SPONTANEOUS MUTAGENESIS OF TRIPLET REPEATS

在过去的三年里，七种人类基因的分子基础 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（SCA1）和齿状核红核苍白球路易氏体萎缩， 介绍了 这些遗传性疾病的相似之处在于， 遗传预测，其中疾病的严重程度增加， 发病年龄在连续几代中降低。 的分子基础 因为预期似乎是三重重复序列的扩展， (CGG在FRAXA和FRAXE的情况下，以及在其他五种疾病中的CTG）。 在脆性X综合征和强直性肌营养不良中，三联体重复序列的长度 （正常个体中通常少于40个重复）可以扩展到 在严重受影响的个体中有数千次重复。 DNA中的大小异质性和特别是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， 重组 人类三重重复序列的显著趋势是， 扩展代表了一种新的遗传现象，以前没有 介绍了 本项目将确定复制和/或 基于重组的错误导致了这种不寻常的遗传 现象。 使用含有来自以下的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）以及 作为长时间不间断的重复，我们将研究重复的效果 DNA聚合酶诱导的滑移和/或链上的序列和长度 位移 我们将检验这样一个假设，即在 三联体重复诱导重复合成，导致扩增。 重复序列的复制将在非洲爪蟾卵和卵提取物中进行研究， 在早期开发中模仿复制的系统（其中 一些疾病被认为会发生）。 三联体的遗传稳定性 重复将在E中确定。大肠杆菌，酵母，中国仓鼠卵巢（CHO）， 小鼠胚胎干（ES）和野生型或正常的人细胞 在含有影响DNA复制的突变的细胞中， 修复和基因重组。 酵母中的选择将使用ura 3。 在CHO、ES和人类细胞中，选择将利用外显子跳跃插入细胞中， APRT或HPRT基因。 基因修饰的ES细胞，具有三联体重复序列， HPRT基因中的第二内含子或人工第三外显子中的第二内含子将是 用于测量与重组相关的重组事件的频率。 重复。 胚胎干细胞和转基因缺陷rep3，MutS错配 修复同源物，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 将研究重复序列的DNA复制的保真度。

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.