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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

Constructs and chromosomal locations of fluorescent-tagged sites. A. T-DNA constructs for fluorescent tagging. Construct 16 is based on red fluorescent protein (R). Construct 25 is based on enhanced green fluorescence protein (G). Both constructs use the lacOs and the LacI (L) fused to R and G. Genes encoding RL and GL are under the control of the 35S promoter of CaMV (35Sp). B. Chromosomal positions of tagged sites. The insertion sites have been reported previously [8,11]. Lines are named according to the construct used (16 or 25) followed by the line number (26, 79, 101, 107, 112). C. Constructs containing the RPS5 promoter to drive expression of genes encoding the EGFP-LacI fusion protein (GL) and histone H2B fused to monomeric DsRed (H2BmR). D. Constructs containing the GC1 promoter to drive expression of GL. Details of construct assembly using the modules shown in C and D are in the Methods section. Gene units are boxed; heavy outlines indicate genes encoding fluorescent fusion proteins (red, green) and lacOs (black). Arrows indicate the directions of transcription. Additional abbreviations: lb, T-DNA left border; rb, T-DNA right border; Np, NOS promoter; red 'R', DsRed2; green 'G', EGFP; t, transcriptional terminator from the 35S transcript of CaMV; nptIIb, neomycin phosphotransferase II for selection of bacteria on kanamycin; nt, NOS terminator; Mp, mannopine synthase promoter; ot, octopine synthase terminator; tag, 300 bp filler sequence; 19Sp, 19S promoter of CaMV; pDH51, pUC18 containing the 35S promoter-35S terminator cassette [33]; pBC, Bluescript plasmid encoding chloramphenicol resistance.
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Figure 1: Constructs and chromosomal locations of fluorescent-tagged sites. A. T-DNA constructs for fluorescent tagging. Construct 16 is based on red fluorescent protein (R). Construct 25 is based on enhanced green fluorescence protein (G). Both constructs use the lacOs and the LacI (L) fused to R and G. Genes encoding RL and GL are under the control of the 35S promoter of CaMV (35Sp). B. Chromosomal positions of tagged sites. The insertion sites have been reported previously [8,11]. Lines are named according to the construct used (16 or 25) followed by the line number (26, 79, 101, 107, 112). C. Constructs containing the RPS5 promoter to drive expression of genes encoding the EGFP-LacI fusion protein (GL) and histone H2B fused to monomeric DsRed (H2BmR). D. Constructs containing the GC1 promoter to drive expression of GL. Details of construct assembly using the modules shown in C and D are in the Methods section. Gene units are boxed; heavy outlines indicate genes encoding fluorescent fusion proteins (red, green) and lacOs (black). Arrows indicate the directions of transcription. Additional abbreviations: lb, T-DNA left border; rb, T-DNA right border; Np, NOS promoter; red 'R', DsRed2; green 'G', EGFP; t, transcriptional terminator from the 35S transcript of CaMV; nptIIb, neomycin phosphotransferase II for selection of bacteria on kanamycin; nt, NOS terminator; Mp, mannopine synthase promoter; ot, octopine synthase terminator; tag, 300 bp filler sequence; 19Sp, 19S promoter of CaMV; pDH51, pUC18 containing the 35S promoter-35S terminator cassette [33]; pBC, Bluescript plasmid encoding chloramphenicol resistance.

Mentions: For the tagged lines developed in our laboratory, a possible reason for low nuclear fluorescence is silencing of the gene encoding the RP-FP fusion protein, which is adjacent to the operator repeats and under the control of the 35S promoter on the original T-DNA constructs (Fig. 1A). To test the occurrence and basis of epigenetic silencing, we introgressed mutations in four epigenetic silencing factors into five tagged lines (Fig. 1B). The epigenetic factors were chosen because of their involvement in different types of gene silencing and/or epigenetic modifications: (1) DDM1 is a chromatin remodelling ATPase important for repeat-induced heterochromatin formation and DNA methylation [12]; (2) DRD1 is a chromatin remodelling ATPase required for RNA-directed DNA methylation and transcriptional gene silencing (TGS) [12]; (3) RDR6 is an RNA-dependent RNA polymerase required for post-transcriptional gene silencing (PTGS), which is often observed with 35S promoter-driven transgenes [13]; (4) MOM1 is an unusual putative chromatin remodelling factor needed for TGS that is independent of DNA methylation [14].


