MOLECULAR GENETICS AND EPIGENETICS OF FRAGILE X SYNDROME

脆性 X 综合征的分子遗传学和表观遗传学

• 批准号: 6181056
• 负责人: CHARLES D LAIRD
• 金额: $ 30.09万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181056
• 关键词: CpG islands; DNA methylation; DNA replication; SDS polyacrylamide gel electrophoresis; cell cycle; cytogenetics; family genetics; flow cytometry; fragile X syndromes; gene expression; gene mutation; gene rearrangement; genetic translation; genomic imprinting; human subject; nucleic acid sequence; polymerase chain reaction; tissue /cell culture; western blottings

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.

描述：（改编自研究者的摘要）该项目调查 与脆性 X 相关的 DNA 甲基化和复制的几个方面 综合症。申请人和同事观察到，通过重复扩展 FMR1 基因异常甲基化（因此转录 失活）并且其复制异常延迟。甲基化 通过防止亚硫酸氢盐转化确定的模式显示出 显着的二分法：等位基因要么高度甲基化，要么不高度甲基化，很少有 中间体。然而，某些位点的甲基化程度有所不同 （显然是转录因子的结合位点）甲基化程度低于 其他的。甲基化模式在细胞分裂过程中相对保守。 DNA 复制，通过 DNA 分选的 BrdU 掺入细胞来确定 含量和细胞周期蛋白 B1 染色，发生在细胞周期的晚期，在 90 年内 FMR1 有丝分裂分钟数和总基因组 DNA 的 1%。这 观察细胞对传统概念之间的间隙（G2）提出质疑 DNA复制和有丝分裂的染色体浓缩，它可以解释 染色体脆性位点。 在续展期间，申请人将进一步追求DNA现象 甲基化和延迟复制。他们将表征甲基化 FMR1区域在不同细胞类型、不同阶段的模式 细胞周期。他们将检查甲基化模式的克隆保守性 通过细胞分裂。他们将研究分选细胞中的晚期 DNA 复制 使用磷酸化组蛋白 H3 的抗体并在促进的条件下 染色体脆弱性。最后他们将进一步开发和使用一项技术 称为“回旋镖 PCR”，用于确定相关 DNA 合成的方向 至甲基化。