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.
Disruption of pathways that buffer fluctuations in nutrient availability leads to diabetes, which affects approximately 11% of American adults. Cancer can also result when pathways that keep growth in check during nutrient limitation are disrupted. By determining the genetic basis of starvation resistance, this project will increase fundamental understanding of such pathways, contributing to novel diagnostics, preventative strategies and therapeutic targets.
