A Study of Diploid Interphase and Anaphase Chromosomes

描述(申请人提供)：拟议的研究建立在对我们硬件和软件的最新扩展的基础上，允许以广义分辨率重叠的方式在光学和电子显微镜水平上重建亚细胞结构的三维。我们将这种方法应用于利用果蝇独特的间期和后期染色体来分析高阶染色体的结构和功能。利用结构照明光学显微镜(SIM)和电子显微镜断层扫描(EMT)的新技术使人们对三维染色体结构有了新的见解。三维条形码提供了一种在逐个细胞的基础上研究细胞核内三维二倍体间期染色体拓扑学的系统方法。现在可以使用绿色荧光蛋白(GFP)标记特定的染色体位点来研究活核中的染色体动力学。这项建议的具体目的是：a)使用三维条形码方法确定果蝇早期胚胎细胞核的三维二倍体染色体拓扑图。B)使用新的GFP染色体位置标记方法研究二倍体染色体行为的4维动力学。C)继续使用SIM和EMT及其延伸技术确定后期染色体的三维结构。

DESCRIPTION (provided by applicant): The proposed research builds on recent extensions to our hardware and software that allows, in a generalized resolution overlapping fashion, 3-dimensional reconstructions of subcellular structures at both the light and electron microscopic levels. We apply this methodology to the analysis of higher-order chromosome structure and function using the unique interphase and anaphase chromosomes of Drosophila melanogaster. New technology utilizing structured illumination optical microscopy (SIM) and electron microscopy tomography (EMT) allows novel new insights into 3-dimensional chromosome structure. A 3-dimensional Bar Code provides for a systematic approach to the study of 3 dimension diploid interphase chromosome topography in the nucleus on a cell-by-cell basis. Chromosome dynamics, in living nuclei, can now be studied using green fluorescent protein (GFP) to label specific chromosomal loci. The specific aims of this proposal are: A) Determine the 3 dimensional diploid chromosome topography in Drosophila early embryo nuclei using the 3-dimensional Bar Code methodology. B) Study the 4-dimensional dynamics of diploid chromosome behavior using the newly available GFP chromosome locus labeling methodology. C) Continue the determination of the 3-dimensional structure of anaphase chromosomes using SIM and EMT and their extensions.