsen T. S., Benbow R. M. Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro. Biochemistry. 1988 May 3;27(9):3216–3222. doi: 10.1021/bi00409a014. [DOI] [PubMed] [Google Scholar]
  26. Krieg P. A., Melton D. A. Developmental regulation of a gastrula-specific gene injected into fertilized Xenopus eggs. EMBO J. 1985 Dec 16;4(13A):3463–3471. doi: 10.1002/j.1460-2075.1985.tb04105.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Marini N. J., Etkin L. D., Benbow R. M. Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis. Dev Biol. 1988 Jun;127(2):421–434. doi: 10.1016/0012-1606(88)90328-4. [DOI] [PubMed] [Google Scholar]
  30. Marini N. J., Hiriyanna K. T., Benbow R. M. Differential replication of circular DNA molecules co-injected into early Xenopus laevis embryos. Nucleic Acids Res. 1989 Jul 25;17(14):5793–5808. doi: 10.1093/nar/17.14.5793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McMahon A. P., Flytzanis C. N., Hough-Evans B. R., Katula K. S., Britten R. J., Davidson E. H. Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev Biol. 1985 Apr;108(2):420–430. doi: 10.1016/0012-1606(85)90045-4. [DOI] [PubMed] [Google Scholar]
  32. McTiernan C. F., Stambrook P. J. Initiation of SV40 DNA replication after microinjection into Xenopus eggs. Biochim Biophys Acta. 1984 Jul 18;782(3):295–303. doi: 10.1016/0167-4781(84)90065-4. [DOI] [PubMed] [Google Scholar]
  33. Montag M., Spring H., Trendelenburg M. F. Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos. Chromosoma. 1988;96(3):187–196. doi: 10.1007/BF00302357. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Méchali M., Kearsey S. Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast. Cell. 1984 Aug;38(1):55–64. doi: 10.1016/0092-8674(84)90526-9. [DOI] [PubMed] [Google Scholar]
  36. Newport J. W., Kirschner M. W. Regulation of the cell cycle during early Xenopus development. Cell. 1984 Jul;37(3):731–742. doi: 10.1016/0092-8674(84)90409-4. [DOI] [PubMed] [Google Scholar]
  37. Newport J., Kirschner M. A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell. 1982 Oct;30(3):675–686. doi: 10.1016/0092-8674(82)90272-0. [DOI] [PubMed] [Google Scholar]
  38. Newport J., Kirschner M. A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell. 1982 Oct;30(3):687–696. doi: 10.1016/0092-8674(82)90273-2. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Rusconi S., Schaffner W. Transformation of frog embryos with a rabbit beta-globin gene. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5051–5055. doi: 10.1073/pnas.78.8.5051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ryoji M., Tominna E., Yasui W. Minichromosome assembly accompanying repair-type DNA synthesis in Xenopus oocytes. Nucleic Acids Res. 1989 Dec 25;17(24):10243–10258. doi: 10.1093/nar/17.24.10243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ryoji M., Worcel A. Chromatin assembly in Xenopus oocytes: in vivo studies. Cell. 1984 May;37(1):21–32. doi: 10.1016/0092-8674(84)90297-6. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Shiokawa K., Sameshima M., Tashiro K., Miura T., Nakakura N., Yamana K. Formation of nucleus-like structure in the cytoplasm of lambda-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis. Dev Biol. 1986 Aug;116(2):539–542. doi: 10.1016/0012-1606(86)90155-7. [DOI] [PubMed] [Google Scholar]
  45. Trendelenburg M. F., Oudet P., Spring H., Montag M. DNA injections into Xenopus embryos: fate of injected DNA in relation to formation of embryonic nuclei. J Embryol Exp Morphol. 1986 Oct;97 (Suppl):243–255. [PubMed] [Google Scholar]
  46. Watanabe S., Taylor J. H. Cloning of an origin of DNA replication of Xenopus laevis. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5292–5296. doi: 10.1073/pnas.77.9.5292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wilson C., Cross G. S., Woodland H. R. Tissue-specific expression of actin genes injected into Xenopus embryos. Cell. 1986 Nov 21;47(4):589–599. doi: 10.1016/0092-8674(86)90623-9. [DOI] [PubMed] [Google Scholar]
  48. Woodland H. R., Adamson E. D. The synthesis and storage of histones during the oogenesis of Xenopus laevis. Dev Biol. 1977 May;57(1):118–135. doi: 10.1016/0012-1606(77)90359-1. [DOI] [PubMed] [Google Scholar]
  49. Zierler M. K., Marini N. J., Stowers D. J., Benbow R. M. Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis. J Biol Chem. 1985 Jan 25;260(2):974–981. [PubMed] [Google Scholar]
  50. Zuber M., Yasui W., Tan E. M., Ryoji M. Quantitation and subcellular localization of proliferating cell nuclear antigen (PCNA/cyclin) in oocytes and eggs of Xenopus laevis. Exp Cell Res. 1989 Jun;182(2):384–393. doi: 10.1016/0014-4827(89)90243-7. [DOI] [PubMed] [Google Scholar]
  51. van der Velden H. M., Wanka F. The nuclear matrix--its role in the spatial organization and replication of eukaryotic DNA. Mol Biol Rep. 1987;12(2):69–77. doi: 10.1007/BF00368873. [DOI] [PubMed] [Google Scholar]

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