CHROMATIN STRUCTURE IN LIVING CELLS

活细胞中的染色质结构

• 批准号: 2750029
• 负责人: ROBERT T SIMPSON
• 金额: $ 21.67万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2750029
• 关键词: 5 methylcytosine; DNA methylation; Saccharomyces cerevisiae; chemical structure; chimeric proteins; chromatin; chromosomes; cytogenetics; deoxyribonuclease I; dinucleotide; fungal genetics; gamma radiation; gene expression; genetic promoter element; genetic strain; hydroxyl radical; method development; methyltransferase; molecular cloning; nucleic acid sequence; nucleic acid structure; nucleosomes; plant virus; structural biology; transfection

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.

作为DNA调节剂，染色质结构已受到越来越多的关注 转录，复制，重组和修复的功能。 最多 映射染色质的方法需要分离成核的细胞核 在细胞器制备过程中，I变化I结构的可能性。 作为 一个例子，酵母α2抑制剂在期间丢失了染色质 核的制备。 我们提出了一系列调查以开发 在活细胞中绘制染色质结构的方法。 我们有 以前利用原核大坝甲基转移酶定义 染色质的特征，无法访问酶。 最近，我们 使用了由受控的 发起人，也修改了GATC。 5 C的基因组测序方法 已改编成阳性化学检测，进行定量 对扩展区域的分析。 我们将开发甲基化方法 使用更多混杂酶。 修饰的SSS I甲基转移酶 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。 这些甲基转移酶一起将允许用A映射染色质 大约七个基础对一个站点的分辨率。 dnase我是第一个 酶指出要识别染色质结构的独特特征 与DNA功能相关。 我们过去曾尝试过表达dnase i 酵母在体内绘制染色质。这些尝试失败了，可能是由于 来自泄漏的受控启动子的核酸酶表达的致死性。 我们有 设计了几种应允许核酸酶表达的策略 只有在需要并实施时才能获得酵母菌菌株 允许在活细胞中绘制核酸酶超敏部位 检测核小体的旋转定位。 最高 分辨率，最小序列特异性技术用于映射染色质 体外使用羟基自由基。 我们提出羟基的发展 使用伽马辐射生成染色质的染色质，以生成 激进分子。 以前的表征将促进研究 固定在酿酒酵母中的α2抑制剂的核小体位置 STE6的微型染色体和抑制染色质结构域 染色体基因。 我们预计将开发到的方法扩展到 在我们的 实验室。 在进行方法论开发研究的同时 他易于易于酿酒酵母，没有理由 方法不能导出到较高的真核细胞进行研究 发育期间和疾病状态中的染色质。