Triplet Repeat Instability in Yeast and Human Cells

酵母和人类细胞中的三联体重复不稳定性

• 批准号: 6916450
• 负责人: Robert S Lahue
• 金额: $ 28.67万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6916450
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; Saccharomyces cerevisiae; astrocytes; biochemistry; chemical stability; enzyme activity; fungal genetics; fungal proteins; gene mutation; genetic regulatory element; helicase; nucleic acid repetitive sequence; point mutation; protein purification; protein structure function; tissue /cell culture; transcription factor

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.

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——导致亨廷顿舞蹈症和其他 14 种神经退行性疾病。第一次导致疾病的 TNR 扩增直到 1991 年才被报道，因此关于其机制根源仍有很多东西需要了解。除了医学相关性之外，三核苷酸重复序列 (TNR) 的遗传学也是独特且复杂的。现在已经清楚，TN​​R 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，以及可能的基因转换。这项工作的目标是更彻底地定义 DNA 复制和修复如何导致酵母和灵长类（人类和猿）细胞中的 TNR 不稳定。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，影响 TNR 的酵母遗传途径的鉴定应有助于阐明同源人类途径的作用。酵母细胞和灵长类细胞之间的差异可能有助于解决某些问题，例如人类的强烈扩张倾向，而这一点尚未在模型系统中重现。我们提案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发生显着变化。我们检测到酵母和灵长类细胞中的阈值，并建议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 扩展或收缩的基因检测方法。将重复序列插入到启动子-报告基因构建体中，以便 TNR 长度决定报告基因的表达。 TNR 长度的变化表现为报告基因表型的变化。这些选择性测定具有几个主要优点，包括灵敏度、定量和灵活性。我们相信，我们的基因检测的创新性，以及我们将这些检测应用于重要模型系统，将继续帮助推进 TNR 遗传学领域的发展。