Circadian Gating of Signals by EARLY FLOWERING 3

描述（由申请人提供）：细胞保持时间超过24小时的能力归功于内源性分子振荡器，即昼夜节律钟，其节奏在缺乏环境提示的情况下持续存在。在所有高等真核生物中，生物钟调节着一系列代谢、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，时钟采用一种称为“门控”的机制，以相位依赖的方式缓冲输入信号。拟南芥早花3 （ELF3）功能的缺失导致核心振荡器对光信号重置异常敏感。本研究旨在从分子水平上了解ELF3如何控制拟南芥生物钟的光携动。我们的假设是ELF3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ELF3功能至关重要。先前的研究表明，ELF3在体外直接与红光光感受器phyB相互作用，然而，这种相互作用在很大程度上仍未被表征。ELF3中缺乏与phyB相互作用能力的突变体，称为相互作用缺陷ELF3 (ELFSdi)，将被构建并引入ELF3植物中，以测定突变表型的互补性。2)识别新的ELF3交互伙伴。利用酵母双杂交技术筛选ELF3的潜在分子伴侣。新的相互作用克隆将通过共免疫沉淀试验得到确认，它们与ELF3和核心振荡子的关系将通过分子和遗传分析在植物中进行研究。3)利用抑制因子筛选确定elf3昼夜表型所需的基因。EMS诱变的elf3-1植株将被筛选部分或完全恢复时钟功能。假定的抑制因子的表型将使用一系列针对生物钟相关表型的测定来表征，突变位点将使用最近开发的利用高密度寡核苷酸阵列的作图协议来鉴定。相关性：在真核生物中，生物钟调节着许多重要的过程。一个有缺陷的时钟会使人容易抑郁、肥胖、不正常的睡眠模式和某些类型的癌症，这一点强调了正确计时的重要性。本研究将探讨真核细胞的昼夜节律过程是如何在分子水平上调控的。

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.