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

描述(申请人提供)：在细胞的生命周期中，复制和环境压力会造成损害，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揭示慢性疾病，如髓系癌和血管疾病，这些疾病分别由造血干细胞老化和内皮细胞老化引起。环境因素对寿命有重大影响，而最有文献记载的延长寿命的扰动之一是卡路里限制(CR)。酵母、蠕虫、苍蝇、小鼠和大鼠在用CR饲料饲养时都活得更长，这是一种至今尚不清楚的机制。到目前为止，对CR反应决定因素的基因组规模筛选还不是很容易；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，使用野生酵母分离株作为模型。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，包括一株在CR条件下寿命明显较长的野生菌株。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。我将通过以下具体目标来验证这一假设：目标1-我将绘制在CR条件下酵母菌株之间寿命差异的遗传基础图。使用一组来自两个野生酵母分离株之间杂交的基因分型后代，我将用成熟的微流控技术来分析每一个的寿命。统计遗传分析将识别与寿命性状可重复分离的基因和多态，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如何调节细胞的增殖和适合性，我将测量目标1中使用的杂交后代在CR条件下的生长特性，并通过统计遗传学方法绘制与生长行为相关联的遗传基因座。对这些屏幕点击的分子验证将建立在细胞水平上调节CR感觉和反应的基因。与目标1的结果进行比较，将能够测试在CR下的寿命和生长在多大程度上受到共同的基因控制。目标3-在系统水平上调查在CR下生长的细胞的状态，我将 从目标1中使用的杂交中对每个菌株进行转录分析。我将绘制与对CR的表达反应共同遗传的序列变体，并在分子实验中验证这些基因座。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，与前两个目标相吻合，并允许对CR期间的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.