Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Jul 1;107(1):33–44. doi: 10.1083/jcb.107.1.33

Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo

PMCID: PMC2115180  PMID: 3392102

Abstract

Biotinylated nucleotides (bio-11-dCTP, bio-11-dUTP, and bio-7-dATP) were microinjected into unfertilized and fertilized Xenopus laevis eggs. The amounts introduced were comparable to in vivo deoxy- nucleoside triphosphate pools. At various times after microinjection, DNA was extracted from eggs or embryos and subjected to electrophoresis on agarose gels. Newly synthesized biotinylated DNA was analyzed by Southern transfer and visualized using either the BluGENE or Detek-hrp streptavidin-based nucleic acid detection systems. Quantitation of the amount of biotinylated DNA observed at various times showed that the microinjected biotinylated nucleotides were efficiently incorporated in vivo, both into replicating endogenous chromosomal DNA and into replicating microinjected exogenous plasmid DNA. At least one biotinylated nucleotide could be incorporated in vivo for every eight nucleotides of DNA synthesized. Control experiments also showed that heavily biotinylated DNA was not subjected to detectable DNA repair during early embryogenesis (for at least 5 h after activation of the eggs). The incorporated biotinylated nucleotides were visualized by electron microscopy by using streptavidin-colloidal gold or streptavidin-ferritin conjugates to bind specifically to the biotin groups projecting from the newly replicated DNA. The incorporated biotinylated nucleotides were thus made visible as electron-dense spots on the underlying DNA molecules. Biotinylated nucleotides separated by 20-50 bases could be resolved. We conclude that nascent DNA synthesized in vivo in Xenopus laevis eggs can be visualized efficiently and specifically using the techniques described.

Full Text

The Full Text of this article is available as a PDF (4.8 MB).

Selected References

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

  1. Benbow R. M., Ford C. C. Cytoplasmic control of nuclear DNA synthesis during early development of Xenopus laevis: a cell-free assay. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2437–2441. doi: 10.1073/pnas.72.6.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benbow R. M., Krauss M. R., Reeder R. H. DNA synthesis in a multi-enzyme system from Xenopus laevis eggs. Cell. 1978 Feb;13(2):307–318. doi: 10.1016/0092-8674(78)90199-x. [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. 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]
  6. 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]
  7. CHAIET L., WOLF F. J. THE PROPERTIES OF STREPTAVIDIN, A BIOTIN-BINDING PROTEIN PRODUCED BY STREPTOMYCETES. Arch Biochem Biophys. 1964 Jul 20;106:1–5. doi: 10.1016/0003-9861(64)90150-x. [DOI] [PubMed] [Google Scholar]
  8. Callan H. G. DNA replication in the chromosomes of eukaryotes. Cold Spring Harb Symp Quant Biol. 1974;38:195–203. doi: 10.1101/sqb.1974.038.01.023. [DOI] [PubMed] [Google Scholar]
  9. Callan H. G. Replication of DNA in eukaryotic chromosomes. Br Med Bull. 1973 Sep;29(3):192–195. doi: 10.1093/oxfordjournals.bmb.a071006. [DOI] [PubMed] [Google Scholar]
  10. Callan H. G. Replication of DNA in the chromosomes of eukaryotes. Proc R Soc Lond B Biol Sci. 1972 Apr 18;181(1062):19–41. doi: 10.1098/rspb.1972.0039. [DOI] [PubMed] [Google Scholar]
  11. Edenberg H. J., Huberman J. A. Eukaryotic chromosome replication. Annu Rev Genet. 1975;9:245–284. doi: 10.1146/annurev.ge.09.120175.001333. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gaudette M. F., Benbow R. M. Replication forks are underrepresented in chromosomal DNA of Xenopus laevis embryos. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5953–5957. doi: 10.1073/pnas.83.16.5953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Green N. M. Avidin. Adv Protein Chem. 1975;29:85–133. doi: 10.1016/s0065-3233(08)60411-8. [DOI] [PubMed] [Google Scholar]
  16. Guinta D. R., Tso J. Y., Narayanswami S., Hamkalo B. A., Korn L. J. Early replication and expression of oocyte-type 5S RNA genes in a Xenopus somatic cell line carrying a translocation. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5150–5154. doi: 10.1073/pnas.83.14.5150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gurdon J. B. Methods for nuclear transplantation in amphibia. Methods Cell Biol. 1977;16:125–139. doi: 10.1016/s0091-679x(08)60096-5. [DOI] [PubMed] [Google Scholar]
  18. Hand R. Eucaryotic DNA: organization of the genome for replication. Cell. 1978 Oct;15(2):317–325. doi: 10.1016/0092-8674(78)90001-6. [DOI] [PubMed] [Google Scholar]
  19. Huberman J. A., Riggs A. D. On the mechanism of DNA replication in mammalian chromosomes. J Mol Biol. 1968 Mar 14;32(2):327–341. doi: 10.1016/0022-2836(68)90013-2. [DOI] [PubMed] [Google Scholar]
  20. Huberman J. A. Visualization of replicating mammalian and T4 bacteriophage DNA. Cold Spring Harb Symp Quant Biol. 1968;33:509–524. doi: 10.1101/sqb.1968.033.01.059. [DOI] [PubMed] [Google Scholar]
  21. Hunting D. J., Dresler S. L., de Murcia G. Incorporation of biotin-labeled deoxyuridine triphosphate into DNA during excision repair and electron microscopic visualization of repair patches. Biochemistry. 1985 Oct 8;24(21):5729–5734. doi: 10.1021/bi00342a007. [DOI] [PubMed] [Google Scholar]
  22. Lang D., Mitani M. Simplified quantitative electron microscopy of biopolymers. Biopolymers. 1970;9(3):373–379. doi: 10.1002/bip.1970.360090310. [DOI] [PubMed] [Google Scholar]
  23. Langer P. R., Waldrop A. A., Ward D. C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6633–6637. doi: 10.1073/pnas.78.11.6633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leary J. J., Brigati D. J., Ward D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4045–4049. doi: 10.1073/pnas.80.13.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Menissier J., Hunting D. J., De Murcia G. Electron microscopic mapping of single-stranded discontinuities in cauliflower mosaic virus DNA by means of the biotin-avidin technique. Anal Biochem. 1985 Aug 1;148(2):339–343. doi: 10.1016/0003-2697(85)90237-4. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Rochaix J. D., Bird A., Barkken A. Ribosomal RNA gene amplification by rolling circles. J Mol Biol. 1974 Aug 15;87(3):473–487. doi: 10.1016/0022-2836(74)90098-9. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. WATSON J. D., CRICK F. H. Genetical implications of the structure of deoxyribonucleic acid. Nature. 1953 May 30;171(4361):964–967. doi: 10.1038/171964b0. [DOI] [PubMed] [Google Scholar]
  33. Wickens M. P., Buell G. N., Schimke R. T. Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I. J Biol Chem. 1978 Apr 10;253(7):2483–2495. [PubMed] [Google Scholar]
  34. Wolf D. P., Hedrick J. L. A molecular approach to fertilization. II. Viability and artificial fertilization of Xenopus laevis gemetes. Dev Biol. 1971 Jul;25(3):348–359. doi: 10.1016/0012-1606(71)90036-4. [DOI] [PubMed] [Google Scholar]
  35. Woodland H. R., Pestell R. Q. Determination of the nucleoside triphosphate contents of eggs and oocytes of Xenopus laevis. Biochem J. 1972 Apr;127(3):597–605. doi: 10.1042/bj1270597. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES