Skip to main content

Fluorescent Transgenes to Study Interphase Chromosomes in Living Plants

  • Protocol
The Nucleus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 463))

Abstract

Fluorescence tagging of genomic sites through the use of bacterial operator/repressor systems combined with fluorescent proteins permits high-resolution analysis of interphase chromosomes in living cells. This technique has been used to study interphase chromosome arrangement and dynamics in yeast, Drosophila, and mammalian cells, but is only beginning to be exploited in plant systems. In this chapter, we describe methods for producing and identifying Arabidopsis thaliana plants harbouring fluorescence-tagged transgenes. The use of these plants to analyze various aspects of interphase chromosome organization and dynamics in living cells using 3D wide-field fluorescence microscopy is discussed. Potential problems encountered when utilizing this technology in plants are considered.

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

Access this chapter

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 109.00
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. Misteli, T. (2004) Spatial positioning: a new dimension in genome function.Cell 119, 153–156.

    Article  PubMed  CAS  Google Scholar 

  2. Gasser, S. (2002) Visualizing chromatin dynamics in interphase nuclei.Science 296, 1412–1416.

    Article  PubMed  CAS  Google Scholar 

  3. Spector, D.L. (2003) The dynamics of chromosome organization and gene regulation.Annu. Rev. Biochem. 72, 573–608.

    Article  PubMed  CAS  Google Scholar 

  4. Kato, N. and Lam, E. (2001) Detection of chromosomes tagged with green fluorescent protein in liveArabidopsis plants.Genome Biol 2, research0045.

    Google Scholar 

  5. Esch, J.J., Chen, M., Sanders, M., Hillestad, M., Ndkium, S., Idelkope, B., Neizer, J., and Marks, M.D. (2003) A contradictory GLABRA3 allele helps define gene interactions controlling trichome development inArabidopsis.Development 130, 5885–5894.

    Article  PubMed  CAS  Google Scholar 

  6. Lam, E., Kato, N., and Watanabe, K. (2004) Visualizing chromosome structure/ organization.Annu. Rev. Plant Biol. 55, 537–554.

    Article  PubMed  CAS  Google Scholar 

  7. Matzke, A.J.M., Huettel, B., van der Winden, J., and Matzke, M. (2005) Use of two color flu-orescently-tagged transgenes to study interphase chromosomes in living plants.Plant Physiol. 139, 1586–1596.

    Article  PubMed  CAS  Google Scholar 

  8. Hood, E.E., Nilsson, O., Wu, E., Wolfe, D.S., and Weigel, D. (1998) Genetic ablation of flowers in transgenicArabidopsis.Plant J. 15799–804.

    Article  Google Scholar 

  9. Matzke, A.J.M. and Matzke, M.A. (1986) A set of novel Ti plasmid-derived vectors for the production of transgenic plants.Plant Mol. Biol. 7, 357–365.

    Article  CAS  Google Scholar 

  10. Matzke, A.J.M., Stöger, E.M., Schernthaner, J.P., and Matzke, M.A. (1990) Deletion analysis of a zein gene promoter in transgenic tobacco plants.Plant Mol. Biol. 14, 323–332.

    Article  PubMed  CAS  Google Scholar 

  11. Matzke, A.J.M., van der Winden, J., and Matzke, M. (2003) Tetracycline operator/ repressor system to visualize fluorescence-tagged T-DNAs in interphase nuclei ofArabidopsis.Plant Mol. Biol. Rep. 21, 9–19.

    Article  CAS  Google Scholar 

  12. Clough, S.J. and Bent, A. (1998) Floral dip: A simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. Plant J. 16, 735–743.

    Article  PubMed  CAS  Google Scholar 

  13. Dhaese, P., De Greve, H., Decraemer, H., Schell, J., and Van Montagu, M. (1979) Rapid mapping of transposon insertion and deletion mutations in the large Ti plasmids of Agrobacterium tumefaciens.Nucleic Acids Res. 7, 1837–1849.

