TRI-NUCLEOTIDE REPEAT AND FRAGILE SITES IN YEAST

酵母中的三核苷酸重复位点和脆弱位点

• 批准号: 2882525
• 负责人: VIRGINIA A. ZAKIAN
• 金额: $ 31.14万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-29 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2882525
• 关键词: DNA replication; Saccharomyces cerevisiae; biological models; fungal genetics; gene mutation; genetic techniques; model design /development; nucleic acid repetitive sequence

DESCRIPTION (adapted from investigator's abstract): An ever-increasing number of human genetic diseases are attributed to expansion of trinucleotide repeats (TNRs). For example fragile X syndrome, the second leading cause of mental retardation, is due to expansion of a CGG tract and myotonic muscular dystrophy, is due to expansion of a CTG tract. Moreover, expansion of CGG tracts induce breakage at five known human chromosomal loci, some of which correlate with human disease. Chromosome breakage at fragile site is also implicated in the chromosomal rearrangements characteristic of human tumors. TNRs associated with human disease are rare. TNR and fragile site research would be greatly aided by genetic assays for tract expansion and/or chromosome fragility. The goal of this grant is to develop Saccharomyces cerevisiae as a model for TNR expansion and triplet-mediate chromosome fragility. The investigator has inserted 130 repeats of CTG onto a yeast chromosome. Large expansion of this tract were obtained, the first large expansion reported outside of humans. CTG tracts may also be length-dependent fragile sites in yeast. This observation provides the basis for a simple, genetic assay for tract expansion and chromosome fragility. The assays will be used to identify yeast and human genes whose mutation or over-expression increases CTG expansion and/or fragility. A premise of the proposed work is that most genes that affect TNR stability will encode proteins involved in replication, repair, or chromatin structure, many of which are conserved from yeast to humans. The behavior of CGG tracts will also be studied. Nucleases and DNA methylases will be used to analyze the chromatin structure of CTG and CGG tracts as both DNAs display unusual nucleosome forming properties in vitro. Finally, fragile sites are thought to be caused by late replication of TNR DNA. This model will be tested using density transfer and two-dimensional gel electrophoresis to determine if replication timing or replication fork progression is affected by the presence of TNRs.

描述（改编自研究者摘要）：不断增加的 许多人类遗传疾病是由于 三核苷酸重复序列（TNR）。 例如脆性X综合征， 精神发育迟滞的主要原因，是由于CGG道的扩张， 强直性肌营养不良是由于CTG束的扩张。 此外，委员会认为， CGG束的扩张诱导五个已知的人类染色体断裂 基因座，其中一些与人类疾病相关。 染色体断裂， 脆性位点也与染色体重排有关 人类肿瘤的特征。 与人类疾病相关的TNR是 罕见. 遗传学方法将极大地帮助TNR和脆性位点研究， 用于束扩张和/或染色体脆性的测定。 这个目标 一项赠款是开发酿酒酵母作为TNR扩展的模型 和三联体介导的染色体脆性。 研究者将130个重复的CTG插入到酵母染色体上。 这一区域获得了大规模扩张， 据报道，在人类之外。 CTG束也可能是长度依赖性脆弱的 酵母中的位点。 这一观察结果为一个简单的遗传学的 用于染色体束扩展和染色体脆性的测定。 将使用这些测定 来鉴定酵母和人类基因的突变或过度表达 增加CTG膨胀和/或脆弱性。 拟议工作的前提是 大多数影响TNR稳定性的基因编码的蛋白质参与了 复制、修复或染色质结构，其中许多是保守的 从酵母到人类 CGG束的行为也将被研究。 核酸酶和DNA甲基化酶将用于分析染色质结构 CTG和CGG束的DNA显示不寻常的核小体形成 体外性能 最后，脆弱的网站被认为是由 TNR DNA的晚期复制。 将使用密度测试此模型 转移和二维凝胶电泳，以确定是否复制 TNR的存在会影响复制叉进展的时间。