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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- ECFP:
-
enhanced cyan fluorescent protein
- EGFP:
-
enhanced green fluorescent protein
- EYFP:
-
enhanced yellow fluorescent protein
- NOS:
-
nopaline synthase
- tet :
-
tetracycline
References
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.
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.
Belmont A (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11: 250–257.
Belmont A and Straight A (1998) In vivo visualization of chromosomes usinglac operator-repressor binding. Trends Cell Biol 8: 121–124.
Clough SJ and Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana. Plant J 16: 735–743.
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.
Gasser S (2002) Visualizing chromatin dynamics in interphase nuclei. Science 296: 1412–1416.
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.
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.
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.
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.
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.
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.
Michaelis C, Ciosk R, and Nasmyth K (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91: 35–45.
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.
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.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Matzke, A.J.M., van der Winden, J. & Matzke, M. Tetracycline operator/repressor system to visualize fluorescence-tagged T-DNAs in interphase nuclei ofArabidopsis . Plant Mol Biol Rep 21, 9–19 (2003). https://doi.org/10.1007/BF02773392
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF02773392