Volatility of mutator phenotypes at single cell resolution

单细胞分辨率下突变表型的波动性

• 批准号: 9295042
• 负责人: ALAN J HERR
• 金额: $ 30.9万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-13 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9295042
• 关键词: Adopted; Affect; Agar; Alleles; Candidate Disease Gene; Cell Count; Cell Lineage; Cell division; Cells; Clone Cells; DNA biosynthesis; Data; Daughter; Defect; Diploidy; Drug resistance; Engineering; Evolution; Exhibits; Future; Genes; Genome; Growth; Haploidy; Human; Individual; Inherited; Lead; Malignant Neoplasms; Mammalian Cell; Maps; Measurement; Measures; Methods; Microbe; Micromanipulation; Modeling; Monitor; Mothers; Mutagenesis; Mutation; Mutation Fixation; Neoplastic Cell Transformation; Phenotype; Phosphotransferases; Poisson Distribution; Polymerase; Population; Process; Publications; Publishing; Regulation; Replication Error; Resolution; S Phase; Saccharomyces cerevisiae; Saccharomycetales; Source; Surface; Technology; Testing; Therapeutic; Work; Yeasts; cancer cell; daughter cell; experimental study; human disease; microbial; mutant; next generation sequencing; public health relevance; sample fixation; segregation; whole genome

 DESCRIPTION (provided by applicant): Elevated mutation rates caused by mutator phenotypes can fuel microbial evolution and neoplastic transformation of mammalian cells. New mutations are assumed to arise at a constant rate in each cell division and segregate equally between the resulting cells. If one or both assumptions are false, an expanding mutator population may contain subclones with widely divergent rates of evolution. Testing these assumptions has not been possible in the past because most mutation rate measurements rely on scoring rare mutations in a small target, which requires large populations of cells. Next Generation Sequencing (NGS) technologies, which permit an entire genome to be used to score mutations, allows us to test the fundamental assumptions about mutation rates by sequencing clones derived from sequential cell divisions. Our strategy is to isolate single-cell lineages fro mutator "mother" yeast cells, including all daughter cells, the first two grand-daughters born to each daughter, and the first great-granddaughter born to the first grand-daughter. We will grow all isolated cells into clones and then sequence the genomes using NGS. From these data, we will determine the fidelity of individual replication cycles and whether replication errors segregate evenly. In work published this year, we monitored the fixation of mutations in just haploid mutator mother cells and found evidence for distinct mutagenic states. Our first specific aim is to monitor the fixation of all replication errors in both daughter and mother cells. We will use the same haploid strain as well as several diploid mutator strains with higher mutation rates. In another publication this year we found mutations that perturb dNTP levels modulate mutator phenotypes. Thus, in our second specific aim, we will investigate whether mutator volatility is due to dNTP pool regulation. Our third specific aim seeks to identify additional factors that may influence mutator volatility through the isolation of antimutator mutations. This proposal will lea to future studies to investigate whether mammalian mutator cells exhibit distinct mutagenic states and whether factors that influence mutator volatility may be used to modulate mutator phenotypes for therapy.

 描述（由申请人提供）：增变基因表型引起的突变率升高可促进哺乳动物细胞的微生物进化和肿瘤转化。假设新的突变在每次细胞分裂中以恒定的速率出现，并且在所产生的细胞之间均等地分离。如果一个或两个假设是错误的，一个不断扩大的增变基因群体可能包含进化速度差异很大的亚克隆。在过去，测试这些假设是不可能的，因为大多数突变率测量依赖于对小目标中的罕见突变进行评分，这需要大量的细胞。下一代测序（NGS）技术允许使用整个基因组来对突变进行评分，使我们能够通过对来自连续细胞分裂的克隆进行测序来测试关于突变率的基本假设。 我们的策略是从突变体“母亲”酵母细胞中分离出单细胞谱系，包括所有的子细胞，每个子细胞所生的前两个孙女，以及第一个孙女所生的第一个曾孙女。我们将所有分离的细胞培养成克隆，然后使用NGS对基因组进行测序。从这些数据中，我们将确定单个复制周期的保真度以及复制错误是否均匀分离。在今年发表的工作中，我们监测了仅在单倍体突变母细胞中的突变固定，并发现了不同致突变状态的证据。我们的第一个具体目标是监测子细胞和母细胞中所有复制错误的固定。我们将 使用同一个单倍体菌株以及几个突变率较高的二倍体突变菌株。在今年的另一篇文章中，我们发现了干扰dNTP水平的突变可以调节增变因子的表型。因此，在我们的第二个具体目标中，我们将研究增变因子的波动是否是由于dNTP池调节。我们的第三个具体目标是通过分离抗突变子突变来确定可能影响突变子波动性的其他因素。这一建议将莱亚未来的研究，以调查是否哺乳动物增变细胞表现出不同的致突变状态，以及是否影响增变波动的因素，可用于调节增变表型的治疗。