Environmental and genetic regulation of copy number variation (CNV)

拷贝数变异 (CNV) 的环境和遗传调控

基本信息

批准号：
7813317
负责人：
THOMAS PETES
金额：
$ 50万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-26 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7813317
关键词：
Address Affect Agrochemicals Aneuploidy Area Background Radiation Biological Assay Cell Cycle Cell Cycle Stage Cells Chromosomes Collection Copper Copy Number Polymorphism DNA DNA Damage DNA Microarray Chip DNA Repair DNA Sequence DNA Sequence Rearrangement DNA biosynthesis DNA damage checkpoint Detection Development Diploidy Disease Dose Environmental Exposure Environmental Pollution Environmental Risk Factor Event Formaldehyde Frequencies Fungal Genome Gel Gene Dosage Genes Genetic Genetic Recombination Genome Genomics Haploidy Human Investigation Ionizing radiation Karyotype Libraries Life Cycle Stages Link Measures Mediating Meiosis Metabolism Methods Mitosis Mitotic Cell Cycle Modeling Molecular Molecular Analysis Mutation Nonhomologous DNA End Joining Pharmacologic Substance Ploidies Population Positioning Attribute Prevalence Process Property Pulsed-Field Gel Electrophoresis Radiation Regulation Reporter Resistance Role Saccharomyces cerevisiae Screening procedure Site Source Staging Stress System Yeasts autism spectrum disorder base comparative genomic hybridization design dosage high throughput screening homologous recombination human disease insight mutant prototype research study response small molecule libraries

项目摘要

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (08), "Genomics", and the specific Challenge Topic (08-ES-106), "The role of environmental exposure in copy number variation (CNV)". CNV events are deletions or duplications that are too small to be recognized by karyotypic analysis. Spontaneous CNVs have been recently recognized as a major source for sporadic disease in human populations, perhaps most notably in Autism Spectrum Disorders (ASD). The mechanisms underlying the formation of CNVs are not known, although it is likely that both environmental and intrinsic cellular factors affect the rate of CNVs. In this proposal, we describe the use of the yeast Saccharomyces cerevisiae as a model to find out how environmental contaminants and various mutations control the rate of CNVs. We will examine both large (>1kb deletions or duplications) and small (1- 1000 bp) CNV events. To investigate the effect of environmental factors and contaminants on large CNV events (Specific Aim 1), we will use a reporter in which duplications can be selected and deletions can be detected by screening. We will use a cassette that contains two yeast genes SFA1 and CUP1 that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. Using yeast strains in which this cassette is integrated into various positions, we will measure the rate of cassette amplifications in a wild-type strain growing under standard lab conditions. We will then determine the rate of amplifications in response to various DNA damaging agents, and common agrochemicals. We will also optimize a high-throughput version of the formaldehyde-copper resistance assay for use in preliminary screens of diverse chemical libraries to identify unknown environmental contaminants associated with CNV stimulation. To determine which mechanisms are involved in the amplifications (homologous recombination between dispersed repeats, microhomology- mediated recombination, non-homologous end joining or aneuploidy), we will characterize the strains with CNV events using DNA microarrays and a gel system that separates intact chromosomal DNAs (CHEF gels). In Specific Aim 2, we will conduct an investigation of cellular mechanisms linked to CNV formation, focusing on spontaneous events and those stimulated by low-dose ionizing radiation. We will determine the rates of CNV and the types of alterations in cells exposed to radiation at different stages of the cell cycle. In addition, we will examine spontaneous CNVs in meiotic cells. A set of mutant strains with compromised DNA repair and replication will also be examined to characterize the genetic control of CNV formation. In Specific Aim 3, we will investigate a different class of CNV events, those that duplicate or delete less than 1000 bp. For this analysis, we will use high-throughput DNA sequencing of sub-cultured untreated yeast strains and of strains treated with low levels of radiation. The analysis should provide much needed insight into the prevalence of this class of CNV, as well as into the mechanism of its formation. In this proposal, we examine the effects of environmental stresses and alterations of the genetic background on the frequency of de novo copy number variation (deletions and duplications of DNA sequences) in yeast. During the past few years, it has become increasingly clear that copy number variation (CNV) is associated with many human diseases. Thus, factors that elevate the rate of CNV are likely to elevate the frequency of those diseases.

描述（由申请人提供）：该应用程序解决了广泛的挑战领域（08）“基因组学”和特定的挑战主题（08-ES-106）“环境暴露在拷贝数变异（CNV）中的作用”。 CNV 事件是指太小而无法被核型分析识别的缺失或重复。自发性 CNV 最近被认为是人群散发性疾病的主要来源，其中最引人注目的可能是自闭症谱系障碍 (ASD)。尽管环境和内在细胞因素可能都会影响 CNV 的发生率，但 CNV 形成的机制尚不清楚。在本提案中，我们描述了使用酿酒酵母作为模型来找出环境污染物和各种突变如何控制 CNV 的发生率。我们将检查大型（>1kb 删除或重复）和小型（1-1000 bp）CNV 事件。为了研究环境因素和污染物对大型 CNV 事件的影响（具体目标 1），我们将使用一种报告基因，可以通过筛选选择重复并检测缺失。我们将使用包含两个酵母基因 SFA1 和 CUP1 的盒，这两个基因分别赋予基因对甲醛和铜的剂量依赖性耐受性。使用将该盒整合到各个位置的酵母菌株，我们将测量在标准实验室条件下生长的野生型菌株的盒扩增率。然后我们将确定对各种 DNA 损伤剂和常见农用化学品的反应扩增率。我们还将优化甲醛-铜抗性测定的高通量版本，用于多种化学库的初步筛选，以识别与 CNV 刺激相关的未知环境污染物。为了确定扩增涉及哪些机制（分散重复之间的同源重组、微同源介导的重组、非同源末端连接或非整倍性），我们将使用 DNA 微阵列和分离完整染色体 DNA 的凝胶系统来表征具有 CNV 事件的菌株（CHEF 凝胶）。在具体目标 2 中，我们将研究与 CNV 形成相关的细胞机制，重点关注自发事件和低剂量电离辐射刺激的事件。我们将确定细胞周期不同阶段暴露于辐射的细胞的 CNV 率和变化类型。此外，我们将检查减数分裂细胞中的自发 CNV。还将检查一组 DNA 修复和复制受损的突变株，以表征 CNV 形成的遗传控制。在具体目标 3 中，我们将研究不同类别的 CNV 事件，即那些复制或删除少于 1000 bp 的事件。对于此分析，我们将对传代培养的未经处理的酵母菌株和经过低水平辐射处理的菌株进行高通量 DNA 测序。该分析应该能够深入了解此类 CNV 的流行情况及其形成机制。在本提案中，我们研究了环境压力和遗传背景的改变对酵母中从头拷贝数变异（DNA 序列的删除和重复）频率的影响。在过去的几年中，人们越来越清楚地发现拷贝数变异（CNV）与许多人类疾病有关。因此，提高 CNV 发生率的因素可能会提高这些疾病的发生率。