Chromatin Charting: Organization and Dynamics of Plant Nuclear DNA in situ

细胞核是亚细胞器，真核细胞内的大部分基因组信息都是在细胞核中组织的。从使用杂交技术的研究和固定细胞的连续或光学切片的显微镜工作中，已经出现了一幅有组织的亚核结构的图景。最近，绿色荧光蛋白(GFP)作为基因组DNA的活体标记的应用使动物和真菌的活细胞中染色质的可视化成为可能。基于三维荧光显微镜的研究，除了可以归因于布朗运动的缓慢运动外，间期核中的染色体被认为具有相对静止的有序排列。然而，同样清楚的是，在已知的核内发生的其他现象中，如平移和重组，染色质的相对较大和较远的移动必须是可能的。这个项目的目标是通过绘制活植物细胞中每条染色体序列的相对物理位置和运动图，有助于对亚核结构的总体理解。为了达到可视化和绘制拟南芥所有染色体序列的目的，GFP和该蛋白的两个不同的颜色变体将作为体内标签部署在拟南芥5条染色体上约1000个分散的位置。对基因组特定区域在空间和时间上的相对位置的比较分析将提供关于控制染色质结构和动态的组织原则的新信息。在光学研究追踪基因组不同区域的相对亚核位置和运动的同时，基因组位置对报告基因转录潜力的影响将被量化。总之，这些研究应该为基因组提供第一个全面的3-D物理和转录活动图，并应该有助于理解亚核定位在控制基因表达中可能发挥的作用。这项研究应该会在基因组中的特定位置产生1,000多个具有3个不同的和光学容易处理的GFP标签的拟南芥的作图插入线。这些材料对于表征影响基因表达和发育的染色质相关突变应该是非常有价值的。由于过去两年由NSF资助的几个基因组项目的努力，此类突变的数量可能会迅速增加。该项目产生的分子工具也将应用于玉米等重要农作物。玉米的尖端成像技术与经典和现代细胞遗传学的丰富融合，应该为遗传信息的全球控制以及参数突变等表观遗传现象提供新的视角。这些新的见解将有助于理解基因组信息在植物中是如何组织的，以及如何在全球范围内调节基因表达。因此，这项拟议工作产生的工具和知识应有助于今后提高作物质量和产量的努力。交付成果：1.约5,000个紫胶操作员标记(信标)的拟南芥品系。2.在整个基因组中具有最大分散度的1,000个选定信标插入的3-D坐标图。3.显示信标的3-D坐标和荧光素酶标记表达水平的全球基因表达图。这些材料和信息将在http://aesop.rutgers.edu/~lamlab/ccharting.html上提供

The nucleus is the subcellular organelle in which the bulk of the genomic information within an eukaryotic cell is organized. From studies using hybridization technologies and microscopy work with serial or optical sections of fixed cells, a picture of an organized subnuclear structure has emerged. More recently, the application of the Green Fluorescent Protein (GFP) as an in vivo tag of genomic DNA has allowed the visualization of chromatin in live cells of animals and fungi. Based on studies using 3-D fluorescence microscopy, the chromosomes within an interphase nuclei are perceived to have an ordered arrangement that is relatively static except for slow motions that can be attributed to Brownian movement. However, it is also clear that during other phenomena that are known to occur within a nucleus, such as transvection and recombination, that relatively large and long-range movement of chromatin must be possible. The goal in this project is to contribute to the general understanding of subnuclear architecture by charting the relative physical position and movement of sequences for each of the chromosomes in cells of living plants. To achieve the objective of visualizing and charting the sequences for all the chromosomes of Arabidopsis, GFP and two different color variants of this protein will be deployed as in vivo tags for about 1,000 dispersed sites within the 5 chromosomes of Arabidopsis. Comparative analyses of the relative positions between defined regions of the genome in space and time will provide novel information about the organization principles that control the structure and dynamics of chromatin. Concurrent with optical studies to track the relative subnuclear location and movement for distinct regions of the genome, the effects of genome location on transcription potential of a reporter gene will be quanitified. Together, these studies should provide the first comprehensive 3-D physical and transcription activity maps for a genome and should contribute significantly to understanding the roles that subnuclear location may play in controlling gene expression. This study should generate more than 1,000 mapped insertion lines of Arabidopsis with 3 distinct and optically tractable GFP-tags at defined locations within the genome. These materials should be invaluable for the characterization of chromatin-related mutations that affect gene expression and development. The number of such mutations are likely to rapidly increase due to the efforts of several genome projects that have been funded by the NSF in the past two years. Molecular tools generated from this project will also be applied to an important crop plant such as maize. The fusion of cutting edge imaging technology with the wealth of classical and modern cytogenetics in maize should provide new perspectives on global control of genetic information as well as epigenetic phenomena such as paramutation. These new insights will facilitate understanding of how genomic information is organized in plants and how gene expression can be regulated at a global scale. As such, the tools and knowledge generated by this proposed work should benefit future efforts to improve the quality and yield of crop plants. Deliverables:1. About 5,000 lac-operator-tagged (beacon) Arabidopsis thaliana lines. 2. 3-D coordinate maps of 1,000 selected beacon insertions with maximal dispersion across the whole genome. 3. A global gene expression map that shows the 3-D coordinates and the luciferase marker expression levels of the beacons. These materials and information will be available at http://aesop.rutgers.edu/~lamlab/ccharting.html