Fluorescently tagged T-DNAs and plant interphase chromatin

Although the genome sequences of several organisms, including Arabidopsis thaliana, are now available, this information reveals little about the three-dimensional structure of genomes or the possible influence of nuclear architecture on gene expression. Conceivably, both the position of a chromosome within the nucleoplasm as well as physical interactions with other chromosomes could affect gene activity. Recently developed methods for visualizing specific genetic loci in nuclei of living cells are revolutionizing the study of interphase chromatin organization and dynamics. One technique, which has been used so far primarily in mammalian, yeast and Drosophila cells, exploits bacterial operator/repressor interactions in conjunction with naturally fluorescing proteins to tag specific chromosomal sites. In the fluorescence microscope, tagged DNA regions appear as fluorescent dots in interphase nuclei of living, unfixed cells. The potential of this approach for studying chromatin dispostion, movement and interactions in living plant cells has not yet been fully realized. For these purposes, we have developed vectors based on the tetracycline operator/repressor system and enhanced yellow fluorescent protein (EYFP) (tetYFP construct) as well as the lac operator/repressor system and red fluorescent protein (DsRed2) (lacRed construct). A number of transgenic Arabidopsis lines showing nuclear fluorescent dots have been generated with the tetYFP construct; lacRed lines are currently being screened for appropriate signals. A collection of transgenic lines in which each Arabidopsis chromosome is tagged at least once will be used to study interphase nuclear position and relative placement of allelic and nonallelic chromatin sites over time and in different plant tissues. These aspects of interphase chromatin organization will be correlated with the expression of reporter genes.

The aim of this project was to initiate a study on chromosome organization and dynamics in living plants by producing Arabidopsis thaliana plants that have specific sites of their genome labelled with a fluorescent tag. Arabidopsis is a model plant used for many studies on plant molecular biology. For this, we produced 16 lines of Arabidopsis that carry in their genome DNA repeats inserted into distinct chromosome sites. Fluorescent proteins, which are also made in these plant lines, bind with high affinity to the DNA repeats. The chromosome sites containing the repeats with the bound fluorescent protein are visible under a fluorescence microscope as bright fluorescent dots in nuclei of living, untreated plants. With appropriate microscope filters and computer software, we can observe and measure the arrangement and dynamics of the fluorescent-tagged chromosome sites in living plants. This information allows us to begin to address questions about how the organization and dynamics of chromosomes influence how the genetic information is expressed in different cells types and under varying environmental conditions. Using the 16 fluroescence-tagged Arabidopsis lines, we determined: (1) distances between fluorescently tagged positions in the genome as described above during interphase; (2) distances between positions on different chromosomes; (3) distances between positions on the same chromosome chain; (4) the mobility of selected positions in nuclei; (5) the 3-dimensional arrangement of various independent tagged sites relative to each other. On the basis of the data obtained so far, we conclude that in root cells (our measurements were limited to this cell type), Arabidopsis chromosomes are arranged in a largely random, static fashion. The Arabidopsis lines produced in this project provide unique tools to study the organisation, expression and dynamic behavior of chromosomes during interphase in living plants. Our work on these lines has generated considerable interest in the plant molecular biology community, as indicated by the numerous requests for these lines from many labs all over the world.