20 - Breakthrough Technologies Use of Two-Color...

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Unformatted text preview: Breakthrough Technologies Use of Two-Color Fluorescence-Tagged Transgenes to Study Interphase Chromosomes in Living Plants 1[W] Antonius J.M. Matzke*, Bruno Huettel, Johannes van der Winden, and Marjori Matzke Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A–1030 Vienna, Austria Sixteen distinct sites distributed on all five Arabidopsis ( Arabidopsis thaliana ) chromosomes have been tagged using different fluorescent proteins and one of two different bacterial operator-repressor systems: (1) a yellow fluorescent protein-Tet repressor fusion protein bound to tet operator sequences, or (2) a green or red fluorescent protein-Lac repressor fusion protein bound to lac operator sequences. Individual homozygous lines and progeny of intercrosses between lines have been used to study various aspects of interphase chromosome organization in root cells of living, untreated seedlings. Features reported here include distances between transgene alleles, distances between transgene inserts on different chromosomes, distances between transgene inserts on the same chromatin fiber, alignment of homologous chromosomes, and chromatin movement. The overall findings are consistent with a random and largely static arrangement of interphase chromosomes in nuclei of root cells. These transgenic lines provide tools for in-depth analyses of interphase chromosome organization, expression, and dynamics in living plants. Although the arrangement of interphase chromo- somes is thought to be important for regulating nuclear function and gene expression (Misteli, 2004; Bolzer et al., 2005; Wegel and Shaw, 2005), little in- formation is currently available about interphase chro- mosome organization in living plant cells (Lam et al., 2004; Tessadori et al., 2004). Most studies so far have used fluorescence in situ hybridization (FISH) to visualize interphase chromosomes in nonliving, fixed material (e.g. Fransz et al., 2002; Pecinka et al., 2004). As an alternate technique, bacterial operator-repressor systems combined with fluorescent proteins offer a unique opportunity to visualize fluorescence-tagged loci in nuclei of living, unfixed cells. The operator re- peats are integrated into the genome as a transgene array, which then specifically binds the respective nuclear-localized repressor protein that is fused with a fluorescent protein such as green fluorescent protein (GFP). The tagged loci appear as bright fluorescent dots when viewed with appropriate filters undera fluo- rescence microscope. Using a fluorescence microscope equipped with a motorized z axis and image-processing software, it is possible to make optical sections through nuclei and reconstruct them in three dimensions to determine spatial relationships among fluorescence- tagged loci. This technique has been employed in yeast, Drosophila, and mammalian cells to analyze interphase chromosome organization and dynamics (Gasser, 2002; Spector, 2003), but so far has been used...
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This note was uploaded on 08/01/2009 for the course 1 1 taught by Professor Maz during the Spring '01 term at Abilene Christian University.

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20 - Breakthrough Technologies Use of Two-Color...

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