Genetic Analysis of C. elegans fat regulatory network

描述(由申请人提供)： 越来越多的证据表明，脂肪调节涉及神经系统中的摄食调节中心与储存脂肪的调节运输、分割、储存和利用之间的复杂相互作用。据认为，这种动态平衡系统的进化是为了通过维持能量储备来确保生存。然而，在营养供应充足的时候，这种动态平衡系统可能会导致有维持大量能量储备倾向的个人肥胖。肥胖与世界范围内的主要健康流行病有关。线虫为鉴定和分析脂肪调节基因提供了机会。重要的是，这些分析是在自我平衡网络的背景下在完整的动物身上进行的。此前，全基因组RNAi方法确定了400多个基因，当被灭活时，会影响线虫体内的脂肪含量。这些包括神经内分泌信号、转录和新陈代谢的组成部分。这些发现突出了线虫和哺乳动物脂肪调节的共同祖先，并确定了许多新的途径。基于上位性分析和哺乳动物同源搜索，已经确定了120个优先候选基因。为了了解这些基因在完整生物体中的功能和调控模式，i)将确定暴露于RNAi的动物的摄食和运动速度，ii)将使用定量RT-PCR方法来监测暴露于RNAi的动物中一组脂肪酸、甾醇和糖代谢酶的表达水平，iii)将被发现影响类似过程的基因将被归类到遗传途径中，iv)脂肪基因及其产物的功能位点将通过GFP标签融合来确定，iv)这些GFP-Tag报告和RT-PCR将被用于确定影响其他脂肪调控基因表达水平和/或定位的脂肪基因失活。同样，将评估饥饿或胰岛素和5-羟色胺信号通路缺陷对脂肪调节基因的影响。

DESCRIPTION (provided by applicant): There is mounting evidence that fat regulation involves a complex interplay between the feeding regulatory centers in the nervous system and the regulated transport, partitioning, storage, and utilization of stored fat. It is thought that this homeostatic system has evolved to ensure survival by maintaining energy reservoirs. However, in times of plentiful nutrient supplies, this homeostatic system can cause obesity in individuals with propensity for maintaining large energy stores. Obesity is associated with major heath epidemics worldwide. C. elegans provides an opportunity to identify and analyze fat regulatory genes. Importantly, these analyses are conducted in intact animals in the context of the homeostatic network. Previously, genome-wide RNAi approaches identified over 400 genes that, when inactivated, affect body fat content in C. elegans. These include components of neuroendocrine signaling, transcription, and metabolism. These findings highlight the shared ancestry of C. elegans and mammalian fat regulation and identify many novel pathways. Based on epistasis analyses and mammalian homology searches, 120 priority candidates have been identified. To understand the modes of function and regulation of these genes in intact organisms, i) feeding and locomotary rates of RNAi exposed animals will be determined, ii) quantitative RT-PCR assays will be used to monitor expression levels of a panel of fatty acid, sterol, and sugar metabolic enzymes in RNAi exposed animals, iii) genes identified to affect similar processes will be grouped into genetic pathways, iv) sites of function of fat genes and their products will be determined by GFP tag fusions, iv) these GFP-tag reporters and RT-PCR assays will be used to identify the fat gene inactivations that affect expression levels and/or localizations of the other fat regulatory genes. Similarly, impact of starvation or defects in insulin and serotonin signaling pathways on the fat regulatory genes will be assessed.