Genome-wide quantitative genetic analysis of growth and starvation survival

描述(由申请人提供)： 阻止生长和饥饿生存的能力是与癌症、糖尿病和衰老相关的基本特征。生长和生育所需的后生动物基因已被全面识别，但已知的饥饿生存所必需的基因相对较少，而且只有少数几个基因能增加存活率。目前还不清楚哪些途径是中心的，哪些是缺失的，以及这些途径是如何整合起来的，以产生对养分可获得性的协调反应。该项目的长期目标是确定作为数量性状的抗饥性的遗传结构。这包括全面识别影响饥饿存活的基因以及它们的成对上位性相互作用。然而，现有的后生动物遗传分析技术相对费力且不是定量的，上位性分析方法通常侧重于个体遗传交互作用。我们正在开发一种大规模并行的方法来测量基因组中每个基因对线虫感兴趣的表型的功能贡献。其基本原理是使用转座子对大量群体进行插入突变，针对感兴趣的表型(饥饿存活)进行选择，并使用深层序列转座子侧翼来测量等位基因频率。线虫是这个项目的理想选择，因为线虫能够适应大餐和饥荒的循环，有丰富的遗传工具包可以用于这种创新方法，基因组紧凑，可以培养极大的种群，并且已经全面确定了必要的基因，便于验证。我们使用的Mos1转座子是理想的，因为它在没有转座酶的情况下是稳定的，它的突变率自然很低，并且是可优化的，并且经典的正向遗传方案已经到位。使用基于模型的模拟的初步结果表明，我们将能够在一次试验中对几乎每个基因进行多次突变，并有能力检测出种群之间等位基因频率的微小差异。这项建议的目标是开发和验证这一方法，并全面识别增强或降低抗饥性的基因。我们假设，许多在传统筛选中没有明显表型的基因将是饥饿生存所必需的，但相对较少的基因将提高存活率。我们将通过以下三个具体目标来实现我们的目标：1)在线虫中建立转座子介导的遗传学(Gen-Seq)，2)通过全面鉴定必需基因来验证Gen-Seq，3)全面鉴定改变抗饥性的非必需基因。我们给出了初步的建模结果和协议设计，以支持可行性。这项提议对于利用下一代测序和模型系统遗传学的力量来开发一种将极大地加快基因分析的方法具有创新性。这一结果将是重要的，因为饥饿生存的遗传基础在后生动物中尚未确定，尽管这是一个与巨大的疾病相关的基本特征。未来的工作将利用Gen-Seq在全基因组范围内鉴定抗饥性的遗传相互作用。

DESCRIPTION (provided by applicant): The ability to arrest growth and survive starvation is a fundamental trait relevant to cancer, diabetes and aging. Metazoan genes required for growth and fertility have been comprehensively identified, but relatively few genes essential to starvation survival are known, and only a handful that increase survival are known. It is un- clear which pathways are central, which are missing, and how these pathways are integrated to produce coordinated responses to nutrient availability. The long-term goal of this project is to determine the genetic architecture f starvation resistance as a quantitative trait. This includes comprehensive identification of genes that influence starvation survival as well as their pair-wise epistatic interactions. However, existing technologies for metazoan genetic analysis are relatively laborious and not quantitative, and approaches to epistasis analysis generally focus on individual genetic interactions. We are developing a massively parallel method to measure the functional contribution of each gene in the genome to a phenotype of interest in C. elegans. The rationale is to use a transposon for insertional mutagenesis of a large population, select for the phenotype of interest (starvation survival), and deep sequence transposon flanks to measure allele frequencies. C. elegans is ideal for this project since nematodes are adapted to survive cycles of feast and famine, there is a rich genetic toolkit that can be leveraged for this innovative approach, the genome is compact, extremely large populations can be cultured, and essential genes have been comprehensively identified facilitating validation. The Mos1 transposon we are using is ideal in that it is stable n the absence of transposase, its mutation rate is naturally low and optimizable and classic forward genetic protocols are in place. Preliminary results using model-based simulation suggest we will be able to mutate almost every gene multiple times in a single trial and have the power to detect small differences in allele frequency between populations. The objectives of this proposal are to develop and validate this approach and to comprehensively identify genes that increase or decrease starvation resistance. We hypothesize that many genes with no apparent phenotype in traditional screens will be essential to survive starvation but that relatively few genes will increase survival. We will accomplish our objectives with the following three specific aims: 1) Develop transposon-mediated genetics by sequencing (Gen-Seq) in C. elegans, 2) Validate Gen-Seq by comprehensively identifying essential genes and 3) Comprehensively identify non-essential genes that modify starvation resistance. We present preliminary modeling results and protocol designs in support of feasibility. This proposal is innovative for leveraging the power of next-generation sequencing and model system genetics to develop a methodology that will greatly expedite genetic analysis. The results will be significant since the genetic basi of starvation survival has not been determined in a metazoan, though it is a fundamental trait with tremendous disease relevance. Future work will take advantage of Gen- Seq for genome-wide identification of genetic interactions underlying starvation resistance.