Regulation of DNA Synthesis at the First Cell Cycle in the Sea Urchin In Vivo

  • Jolanta Kisielewska
  • Michael Whitaker
Part of the Methods in Molecular Biology book series (MIMB, volume 1128)


Using fluorescent and non-fluorescent recombinant proteins has proved to be a very successful technique for following postfertilization events, in both male and female pronuclei during the first cell cycle of sea urchin in vivo. Proteins and dyes are introduced by microinjection into the unfertilized egg, and their function can be monitored by fluorescence or confocal/two-photon (2P) and transmitted light microscopy after insemination. Here, we describe expression and purification of GFP/RFP-tagged proteins involved in regulation of DNA replication. We also explain the techniques used to introduce recombinant proteins and fluorescent tubulin into sea urchin eggs and embryos.

Key words

Fluorescent proteins PCNA GFP/RFP Microinjection Confocal/2P imaging Male and female pronuclei 


  1. 1.
    Aze A et al (2010) Replication origins are already licensed in G1 arrested unfertilized sea urchin eggs. Dev Biol 340:557–570PubMedCrossRefGoogle Scholar
  2. 2.
    Stricker SA (1999) Comparative biology of calcium signalling during fertilization and egg activation in animals. Dev Biol 211:157–176PubMedCrossRefGoogle Scholar
  3. 3.
    Kisielewska J, Lu P, Whitaker M (2005) GFP-PCNA as an S-phase marker in embryos during the first and subsequent cell cycles. Biol Cell 97:221–229PubMedCrossRefGoogle Scholar
  4. 4.
    Philipova R et al (2005) ERK activation is required for S-phase onset and cell cycle progression after fertilization in sea urchin embryos. Development 132:579–589PubMedCrossRefGoogle Scholar
  5. 5.
    Beach D, Durkacz B, Nurse P (1982) Functionally homologous cell cycle control genes in budding and fission yeast. Nature 300:706–709PubMedCrossRefGoogle Scholar
  6. 6.
    Kisielewska J et al (2009) MAP kinase dependent cyclin E/cdk2 activity promotes DNA replication in early sea urchin embryos. Dev Biol 334:383–394PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Leonhardt H et al (2000) Dynamics of DNA replication factories in living cells. J Cell Biol 149:271–279PubMedCrossRefGoogle Scholar
  8. 8.
    Das-Bradoo S, Nguyen HD, Bielinsky AK (2010) Damage-specific modification of PCNA. Cell Cycle 9:3674–3679PubMedCrossRefGoogle Scholar
  9. 9.
    Essers J et al (2005) Nuclear dynamics of PCNA in DNA replication and repair. Mol Cell Biol 25:9350–9359PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Bell SP, Dutta A (2001) DNA replication in eukaryotic cells. Annu Rev Biochem 71:333–374PubMedCrossRefGoogle Scholar
  11. 11.
    Schnackenberg BJ, Marzluff WF (2002) Novel localization and possible functions of cyclin E in early sea urchin development. J Cell Sci 115:113–121PubMedGoogle Scholar
  12. 12.
    Philipova R, Whitaker M (1998) MAP kinase activity increases during mitosis in early sea urchin embryos. J Cell Sci 111:2497–2505PubMedGoogle Scholar
  13. 13.
    Mazia D, Schatten G, Sale W (1975) Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy. J Cell Biol 66:198–200PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Jolanta Kisielewska
    • 1
  • Michael Whitaker
    • 1
  1. 1.Institute of Cell and Molecular Biosciences, Faculty of Medical Sciences, The Medical SchoolNewcastle UniversityNewcastle upon TyneUK

Personalised recommendations