    Article  PubMed  CAS  Google Scholar 

  14. Maniatis, T. Fritsch, E.F., and Sambrook, J. (eds.) (1982)Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, NY.

    Google Scholar 

  15. Bystricky, K., Heun, P., Gehlen, L., Langowski, J., and Gasser, S.M. (2004) Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high resolution imaging techniques.Proc. Natl. Acad. Sci. USA 101, 16495–16500.

    Article  PubMed  CAS  Google Scholar 

  16. Pecinka, A., Kato, N., Meister, A., Probst, A.V., Schubert, I., and Lam, E. (2005) Tandem repetitive transgenes and fluorescent chromatin tags alter local interphase chromosome arrangement in Arabidopsis thaliana.J. Cell Sci. 118, 3751–3758.

    Article  PubMed  CAS  Google Scholar 

  17. Watanabe, K., Pecinka, A., Meister, A., Schubert, I., and Lam, E. (2005) DNA hypomethyla-tion reduces homologous pairing of inserted tandem repeat arrays in somatic nuclei ofArabidopsis thaliana.Plant J. 44, 531–540.

    Article  PubMed  CAS  Google Scholar 

  18. Fuchs, J., Lorenz, A., and Loidl, J. (2002) Chromosome associations in budding yeast caused by integrated tandemly repeated transgenes.J. Cell Sci. 15, 1213–1220.

    Google Scholar 

  19. Vasquez, J., Belmont, A.S., and Sedat, J.W. (2002) The dynamics of homologous chromosome pairing in maleDrosophila meiosis.Curr. Biol. 12, 1473–1483.

    Article  Google Scholar 

  20. Fransz, P., de Jong, J.H., Lysak, M., Castiglione, M.R., and Schubert, I. (2002) Interphase chromosomes inArabidopsis are organized as well defined chromocenters from which euchromatin loops emanate.Proc. Natl. Acad. Sci. USA 99, 14584–14589.

    Article  PubMed  CAS  Google Scholar 

  21. Pecinka, A., Schubert, V., Meister, A., Kreth, G., Klatte, M., Lysak, M.A., Fuchs, J., and Schubert, I. (2004) Chromosome territory arrangement and homologous pairing in nuclei ofArabidopsis thaliana are predominantly random except of NOR-bearing chromosomes.Chromosoma 113, 258–269.

    Article  PubMed  CAS  Google Scholar 

  22. Chuang, C.H., Carpenter, A.E., Fuchsova, B., Johnson, T., de Lanerolle, P., and Belmont, A.S. (2006) Long-range directional movement of an interphase chromosome site.Curr. Biol. 16, 825–831.

    Article  PubMed  CAS  Google Scholar 

  23. Kato, N. and Lam, E. (2003) Chromatin of endoreduplicated pavement cells has greater range of movement than that of diploid guard cells inArabidopsis thaliana.J. Cell Sci. 116, 2195–2201.

    Article  PubMed  CAS  Google Scholar 

  24. Chytilova, E., Macas, J., Sliwinska, E., Rafelski, S.M., Lambert, G.M., and Galbraith, D.W. (2008) Nuclear dynamics inArabidopsis thaliana.Mol. Biol. Cell 11, 2733–2741.

    Google Scholar 

  25. Meier, I. (2007) Composition of the plant nuclear envelope: theme and variations. J. Exp. Bot. 58, 27–34.

    Article  PubMed  CAS  Google Scholar 

  26. Aufsatz, W. Mette, M.F., van der Winden, J., Matzke, M., and Matzke, A.J.M. (2002) HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double stranded RNA. EMBO J. 21, 6832–6841.

    Article  PubMed  CAS  Google Scholar 

  27. Pietrzak, M., Shillito, R.D., Hohn, T., and Potrykus, I. (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector.Nucleic Acids Res. 14, 5857–5868.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung (grant no. P 16545-B12).

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press

About this protocol

Cite this protocol

Matzke, A.J.M., Huettel, B., van der Winden, J., Matzke, M. (2008). Fluorescent Transgenes to Study Interphase Chromosomes in Living Plants. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 463. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-406-3_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-406-3_16

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-977-2

  • Online ISBN: 978-1-59745-406-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics