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 有丝分裂重组导致的杂合性丢失（洛）是无性二倍体不可避免的结果 繁殖，它已被普遍观察到的实验和自然种群的克隆 单细胞病原体然而，目前还不清楚这些复制错误是否经常成为适应性疾病的来源。 变化量特别是，由于没有估计的分布的健身效果（DFE）的自发 洛缺失事件，尚不清楚群体水平的频繁洛缺失是否表明基因型 有益的、有害的或中性的变化。该提案的目标是直接解决 洛缺失促进单细胞二倍体病原体快速适应的潜力和局限性， 估计洛缺失事件的适合度和多效性。因为洛缺失是通过多种 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洛缺失事件与拮抗性 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 由于对有丝分裂的丰富知识， 重组和基因功能的物种和大量的遗传工具可用。该物种是一个 机会致病菌在临床上越来越重要，可以使用简单的无脊椎动物进行研究 感染的模型。为了估计洛缺失事件的DFE，将随机整合转座子盒。 用于触发整个基因组的双链DNA断裂，刺激有丝分裂修复， 重组和洛缺失。在使用基因组重测序表征洛缺失事件后， 所得转化体将使用竞争测定法进行评估。这些试验将分两次进行 动物模型（蜡虫和线虫）和多重应激和化学变化的纯培养物 环境，并通过与完全杂合的基因组相比，特定的洛合事件的健身效果 估算这些数据将是任何物种中洛缺失的第一个稳健的DFE，允许第一近似值。 潜在的洛缺失驱动进化。本实验还将通过检测来探讨洛缺失的适应限度 不同环境中碱基对洛缺失大小与适合度效应关系 多效性在动物模型和应激营养条件下， 揭示了S.酿酒酵母和相关真菌。成功实施，将 这是对洛缺失对无性二倍体快速进化的影响的最详细的观察， 在临床上的重要性。