Isolating the phenotypic effects of individual loss of heterozygosity events in a pathogenic yeast model system

项目总结 有丝分裂重组导致的杂合性缺失是无性二倍体的必然结果 在克隆的实验种群和自然种群中已经普遍观察到 单细胞病原体。然而，目前还不清楚这些复制错误作为适应性的来源的频率有多高 变种。特别是，因为没有对自发健康效应(DFE)的分布进行估计 LOH事件，尚不清楚频繁LOH的人群水平观察是否表明存在基因型别 有益、有害或中性的变化。这项提案的目标是直接解决 LOH通过以下途径促进单细胞二倍体病原体快速适应的潜力和局限性 评估LOH事件的适合度和多效性。因为LOH是通过一系列不同的 机制，其中一些是精确的，如基因转换，而另一些是不精确的，如 不分离，一个中心假设是长距离杂合事件与拮抗有关。 多效性，限制了对不同环境和宿主的适应性。酿酒酵母是理想的 基于丰富的有丝分裂知识研究杂合性缺失的进化影响模型 物种中的重组和基因功能，以及可用的过多遗传工具。该物种是一种 临床上越来越重要的可用简单无脊椎动物研究的条件致病菌 感染的模型。为了估计LOH事件的DFE，一个随机整合的转座子盒将是 用于在整个基因组中触发双链DNA断裂，从而刺激有丝分裂的修复 重组和LOH。在使用基因组重测序来表征LOH事件之后， 所产生的转化子将使用竞争分析进行评估。这些化验将分两次进行 动物模型(蜡虫和线虫)和多重应激和化学变化的纯培养 环境，以及通过与完全杂合性基因组的比较，特定杂合性缺失事件的适合性影响 估计。这些数据将是任何物种中第一个稳健的杂合性缺失的DFE，允许一级近似 LOH有可能推动进化。实验还将通过测试来探索陆恭蕙的适应限度。 检测拮抗剂时碱基对LOH大小与不同环境适应性的关系 多效性。动物模型和紧张的营养条件之间的健身效果呈正相关 告知酿酒酵母和相关真菌致病的遗传基础。如果成功实施，这将 最详细地观察杂合性缺失对无性二倍体快速进化的影响，一组不断增加的 在临床上的重要性。

PROJECT SUMMARY Loss of heterozygosity (LOH) by mitotic recombination is an inevitable outcome of asexual diploid reproduction, and it has been ubiquitously observed in experimental and natural populations of clonal unicellular pathogens. Yet, it is unclear how often these replication errors serve as a source of adaptive variation. In particular, because there is no estimate of the distribution of fitness effects (DFE) of spontaneous LOH events, it is unclear whether the population-level observations of frequent LOH indicate genotypic changes that were beneficial, deleterious, or neutral. The goal of this proposal is to directly address the potential and limitations of LOH to facilitate rapid adaptation of unicellular diploid pathogens by estimating the fitness and pleiotropy of LOH events. Because LOH occurs through a diversity of mechanisms, some of which are precise, such as gene conversion, while others are imprecise, such as nondisjunction, a central hypothesis is that long-distance LOH events are associated with antagonistic pleiotropy that limits adaptability to diverse environments and hosts. Saccharomyces cerevisiae is the ideal model for the study of the evolutionary impacts of LOH because of the wealth of knowledge on mitotic recombination and gene function in the species and the plethora of genetic tools available. The species is an opportunistic pathogen of increasing significance in the clinic that can be studied using simple invertebrate models of infection. To estimate the DFE of LOH events, a randomly-integrated transposon cassette will be used to trigger double strand DNA breaks throughout the genome that stimulate repair by mitotic recombination and LOH. After LOH events are characterized using genome resequencing, the fitness of the resulting transformants will be estimated using competition assays. These assays will be conducted in two animal models (waxworm and nematode) and multiple stressful and chemically varying pure culture environments, and by comparison to fully heterozygous genomes the fitness effect of specific LOH events estimated. These data will be the first robust DFE of LOH in any species, allowing a first approximation of the potential for LOH to drive evolution. The experiment will also explore the limits of adaptation by LOH by testing the relationship between LOH size in base pairs and fitness effect across environments to detect antagonistic pleiotropy. Positively correlated fitness effects across animal models and stressful nutrient conditions will inform the genetic bases of pathogenicity in S. cerevisiae and related fungi. Successfully implemented, this will be the most detailed look at the impact of LOH on rapid evolution of asexual diploids, a group of increasing importance in the clinic.