POLYTENE AND DIPLOID INTERPHASE CHROMOSOMES

提出的研究建立在最近对我们的硬件和软件的扩展之上，这些扩展允许在广义分辨率重叠的方式下，在光和电子微观水平上对亚细胞结构进行三维重建。我们将这种方法应用于高阶染色体结构和功能的分析，利用果蝇独特的间期多烯染色体。我们专注于细胞学鉴定的多烯染色体带，带间和泡芙（转录活性，形态独特的染色体位点）。我们利用由昆虫激素蜕皮激素引发的果蝇变态，作为一种靶向特征性转录级联的方式。三维条形码技术为研究细胞核内细胞间期染色体的三维形态提供了一种系统的方法。染色体动力学，在活核，现在可以研究使用绿色荧光蛋白（GFP）标记特定的染色体位点。本提案的具体目的是：a)继续使用电子显微镜断层扫描测定特定多烯染色体带、带间和泡的三维结构。b .)研究脉冲标记DNA复制位点在蜕皮激素诱导的泡芙形成和消退后的体内结构动力学（定量运动分析）。c .)利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地形。d .)分析二倍体染色体核地形在PEV遗传扰动的细胞逐细胞的基础上，在眼睛成像盘。大肠)利用最新的GFP染色体位点标记方法研究二倍体染色体行为的四维动态。

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.