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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要有丝分裂重组导致的杂合性丢失（LOH）是无性二倍体不可避免的结果繁殖，并且在克隆的实验和自然群体中普遍观察到单细胞病原体。然而，尚不清楚这些复制错误多久会成为适应性的来源。变化。特别是，因为没有对自发的适应度效应（DFE）的分布进行估计 LOH 事件，尚不清楚频繁 LOH 的群体水平观察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 LOH促进单细胞二倍体病原体快速适应的潜力和局限性估计 LOH 事件的适应性和多效性。因为 LOH 通过多种方式发生机制，其中一些是精确的，例如基因转换，而另一些则是不精确的，例如不分离，一个中心假设是长距离 LOH 事件与拮抗相关多效性限制了对不同环境和宿主的适应性。酿酒酵母是理想的由于有丝分裂方面的知识丰富，用于研究 LOH 的进化影响的模型物种中的重组和基因功能以及大量可用的遗传工具。该物种是一个在临床中具有越来越重要意义的机会性病原体，可以使用简单的无脊椎动物进行研究感染模型。为了估计 LOH 事件的 DFE，将随机集成转座子盒用于触发整个基因组的双链 DNA 断裂，从而刺激有丝分裂修复重组和LOH。使用基因组重测序对 LOH 事件进行表征后，将使用竞争测定来估计所得转化体。这些检测将分两次进行动物模型（蜡虫和线虫）和多种应激和化学变化的纯培养物环境，并通过与完全杂合基因组进行比较，了解特定 LOH 事件的适应性效应估计的。这些数据将是任何物种中 LOH 的第一个稳健的 DFE，从而可以对 LOH 驱动进化的潜力。该实验还将通过测试来探索LOH的适应极限碱基对中的 LOH 大小与跨环境的适应度效果之间的关系以检测拮抗多效性。动物模型和应激营养条件之间的健康效应呈正相关了解酿酒酵母和相关真菌致病性的遗传基础。成功实施后，这将最详细地研究了 LOH 对无性二倍体快速进化的影响，无性二倍体是一组不断增加的在临床中的重要性。