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

分离致病酵母模型系统中个体杂合性丢失事件的表型效应

• 批准号: 10621822
• 负责人: Tim James
• 金额: $ 19.11万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-13 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10621822
• 关键词: Address; Animal Model; Automobile Driving; Bar Codes; Base Pairing; Biological Assay; Biological Models; Biology; Candida albicans; Chemicals; Clinic; Clonal Evolution; Costs and Benefits; Cryptococcus neoformans; DNA Double Strand Break; DNA Resequencing; DNA lesion; Data; Diploidy; Elements; Environment; Eukaryota; Event; Evolution; Frequencies; Funding Mechanisms; Fungal Drug Resistance; Future; Gene Conversion; Genes; Genetic; Genetic Crossing Over; Genetic Nondisjunction; Genetic Recombination; Genome; Genomics; Genotype; Goals; Heterozygote; Hybrids; In Vitro; Individual; Infection; Invertebrates; Knowledge; Larva; Lead; Libraries; Link; Loss of Heterozygosity; Malignant Neoplasms; Measurement; Measures; Meiosis; Mitosis; Mitotic; Mitotic Recombination; Modeling; Mutagenesis; Mutation; Nature; Nematoda; Nutrient; Organism; Outcome; Pathogenicity; Phenotype; Play; Population; Replication Error; Reproduction; Rewards; Role; Saccharomyces cerevisiae; Serial Passage; Source; Study models; Testing; Variant; Virulence; Work; Yeast Model System; Yeasts; asexual; base; ds-DNA; experimental study; fitness; fungus; gene function; genome sequencing; genome-wide; high risk; in vivo; insight; opportunistic pathogen; pathogen; pathogenic fungus; pleiotropism; repaired; tool; trait; yeast genetics

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.

项目摘要 有丝分裂重组失去杂合性（LOH）是无性二倍体的必然结果 繁殖，并且在克隆的实验和自然种群中已无处不在 单细胞病原体。但是，目前尚不清楚这些复制错误的频率是适应性的来源 变化。特别是因为没有自发的健身效应分布（DFE）的估计值 LOH事件，尚不清楚频繁的LOH的种群水平观察是否指示基因型 有益，有害或中立的变化。该提议的目的是直接解决 LOH的潜力和局限 估计LOH事件的适应性和多效性。因为loh是通过多样性发生的 机制，有些是精确的，例如基因转化，而其他机制则不精确，例如 非分离，一个中心假设是长途LOH事件与拮抗作用有关 限制对不同环境和宿主的适应性的多效性。酿酒酵母是理想的 研究LOH进化影响的模型，因为有丝分裂知识的丰富 可用的物种和大量遗传工具中的重组和基因功能。该物种是 可以使用简单无脊椎动物研究的诊所中提高显着性的机会性病原体 感染模型。为了估计LOH事件的DFE，将随机整合的转座盒将是 用于在整个基因组中触发双链DNA断裂，从而通过有丝分裂刺激修复 重组和LOH。使用基因组重新方程来表征LOH事件后， 将使用竞争测定法估算产生的转化体。这些测定将分为两次 动物模型（Waxworm和线虫）和多种压力和化学变化的纯培养物 环境以及与完全杂合基因组相比，特定LOH事件的适应性效应 估计的。这些数据将是任何物种中LOH的第一个强大DFE，允许第一个近似 LOH推动进化的潜力。该实验还将通过测试探索LOH适应的极限 基本对中的LOH大小与环境之间的适应性效应之间的关系以检测拮抗作用 多效性。动物模型之间的正相关效应和压力性营养条件的正相关效应将 告知酿酒酵母和相关真菌中致病性的遗传基础。成功实施，这将 最详细地了解LOH对无性二倍体快速发展的影响，这是一组增加 在诊所的重要性。