CONTROL OF DNA METHYLATION IN EUKARYOTES

真核生物中 DNA 甲基化的控制

• 批准号: 3023446
• 负责人: Eric U. SELKER
• 金额: $ 4.32万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-13 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3023446
• 关键词: DNA methylation; Neurospora; chromatin; eukaryote; genetic regulation; nucleic acid sequence; point mutation

The goal of the research is to understand the function and control of DNA methylation in eukaryotes. We already know that methylation 1) can affect gene expression and 2) can cause mutations. The proposed collaboration will combine the biochemical expertise of my laboratory to dissect the mechanism and function of DNA methylation in Neurospora crassa, an organism well suited for both biochemical and genetic studies. Specific aims of the planned experiments are: 1. To define what constitutes a methylation "signal" in Neurospora. Discovery and characterization of RIP (repeat-induced point mutation) led to the conclusion that a sprinkling of G:C to A:T mutations can induced methylation. The proposed research will test a rapid in vivo assay for de novo methylation and apply it in experiments: a) to look for short sequences capable of preventing or inducing methylation of surrounding sequences, b) to determine how many G:C to A:T mutations are required to trigger methylation of a chromosomal region and which positions are critical, and c) to ascertain if various types of mutations (e.g., polarized transitions vs. transversions and frameshifts) are equally effective in triggering methylation. 2. To look for evidence of maintenance methylation in Neurospora. Evidence for cooperativity in methylation of opposite chains in Neurospora DNA will be sought and, if feasible, maintenance of methylation patterns will be directly assessed. 3. To determine whether methylated and unmethylated sequences of Neurospora are in different chromatin forms. A methyl-CpG binding protein available in the Bird laboratory will be employed to affinity-purify oligonucleosomes containing methylated sequences. This chromatin will be characterized with respect to acetylation state of histones H3 and H4 and presence of histone H1. 4. To determine whether Neurospora has a methyl-C binding protein. 5. To determine whether DNA methylation is associated with DNA replication in Neurospora.

这项研究的目的是了解DNA的功能和控制 真核生物中的甲基化 我们已经知道甲基化1）可以影响 基因表达和2）可能导致突变。 的拟议合作 我将联合收割机结合我实验室的生化专业知识， 粗糙脉孢菌DNA甲基化机制及其功能 非常适合生物化学和遗传学研究。 的具体目标 计划的实验是： 1. 定义链孢菌中甲基化“信号”的构成。 RIP（重复诱导的点突变）的发现和表征 得出的结论是，可以诱导少量G：C到A：T的突变， 甲基化 拟议的研究将测试一种快速的体内检测方法， novo甲基化并将其应用于实验：a）寻找短 能够防止或诱导周围细胞甲基化的序列 序列，B）以确定需要多少G：C至A：T突变来 触发染色体区域的甲基化， 关键的，和c）确定是否各种类型的突变（例如， 极化转换与颠换和移码）是平等的 有效地触发甲基化。 2. 寻找脉孢菌中甲基化维持的证据。 脉孢菌相对链甲基化协同作用的证据 将寻找DNA，如果可行，将维持甲基化模式 将直接评估。 3. 为了确定是否甲基化和非甲基化的序列， 脉孢菌具有不同的染色质形式。 甲基-CpG结合蛋白 将采用Bird实验室中可用的方法进行亲和纯化 含有甲基化序列的寡核小体。 这个染色质将是 特征在于组蛋白H3和H4的乙酰化状态， 组蛋白H1。 4. 确定脉孢菌是否具有甲基-C结合蛋白。 5. 确定DNA甲基化是否与DNA复制有关 在脉孢菌属。