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High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants.

Matzke AJ, Watanabe K, van der Winden J, Naumann U, Matzke M - Plant Methods (2010)

Bottom Line: First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence.The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle.These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria.

ABSTRACT

Background: Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells.

Results: We used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B.

Conclusions: The results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending on the promoter used to drive expression of the RP-FP fusion protein gene, the fluorescent tagged sites can be visualized at high frequency in different cell types. The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle. These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

No MeSH data available.


Related in: MedlinePlus

Range of inter-allelic distances in cells of root division zone. 3D inter-allelic distances in interphase nuclei of the root division zone (shown in Fig. 3) were measured using Imaris software as described in the Method section. The values were entered into Excel and the graph was produced by sorting in descending order. For nuclei numbered 86 to 100, two distinct dots were not distinguished by Imaris software. These are displayed on this graph as 'zero' and indicated by white arrows in Fig. 3. Possible explanations for this are discussed in the text.
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Figure 5: Range of inter-allelic distances in cells of root division zone. 3D inter-allelic distances in interphase nuclei of the root division zone (shown in Fig. 3) were measured using Imaris software as described in the Method section. The values were entered into Excel and the graph was produced by sorting in descending order. For nuclei numbered 86 to 100, two distinct dots were not distinguished by Imaris software. These are displayed on this graph as 'zero' and indicated by white arrows in Fig. 3. Possible explanations for this are discussed in the text.

Mentions: High frequency and uniform expression of the RP-FP fusion protein through the use of cell type-specific promoters allows the measurement of significant numbers inter-allelic 3D distances in neighbouring cells of homozygous tagged lines. In cells of the root division zone, inter-allelic distances comprise a continuum of values, with most ranging between 1 μm to 5 μm (Fig. 5). The rather wide range of inter-allelic distances is consistent with previous results on smaller sample sizes from other cell types and suggests an essentially random arrangement of interphase chromosomes [11,22].


High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants.

Matzke AJ, Watanabe K, van der Winden J, Naumann U, Matzke M - Plant Methods (2010)

Range of inter-allelic distances in cells of root division zone. 3D inter-allelic distances in interphase nuclei of the root division zone (shown in Fig. 3) were measured using Imaris software as described in the Method section. The values were entered into Excel and the graph was produced by sorting in descending order. For nuclei numbered 86 to 100, two distinct dots were not distinguished by Imaris software. These are displayed on this graph as 'zero' and indicated by white arrows in Fig. 3. Possible explanations for this are discussed in the text.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2820019&req=5

Figure 5: Range of inter-allelic distances in cells of root division zone. 3D inter-allelic distances in interphase nuclei of the root division zone (shown in Fig. 3) were measured using Imaris software as described in the Method section. The values were entered into Excel and the graph was produced by sorting in descending order. For nuclei numbered 86 to 100, two distinct dots were not distinguished by Imaris software. These are displayed on this graph as 'zero' and indicated by white arrows in Fig. 3. Possible explanations for this are discussed in the text.
Mentions: High frequency and uniform expression of the RP-FP fusion protein through the use of cell type-specific promoters allows the measurement of significant numbers inter-allelic 3D distances in neighbouring cells of homozygous tagged lines. In cells of the root division zone, inter-allelic distances comprise a continuum of values, with most ranging between 1 μm to 5 μm (Fig. 5). The rather wide range of inter-allelic distances is consistent with previous results on smaller sample sizes from other cell types and suggests an essentially random arrangement of interphase chromosomes [11,22].

Bottom Line: First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence.The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle.These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria.

ABSTRACT

Background: Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells.

Results: We used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B.

Conclusions: The results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending on the promoter used to drive expression of the RP-FP fusion protein gene, the fluorescent tagged sites can be visualized at high frequency in different cell types. The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle. These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

No MeSH data available.


Related in: MedlinePlus