MOLECULAR GENETICS AND EPIGENETICS OF FRAGILE X SYNDROME

描述：（改编自研究者摘要）本项目研究 DNA甲基化和复制与脆性X染色体相关的几个方面 综合征申请人及其同事观察到，随着重复扩增， FMR 1基因异常甲基化（从而在转录上 失活），并且其复制异常延迟。甲基化 如通过保护免于亚硫酸氢盐转化所确定的，图案显示出 值得注意的二分法：等位基因要么高度甲基化，要么没有，很少有甲基化。 中间体的然而，甲基化的程度随着某些位点而变化， （转录因子的明显结合位点）甲基化程度低于 他人甲基化模式在细胞分裂中相对保守。 DNA复制，如通过DNA分选的BrdU掺入细胞中所确定的 含量和细胞周期蛋白B1染色，发生在细胞周期的相当晚，在90 FMR 1和多达1%的总基因组DNA的有丝分裂分钟。这 观察细胞质疑传统的概念之间的差距（G2） DNA复制和染色体凝聚的有丝分裂，它可以解释 染色体脆性的位点。 在续签期间，申请人将进一步追究DNA的现象 甲基化和延迟复制。他们将描述甲基化 FMR 1区在不同细胞类型中的模式在不同的阶段， 细胞周期他们将研究甲基化模式的克隆保守性 通过细胞分裂。他们将研究细胞中的晚期DNA复制， 与磷酸化组蛋白H3的抗体，并在促进 染色体脆性最后，他们将进一步开发和使用一种技术， 被称为“回旋PCR”，以确定DNA合成的方向， 甲基化。

DESCRIPTION: (Adapted from investigator's abstract) This project investigates several aspects of DNA methylation and replication related to fragile X syndrome. The applicant and colleagues have observed that with repeat expansion the FMR1 gene is abnormally methylated (and thereby transcriptionally inactivated) and that its replication is abnormally delayed. The methylation pattern, as determined by protection from bisulfite conversion, showed a remarkable dichotomy: alleles are either highly methylated or not, with few intermediates. The degree of methylation varies, however, with some sites (apparently binding sites for transcription factors) less methylated than others. The methylation pattern is relatively conserved through cell division. DNA replication, as determined by BrdU incorporation in cells by sorted by DNA content and cyclin B1 staining, occurs quite late in the cell cycle, within 90 minutes of mitosis for FMR1 and as much as 1% of total genomic DNA. This observation cells into question the traditional concept of a gap (G2) between DNA replication and chromosome condensation for mitosis, and it may explain sites of chromosome fragility. In the renewal period, the applicants will further pursue the phenomena of DNA methylation and delayed replication. They will characterize the methylation pattern of the FMR1 region in different cell types at different stages of the cell cycle. They will examine clonal conservation of methylation patterns through cell division. They will study late DNA replication in cells sorted with an antibody to phosphorylated histone H3 and in conditions that promote chromosome fragility. Finally they will further develop and use a technique called "boomerang PCR" to determine the direction of DNA synthesis in relation to methylation.