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

细胞核是亚细胞细胞器，真核细胞内的大部分基因组信息被组织在其中。通过使用杂交技术和显微镜对固定细胞进行连续或光学切片的研究，出现了一幅有组织的亚核结构的图像。最近，绿色荧光蛋白（GFP）作为基因组DNA的活体标记的应用，使动物和真菌活细胞中的染色质可视化成为可能。基于使用三维荧光显微镜的研究，间期细胞核内的染色体被认为具有相对静态的有序排列，除了可以归因于布朗运动的缓慢运动。然而，同样清楚的是，在已知发生在细胞核内的其他现象中，如横切和重组，染色质的相对较大和较远的运动必须是可能的。该项目的目标是通过绘制活植物细胞中每条染色体的相对物理位置和序列运动来促进对亚核结构的一般理解。为了实现拟南芥所有染色体序列的可视化和图表化，GFP和该蛋白的两种不同颜色变体将被部署为拟南芥5条染色体内约1000个分散位点的体内标签。对基因组特定区域在空间和时间上的相对位置进行比较分析，将提供有关控制染色质结构和动力学的组织原理的新信息。通过光学研究来追踪基因组不同区域的相对亚核位置和运动，基因组位置对报告基因转录潜能的影响将被量化。总之，这些研究将提供基因组的第一个全面的3-D物理和转录活性图谱，并将对理解亚核位置在控制基因表达方面可能发挥的作用做出重大贡献。这项研究将在基因组的指定位置生成1000多个具有3个不同且光学可处理的gfp标签的拟南芥插入系。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是无价的。由于NSF在过去两年中资助的几个基因组项目的努力，这种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作物。尖端成像技术与丰富的经典和现代玉米细胞遗传学的融合，将为遗传信息的全局控制以及参数化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，以及基因表达如何在全球范围内被调节。因此，这项拟议工作所产生的工具和知识应该有利于未来提高作物质量和产量的努力。可交付成果:1。约5000个lac-算子标记（信标）拟南芥系。2. 1000个选定的信标插入的三维坐标图，在整个基因组中有最大的分散。3. 全球基因表达图谱，显示了信标的三维坐标和荧光素酶标记表达水平。这些材料和信息可在http://aesop.rutgers.edu/~lamlab/ccharting.html上获得

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