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

描述（由申请人提供）：细胞一生中持续的复制和环境压力会导致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一个主要问题。对细胞衰老的全面了解将有助于研究骨髓癌和血管疾病等慢性疾病，它们分别是由造血干细胞衰老和内皮细胞衰老引起的。环境因素对寿命有显著的影响，其中一个记录最充分的延长寿命的扰动是热量限制（CR）。酵母、蠕虫、苍蝇、小鼠和大鼠在用CR饮食喂养时都能活得更长，其机制尚不清楚。迄今为止，对CR反应决定因素的基因组级筛选还不容易处理；我建议利用野生酵母分离物作为模型，利用CR反应中的自然遗传变异来应对这一挑战。我的初步研究显示，在CR期间，酵母菌株之间的寿命发生了强劲的变化，包括在CR条件下具有明显较长寿命的野生分离株。我假设野生酵母在CR条件下的寿命延长、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。我将以以下具体目标来检验这一假设：目标1-我将绘制CR条件下酵母菌株寿命差异的遗传基础。使用两种野生酵母菌分离株杂交的基因分型后代，我将用已建立的微流体技术测定每种酵母菌的寿命。统计遗传学分析将识别与寿命特征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。目标2-为了了解CR如何调节细胞增殖和适应性，我将测量目标1中使用的杂交后代在CR条件下的生长性状，并将通过统计遗传学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在细胞水平上建立介导对CR的感知和反应的基因。与Aim 1的结果进行比较，可以测试CR下的寿命和生长受共同遗传控制的程度。目的3-研究在CR下生长的细胞在系统水平上的状态

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.