Stability & Fragility of Trinucleotide Repeats in Yeast

稳定

• 批准号: 6322261
• 负责人: CATHERINE H FREUDENREICH
• 金额: $ 22.98万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6322261
• 关键词: DNA damage; DNA replication; Saccharomyces cerevisiae; artificial chromosomes; chemical stability; fungal genetics; genetic screening; mutant; nucleic acid repetitive sequence; polymerase chain reaction

DESCRIPTION (Verbatim from the applicant's abstract): Expansion of trinucleotide repeat (TNR) sequences is the causative mutation for a number of hereditary diseases, including myotonic dystrophy, the most common dystrophy in adults, Fragile X syndrome, the most common form of inherited mental retardation, and neurodegenerative diseases such as Huntington's and the spinocerebellar ataxias. The mechanism of TNR instability is interesting both for understanding the etiology and inheritance of the triplet repeat diseases, and for a basic understanding of genome stability in humans. In addition, expanded CGG/CCG and CTGICAG sequences are sites of chromosome fragility, areas prone to breakage in vivo. Chromosome breakage is implicated in the generation of translocations and deletions found in many types of cancer. The aim of this proposal is to elucidate the mechanisms involved in TNR instability and fragility, and determine how these two unusual characteristics are interrelated using Saccharomyces cerevisiae. A novel genetic assay has been developed that produces a selectable phenotype when a TNR tract expands or breaks. This assay will be used to screen for proteins whose over-expression influences TNR expansion or fragility. The proteins found to influence TNRs will be characterized to determine both their normal cellular functions and their influence on repeat maintenance. In addition, the hypothesis that TNR expansions occur by aberrant lagging strand replication will be tested by analyzing tract stability (by PCR) and fragility (by genetic and physical analysis) in specific yeast replication mutants. The role of the G2IM checkpoint in detecting TNR tract damage and preventing chromosome breakage will be investigated by comparing rates of TNR tract breakage in wild-type and cheokpoint-defective cells. Lastly, these analyses will be extended to other types of minisatellite sequences that act as fragile sites in human cells. The proposed experiments are designed to elucidate not only how simple repeats expand to cause human disease, but also the consequences of and cellular response to expanded tracts, with the goal of understanding how genomic instability can affect human health.

描述(逐字摘自申请人的摘要)：扩展 三核苷酸重复(TNR)序列是许多 遗传性疾病，包括强直性肌营养不良，是 成人，脆性X综合征，最常见的遗传性精神疾病 发育迟缓和神经退行性疾病，如亨廷顿氏症和 脊髓小脑性共济失调。TNR不稳定的机制是有趣的 为了了解三联体重复疾病的病因和遗传， 并对人类基因组稳定性有一个基本的了解。此外, 扩展的CGG/CCG和CTGICAG序列是染色体脆性部位，区域 在活体内容易破裂。染色体断裂与这一代有关 在许多类型的癌症中发现的易位和缺失。这样做的目的是 建议澄清TNR不稳定和TNR不稳定的机制 脆弱性，并确定这两个不同寻常的特征是如何相互关联的 使用酿酒酵母。一种新的遗传分析已经被开发出来 当TNR束扩张或断裂时，产生一种可选择的表型。这个化验 将用于筛选其过度表达影响TNR的蛋白质 扩张或脆弱。已发现的影响TNRs的蛋白质将是 其特征是确定它们的正常细胞功能和它们的 对重复维护的影响。此外，TNR的假设是 由于异常滞后的链复制而发生的扩展将通过 分析肠道的稳定性(通过聚合酶链式反应)和脆弱性(通过遗传和生理因素 分析)在特定酵母复制突变体中。G2IM的作用 检查点在检测TNR束损伤和防止染色体断裂中的作用 将通过比较野生型和野生型TNR束断裂率进行调查 Checokpoint-有缺陷的细胞。最后，这些分析将扩展到其他 在人类细胞中充当脆弱位置的小卫星序列的类型。这个 拟议的实验不仅是为了阐明如何简单地重复 扩张会导致人类疾病，而且还会造成细胞和 对扩大的区域的反应，目的是了解基因组如何 不稳定会影响人类健康。