EXISTENCE OF TORSIONAL TENSION IN EUKARYOTIC DNA IN VIVO

真核生物和原核生物染色体中的DNA都被组织成 超螺旋拓扑结构域 在原核生物中， 在DNA中被平衡为不受限制的扭转张力。 这种应变 已经显示在控制基因表达的机制中是重要的。 在真核生物中，大多数超螺旋受到核小体的限制， 平均而言，DNA螺旋不受扭转张力的影响。 虽然 没有直接证据表明真核DNA中存在无限制的超螺旋， 目前的基因调控模型表明， 涉案 一个模型表明，真核生物中的扭转张力 染色质可能是由RNA聚合酶的运动产生的， 转录。 扭转张力可以促进DNA的呼吸， 形成十字形，形成三链DNA，或形成 左手Z基因 该提案旨在确定扭转 真核生物DNA中存在张力。 我们的起源体内试验， 无限制的扭转张力，包括测量 光结合到DNA的三甲基胡椒烯。 这提供了一个平均值 如果存在于较小的 染色体的一部分，如在活跃的基因或在特定的 DNA中的定位位点。 将对该试验进行修改，以定量速率 通过与选择的DNA限制性片段交联， 测量DNA的选定区域内的扭转应变。 我们将 合成、表征和应用十字形和Z-DNA "扭转调谐 探针"用于体内无限制的超螺旋。 这涉及应用 我们已经开发了一种基于peptide的检测方法， 活细胞中潜在的十字形和Z-DNA序列的构象。 这些交替的螺旋结构从精确的最小值（调谐） 超螺旋密度允许测量超螺旋的水平， 在DNA中的特定位点。 将应用交联试验 以确定SV40微型染色体的选定区域或各种 果蝇的基因在生活中受到无限制的扭转张力的缠绕 细胞 此外，我们将插入"扭转调谐探针"序列 插入SV40 DNA的控制区，位于诱导基因的两端， 一个稳定的myc表染色体载体，与果蝇hsp70热邻近 休克基因，以询问是否可以检测到无限制的DNA超螺旋 组成型或与体内转录相关。

DNA in both eukaryotic and prokaryotic chromosomes is organized into supercoiled topological domains. In prokarytotes about half the supercoils are equilibrated as unrestrained torsional tension in the DNA. Such strain has been shown to be important in mechanisms controlling gene expression. In eukaryotes, most supercoils are restrained by nucleosomes and,on average, the DNA helix is not wound with torsional tension. Although there is no direct evidence for unrestrained supercoiling in eukaryotic DNA, some current gene regulatory models suggest that a torsionally strained helix is involved. One model suggests that torsional tension in eukaryotic chromatin may be generated by the movement of RNA polymerase during transcription. Torsional tension could promote breathing the DNA, formation of cruciforms, formation of triple stranded DNA, or formation of left-handed Z-DNA. The proposal is designed to determine if torsional tension exists in eukaryotic DNA. Our origin in vivo assay for unrestrained torsional tension involved measurement of rates of trimethylpsoralen photobinding to DNA. This provided an averaging measurement which would not detect torsional tension if present in a small fraction of the chromosome, such as in active genes or at a specific localized site in the DNA. This assay will be modified to quantitate rates of psoralen cross-linking to selected restriction fragments of DNA to measure torsional strain within a selected region of DNA. We will synthesize, characterize, and apply cruciform and Z-DNA "torsionally tuned probes" for unrestrained supercoiling in vivo. This involves application of psoralen-based assays we have developed that distinguish the conformation of potential cruciforms and Z-DNA sequences in living cells. These alternate helical structures from at precise minimum (tuned) superhelical densities allowing measurement of the level of supercoiling in vivo at specific sites in the DNA. The cross-linking assay will be applied in vivo to determine if selected regions of SV40 minichromosomes or various Drosophila genes are wound with unrestrained torsional tension in living cells. In addition we will insert the "torsionally tuned probe" sequences into the control region of SV40 DNA, on either end of an inducible gene in a stable myc epichromosomal vector, and adjacent to Drosophila hsp70 heat shock genes to ask if unrestrained DNA supercoiling can be detected constitutively or in association with transcription in vivo.