Circadian Gating of Signals by EARLY FLOWERING 3

描述（由申请人提供）：细胞保持时间超过约24小时的能力归因于称为生物钟的内源性分子振荡器，其节律在没有环境线索的情况下持续存在。生物钟在所有高等真核生物中调节一系列代谢、生理和行为活动。为了保护核心振荡器免受虚假复位信号的影响，时钟使用一种称为“门控”的机制，以相位相关的方式缓冲输入提示。拟南芥中EARLY FLOWER 3（ELF 3）功能的丧失使得核心振荡器对光信号的重置高度敏感。本研究旨在从分子水平上了解ELF 3如何调控拟南芥生物钟的光诱导。我们的假设是，ELF 3通过与其他蛋白质相互作用并改变其活性来发挥其作用。本提案的具体目的是：1）确定与光敏色素B（phyB）的物理相互作用是否对ELF 3功能至关重要。以前的工作已经证明，ELF 3直接与红光感光体phyB在体外相互作用，然而，这种相互作用仍然在很大程度上未被表征。将构建缺乏与phyB相互作用能力的ELF 3突变体，称为相互作用缺陷型ELF 3（ELFSdi），并将其引入elf 3植物中以测定突变体表型的互补。2)识别新的ELF 3相互作用伙伴。利用酵母双杂交技术筛选ELF 3的潜在分子伴侣。新的相互作用的克隆将通过免疫共沉淀试验进行确认，它们与ELF 3和核心振荡器的关系将通过分子和遗传分析在植物中进行研究。3)用抑制筛选鉴定elf 3昼夜节律表型所需的基因。筛选EMS诱变的elf 3 -1植物的时钟功能的部分或完全恢复。推定的抑制基因的表型将使用一组针对昼夜节律钟相关表型的测定来表征，并且将使用最近开发的利用高密度寡核苷酸阵列的作图方案来鉴定突变位点。生物钟调节真核生物的许多重要过程。一个有缺陷的生物钟会使人容易患上抑郁症、肥胖症、不正常的睡眠模式和某些类型的癌症，这强调了正确守时的重要性。本研究将探讨真核细胞中昼夜节律过程是如何在分子水平上调节的。

DESCRIPTION (provided by applicant): The ability of cells to keep time over approximately 24 hours owes to an endogenous molecular oscillator known as the circadian clock, whose rhythms persist in the absence of environmental cues. The circadian clock regulates a range of metabolic, physiological, and behavioral activities in all higher eukaryotes. To protect the core oscillator from spurious resetting signals, the clock uses a mechanism called "gating" to buffer input cues in a phase-dependent manner. Loss of EARLY FLOWERING 3 (ELF3) function in Arabidopsis thaliana renders the core oscillator hypersensitive to resetting light signals. This investigation aims understand at the molecular level how ELF3 gates light entrainment of the Arabidopsis circadian clock. Our hypothesis is that ELF3 exerts its effects by interacting with other proteins and altering their activity. The specific aims of this proposal are to: 1) Determine whether physical interaction with phytochrome B (phyB) is critical to ELF3 function. Previous work has demonstrated that ELF3 directly interacts with the red light photoreceptor phyB in vitro, however, this interaction remains largely uncharacterized. Mutants in ELF3 lacking the capacity to interact with phyB, called interaction defective ELF3 (ELFSdi), will be constructed and introduced into elf3 plants to assay for complementation of the mutant phenotype. 2) Identify novel ELF3 interacting partners. Potential molecular partners of ELF3 will be identified using yeast two-hybrid by screening. Novel interacting clones will be confirmed by co-immunoprecipitation assays, their relationships to ELF3 and the core oscillator will be investigated in plants by molecular and genetic analyses. 3) Identify genes required for the elf3 circadian phenotype using a suppressor screen. EMS mutagenized elf3-1 plants will be screened for partial or complete restoration of clock function. The phenotypes of putative suppressors will be characterized using a battery of assays aimed at circadian clockrelated phenotypes, and the mutant locus will be identified with recently developed mapping protocols making use of high density oligonucleotide arrays. Relevance: The circadian clock regulates numerous important processes in eukaryotic organisms. That a defective clock can predispose humans to depression, obesity, abnormal sleep patterns, and certain types of cancer emphasizes the importance of proper time keeping. This research will investigate how circadian processes are regulated at the molecular level in eukaryotic cells.