DNA CONFORMATION, TRIPLET EXPANSION, AND HUMAN DISEASE

在过去的三年里，七种人类基因突变 疾病；包括脆性X综合征(FRAXA和FRAXE)、肌强直 营养不良、肯尼迪病、亨廷顿病、脊髓小脑性共济失调 1型(SCA1)和齿状核苍白球萎缩 描述。其中几种遗传性疾病显示出预期，在 疾病的严重程度增加和/或发病年龄 一代又一代地减少。在这些基因中发现的每一个突变 疾病包括三核苷酸重复长度的增加(CGG和CTG) 这些基因是不稳定的。重复序列的不稳定性很可能 解释了观察到的遗传预期。扩展的重复长度 在受影响更严重的患者中发现；因此，重要的是 了解重复序列扩张所涉及的分子机制。 该计划项目拨款将检验扩张的几个假设 三胞胎重复序列。方案I介绍初步调查 证明(CTG)n三联体重复序列采用非B DNA结构 这种重复序列的滑移发生在大肠杆菌中。由这十个人组成的结构 三核苷酸重复序列的特征，以及它们参与的 将调查复制暂停和重组。方案II 将调查潜在的错配结构，这些结构很可能 在这些序列中形成。与三联体特异结合的蛋白质 重复将被描述为特征。扩增的三联体重复序列对小鼠的影响 将研究染色质结构和基因表达。计划III将 体外和体内复制过程中的检测扩增(包括非洲爪哇 鸡蛋和提取物)，并检验复制阻断在 重复导致叠加性(扩张性)合成。的稳定性 在大肠杆菌、酵母、CHO、小鼠胚胎干细胞(ES)中有三联体重复 人类细胞将被研究。基因重组在重复序列中的作用 不稳定将在正常和错配修复缺陷(REP3)中进行检查 ES细胞。计划IV将利用人类DNA聚合酶并测试 假设启动子的重新定位会导致扩张。这个项目将 还研究了人体细胞提取液中的复制情况 受预期相关疾病影响，以及 缺乏DNA修复、错配修复或DNA复制的细胞。 方案V将利用ES细胞开发一个模型系统来检查 正常和非正常序列中三联体重复序列的稳定性 以确定这种不稳定性是否是重复所固有的 独自一人。此外，该项目还将提供与生物相关的材料 包括正常个体和受影响个体的序列。 这组经验丰富的调查人员有经过证实的 成就融合了大量的专业知识来调查问题 分子医学具有重要的遗传学和临床意义。如此之高 聚焦的项目将通过提供关于 几个重要人类基因突变事件的分子基础 疾病。

Within the last three years mutations responsible for seven human genetic diseases; including Fragile X-syndrome (FRAXA and FRAXE), myotonic dystrophy, Kennedy's disease, huntington's disease, spinocerebellar ataxia type 1 (SCA1), and dentatorubral pallidoluysian atrophy, have been described. Several of these hereditary diseases show anticipation, in which the severity of the disease increases and/or the age of onset decreases in successive generations. The mutations found in each of these disorders involve increased lengths of trinucleotide repeats (CGG and CTG) which are genetically unstable. The instability of the repeats likely accounts for the genetic anticipation observed. Expanded repeats lengths are found in more severely affected patients; thus it is important to understand the molecular mechanisms involved in repeat expansion. This Program Project Grant will test several hypothesis for the expansion of triplet repeats. Program I presents preliminary investigations demonstrating that (CTG)n triplet repeats adopt non-B DNA structures and that slippage at repeats occurs in E. coli. Structures formed by all ten trinucleotide repeats will be characterized, and their involvement in replication pausing and recombination will be investigated. Program II will investigate potential slipped mis-paired structures that are likely to form in these sequences. Proteins that bind specifically to triplet repeats will be characterized. The effect of expanded triplet repeats on chromatin structure and gene expression will be studied. Program III will assay expansion during replication in vitro and in vivo (including Xenopus eggs and extracts), and test the hypothesis that a replication block at repeats induces reiterative (expansive) synthesis. The stability of triplet repeats in E. coli, yeast, CHO, mouse embryonic stem (ES), and human cells will be studied. The role of genetic recombination in repeat instability will be examined in normal and mismatch repair deficient (rep3) ES cells. Program IV will utilize human DNA polymerases and test the hypothesis that primer relocation leads to expansion. This project will also investigate replication in extracts of human cells from individuals affected with anticipation-associated diseases, as well in extracts from cells deficient in DNA repair, mismatch repair, or DNA replication. Program V will develop a model system utilizing ES cells for examining the stability of triplet repeats in sequences from normal and affected individuals to determine if the instability is inherent for the repeat alone. Moreover, this project will provide biologically relevant materials to the project, including sequences from normal and affected individuals. This group of experienced investigators with proven records of accomplishments blends substantial expertise to investigate a problem in molecular medicine of great genetic and clinical importance. This highly focused project will fill a void by providing new information on the molecular basis for mutational events underlying several important human diseases.