Skip to main content
SpringerLink
Log in

Methods for Detecting Cells in S Phase

  • Protocol
Cell Cycle Checkpoint Control Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 241))

  • 1266 Accesses

Abstract

S phase is that period of time in the cell-division cycle during which nuclear chromosomal deoxyribonucleic acid (DNA) is replicated (1,2). The time required for S phase depends on the size of the genome, the organism, and its developmental state. DNA replication requires only 15 to 20 min in budding yeast, but 6 to 7 h in mammalian cells. In organisms, such as frogs, fish, echinoderms, and flies that undergo rapid cell cleavage events at the beginning of their development, S phase takes only a few minutes during these initial cell cleavage events, but it takes several hours in late-stage embryos, adult animals, or cells cultured in vitro.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. DePamphilis, M. L. (ed.) (1996) DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Plainview, NY.

    Google Scholar 

  2. Kornberg, A. and Baker, T. (1992) DNA Replication. W. H. Freeman, New York.

    Google Scholar 

  3. DePamphilis, M. L. (1995) Specific labeling of newly replicated DNA. Methods Enzymol. 262, 628–669.

    Article  PubMed  CAS  Google Scholar 

  4. DePamphilis, M. L. (ed.) (1997) Identification and Analysis of Replication Origins in Eukaryotic Cells, Vol. 13. Methods. Academic, New York.

    Google Scholar 

  5. Jackman, J. and O’Connor, P. M. (2002) Methods for synchronizing cells at specific stages of the cell cycle, In: Current Protocols in Cell Biology (Bonifacino J. S., et al., ed.), Wiley, New York, Unit 8.3.

    Google Scholar 

  6. Taylor, J. H., Myers, T. L., and Cunningham, H. L. (1971) Programmed synthesis of deoxyribonucleic acid during the cell cycle. In Vitro 6, 309–321.

    Article  PubMed  CAS  Google Scholar 

  7. Gilbert, D. M., Miyazawa, H., and DePamphilis, M. L. (1995) Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure. Mol. Cell. Biol. 15, 2942–2954.

    PubMed  CAS  Google Scholar 

  8. Brooks, R. F. (1975) The kinetics of serum-induced initiation of DNA synthesis in BHK 21/C13 cells, and the influence of exogenous adenosine. J. Cell. Physiol. 86(2 Pt 2 Suppl 1), 369–377.

    Article  PubMed  CAS  Google Scholar 

  9. Hola, M., Castleden, S., Howard, M., and Brooks, R. F. (1994) Initiation of DNA synthesis by nuclei from scrape-ruptured quiescent mammalian cells in high-speed supernatants of Xenopus egg extracts. J. Cell Sci. 107(Pt 11), 3045–3053.

    PubMed  CAS  Google Scholar 

  10. Rogers, M. (2002) Detection of hybridized probe, In: Current Protocols in Molecular Biology (Ausubel F. M., et al., ed.). Wiley, New York, Unit 14.4.

    Google Scholar 

  11. Taylor, J. H. (1968) Rates of chain growth and units of replication in DNA of mammalian chromosomes. J. Mol. Biol. 31, 579–594.

    Article  PubMed  CAS  Google Scholar 

  12. Shewach, D. S., Ellero, J., Mancini, W. R., and Ensminger, W. D. (1992) Decrease in TTP pools mediated by 5-bromo-2-deoxyuridine exposure in a human glioblastoma cell line. Biochem. Pharmacol. 43, 1579–1585.

    Article  PubMed  CAS  Google Scholar 

  13. Nakamura, H., Morita, T., and Sato, C. (1986) Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. Exp. Cell Res. 165, 291–297.

    Article  PubMed  CAS  Google Scholar 

  14. Jackson, D. A. and Pombo, A. (1998) Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–1295.

    Article  PubMed  CAS  Google Scholar 

  15. Herman, B. (2002) Fluorescence microscopy, In: Current Protocols in Cell Biology (Bonifacino J. S., et al., ed.), Wiley, New York, Unit 4.2.

    Google Scholar 

  16. Bouniol-Baly, C., Nguyen, E., Besombes, D., and Debey, P. (1997) Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation. Exp. Cell Res. 236, 201–211.

    Article  PubMed  CAS  Google Scholar 

  17. Anderson, S. and DePamphilis, M. L. (1979) Metabolism of Okazaki fragments during simian virus 40 DNA replication. J. Biol. Chem. 254, 11,495–11,504.

    PubMed  CAS  Google Scholar 

  18. Heintz, N. H. and Stillman, B. W. (1988) Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo. Mol. Cell. Biol. 8, 1923–1931.

    PubMed  CAS  Google Scholar 

  19. Dimitrova, D. S. and Gilbert, D. M. (1998) Regulation of mammalian replication origin usage in Xenopus egg extract. J. Cell Sci. 111(Pt 19), 2989–2998.

