Volatility of mutator phenotypes at single cell resolution

 描述(由申请人提供)：突变子表型引起的突变率升高可以促进微生物进化和哺乳动物细胞的肿瘤转化。新的突变假设在每个细胞分裂中以恒定的速度出现，并在所产生的细胞之间平均分离。如果其中一个假设或两个假设都是错误的，则不断扩大的突变子种群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在一个小目标中获得罕见的突变，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，允许我们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。我们的策略是分离突变“母”酵母细胞的单细胞谱系，包括所有的子代细胞，每个子代所生的前两个孙女，以及第一个孙女所生的第一个曾孙女。我们将把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。根据这些数据，我们将确定各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，我们监测了单倍体突变子母细胞中突变的固定，并发现了不同突变状态的证据。我们的第一个具体目标是监测子代和母体细胞中所有复制错误的固定。我们会 使用相同的单倍体菌株以及具有较高突变率的几个二倍体突变菌株。在今年的另一篇文章中，我们发现了扰乱dNTP水平的突变，这些突变调节了突变子的表型。因此，在我们的第二个特定目标中，我们将调查突变体的波动性是否源于dNTP池调控。我们的第三个具体目标是通过分离抗突变体突变来确定可能影响突变体挥发性的其他因素。这一建议将有助于未来的研究，以调查哺乳动物突变细胞是否表现出明显的诱变状态，以及影响突变子挥发性的因素是否可用于调节突变子的表型以用于治疗。

 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.