EXISTENCE OF TORSIONAL TENSION IN EUKARYOTIC DNA IN VIVO

真核染色体中的DNA被组织成拓扑域 超螺旋DNA。在原核生物中，这种超级卷曲的大部分是平衡的 DNA螺旋中的不受限制的扭转张力。在细菌中这是 扭转应变已被证明在机械控制中是重要的。 基因表达。虽然真核DNA是超螺旋的，但大多数超螺旋 在核小体中受到抑制，平均而言，扭转张力不受 与DNA螺旋保持平衡。虽然没有直接证据， 目前真核生物中的基因调控模型引发了扭曲的 可以促进DNA呼吸或形成的拉伸螺旋 左撇子Z-DNA。自由扭转的体内检测方法研究进展 张力包括光结合速率的测量 4，5‘，8-三甲基补骨脂素对DNA的作用。此过程提供平均 测量不会检测扭转张力，如果存在于小型 染色体的片段，如在活跃的基因中。这个化验结果将是 改良以定量补骨脂素交联率至选定 DNA的限制性片段可作为扭转应变的指示。一个 核酸外切酶III-光逆转试验将被开发用于定量 补骨脂素结合在碱基对分辨水平。这些化验结果将 然后在体内应用以确定SV40的选定区域 小染色体或果蝇热休克基因被自由缠绕 扭转张力。此外，我们还将在体内应用最新开发的 用十字形和Z-DNA作为无拘束的活体探针 紧张。潜在的十字形和Z-DNA形成序列，形成 在精确的超级卷曲水平上的替代构象将是 合成的。将这些序列插入到SV40 DNA及其附近 通过P元件转化的果蝇热休克基因将提供 用于不同水平的自由DNA的扭转调谐探针 活体内的超级盘绕。

DNA in eukaryotic chromosomes is organized into topological domains of supercoiled DNA. In prokaryotes much of this supercoiling is equilibrated as unrestrained torsional tension in the DNA helix. In bacteria this torsional strain has been shown to be important in mechanisms controlling gene expression. Although eukaryotic DNA is supercoiled, most supercoils are restrained in nucleosomes and, on average, torsional tension is not equilibrated with the DNA helix. Although there is no direct evidence, current models for gene regulation in eukaryotes invoke a torsionally strained helix which could promote breathing of the DNA or formation of left-handed Z-DNA. The present in vivo assay for unrestrained torsional tension involves measurement of rates of photobinding of 4,5',8-trimethylpsoralen to DNA. This procedure provides averaging measurements which would not detect torsional tension if present in a small fraction of the chromosome, such as in active genes. This assay will be modified to quantitate rates of psoralen cross-linking to selected restriction fragments of DNA as an indication of torsional strain. An Exonuclease III-photoreversal assay will be developed to quantitate psoralen binding at the level of base pair resolution. These assays will then be applied in vivo to determine if selected regions of SV40 minichromosomes or Drosophila heat shock genes are wound with unrestrained torsional tension. In addition, we will apply recently developed in vivo assays for cruciform and Z-DNA formation as in vivo probes for unrestrained tension. Potential cruciform and Z-DNA forming sequences that form alternate conformations at precise levels of supercoiling will be synthesized. Insertion of these sequences into SV40 DNA and near Drosophila heat shock genes via P-element transformation will provide torsionally tuned probes for various levels of unrestrained DNA supercoiling in vivo.