GENETIC DISSECTION OF NATURAL VARIATION IN CALORIC RESTRICTION-INDUCED CELLULAR L

描述（由申请人提供）：在细胞的生命周期中持续的复制和环境压力会造成损伤，并最终导致不同类型的细胞死亡。了解细胞衰老的精确分子机制是现代生物学尚未解决的主要问题。对细胞衰老的全面了解将有助于阐明慢性疾病，如骨髓癌和血管疾病，这分别是造血干细胞衰老和内皮细胞衰老的结果。环境因素对寿命有显著影响，最有记录的延长寿命的干扰之一是热量限制（CR）。酵母、蠕虫、苍蝇、小鼠和大鼠在用CR饮食饲养时都活得更长，其机理仍然知之甚少。迄今为止，基因组规模的CR反应的决定因素筛选还不容易处理;我建议通过利用CR反应中的自然遗传变异来应对这一挑战，使用野生酵母菌株作为模型。我的初步研究显示，在CR期间，酵母菌株的寿命发生了显著变化，包括在CR条件下具有明显长寿命的野生分离株。我假设，在CR条件下野生酵母菌的寿命延长、生长行为和调控特征的遗传决定因素包括CR反应的新决定因素。我将测试这一假设与以下具体目标：目标1-我将映射酵母菌株之间的差异，在CR条件下的寿命的遗传基础。使用一组来自两个野生酵母分离株之间杂交的基因型后代，我将用已建立的微流控技术测定每个菌株的寿命。统计遗传学分析将确定与寿命性状可重复分离的基因和多态性，这些因素在寿命中的作用将在独立的分子遗传学实验中得到验证。目的2-为了了解CR如何调节细胞增殖和适应性，我将在CR条件下测量目标1中使用的杂交后代的生长性状，并通过植物遗传方法绘制与生长行为相关的遗传位点。这些筛选命中的分子验证将建立在细胞水平上介导CR的感测和应答的基因。与目标1的结果进行比较，将能够检验在CR下的寿命和生长在多大程度上受到共同的遗传控制。目的3-为了在系统水平上研究CR下生长的细胞的状态，我将 对来自Aim 1中使用的杂交的每个菌株进行转录谱分析。我将绘制与CR表达反应共同遗传的序列变异，并在分子实验中验证这些位点。研究结果将确定基因网络的组成部分和连接，该基因网络调节细胞状态以响应营养环境，与前两个目标相一致，并允许在CR期间对生长和寿命决定进行基因组规模的分子解剖。

DESCRIPTION (provided by applicant): Replication and environmental stresses sustained over a cell's lifetime cause damage and ultimately lead to different types of cell death. Understanding the precise molecular mechanisms by which cells age is a major unsolved problem of modern biology. A comprehensive understanding of cellular aging will shed light on chronic diseases such as myeloid cancer and vascular disease, which result from hematopoetic stem cell aging and endothelial cell aging, respectively. Environmental factors have a significant impact on longevity, and one of the best-documented lifespan-extending perturbations is caloric restriction (CR). Yeast, worms, flies, mice, and rats all live longer when reared on CR diets, via a mechanism that remains poorly understood. Genome-scale screens for determinants of the CR response have not been tractable to date; I propose to meet this challenge by harnessing natural genetic variation in the CR response, using wild yeast isolates as a model. My preliminary studies have revealed robust changes between yeast strains in lifespan during CR, including a wild isolate with a distinctly long lifespan under CR conditions. I hypothesize that th genetic determinants of the lifespan extension, growth behavior, and regulatory profile of wild yeast under CR conditions include novel determinants of the CR response. I will test this hypothesis with the following specific aims: Aim 1- I will map the genetic basis of differences between yeast strains in lifespan under CR conditions. Using a panel of genotyped progeny from a cross between two wild yeast isolates, I will assay the lifespan of each with established microfluidic techniques. Statistical-genetic analyses will identify genes and polymorphisms that reproducibly segregate with the lifespan trait, and the role of these factors in lifespan will be validated in independent molecular-genetic experiments. Aim 2- To understand how CR modulates cell proliferation and fitness, I will measure growth traits under CR conditions in the progeny from the cross used in Aim 1, and I will map genetic loci linked to growth behaviors via statistical-genetic methods. Molecular validation of these screen hits will establish genes that mediate the sensing of and response to CR at the cellular level. Comparison to the results of Aim 1 will enable a test of the degree to which lifespan and growth under CR are under shared genetic control. Aim 3- To investigate at a systems level the state of cells grown under CR, I will transcriptionally profile each strain from the cross used in Aim 1. I will map sequence variants that co-inherit with the expression response to CR, and validate these loci in molecular experiments. The results will identify components and connections of the gene network that regulates cell state in response to the nutritional environment, dovetailing with the first two Aim and allowing a genome-scale molecular dissection of growth and longevity decisions during CR.