DNA TOPOISOMERASE II AND CHROMOSOME STRUCTURE

这项研究的长期目标是确定潜在的 区分真核生物DNA的生物化学/遗传特性 染色体分为不同的类别-常染色质和 异染色质 常染色质和异染色质在 含有常染色质的基因的基本特征 不同的表型效应，异染色质，最初 在细胞学上被定义为高度浓缩的DNA， 重复的非编码DNA序列。 尽管异染色质DNA具有 在很大程度上未知的功能，它是必不可少的正常分工， 减数分裂和有丝分裂过程中的遗传物质。 因此 很可能一些或大多数异染色质DNA不是“垃圾”，而是 一组序列通过非转录方式 机制等 这项研究将阐明这种重复的一个方面， 已被认为在引起异染色质 与拓扑异构酶II（topo II）相互作用。 有一种假说认为，异色性行为是 它与DNA修饰蛋白Topo II的相互作用。 Topo II，an 果蝇核基质的重要组成部分，已被证明 在许多通常与之相关的特征中， 异染色质从DNA凝聚到影响的频率， 重组 Topo II毒药也是有效的抗肿瘤药物。 初步证据表明异染色质高度富集 对于topo II功能至关重要的位点，导致假设 染色质结构可以用不同密度的 拓扑二网站。 这个假设主要是基于概念化 从基因库中提取的DNA序列。 因此 充分测试这个想法，有必要确定的一般性， 观察.这将使用两种实验方法来完成。 首先，将进行计算机分析的序列克隆， 用于体外分析以测试topo II结合和切割。 来自异染色质区的克隆应该具有更高的 合法的拓扑二型网站 二、功能拓扑分布 将在体内比较常染色质和异染色质中的II位点 使用细胞系。 一种拓扑异构酶II抑制剂VP-16， 可辨别的topo II切割将应用于果蝇细胞 然后，培养物和DNA将作为topo II的功能进行分级 乳沟 DNA的Southern印迹，来自经抑制剂处理的细胞， 标准和脉冲场凝胶都将用大的PI克隆进行探测 从常染色质和异染色质的位置。 的假设 预测异染色质区域将包含更多的活性物质， topo II位点，如由每个 放轻松

The long-term goal of this research is to determine the underlying biochemical/genetic properties that differentiate the DNA on eukaryotic chromosomes into its distinct classes - euchromatin and heterochromatin. Euchromatin and heterochromatin differ in very fundamental characteristics with euchromatin containing genes that have distinct phenotypic effects, and the heterochromatin, originally defined cytologically as highly condensed DNA, being a repository of repeated, non-coding DNA sequences. Although heterochromatic DNA has largely unknown function, it is essential for the normal division of the genetic material during meiosis and mitosis. It is therefore likely that some or most heterochromatic DNA is not "junk" but rather a collection of sequences that function via non-transcriptional mechanisms. This research will elucidate one aspect of this repeated DNA that has been proposed as important in causing heterochromatin to behave as it does - interactions with topoisomerase II (topo II). One hypothesis proposes that heterochromatic behavior is a function of its interactions with the DNA modifying protein, topo II. Topo II, an important component of the nuclear matrix in Drosophila, has been shown to be important in many of the features usually associated with heterochromatin from DNA condensation to affecting the frequency of recombination. Topo II poisons are also potent antitumor drugs. Preliminary evidence indicates that heterochromatin is highly enriched for sites essential for topo II function, leading to the hypothesis that chromatin structure can be explained by the differing density of topo II sites. This hypothesis is primarily based on conceptualization of DNA sequences extracted from the GenBank database. Therefore, to fully test this idea it is necessary to determine the generality of the observation. This will be done using two experimental approaches. First, clones of the sequences subjected to computer analysis will be used for in vitro analysis to test for topo II binding and cutting. Clones from heterochromatic regions should have a higher density of legitimate topo II sites. Second, the distribution of functional topo II sites in euchromatin and heterochromatin will be compared in vivo using cell lines. A topo II inhibitor, VP-16, that causes easily discernible topo II cleavage will be applied to Drosophila cell cultures and DNA will then be fractionated as a function of topo II cleavage. Southern blots of the DNA from inhibitor treated cells from both standard and pulse field gels will be probed with large PI clones from euchromatic and heterochromatic locations. The hypothesis predicts that heterochromatic regions will contain many more active topo II sites as determined by the number of cleavable sites per kilobase.