Triplet Repeat Instability in Yeast and Human Cells

描述(申请人提供)：一种不寻常的基因突变类型--三核苷酸重复扩大--导致亨廷顿氏病和其他14种神经退行性疾病。第一次致病的TNR扩张是在1991年才被报道的，所以关于它们的机制根源仍然有很多需要了解。除了医学上的相关性，三核苷酸重复序列(TNR)的遗传学也是独特而复杂的。现在已经很清楚，TNR的扩张和收缩是通过多种遗传机制发生的，包括异常的DNA复制、修复和可能的基因转换。这项工作的目标是更彻底地确定DNA复制和修复如何导致酵母和灵长类(人和猿猴)细胞中TNR的不稳定。酵母和灵长类细胞之间的机械相似性将有助于描述支配三联体重复改变的重要基本性质。例如，识别影响TNRs的酵母遗传途径应该有助于阐明同源人类途径的作用。酵母和灵长类细胞之间的差异可能有助于解决某些问题，例如人类强烈的扩张趋势，这一趋势尚未在模型系统中概括。我们提议的一个独特方面是更好地理解门槛。阈值是TNRs的一个独特而神秘的特征，在这种情况下，不稳定程度在很小的范围内变化很大。我们在酵母和灵长类细胞中都检测到了阈值，并建议从基因上剖析它们。为了帮助实现我们的目标，我们开发了直接选择TNR扩张或收缩的遗传分析方法。重复序列被插入到启动子-报告结构中，从而TNR长度决定报告基因的表达。TNR长度的变化表现为报告表型的变化。这些选择性分析提供了几个主要的优点，包括灵敏度、定量和灵活性。我们相信，我们的基因分析的创新性，以及我们将这些分析应用于重要的模型系统，将继续帮助推动TNR遗传学领域的发展。

DESCRIPTION (provided by applicant): An unusual type of genetic mutation-trinucleotide repeat expansion-causes Huntington's disease and 14 other neurodegenerative disorders. The first disease-causing TNR expansions were reported only in 1991, so there is still much to learn about their mechanistic roots. In addition to their medical relevance, the genetics of trinucleotide repeats (TNRs) are unique and complex. It is now clear that TNR expansions and contractions occur by multiple genetic mechanisms, including aberrant DNA replication, repair, and possibly gene conversion. The goal of this work is to define more thoroughly how DNA replication and repair contribute to TNR instability in yeast and in primate (human and simian) cells. Mechanistic similarities between yeast and primate cells will help delineate important fundamental properties that govern triplet repeat alterations. For example, the identification of yeast genetic pathways affecting TNRs should help clarify the roles of homologous human pathways. Differences between yeast and primate cells may help resolve certain issues, such as the strong tendency towards expansions in humans which has not been recapitulated in model systems. One unique facet of our proposal is to better understand thresholds. The threshold is a distinctive but enigmatic feature of TNRs where instability changes dramatically over a narrow range of tract lengths. We detected thresholds in both yeast and primate cells, and we propose to dissect them genetically. To help achieve our goal, we developed genetic assays for the direct selection of TNR expansions or contractions. Repeats are inserted into a promoter-reporter construct such that the TNR length determines reporter gene expression. Variations in TNR length are revealed as changes in the reporter phenotype. These selective assays provide several major advantages, including sensitivity, quantitation, and flexibility. We believe the innovative nature of our genetic assays, and our application of those assays to important model systems, will continue to help advance the field of TNR genetics.