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.

在过去三年内，七个人类遗传的分子基础 疾病；包括脆弱的X-Syndrome（Fraxa和Fraxe），Myotonic 营养不良，肯尼迪氏病，亨廷顿氏病，脊髓疾病共济失调 1型（SCA1）和dentatorubral pallidoluysian萎缩已是 描述。 这些遗传性疾病是相似的 遗传预期，疾病的严重程度增加， 连续几代人的发作年龄降低。 分子基础 为了预期似乎是三胞胎重复段的扩展 （在其他五种疾病中，在Fraxa，Fraxe和CTG的情况下）。 在脆弱的X综合体和肌发达症中，三重态的长度重复 （通常在正常人中少于40次重复）可以扩展到 受影响的个体中成千上万的重复。 大小异质性，尤其是DNA中重复序列的缺失很常见，并且 大概是由于在DNA复制过程中未对准或 重组。 人类重复三胞胎的显着趋势 扩展代表了一种新的遗传现象，以前尚未 描述。 该项目将确定复制和/或是否 基于重组的错误是这种不寻常遗传的原因 现象。 使用包含来自中断的三重序列重复序列 正常脆弱的X（CGG重复序列）和SCA1个体（CTG重复序列） 只要不间断的重复，我们将研究重复的效果 DNA聚合酶的序列和长度诱导的滑动和/或链 位移。 我们将测试以下假设 三重态重复​​诱导重复合成，导致膨胀。 重复的复制将在Xenopus卵和鸡蛋提取物中研究 模仿早期开发中复制的系统（其中扩展 人们认为某些疾病会发生）。 三胞胎的遗传稳定性 重复将在大肠杆菌，酵母，中国仓鼠卵巢（CHO）中确定， 小鼠胚胎干（ES）和野生型或正常细胞中的人类细胞 在包含影响DNA复制的突变的细胞中，不匹配 修复和遗传重组。 酵母中的选择将采用URA3。 在CHO，ES和人类细胞中选择将利用外显子跳过INT EH APRT或HPRT基因。 基因修饰的ES细胞，三联体重复 HPRT基因中的第二个内含子或人造第三个外显子将是 用于测量与 重复。 ES细胞和转基因缺乏REP3，A MUT不匹配 维修同源物将进行并测试三重态重复​​稳定性。 环境诱变剂，致癌物和化学物质对 将研究重复的DNA复制的保真度。