    PubMed  CAS  Google Scholar 

  20. Krude, T. (2000) Initiation of human DNA replication in vitro using nuclei from cells arrested at an initiation-competent state. J. Biol. Chem. 275, 13,699–13,707.

    Article  PubMed  CAS  Google Scholar 

  21. Graham, J. M. (2002) Isolation of nuclei and nuclear membranes from animal tissues, In: Current Protocols in Cell Biology (Bonifacino J. S. et al.,, ed.). Wiley, New York, Unit 3.10.

    Google Scholar 

  22. Fisher, P. A. and Berrios, M. (1998) Cell-free nuclear assembly and disassembly in Drosophila. Methods Cell Biol. 53, 397–416.

    Article  PubMed  CAS  Google Scholar 

  23. Berrios, M. (1998) Isolation and characterization of karyoskeletal protein-enriched fractions from vertebrate livers. Methods Cell Biol. 53, 3–22.

    Article  PubMed  CAS  Google Scholar 

  24. Dove, J. E., Brockenbrough, J. S., and Aris, J. P. (1998) Isolation of nuclei and nucleoli from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 53, 33–46.

    Article  PubMed  CAS  Google Scholar 

  25. Chong, J. P., Thommes, P., Rowles, A., Mahbubani, H. M., and Blow, J. J. (1997) Characterization of the Xenopus replication licensing system. Methods Enzymol. 283, 549–564.

    Article  PubMed  CAS  Google Scholar 

  26. Mills, A. D., Coleman, N., Morris, L. S., and Laskey, R. A. (2000) Detection of S-phase cells in tissue sections by in situ DNA replication. Nat. Cell Biol. 2, 244–245.

    Article  PubMed  CAS  Google Scholar 

  27. Sun, W., Hola, M., Pedley, K., et al. (2000) The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins. J. Cell Sci. 113(Pt 4), 683–695.

    PubMed  CAS  Google Scholar 

  28. Blow, J. J. and Laskey, R. A. (1986) Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell 47, 577–587.

    Article  PubMed  CAS  Google Scholar 

  29. Hutchison, C. J., Cox, R., Drepaul, R. S., Gomperts, M., and Ford, C. C. (1987) Periodic DNA synthesis in cell-free extracts of Xenopus eggs. EMBO J. 6, 2003–2010.

    PubMed  CAS  Google Scholar 

  30. Bravo, R. (1986) Synthesis of the nuclear protein cyclin (PCNA) and its relationship with DNA replication. Exp. Cell Res. 163, 287–293.

    Article  PubMed  CAS  Google Scholar 

  31. Kill, I. R., Bridger, J. M., Campbell, K. H., Maldonado-Codina, G., and Hutchison, C. J. (1991) The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts. J. Cell Sci. 100(Pt 4), 869–876.

    PubMed  Google Scholar 

  32. Dimitrova, D. S., Todorov, I. T., Melendy, T., and Gilbert, D. M. (1999) Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex. J. Cell Biol. 146, 709–722.

    Article  PubMed  CAS  Google Scholar 

  33. Iftode, C., Daniely, Y., and Borowiec, J. A. (1999) Replication protein A (RPA): the eukaryotic SSB. Crit. Rev. Biochem. Mol. Biol. 34, 141–180.

    Article  PubMed  CAS  Google Scholar 

  34. Todorov, I. T., Attaran, A., and Kearsey, S. E. (1995) BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. J. Cell Biol. 129, 1433–1445.

    Article  PubMed  CAS  Google Scholar 

  35. Gray, J. W. and Coffino, P. (1979) Cell cycle analysis by flow cytometry. Methods Enzymol. 58, 233–248.

    Article  PubMed  CAS  Google Scholar 

  36. Darzynkiewicz, Z., Juan, G., and Bedner, E. (2002) Cell cycle analysis, In: Current Protocols in Cell Biology (Bonifacino J. S., et al., ed.). Wiley, New York, Unit 8.4.

    Google Scholar 

  37. Dean, P. N. (2002) Flow cytometry instrumentation, In: Current Protocols in Cytometry (Robinson, J. P., ed.). Wiley, New York, Unit 1.1–1.18.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. National Institute of Mental Health, National Institutes of Health, Bethesda, MD

    Wei-Hsin Sun

  2. National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD

    Melvin L. DePamphilis

Authors
  1. Wei-Hsin Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Melvin L. DePamphilis
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Center for Radiological Research, Columbia University, New York, NY

    Howard B. Lieberman

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Sun, WH., DePamphilis, M.L. (2004). Methods for Detecting Cells in S Phase. In: Lieberman, H.B. (eds) Cell Cycle Checkpoint Control Protocols. Methods in Molecular Biology™, vol 241. Humana Press. https://doi.org/10.1385/1-59259-646-0:37

Download citation

  • DOI: https://doi.org/10.1385/1-59259-646-0:37

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-115-8

  • Online ISBN: 978-1-59259-646-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation