CHROMATIN STRUCTURE IN LIVING CELLS

染色质结构作为DNA的调节剂受到越来越多的关注 在转录、复制、重组和修复中发挥作用。多数 绘制染色质图谱的方法需要分离提高 细胞器制备过程中结构改变的可能性。AS 一个例子是，酵母α2抑制子在 原子核的制备。我们建议开展一系列调查 在活细胞中定位染色质结构的方法。我们有 以前利用原核细胞DAM甲基转移酶来定义 无法获得酶的染色质的特性。最近，我们 使用了胞嘧啶甲基转移酶，从受控的 启动子，这也修改了GATC。5C的基因组测序方法 已被改装用于阳性化学检测，使定量 可能对扩展区域进行分析。我们将开发甲基化方法 使用更多混杂的酶。修饰SSS I甲基转移酶 CpG序列可作为克隆基因使用。我们将克隆并表达 识别CPC和RpCpY序列的小球藻病毒酶基因。 这些甲基转移酶结合在一起，将使染色质与 大约每七个碱基对就有一个碱基的分辨。DNase I是第一个 识别染色质结构的独特特征的酶 与DNA功能相关。我们在过去曾尝试在 酵母菌在体内绘制染色质图谱。这些尝试失败了，可能是因为 来自泄漏控制启动子的核酸酶表达的致命性。我们有 设计了几种策略来允许核酸酶的表达 只有在需要的时候才会实施，然后才能获得 允许绘制活细胞中核酸酶敏感部位的图谱，以及 检测核小体的旋转定位。最高的 分辨率，最小序列特异性技术，用于定位染色质 体外实验使用羟基自由基。提出了羟基自由基的发展方向。 细胞中染色质的作图，使用伽马辐射产生 激进分子。这些研究将通过先前的特征描述来促进 酿酒酵母中与α2抑制子相邻的核小体定位 微染色体，以及STE6的抑制染色质结构域 染色体基因。我们期待着将开发的方法扩展到 我国酵母III号染色体30kb结构的平行研究 实验室。而方法论的发展研究是在INT 酿酒酵母的易驯化环境，没有理由这些 方法不能输出到高等真核细胞进行研究 染色质在发育和疾病状态下。

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.