Environmental Factors Influencing Minisatellite Stability in Yeast

影响酵母小卫星稳定性的环境因素

• 批准号: 7953099
• 负责人: David T. Kirkpatrick
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7953099
• 关键词: Acrylamides; Address; Affect; Aluminum; Asthma; Attention; Attention deficit hyperactivity disorder; Biological Assay; Cadmium; Caffeine; Calcium; Camptothecin; Cell Cycle; Cells; Color; Complex; Copper; DNA; DNA Damage; DNA Fingerprinting; DNA Repair; DNA Sequence; DNA Sequence Rearrangement; Data; Detection; Diabetes Mellitus; Diamide; Disease; Environmental Risk Factor; Epilepsy; Essential Genes; Event; Exposure to; Failure; Ficusin; Funding; Fungal Genome; Genes; Genetic; Genome; Genomic Instability; High temperature of physical object; Homeostasis; Human; Hydrogen Peroxide; Induced Mutation; Insulin-Dependent Diabetes Mellitus; Ions; Iron; Knowledge; Libraries; Link; Longevity; Magnesium; Malignant Neoplasms; Manganese; Mediating; Metals; Microbiological Techniques; Microsatellite Repeats; Minisatellite Repeats; Mitotic; Morphology; Mutation; Nature; Nucleotides; Oncogenic; Paraquat; Pathway Analysis; Pathway interactions; Phase; Potassium; Progressive Myoclonic Epilepsies; Psoralens; Relative (related person); Repetitive Sequence; Saccharomyces cerevisiae; Saccharomycetales; Screening procedure; Sirolimus; Source; Surveys; Syndrome; System; Temperature; Testing; Ulcerative Colitis; Ultraviolet Rays; Variant; Vitamins; Work; Yang; Yeasts; Zinc; cancer cell; cold temperature; high throughput screening; human disease; irradiation; novel; public health relevance; repaired; research study; response

DESCRIPTION (provided by applicant): Altered minisatellite DNA tracts have been linked to many human diseases, including HRAS1-related cancers, progressive myoclonus epilepsy, insulin-dependent diabetes mellitus, asthma, ulcerative colitis and even ADHD. Unfortunately very little is known about the factors, both environmental and genetic, that regulate minisatellite stability. Progress in identifying the factors that cause alterations in repetitive minisatellite DNA tracts has been slowed by the lack of strong assay systems. We recently developed a robust assay system in the budding yeast S. cerevisiae that readily detects tract alterations as changes in colony color and morphology, allowing for simple, rapid screening. Importantly, we used our assay to demonstrate that minisatellite tract alterations result from perturbations in the level of zinc, demonstrating that our novel assay is an ideal means to determine the effect of exposure to environmental conditions and compounds on minisatellite stability. Equally importantly, disruption of zinc homeostasis causes minisatellite rearrangements, but only when the cells are in a post-mitotic, quiescent state. Our understanding of the factors inducing genetic rearrangement in post-mitotic cells is extremely limited, unlike the situation with replicating cells, where a large amount of effort has been expended on determining the mechanisms that alter the genome during a typical cell cycle. The majority of human cells are quiescent, spending most of their lifespan in that state. Importantly, the initial oncogenic mutations that generate a cancer cell occur in these quiescent cells. We hypothesize that these mutations result from a failure of repair factors to identify or repair DNA damage in the quiescent cell. However, the nature of the mutation-inducing agents and the DNA repair systems these agents activate have not been identified in quiescent cells. Identification of these environmental and genetic factors in quiescent cells is vital to our understanding of early oncogenic events. Therefore, to address all of these significant knowledge gaps, we will identify compounds and environmental conditions that influence minisatellite repeat stability in quiescent cells, using our well-characterized assay system in combination with rapid high- throughput microbiological techniques and whole-genome analysis. Importantly, our assay system uniquely allows us to differentiate between events occurring in actively dividing cells and in post-mitotic cells, guaranteeing that we are surveying the complete spectrum of possible alterations. Once we have identified conditions or compounds that affect minisatellite repeat stability, we will screen the entire yeast genome to identify all of the genes required for the effect. Therefore, the experiments described in this project allow us to identify environmental conditions and compounds that affect one of the most common repetitive DNA types and to determine the genes mediating the effects, data that will significantly impact our understanding of such diverse diseases as cancers, especially initial oncogenic events, epilepsy, and diabetes. PUBLIC HEALTH RELEVANCE: Repetitive DNA tracts are a primary source of genome instability; alterations in repetitive minisatellite tracts have been associated with the onset of many human diseases, including cancers, epilepsy and diabetes. We recently constructed a novel and unique assay for minisatellite instability that detects alterations occurring in both actively-growing yeast cells and cells that have ceased growing and entered stationary phase (the state for most human cells). We will use this assay to identify all of the environmental factors that influence minisatellite stability in growing and stationary cells, and identify the genes that control the effect.

