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：
7939784
负责人：
THOMAS PETES
金额：
$ 49.3万
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/7939784
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化（CNV）中的作用”。 CNV事件是删除或重复的，它们太小，无法通过核型分析识别。最近，自发的CNV被公认为是人群中零星疾病的主要来源，也许是自闭症谱系障碍（ASD）。 CNV形成的基础机制尚不清楚，尽管环境和内在细胞因子可能都会影响CNV速率。在此提案中，我们描述了酿酒酵母的使用用作一种模型，以找出环境污染物和各种突变如何控制CNV速率。我们将检查大型（> 1KB缺失或重复）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（特定目标1），我们将使用一个记者，可以选择重复，并通过筛选检测到删除。我们将使用一个包含两个酵母基因SFA1和CUP1的盒式盒子，该盒分别赋予基因剂量依赖于甲醛和铜的耐受性。使用将此盒式集成到各种位置的酵母菌菌株，我们将测量在标准实验室条件下生长的野生型菌株中的盒式放大速率。然后，我们将确定对各种DNA损害剂和常见的农业化学物质的响应的放大速率。我们还将优化甲醛抗抗耐药性测定的高通量版本，用于用于不同化学文库的初步筛选，以识别与CNV刺激相关的未知环境污染物。为了确定哪些机制参与放大（分散重复序列之间的同源重组，微型学介导的重组，非同源末端连接或非整倍性），我们将使用DNA微阵列和凝胶系统来表征CNV事件的菌株，从而分离出完整的色体染色体染色体DNAS（cheagosomal dnas）。在特定目标2中，我们将研究与CNV形成相关的细胞机制，重点是自发事件以及低剂量电离辐射刺激的事件。我们将确定CNV的速率以及在细胞周期不同阶段暴露于辐射的细胞的变化类型。此外，我们将检查减数分裂细胞中的自发CNV。还将检查一组具有受损DNA修复和复制的突变菌株，以表征CNV形成的遗传控制。在特定的目标3中，我们将研究不同类别的CNV事件，这些事件复制或删除少于1000 bp。为了进行此分析，我们将使用亚培养的未处理酵母菌菌株的高通量DNA测序以及辐射水平较低的菌株的菌株。该分析应提供急需的洞察力，以了解这类CNV的普遍性及其形成机理。在此提案中，我们检查了遗传背景的环境应力和遗传背景的变化对酵母中从头拷贝数变化（DNA序列的缺失和重复）频率的改变。在过去的几年中，越来越明显的是，拷贝数变化（CNV）与许多人类疾病有关。因此，提高CNV速率的因素可能会提高这些疾病的频率。