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的生化/遗传学特性 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有很大的不同 常染色质的基本特征包含有 明显的表型效应，和异染色质，最初 细胞学定义为高度浓缩的DNA，是 重复的、非编码的DNA序列。尽管异色DNA具有 很大程度上未知的函数，它对于正常的除法是必不可少的 减数分裂和有丝分裂过程中的遗传物质。因此，它是 很可能某些或大多数异染色质DNA不是“垃圾”，而是 通过非转录途径发挥作用的序列的集合 机械装置。这项研究将阐明这种重复的一个方面 DNA已被认为是导致异染色质 行为方式--与拓扑异构酶II(TOPO II)的相互作用。 一种假说认为，异色行为是 它与DNA修饰蛋白Topo II、Topo II、And 果蝇核基质的重要组成部分，已被证明 在通常与以下内容相关的许多功能中很重要 异染色质从DNA缩合到影响基因频率 重组。Topo II毒药也是有效的抗肿瘤药物。 初步证据表明，异染色质高度浓缩 对于Topo II功能必不可少的位点，导致了假设 这种染色质结构可以用不同密度的 Topo II站点。这一假设主要是基于概念化。 从GenBank数据库中提取的DNA序列。因此，要 充分检验这一想法，有必要确定其通用性 观察。这将使用两种实验方法来完成。 首先，将对经过计算机分析的序列进行克隆 用于体外分析，以测试Topo II的结合和切割。 来自异染色质区域的克隆应该具有更高的密度 合法的TOPO II站点。第二，功能拓朴的分布 常染色质和异染色质中的II位点将在体内进行比较 使用细胞系。一种Topo II抑制剂VP-16，很容易导致 可识别的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。经抑制剂处理的细胞DNA的Southern杂交结果 将用大的PI克隆来探测标准和脉冲场凝胶 从等色线和异色线位置。假说 预测异色区域将包含更活跃的 TOPO II位点由每一个可切割位点的数量确定 千基数。

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.