GENETIC ANALYSIS OF CHEMOSENSATION IN C ELEGANS

这项正在进行的研究项目的广泛目标是使用线虫达尔形成作为一种遗传上易于处理的化学感觉模型。达尔幼虫是一个可有可无的替代第三幼虫阶段，其形成受到化学感官信号和营养状态的共同调节。以前的工作定义了大量调控Dauer形成的Daf-C(Dauer形成构成基因和Daf-d(缺陷)基因)，并定义了它们参与一系列复杂的遗传途径。这些途径在分子和细胞上是不同的。化学感觉神经元使用cGMP介导的初级感觉转导过程来控制Dauer的形成，部分是通过释放一种与转化生长因子相关的配体。下游步骤包括转化生长因子反应通路和与感觉通路整合的胰岛素信号通路。很可能这些通路的主要DAF-12核激素受体的配基启动DAF-12核激素受体的DAF-12配基，启动DAF-12的DAF-12核激素受体的配基，启动DAER分化。我们对达尔形成的遗传方法引导我们研究这一途径的不同元素。在这里，我们建议对我们已经鉴定的一组新的daf基因进行遗传分析，其中大多数似乎在胰岛素信号通路中发挥作用，并对其中一些基因进行分子研究。类似地，我们将分析我们新发现的两个基因，它们可能作用于转化生长因子-β途径的下游步骤。Dauer途径胰岛素信号分支中已知的最后一步是有翼螺旋转录因子daf-16。在两个目标中，我们将研究作用于daf-16附近或下游的基因。在遗传学方法中，我们将分离蛋白激酶PDK-1的Daf-c等位基因的抑制者，该等位基因被认为作用于daf-16的上游。在生化方法中，我们将结合结合位点选择、基因组搜索和基于DNA阵列的转录分析来直接搜索daf-16的转录靶标。最后，我们最近发现，daf-19，一个长期以来被认为调节感觉神经元发育的基因，编码了一种RFX型转录因子，控制着组成核心感觉纤毛的蛋白质的表达。我们将结合基因组搜索、转基因表达测试和DNA阵列分析来进一步研究daf-19的作用。这里研究的所有途径都对应于人类的重要调控途径，其中许多基因在各种遗传性疾病中受到影响。

The broad objective of this ongoing research project is to use C. elegans dauer formation as a genetically tractable model for chemosensation. The dauer larva is a dispensable alternative third larval stage whose formation is regulated by a combination of chemosensory signals and nutritional status. Previous work defined a large number of Daf-C (dauer formation constitutive and Daf-d (defective) genes that regulate dauer formation, and defined their participation in a complex set of genetic pathways. These pathways are molecularly and cellularly diverse. Chemosensory neurons use a cGMP mediated primary sensory transduction process to control dauer formation, partly through release of a TGF-related ligand. Downstream steps include the TGF-response pathway and an insulin- signaling pathway that integrates with the sensory pathways. It is likely that the main dauer-regulating output of these pathways is the ligand for the orphan daf-12 nuclear hormone receptor, which initiates dauer- regulating output of these pathways is the ligand for the orphan daf-12 nuclear hormone receptor, which initiates dauer differentiation. Our genetic approach to dauer formation has lead us to study diverse elements of this pathway. Here, we propose to genetically analyze a set of new daf genes that we have identified, most of which appear to function in the insulin-signaling pathway, and to molecularly study a select few of these genes. We will similarly analyze two genes that we have newly identified that may act at a downstream step in the TGF-beta pathway. The last known step in the insulin-signaling branch of the dauer pathway is the winged-helix transcription factor daf-16. In two aims, we will study genes that act close to or downstream of daf-16. In a genetic approach, we will isolate suppressors of a Daf-c allele of the protein kinase pdk-1, thought to act just upstream of daf-16. In a biochemical approach, we will use a combination of binding site selection, genome searches, and DNA-array based transcript analysis to search directly for transcriptional targets for daf-16. Finally, we have recently found that daf-19, a gene long thought to regulate sensory neuron development, encodes an RFX-type transcription factor that controls expression of proteins that comprise the core sensory cilium. We will use a combination of genome searches, transgenic expression tests, and DNA-array analysis to further investigate the role of daf-19. All of the pathways under study here correspond to important regulatory pathways in humans, and many of the genes are affected in various inherited disorders.