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: 8115131
• 负责人: David T. Kirkpatrick
• 金额: $ 21.66万
• 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 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115131
• 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; HRAS gene; 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; Vitamin E; Work; Yeasts; Zinc; cancer cell; cold temperature; factor A; 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）：改变的迷你卫星DNA界已与许多人类疾病有关，包括与HRAS1相关的癌症，进行性肌阵挛性癫痫，胰岛素依赖性糖尿病，哮喘，哮喘，溃疡性结肠炎，甚至ADHD。不幸的是，对调节微型卫星稳定性的环境和遗传因素知之甚少。缺乏强大的测定系统，确定导致重复性迷你卫星DNA区域改变的因素的进展已减慢。最近，我们在酿酒酵母的萌芽酵母中开发了一个强大的测定系统，该系统很容易检测到菌落色和形态的变化，从而可以进行简单，快速的筛查。重要的是，我们使用我们的测定法证明，微型卫星的改变是由锌水平的扰动引起的，这表明我们的新测定是确定暴露于环境条件和化合物对微型层稳定性的影响的理想方法。同样重要的是，锌稳态的破坏会导致微型卫星重排，但仅当细胞处于有丝分裂后的静态状态时。与复制细胞的情况不同，我们对诱导造成遗传重排的因素的理解非常有限，在这种情况下，在确定在典型细胞周期中改变基因组的机制方面已经花费了大量精力。大多数人类细胞都是静止的，大部分寿命都处于该状态。重要的是，在这些静止的细胞中发生了产生癌细胞的初始致癌突变。我们假设这些突变是由于修复因子未能识别或修复静态细胞中DNA损伤的原因。但是，在静态细胞中尚未鉴定出诱导突变剂和DNA修复系统的性质。静止细胞中这些环境和遗传因素的鉴定对于我们对早期致癌事件的理解至关重要。因此，为了解决所有这些重要的知识差距，我们将使用我们的特征良好的测定系统以及快速的高吞吐量微生物技术和全基因组分析，确定影响静态细胞中微型重复稳定性的化合物和环境条件。重要的是，我们的测定系统唯一地使我们能够区分主动分裂细胞和有丝质细胞中发生的事件，从而确保我们正在调查可能发生的完全改变的频谱。一旦我们确定了影响迷你卫星重复稳定性的疾病或化合物，我们将筛选整个酵母基因组，以识别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确定影响最常见的重复性DNA类型之一的环境条件和化合物，并确定介导的效果的基因，这些数据将极大地影响我们对像癌症一样，尤其是癌症的各种疾病的理解最初的致癌事件，癫痫和糖尿病。 公共卫生相关性：重复的DNA区是基因组不稳定性的主要来源；重复的迷你卫星区的改变与包括癌症，癫痫和糖尿病在内的许多人类疾病的发作有关。我们最近为迷你卫星不稳定性构建了一种新颖而独特的测定法，该测定法检测出在积极生长的酵母细胞和已经停止生长和进入固定相的细胞中发生的变化（大多数人类细胞的状态）。我们将使用该测定法确定影响在生长和固定细胞中影响迷你卫星稳定性的所有环境因素，并确定控制效果的基因。