GENETIC ASPECTS OF DNA METHYLATION

在包括哺乳动物在内的许多生物体的DNA中， 胞嘧啶在DNA复制后被甲基化。虽然有一个链接 在DNA甲基化和失活之间， DNA甲基化的机制仍然不清楚。特别是，几乎没有什么 是什么决定了哪些序列被甲基化。特征 相对简单的真核生物Neospora crassa的研究将有助于 这个和其他甲基化问题一直难以分析 在高等真核生物中。在脉孢菌属中，通常 在重新引入后， 转化成有机体。这一点，加上能力， 通过RIP（重复诱导）诱导选定染色体区域甲基化 点突变）开辟了一条道路，以实验确定什么触发 甲基化最后，脉孢菌的经典遗传技术将 促进DNA甲基化的机制和功能研究。 对RIP改变的序列的分析表明，少量的G：C 到A：T突变可以将正常未甲基化的序列转化为 甲基化区域。计划中的实验应该能阐明 突变和甲基化之间的关系，测试模型， 提供基本信息，以达到对DNA的坚定理解 真核生物中的甲基化体内和体外方法的组合 将揭示哪些位点必须突变（以及各种突变是否 等同）以触发染色体区域的甲基化。根据 “塌陷的染色质”模型，缺乏结合的染色体区域， 非组蛋白染色体蛋白在一定范围内浓缩 自发地，并随后变成甲基化。具体实验将 测试正常甲基化区域（zeta-eta）是否可以保持未甲基化 通过将其放置在活性基因（am）中或靠近与 非组蛋白染色体蛋白（qa-1F）。一般认为甲基化 是默认状态将通过查找短（即，<50 bp） 能够防止或诱导周围细胞甲基化的序列 序列的 将分离突变体并用于研究 甲基化，并进一步探讨其机制。为了测试DNA是否 甲基化对于N.克拉萨， 分离并灭活粗糙脉孢菌DNA甲基转移酶基因 RIP和/或“基因置换”。 其他基因缺陷突变体 将使用两种方法分离甲基化所必需的蛋白质-一种使用 一个选择在初步实验中成功，另一个使用 “蛮力”获得所有可能的突变体。

In the DNA of many organisms, including mammals, a fraction of the cytosines become methylated after DNA replication. Although a link has been demonstrated between methylation and inactivity of DNA, the function and mechanism of DNA methylation remains unclear.In particular, almost nothing is known about what determines which sequences become methylated. Features of the relatively simple eukaryote Neospora crassa will allow progress on this and other methylation problems that have been refractory to analysis in higher eukaryotes. In Neurospora, DNA sequences that are normally methylated are faithfully methylated de novo after they are reintroduced into the organism by transformation. This, together with the ability to induce methylation of selected chromosomal regions by RIP (repeat-induced point mutation) opens the way to determine experimentally what triggers methylation. Finally, classical genetic techniques for Neurospora will facilitate both mechanistic and functional studies of DNA methylation. Analysis of sequences altered by RIP has indicated that a sprinkling of G:C to A:T mutations can convert a normally unmethylated sequence into a methylated region. The planned experiments should elucidate the causal relationship between the mutations and methylation, test a model, and provide basic information essential to reach a firm understanding of DNA methylation in eukaryotes. A combination of in vivo and in vitro approaches will reveal which sites must be mutated (and whether various mutations are equivalent) to trigger methylation of a chromosomal region. According to the "collapsed chromatin" model, chromosomal regions that lack bound non-histone chromosomal proteins over a certain span condense spontaneously, and secondarily become methylated. Specific experiments will test'if a normally methylated region (zeta-eta) can be kept unmethylated by placing it in an active gene (am) or next to a site bound by a nonhistone chromosomal protein (qa-1F). The general idea that methylation is the default state will be addressed by looking for short (ie. <50 bp) sequences capable of preventing or inducing methylation of surrounding sequences. Mutants will be isolated and employed to investigate the function of methylation, and to further explore mechanism. To test whether DNA methylation is essential for viability or normal development of N. crassa, the DNA methyltransferase gene of N.crassa will be isolated and inactivated by RIP and/or "gene replacement". Mutants defective in other genes necessary for methylation will be isolated using two approaches - one using a selection successful in a preliminary experiment, and the other using "brute force" to obtain all possible mutants.