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)

Constructs and chromosomal locations of fluorescent-tagged sites. A. T-DNA constructs for fluorescent tagging. Construct 16 is based on red fluorescent protein (R). Construct 25 is based on enhanced green fluorescence protein (G). Both constructs use the lacOs and the LacI (L) fused to R and G. Genes encoding RL and GL are under the control of the 35S promoter of CaMV (35Sp). B. Chromosomal positions of tagged sites. The insertion sites have been reported previously [8,11]. Lines are named according to the construct used (16 or 25) followed by the line number (26, 79, 101, 107, 112). C. Constructs containing the RPS5 promoter to drive expression of genes encoding the EGFP-LacI fusion protein (GL) and histone H2B fused to monomeric DsRed (H2BmR). D. Constructs containing the GC1 promoter to drive expression of GL. Details of construct assembly using the modules shown in C and D are in the Methods section. Gene units are boxed; heavy outlines indicate genes encoding fluorescent fusion proteins (red, green) and lacOs (black). Arrows indicate the directions of transcription. Additional abbreviations: lb, T-DNA left border; rb, T-DNA right border; Np, NOS promoter; red 'R', DsRed2; green 'G', EGFP; t, transcriptional terminator from the 35S transcript of CaMV; nptIIb, neomycin phosphotransferase II for selection of bacteria on kanamycin; nt, NOS terminator; Mp, mannopine synthase promoter; ot, octopine synthase terminator; tag, 300 bp filler sequence; 19Sp, 19S promoter of CaMV; pDH51, pUC18 containing the 35S promoter-35S terminator cassette [33]; pBC, Bluescript plasmid encoding chloramphenicol resistance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Constructs and chromosomal locations of fluorescent-tagged sites. A. T-DNA constructs for fluorescent tagging. Construct 16 is based on red fluorescent protein (R). Construct 25 is based on enhanced green fluorescence protein (G). Both constructs use the lacOs and the LacI (L) fused to R and G. Genes encoding RL and GL are under the control of the 35S promoter of CaMV (35Sp). B. Chromosomal positions of tagged sites. The insertion sites have been reported previously [8,11]. Lines are named according to the construct used (16 or 25) followed by the line number (26, 79, 101, 107, 112). C. Constructs containing the RPS5 promoter to drive expression of genes encoding the EGFP-LacI fusion protein (GL) and histone H2B fused to monomeric DsRed (H2BmR). D. Constructs containing the GC1 promoter to drive expression of GL. Details of construct assembly using the modules shown in C and D are in the Methods section. Gene units are boxed; heavy outlines indicate genes encoding fluorescent fusion proteins (red, green) and lacOs (black). Arrows indicate the directions of transcription. Additional abbreviations: lb, T-DNA left border; rb, T-DNA right border; Np, NOS promoter; red 'R', DsRed2; green 'G', EGFP; t, transcriptional terminator from the 35S transcript of CaMV; nptIIb, neomycin phosphotransferase II for selection of bacteria on kanamycin; nt, NOS terminator; Mp, mannopine synthase promoter; ot, octopine synthase terminator; tag, 300 bp filler sequence; 19Sp, 19S promoter of CaMV; pDH51, pUC18 containing the 35S promoter-35S terminator cassette [33]; pBC, Bluescript plasmid encoding chloramphenicol resistance.
Mentions: For the tagged lines developed in our laboratory, a possible reason for low nuclear fluorescence is silencing of the gene encoding the RP-FP fusion protein, which is adjacent to the operator repeats and under the control of the 35S promoter on the original T-DNA constructs (Fig. 1A). To test the occurrence and basis of epigenetic silencing, we introgressed mutations in four epigenetic silencing factors into five tagged lines (Fig. 1B). The epigenetic factors were chosen because of their involvement in different types of gene silencing and/or epigenetic modifications: (1) DDM1 is a chromatin remodelling ATPase important for repeat-induced heterochromatin formation and DNA methylation [12]; (2) DRD1 is a chromatin remodelling ATPase required for RNA-directed DNA methylation and transcriptional gene silencing (TGS) [12]; (3) RDR6 is an RNA-dependent RNA polymerase required for post-transcriptional gene silencing (PTGS), which is often observed with 35S promoter-driven transgenes [13]; (4) MOM1 is an unusual putative chromatin remodelling factor needed for TGS that is independent of DNA methylation [14].

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