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

染色质图谱：植物核 DNA 的原位组织和动态

• 批准号: 0077617
• 负责人: Eric Lam
• 金额: --
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-10-01 至 2007-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0077617&HistoricalAwards=false
• 关键词: Chromatin; Charting; Organization; Dynamics; Plant

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

细胞核是亚细胞细胞器，真核细胞内的大量基因组信息在其中组织。通过使用杂交技术和显微镜对固定细胞的连续或光学切片进行的研究，出现了有组织的亚核结构的图片。最近，绿色荧光蛋白 (GFP) 作为基因组 DNA 体内标签的应用使得动物和真菌活细胞中染色质的可视化成为可能。根据使用 3-D 荧光显微镜的研究，间期核内的染色体被认为具有相对静态的有序排列，除了可归因于布朗运动的慢运动之外。然而，同样清楚的是，在已知的细胞核内发生的其他现象中，例如横行和重组，染色质相对较大和长距离的运动必定是可能的。该项目的目标是通过绘制活植物细胞中每条染色体序列的相对物理位置和运动图表，促进对亚核结构的一般理解。为了实现对拟南芥所有染色体序列进行可视化和绘制图表的目标，GFP 和该蛋白质的两种不同颜色变体将被部署为拟南芥 5 条染色体内约 1,000 个分散位点的体内标签。对基因组定义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态的组织原理的新信息。与跟踪基因组不同区域的相对亚核位置和运动的光学研究同时进行，基因组位置对报告基因转录潜力的影响将被量化。总之，这些研究应提供第一个全面的基因组 3D 物理和转录活性图谱，并应为理解亚核定位在控制基因表达中可能发挥的作用做出重大贡献。这项研究应该生成超过 1,000 个拟南芥的映射插入系，在基因组内的指定位置具有 3 个不同且光学可处理的 GFP 标签。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具有无价的价值。由于过去两年美国国家科学基金会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。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应该为遗传信息的全局控制以及副突变等表观遗传现象提供新的视角。这些新见解将有助于理解植物中基因组信息如何组织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该有利于未来提高作物质量和产量的努力。可交付成果：1。大约 5,000 个带有 lac-operator 标签（信标）的拟南芥品系。 2. 1,000 个选定信标插入的 3-D 坐标图，在整个基因组中具有最大分散度。 3. 显示信标的 3-D 坐标和荧光素酶标记表达水平的全局基因表达图。这些材料和信息可在 http://aesop.rutgers.edu/~lamlab/ccharting.html 上获取