POLYTENE AND DIPLOID INTERPHASE CHROMOSOMES

这项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多线染色体来分析高阶染色体的结构和功能。我们专注于细胞学鉴定的多线染色体带、带间带和泡芙(转录活跃、形态独特的染色体位置)。我们利用果蝇的变态，由昆虫激素蜕皮激素触发，作为一种针对特定转录级联的方式。一项名为三维条形码的新技术提供了一种系统的方法，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的形态。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。这项建议的具体目标是：a)继续用电子显微镜断层扫描测定特定多线染色体条带、条带间和泡芙的三维结构。B.)研究脉冲标记的DNA复制部位的体内结构动力学(定量运动分析)，随后是蜕皮激素诱导的气泡形成和消退。(C)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。D.)在眼成像盘中逐个细胞地分析PEV遗传扰动中的二倍体染色体核形态。E.)利用最新的绿色荧光蛋白染色体位点标记方法，研究二倍体染色体行为的4维动力学。

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 polytene chromosomes of Drosophilia melanogaster. We focus on cytologically identified polytene chromosome bands, interbands and puffs (transcriptionally active, morphologically distinctive chromosome sites). We utilize Drosophila metamorphosis, triggered by the insect hormone ecdysone, as a way to target a characteristic transcriptional cascade. New technology, 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.) Continue the determination of the 3-dimensional structure of specific polytene chromosome band, interbands and puffs using electron microscope tomography. B.) Study the in vivo structural dynamics (quantitative motion analysis) of pulse labeled DNA replication sites followed by ecdysone induced puff formation and regression. C.) Determine the 3-dimensional diploid chromosome topography in Drosophila early embryo nuclei using the 3- dimensional Bar Code methodology. D.) Analyze diploid chromosome nuclear topography within a PEV genetic perturbation on a cell- by-cell basis in eye imaginal disks. E.) Study the 4-dimensional dynamics of diploid chromosome behavior using the newly available GFP chromosome locus labeling methodology.