GENETIC ASPECTS OF DNA METHYLATION

描述：调查员提议继续调查 以脉孢菌为靶标的DNA甲基化机制和目的 模型系统。在之前的研究中，研究人员表明， 脉孢子虫的序列，包括一个复制的5S RNA基因和一个5S基因 假基因，在性交过程中被点突变严重改变 生命周期的阶段。这个过程称为RIP(重复诱导 点突变)，导致成熟序列的甲基化。这些 观察结果导致了在上一个资助期进行的研究 关于DNA甲基化的范围、性质和目的 脉孢菌属。 当前提案的总体目标是了解 真核生物模型中DNA甲基化的功能和调控。这个 具体目标包括：(1)确定为什么DNA 甲基化缺陷突变体变成非整倍体，(2)分离新的DNA 甲基化突变体的方法将有助于克隆 基因，(3)分离和鉴定DNA中涉及的基因 甲基化，(4)定义从头甲基化的信号，(5)至 鉴定和纯化与甲基化特异结合的蛋白质 序列，以及(6)探索DNA之间的潜在联系 甲基化和DNA复制。 这一建议源于将基因和基因中的甲基化联系起来的证据 表观遗传现象，如印记。两者之间存在着许多相关性 甲基化和缺乏基因活性已经积累起来，而且有 强有力的证据表明甲基化可以阻止基因表达。发现 真核细胞甲基转移酶优先甲基化半- 甲基化的CPGS为Riggs提出的模型提供了支持 霍利迪认为甲基化是通过一种酶来传播的 使对称部位甲基化。然而，研究表明，这一点已经变得清晰起来 在真菌上甲基化的传播可以发生在不对称的地方 网站。 在脉孢子菌中，甲基化可以从头开始发生，并且以一种 不传播甲基化模式。约1.5%的C 粗糙核糖核酸是甲基化的，但基因组的大部分缺乏 甲基化。详细描述的三个甲基化序列包括 Rdna、1.6kb的zeta-eta区和psi 区域，后者是RIP的残留物，重复诱导点突变。 这是Selker博士发现的一个过程，是一种检测 双核细胞单倍体细胞核中的重复序列 受精。然后，复制的序列的两个副本都变成 G：C到A：T突变的频繁靶点，以至于30%的G：C 配对可以更改。通常，RIP改变的序列会变成 甲基化。RIPED序列的甲基化发生在营养细胞中 对RIP无效，由RIP改变的序列重新甲基化 当通过变形重新引入时。 因此，RIP可以将未甲基化的染色体序列改变为靶标 甲基化。甲基化可以延伸到突变区域之外，并 超出最初复制的线段的边界。 甲基化并不局限于对称部位。修改过的序列 当发生以下情况时，RIP会发生甲基化，但不会发生相应的非甲基化 通过转变重新引入的。因此，甲基化的信号产生了 由RIP提供，是便携的。Riped序列完全重新甲基化 染色体位置检测。一个普遍感兴趣的问题是， RIP突变产生的甲基化信号。

DESCRIPTION: The investigator proposes to continue his investigations on the mechanism and purpose of DNA methylation using Neurospora as a model system. In prior studies, the investigator showed that duplicated sequences in Neurospora, including a duplicated 5S RNA gene and a 5S pseudogene, are heavily altered by point mutations during the sexual phase of the life cycle. This process, called RIP (for repeat-induced point mutation), leads to methylation of the RIPed sequences. These observations led to studies conducted during the last funding period regarding the extent, nature, and purpose of DNA methylation in Neurospora. The overall objective of the current proposal is to understand the function and control of DNA methylation in a model eukaryote. The specific aims include the following: (1) to determine why DNA methylation-deficient mutants become aneuploid, (2) to isolate new DNA methylation mutants by approaches that will facilitate cloning the genes, (3) to isolate and characterize the genes involved in DNA methylation, (4) to define the signals for de novo methylation, (5) to identify and purify proteins that bind specifically to methylated sequences, and (6) to explore the potential connection between DNA methylation and DNA replication. The proposal springs from evidence linking methylation in genetic and epigenetic phenomena such as imprinting. Many correlations between methylation and the lack of gene activity have accumulated and there is strong evidence that methylation prevents gene expression. Findings that eukaryotic methyl transferases preferentially methylate hemi- methylated CpGs have provided support for the models proposed by Riggs and Holliday that methylation is propagated through an enzyme that methylates symmetrical sites. However, it has become clear in studies on fungi that propagation of methylation can occur at non-symmetrical sites. In Neurospora, methylation can take place de novo and in a manner that does not propagate the methylation pattern. About 1.5% of the C's of N. crassa DNA are methylated, but the bulk of the genome is devoid of methylation. Three methylated sequences characterized in detail include the tandemly arranged rDNA, the 1.6 kb zeta-eta region, and the psi region, the latter being relics of RIP, repeat induced point mutation. This is a process discovered by Dr. Selker to be mechanism that detects duplicated sequences in the haploid nuclei of dikaryotic cells after fertilization. Both copies of the duplicated sequence then become frequent targets of G:C to A:T mutation, so much so that 30% of G:C pairs can be altered. Frequently, sequences altered by RIP become methylated. Methylation of RIPed sequences occurs in vegetative cells inactive for RIP and sequences altered by RIP become methylated de novo when reintroduced by transformation. Thus, RIP can change unmethylated chromosomal sequences into targets for methylation. The methylation can extend beyond the mutated region and beyond the boundaries of the segment that was originally duplicated. Methylation is not limited to symmetrical sites. Sequences altered by RIP become methylated but not their nonmethylated counterparts, when reintroduced by transformation. Thus, signals for methylation created by RIP are portable. RIPed sequences become remethylated at all chromosomal positions tested. A question of general interest is how are signals for methylation generated by mutations from RIP.