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: 2904659
• 负责人: CHARLES D LAIRD
• 金额: $ 30.15万
• 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/2904659
• 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：(改编自调查人员摘要)本项目调查 与脆性X相关的DNA甲基化和复制的若干方面 综合症。申请者和同事已经观察到，随着重复扩展 FMR1基因异常甲基化(因此在转录上 未激活)，并且其复制被异常延迟。甲基化 通过防止亚硫酸氢盐转化而确定的图案显示 引人注目的二分法：等位基因要么高度甲基化，要么不高度甲基化，几乎没有 中间体。然而，甲基化的程度在某些部位有所不同 (明显是转录因子的结合部位)甲基化程度低于 其他。甲基化模式在细胞分裂过程中相对保守。 DNA复制，由BrdU掺入细胞通过DNA分选确定 含量和细胞周期蛋白B1染色出现在细胞周期的较晚阶段，在90 FMR1的有丝分裂数分钟，占总基因组DNA的1%。这 观察细胞质疑传统概念之间的差距(G2) DNA复制和染色体凝聚促进有丝分裂，这可能解释了 染色体脆弱的部位。 在续展期间，申请者将进一步追查DNA现象 甲基化和复制延迟。他们将描述甲基化的特征 FMR1区在不同细胞类型和不同发育阶段的分布 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。他们将研究分类细胞中的晚期DNA复制 具有磷酸化组蛋白H3的抗体，并在促进 染色体的脆弱性。最后，他们将进一步开发和使用一种技术 被称为“回旋镖聚合酶链式反应”的DNA合成方向的确定关系 到甲基化。