Triplet Repeat Instability in Yeast and Human Cells

描述（由申请人提供）：一种不寻常的基因突变-三核苷酸重复扩增-导致亨廷顿氏病和其他14种神经退行性疾病。第一次引起疾病的TNR扩张仅在1991年被报道，因此关于其机制根源仍有很多需要了解。除了它们的医学相关性外，三核苷酸重复序列（tnr）的遗传学是独特而复杂的。现在很清楚，TNR的扩张和收缩是由多种遗传机制引起的，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定义DNA复制和修复如何促进酵母和灵长类动物（人类和类人猿）细胞的TNR不稳定性。酵母和灵长类细胞之间的机制相似性将有助于描述控制三联体重复改变的重要基本特性。例如，鉴定影响tnr的酵母遗传途径应该有助于阐明人类同源途径的作用。酵母和灵长类细胞之间的差异可能有助于解决某些问题，例如在模型系统中尚未重现的人类细胞扩增的强烈倾向。我们建议的一个独特方面是更好地理解阈值。阈值是tnr的一个独特但神秘的特征，其中不稳定性在狭窄的束长度范围内急剧变化。我们在酵母和灵长类细胞中都检测到了阈值，我们打算对它们进行基因剖析。为了帮助实现我们的目标，我们开发了直接选择TNR扩增或收缩的遗传分析。重复序列被插入到启动子-报告基因结构中，使得TNR长度决定报告基因的表达。TNR长度的变化显示为报告表型的变化。这些选择性分析提供了几个主要优点，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质，以及我们将这些分析应用于重要的模型系统，将继续帮助推进TNR遗传学领域。

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.