CHROMATIN STRUCTURE IN LIVING CELLS

染色质结构作为DNA的一种调控因子，越来越受到人们的关注 在转录、复制、重组和修复中起作用。 最 绘制染色质的方法需要分离细胞核， 在细胞器制备过程中改变结构的可能性。 作为 一个例子是酵母α 2阻遏物在染色质中丢失， 核的制备。 我们提出了一系列的调查，以发展 绘制活细胞中染色质结构的方法。 我们有 先前利用原核dam甲基转移酶来定义 阻止酶进入的染色质特征。 最近我们 已经使用了胞嘧啶甲基转移酶，从受控的 启动子，其也修饰GATC。 5C基因组测序方法 已被改编为积极的化学检测，使定量 可以对扩展区域进行分析。 我们将开发甲基化方法 使用更混杂的酶。 Sss I甲基转移酶，其修饰 CpG序列可作为克隆基因获得。 我们将克隆并表达 识别CpC和RpCpY序列的小球藻病毒酶的基因。 总之，这些甲基转移酶将允许用一种特异性标记绘制染色质。 大约每七个碱基对一个位点的分辨率。 DNA酶I是第一个 一种能识别染色质结构的独特特征的酶， 与DNA功能有关。 我们过去曾尝试在大肠杆菌中表达DNA酶I， 酵母在体内绘制染色质图谱。这些尝试都失败了，可能是因为 来自泄漏控制启动子的核酸酶表达的致死性。 我们有 设计了几种策略， 只有在需要时才进行，然后进行，以获得酵母菌株， 允许绘制活细胞中的核酸酶超敏感位点，以及 检测核小体的旋转定位。 最高 分辨率，最低序列特异性技术，用于绘制染色质， 体外使用羟基自由基。 我们建议开发羟基自由基 绘制细胞中的染色质，使用伽马辐射产生 根的 这些研究将通过以前的表征得到促进， 定位核小体邻接α 2阻遏物在S.酿酒酵母 微型染色体，以及STE6抑制的染色质结构域 染色体基因 我们预计开发的方法的扩展， 平行研究的结构的30 kb的酵母染色体III在我们的 实验室 在进行方法学发展研究的同时， S.酿酒厂，没有理由，这些 方法不能输出到高等真核细胞的研究 染色质在发育和疾病状态。

Chromatin structure has received increasing attention as a modulator of DNA function in transcription, replication, recombination and repair. Most methods for mapping chromatin require isolation of nuclei raising the possibility of alterations i structure during organelle preparation. As one example, the yeast alpha2 repressor is lost form chromatin during preparation of nuclei. We propose a series of investigations to develop methods for mapping chromatin structure in living cells. We have previously utilized the prokaryotic dam methyltransferase to define features of chromatin which preclude access to the enzyme. Recently, we have used a cytosine methyltransferase, expressed from a controlled promoter, which also modifies GATC. The genomic sequencing method for 5 C has been adapted for positive chemical detection, making quantitative analysis of extended regions possible. We will develop methylation methods using more promiscuous enzymes. The Sss I methyltransferase which modifies CpG sequences is available as a cloned gene. We will clone and express genes for Chlorella virus enzymes which recognize CpC and RpCpY sequences. Together, these methyltransferases will allow mapping chromatin with a resolution of one site about every seven base pairs. DNase I was the first enzyme noted to recognize distinctive features of chromatin structure that correlated with DNA function. We tried in the past to express DNase I in yeast to map chromatin in vivo. These attempts failed, likely due to lethality of nuclease expression from a leaky controlled promoter. We have devised several strategies which should allow expression of the nuclease only when desired and will implement then to obtain yeast strains which allow mapping of nuclease hypersensitive sites in living cells as well as detection of the rotational positioning of nucleosomes. The highest resolution, least sequence-specific technique for mapping chromatin in vitro uses hydroxyl radicals. We propose development of hydroxyl radical mapping for chromatin in cells, using gamma radiation for generation of radicals. The studies will be facilitated by previous characterization of positioned nucleosomes abutting the alpha2 repressor in S. cerevisiae minichromosomes, and of a repressed chromatin domain for the STE6 chromosomal gene. We anticipate extension of the methods developed to parallel studies of the structure of 30 kb of yeast chromosome III in our laboratory. While the methodologic development studies are carried out int he tractable environment of S. cerevisiae, there is not reason that these methods can not be exported to higher eukaryotic cells for study of chromatin during development and in disease states.