Advertisement

Plant Molecular Biology Reporter

, Volume 21, Issue 1, pp 9–19 | Cite as

Tetracycline operator/repressor system to visualize fluorescence-tagged T-DNAs in interphase nuclei ofArabidopsis

  • Antonius J. M. Matzke
  • Johannes van der Winden
  • Marjori Matzke
Commentary

Abstract

The bacterial tetracycline operator/repressor (tetO/TetR) system and enhanced yellow fluorescent protein (EYFP) have been adapted for use inArabidopsis to visualize tagged T-DNAs in interphase nuclei of living cells. The 2-component system was assembled on a single T-DNA construct that contained a gene encoding a nuclear-targeted TetR-EYFP fusion protein under the control of the 35S promoter of the cauliflower mosaic virus together with multiple tandemly arranged copies of thetetO. In a number of independentArabidopsis lines transformed with this construct, bright fluorescent dots corresponding to tagged T-DNAs were observed in interphase nuclei of various cell types using standard fluorescence microscopy. In selfed progeny of a single locus line, hemizygous and homozygous plants were distinguished by having 1 or 2 fluorescent dots, respectively. Low background fluorescence of EYFP in many plant tissues facilitates the visualization of tagged T-DNAs. We compared features of thetetO/TetR-EYFP system to a second system we developed on the basis of the bacteriallac operator/repressor and enhanced green fluorescent protein.

Key words

chromatin dynamics green fluorescent protein interphase nuclei lac operator/repressor tet operator/repressor yellow fluorescent protein 

Abbreviations

ECFP

enhanced cyan fluorescent protein

EGFP

enhanced green fluorescent protein

EYFP

enhanced yellow fluorescent protein

NOS

nopaline synthase

tet

tetracycline

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Abranches R, Santos A, Wegel E, Williams S, Castilho A, Christou P, Shaw P, and Stöger E (2000) Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase. Plant J 24: 1–14.CrossRefGoogle Scholar
  2. Aragón-Alcaide L, Reader S, Beven A, Shaw P, Miller T, and Moore G (1997) Association of homologous chromosomes during floral development. Curr Biol 7: 905–908.PubMedCrossRefGoogle Scholar
  3. Belmont A (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11: 250–257.PubMedCrossRefGoogle Scholar
  4. Belmont A and Straight A (1998) In vivo visualization of chromosomes usinglac operator-repressor binding. Trends Cell Biol 8: 121–124.PubMedCrossRefGoogle Scholar
  5. Clough SJ and Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana. Plant J 16: 735–743.PubMedCrossRefGoogle Scholar
  6. Fuchs J, Lorenz A, and Loidl J (2002) Chromosome associations in budding yeast caused by integrated tandemly repeated transgenes. J Cell Sci 115: 1213–1220.PubMedGoogle Scholar
  7. Gasser S (2002) Visualizing chromatin dynamics in interphase nuclei. Science 296: 1412–1416.PubMedCrossRefGoogle Scholar
  8. ten Hoopen R, Montijn BM, Veuskens JTM, Oud O, and Nanninga N (1999) The spatial localization of T-DNA insertions in petunia interphase nuclei: consequences for chromosome organization and transgene insertion sites. Chromosome Res 7: 611–623.PubMedCrossRefGoogle Scholar
  9. Kato N and Lam E (2001) Detection of chromosomes tagged with green fluorescent protein in liveArabidopsis thaliana plants. Genome Biol 2: 0045.1–0045.10.CrossRefGoogle Scholar
  10. Kato N, Pontier D, and Lam E (2002) Spectral profiling for the simultaneous observation of four distinct fluorescent proteins and detection of protein-protein interaction via fluorescence resonance energy transfer in tobacco leaf nuclei. Plant Physiol 129: 931–942.PubMedCrossRefGoogle Scholar
  11. Matzke AJM and Matzke M (1986) A set of novel Ti plasmid-derived vectors for the production of transgenic plants. Plant Mol Biol 7: 357–365.CrossRefGoogle Scholar
  12. Matzke M, Mette MF, Jakowitsch J, Kanno T, Moscone EA, Van der Winden J, and Matzke AJM (2001) A test for transvection in plants: DNA pairing may lead to transactivation or silencing of complex heteroalleles in tobacco. Genetics 158: 451–461.PubMedGoogle Scholar
  13. Matzke M, Primig M, Trnovsky J, and Matzke AJM (1989) Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. MEBO J 8: 643–649.Google Scholar
  14. Michaelis C, Ciosk R, and Nasmyth K (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91: 35–45.PubMedCrossRefGoogle Scholar
  15. Pietrzak M, Shillito RD, Hohn T, and Potrykus I (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucl Acids Res 14: 5857–5868.PubMedCrossRefGoogle Scholar
  16. Straight A, Belmont A, Robinett C, and Murray A (1996) GFP tagging of budding yeast chromosome reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6: 1599–1608.PubMedCrossRefGoogle Scholar

Copyright information

© International Society for Plant Molecular Biology 2003

Authors and Affiliations

  • Antonius J. M. Matzke
    • 1
  • Johannes van der Winden
    • 1
  • Marjori Matzke
    • 1
  1. 1.Institute of Molecular BiologyAustrian Academy of SciencesSalzburgAustria

Personalised recommendations