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.

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）会影响 基因表达和2）会引起突变。 拟议的合作 将结合我实验室的生化专业知识，以剖析 DNA甲基化在神经孢子虫中的机理和功能，一种有机体 非常适合生化和遗传研究。 特定目标 计划实验是： 1。定义构成神经孢子中甲基化的“信号”。 RIP的发现和表征（重复诱导点突变）LED 得出的结论是，撒上g：c至a：t突变会引起 甲基化。 拟议的研究将测试DE的快速体内测定 Novo甲基化并将其应用于实验：a）寻找短暂的 能够预防或诱导周围甲基化的序列 序列，b）确定需要多少个g：c至a：t突变 触发染色体区域的甲基化，哪些位置是 批判性，c）确定是否存在各种类型的突变（例如， 两极分化的过渡与横向和移架）是同样的 有效触发甲基化。 2。寻找神经孢子中维持甲基化的证据。 神经孢子中相对链甲基化合作的证据 将寻求DNA，如果可行的话，可以维持甲基化模式 将直接评估。 3。确定是否甲基化和未甲基化序列 神经孢子为不同的染色质形式。 甲基-CPG结合蛋白 在鸟类实验室中可用来提供亲和力纯化 含有甲基化序列的寡核小体。 这个染色质将是 根据组蛋白H3和H4的乙酰化状态的特征 组蛋白H1的存在。 4。确定神经孢子是否具有甲基-C结合蛋白。 5。确定DNA甲基化是否与DNA复制有关 在Neurospora。