SPONTANEOUS MUTAGENESIS OF TRIPLET REPEATS

三联体重复的自发诱变

• 批准号: 6204268
• 负责人: RICHARD Rankin SINDEN
• 金额: $ 11.38万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6204268
• 关键词: CHO cells; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; SDS polyacrylamide gel electrophoresis; adenine phosphoribosyltransferase; bacterial genetics; embryonic stem cell; enzyme activity; fungal genetics; gene mutation; genetic recombination; genetically modified animals; hypoxanthine phosphoribosyltransferase; laboratory mouse; molecular cloning; nucleic acid repetitive sequence; nucleic acid structure; plasmids; simian virus 40; virus genetics; yeasts

Within the last three years the molecular basis of 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, has been described. These hereditary diseases are similar in that they show genetic anticipation, in which the severity of the disease increases and the age of onset decreases in successive generations. The molecular basis for anticipation appears to be the expansion of tracts of triplet repeats (CGG in the case of FRAXA and FRAXE and CTG in the other five diseases). In Fragile X-syndrome and myotonic dystrophy, the length of triplet repeat (usually less than 40 repeats in normal individuals) can expand to thousands of repeats in severely affected individuals. Size heterogeneity and especially deletion of repeats in DNA are common and presumably due to slipped misalignment during DNA replication or recombination. The remarkable tendency of triplet repeats in humans to expand represents a novel genetic phenomenon, which has not been previously described. This project will determine if replication and/or recombination-based errors are responsible for this unusual genetic phenomena. Using triplet repeat sequences containing interruptions from normal Fragile X (CGG repeats) and SCA1 individuals (CTG repeats) as well as long uninterrupted repeats, we will investigate the effect of repeat sequence and length on DNA polymerase induced slippage and/or strand displacement. We will test the hypothesis that a block to replication at triplet repeats induces reiterative synthesis resulting in expansion. Replication of repeats will be studied in Xenopus eggs and egg extracts, a system that mimics replication in early development (where expansion in some diseases is thought to occur). The genetic stability of triplet repeats will be determine in E. coli, yeast, Chinese hamster ovary (CHO), mouse embryonic stem (ES), and human cells in wild type or normal) cells and in cells containing mutations affecting DNA replication, mismatch repair, and genetic recombination. Selection in yeast will employ ura3. In CHO, ES, and human cells selection will utilize exon skipping int eh APRT or HPRT gene. Genetically modified ES cells, with triplet repeats in the second intron or in an artificial third exon in the HPRT gene will be used to measure the frequency of recombination events associated with the repeats. ES cells and transgenics deficient in rep3, a MutS mismatch repair homologue, will be made and tested for triplet repeat stability. The effects of environmental mutagens, carcinogens, and chemicals on the fidelity of DNA replication of repeats will be investigated.

在过去的三年里，七种人类遗传的分子基础 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、脊髓小脑共济失调 1 型 (SCA1) 和齿状红核苍白卢伊体萎缩 描述的。 这些遗传性疾病的相似之处在于 遗传预期，其中疾病的严重程度增加 发病年龄逐代减少。 分子基础 预期似乎是三联体重复序列的扩展 （对于 FRAXA 为 CGG，对于其他五种疾病为 FRAXE 和 CTG）。 在脆性 X 综合征和强直性肌营养不良中，三联体重复的长度 （正常个体中通常少于 40 次重复）可以扩展到 在严重受影响的个体中重复了数千次。 大小异质性，尤其是 DNA 中重复序列的缺失是常见的， 可能是由于 DNA 复制过程中的滑动错位或 重组。 人类三联体重复的显着趋势 Expand 代表了一种新的遗传现象，以前从未有过 描述的。 该项目将确定是否复制和/或 基于重组的错误是造成这种不寻常遗传的原因 现象。 使用包含中断的三联体重复序列 正常脆性 X（CGG 重复）和 SCA1 个体（CTG 重复） 只要不间断地重复，我们就会研究重复的效果 DNA 聚合酶诱导的滑移和/或链的序列和长度 位移。 我们将测试以下假设：复制块位于 三联体重复诱导重复合成，从而导致扩展。 将在非洲爪蟾卵和卵提取物中研究重复序列的复制， 模仿早期开发中的复制的系统（其中扩展 据认为会发生某些疾病）。 三联体遗传稳定性 将在大肠杆菌、酵母、中国仓鼠卵巢 (CHO)、 小鼠胚胎干（ES）和人类细胞（野生型或正常）细胞 在含有影响 DNA 复制的突变的细胞中，错配 修复和基因重组。 酵母中的选择将使用ura3。 在 CHO、ES 和人类细胞中，选择将利用外显子跳跃 int eh APRT 或 HPRT 基因。 转基因 ES 细胞，具有三联体重复 HPRT 基因中的第二个内含子或人工第三个外显子将是 用于测量与相关重组事件的频率 重复。 ES 细胞和转基因细胞缺乏rep3（MutS 不匹配） 修复同系物，将被制备并测试三联体重复稳定性。 环境诱变剂、致癌物和化学物质对人体的影响 将研究重复 DNA 复制的保真度。