Circadian Gating of Signals by EARLY FLOWERING 3

描述(申请人提供)：细胞保持时间大约24小时的能力归功于一种被称为生物钟的内源分子振荡器，它的节奏在没有环境提示的情况下保持不变。生物钟调节所有高等真核生物的一系列代谢、生理和行为活动。为了保护核心振荡器不受杂散重置信号的影响，时钟使用一种称为“选通”的机制，以相位相关的方式缓冲输入提示。拟南芥早期开花3(ELF3)功能的丧失使核心振荡器对重置光信号高度敏感。这项研究旨在从分子水平上了解ELF3是如何控制拟南芥生物钟的光携带的。我们的假设是，ELF3通过与其他蛋白质相互作用并改变它们的活性来发挥作用。这项建议的具体目的是：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。以前的工作已经证明ELF3在体外直接与红光感受器PhyB相互作用，然而，这种相互作用在很大程度上仍未被表征。我们将构建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)，并将其导入elf3植物中，以检测突变表型的互补性。2)确定新的ELF3相互作用伙伴。ELF3的潜在分子伴侣将通过筛选利用酵母双杂交进行鉴定。新的相互作用克隆将通过免疫共沉淀实验得到确认，它们与ELF3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)使用抑制子筛选确定elf3昼夜节律表型所需的基因。将对EMS诱变的elf3-1植株进行筛选，以确定是否部分或完全恢复了时钟功能。可能的抑制子的表型将使用一系列针对昼夜节律相关表型的分析来表征，突变基因座将使用最近开发的利用高密度寡核苷酸阵列的作图方法来识别。相关性：生物钟调节着真核生物中许多重要的过程。有缺陷的时钟可能会使人类患上抑郁症、肥胖症、异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。这项研究将探讨在真核细胞中昼夜节律过程是如何在分子水平上被调节的。

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.