DNA CONFORMATION, TRIPLET EXPANSION, AND HUMAN DISEASE

DNA 构象、三联体扩增和人类疾病

• 批准号: 2734769
• 负责人: Robert Dale Wells
• 金额: $ 41.78万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734769
• 关键词: nucleic acid repetitive sequence

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.

在过去的三年里，导致七种人类基因突变的突变 疾病;包括脆性X综合征（FRAXA和FRAXE），肌强直性 营养不良、肯尼迪病、亨廷顿病、脊髓小脑性共济失调 1型（SCA 1）和齿状核红核苍白球路易氏体萎缩， 介绍了 这些遗传性疾病中有几种表现出预期， 疾病的严重程度增加和/或发病年龄 在连续几代人中下降。 在这些基因中发现的突变 涉及三核苷酸重复序列（CGG和CTG）长度增加的疾病 基因不稳定 重复序列的不稳定性 解释了观察到的遗传预测。 扩展重复序列长度 在受影响更严重的患者中发现;因此， 了解重复扩增的分子机制。 这项计划项目赠款将测试几个假设的扩张 三重重复。 方案一介绍了初步调查 证明（CTG）n三联体重复采用非B DNA结构， 在E.杆菌 由这10个组成的结构 三核苷酸重复将被表征，并且它们参与 将研究复制暂停和重组。 方案二 将研究潜在的滑动错配结构， 形成这些序列。 与三联体特异性结合的蛋白质 重复将被表征。 扩增的三联体重复序列对 将研究染色质结构和基因表达。 方案三将 在体外和体内（包括非洲爪蟾）复制期间的测定扩增 鸡蛋和提取物），并测试假设，复制块在 repeats诱导了迭代（扩展）合成。 的稳定性 E.大肠杆菌、酵母、CHO、小鼠胚胎干（ES），以及 将研究人类细胞。 基因重组在重复序列中的作用 将在正常和错配修复缺陷（rep 3）中检查不稳定性 ES细胞。 计划四将利用人类DNA聚合酶和测试 假设引物重新定位导致扩增。 该项目将 我还研究了从个体中提取的人类细胞中的复制 与预期相关的疾病，以及提取物， DNA修复、错配修复或DNA复制缺陷的细胞。 计划五将开发一个利用胚胎干细胞的模型系统， 正常和受累序列中三联体重复序列的稳定性 个人，以确定是否不稳定性是固有的重复 一个人 此外，该项目将提供生物相关材料， 包括正常和受影响个体的序列。 这组经验丰富的调查人员， 成就融合了大量的专业知识，以调查一个问题， 分子医学在遗传学和临床上具有重要意义。 这种高度 重点项目将通过提供有关 几个重要人类基因突变事件的分子基础 疾病