描述（由申请人提供）：改变的小卫星 DNA 束与许多人类疾病有关，包括 HRAS1 相关癌症、进行性肌阵挛癫痫、胰岛素依赖性糖尿病、哮喘、溃疡性结肠炎甚至 ADHD。不幸的是，人们对调节小卫星稳定性的环境和遗传因素知之甚少。由于缺乏强大的检测系统，识别导致重复小卫星 DNA 束改变的因素的进展已经放缓。我们最近在芽殖酵母酿酒酵母中开发了一种强大的检测系统，可以轻松检测菌落颜色和形态变化引起的菌道变化，从而实现简单、快速的筛选。重要的是，我们使用我们的测定法证明了小卫星束的改变是由锌水平的扰动引起的，证明我们的新型测定法是确定暴露于环境条件和化合物对小卫星稳定性影响的理想方法。同样重要的是，锌稳态的破坏会导致小卫星重排，但仅限于细胞处于有丝分裂后静止状态时。我们对有丝分裂后细胞中诱导基因重排的因素的理解极其有限，这与复制细胞的情况不同，在复制细胞中，我们花费了大量的精力来确定在典型细胞周期中改变基因组的机制。大多数人体细胞处于静止状态，其大部分生命周期都处于这种状态。重要的是，产生癌细胞的最初致癌突变发生在这些静止细胞中。我们假设这些突变是由于修复因子未能识别或修复静止细胞中的 DNA 损伤所致。然而，突变诱导剂的性质以及这些剂激活的 DNA 修复系统尚未在静止细胞中得到鉴定。识别静止细胞中的这些环境和遗传因素对于我们了解早期致癌事件至关重要。因此，为了解决所有这些重大的知识差距，我们将使用我们充分表征的测定系统结合快速高通量微生物技术和全基因组分析来确定影响静止细胞中小卫星重复稳定性的化合物和环境条件。重要的是，我们的检测系统独特地允许我们区分活跃分裂细胞和有丝分裂后细胞中发生的事件，保证我们正在调查可能改变的完整范围。一旦我们确定了影响小卫星重复稳定性的条件或化合物，我们将筛选整个酵母基因组，以确定该效果所需的所有基因。因此，该项目中描述的实验使我们能够识别影响最常见的重复DNA类型之一的环境条件和化合物，并确定介导这些影响的基因，这些数据将显着影响我们对癌症等多种疾病的理解，特别是最初的致癌事件、癫痫和糖尿病。 公共卫生相关性：重复 DNA 片段是基因组不稳定的主要来源；重复小卫星束的改变与许多人类疾病的发生有关，包括癌症、癫痫和糖尿病。我们最近构建了一种新颖且独特的小卫星不稳定性检测方法，可检测活跃生长的酵母细胞和已停止生长并进入静止期（大多数人类细胞的状态）的细胞中发生的变化。我们将使用这种测定来识别影响生长和静止细胞中小卫星稳定性的所有环境因素，并识别控制该效应的基因。