H-1 HYPERSITES

描述 最近的发现表明，连接物组蛋白H1与DNA的结合可能 比之前认为的更具体，包括承认任何一种 DNA的某些核苷酸序列或某些结构特征(例如 超级线圈、4路连接)。这位外国通敌者最近发现 两个表现出异常高亲和力的质粒限制片段 H1，基于一种简单的凝胶位移电泳法获得的结果。 对这些H1“超位”的初步研究表明，DNA曲率为 以及特定序列在高亲和力结合中发挥作用。这个 拟议的合作包括验证该假说的实验 含有超硫酸盐的DNA片段形成的环状结构 DNA交叉处或附近的高亲和力序列。 初步实验将验证对DNA曲率的要求 结合H1的DNA片段。环路形成将通过以下方式间接评估 凝胶电泳法和直接扫描力显微镜法。二 将使用各种方法来确定H1与DNA超位的结合是否 是合作的：首先，通过Western blotting定位凝胶位移中的H1 第二，通过分析超速离心法。第三个具体目标 是绘制与紧密H1结合有关的假定的特定序列；这 将尝试使用DNA酶保护分析。最终的具体目标是 在选定的真核基因组区域中使用 凝胶位移分析，并使用所应用的方法来表征这些位置 到含有超抗原的质粒片段。

DESCRIPTION Recent findings suggest that the binding of the linker histone H1 to DNA may be more specific than previously believed, involving recognition of either certain nucleotide sequences or certain structural features of DNA (e.g. supercoils, 4-way junctions). The foreign collaborator recently discovered two plasmid restriction fragments that exhibit unusually high affinity for H1, based on results obtained with a simple gel shift electrophoresis assay. Preliminary studies of these H1 "hypersites" suggest that DNA curvature as well as specific sequences play a role in the high affinity binding. The proposed collaboration includes experiments which will test the hypothesis that the hypersite-containing DNA fragments form loops which place high-affinity sequences at or near a DNA cross-over. Initial experiments will verify the requirement for DNA curvature in H1-binding DNA fragments. Loop formation will be evaluated indirectly by gel electrophoresis and directly by scanning force microscopy. Two approaches will be used to determine if the binding of H1 to DNA hypersites is cooperative: first, by Western blotting to localize H1 in the gel shift assay, and second, by analytical ultracentrifugation. A third specific aim is to map the putative specific sequences involved in tight H1 binding; this will be attempted using a DNase protection assay. The final specific aim is to search for H1 hypersites in selected eukaryotic genome regions using the gel shift assay and to characterize these sites using the approach applied to the hypersite-containing plasmid fragments.