Transcriptional Regulation of pdf in Drosophila

描述(申请人提供)：大多数生命形式的生理和行为的日常节律是由内部昼夜节律起搏器驱动的，它使用身体波动的刺激来测量时间的流逝。生物钟的紊乱会在我们的社会中造成严重的健康问题，包括时差、失眠和与轮班工作相关的疾病。哺乳动物和果蝇(果蝇)中枢起搏功能的分子机制相似，表明果蝇是研究生物时钟的优秀模型系统。然而，中枢振荡器调节公开的生理和行为节律(输出途径)的机制在很大程度上是未知的。我们最近的研究发现了第一个昼夜节律信使；它是一种神经肽，色素分散因子(PDF)。有趣的是，核心时钟转录因子激活了起搏细胞中pdf的表达。这些研究表明，任何影响正常PDF产生的因素都可能导致异常节律。鉴于PDF在时钟输出调控中的重要性，本应用程序的目的是通过执行以下特定目标来研究果蝇pdf基因的转录调控机制： 1.对pdf启动子进行剖析。这将通过pdf增强子的定点突变和随后的体内功能试验来完成。酵母单杂交和EMS-突变筛选将被用来寻找pdf转录调控因子。 2.确定了一种新的节律突变体。被P元件插入破坏的基因将被分离出来，并进行分子特征分析。 3.克隆DV-pdf中的pdf基因。通过RT-PCR或文库筛选，我们将分离出DV-pdf基因及其调控元件。DV-pdf启动子将在黑腹隐翅虫中进行异源检测。

DESCRIPTION (provided by applicant): Daily rhythms of physiology and behavior in most life forms are driven by internal circadian pacemakers which use the physically fluctuating stimuli to measure the passage of time. Disruptions of biological clock cause severe health problems in our society, including jet-lag, insomnia, and shiftwork-related illnesses. Similarities in molecular mechanisms underlying central pace-making functions in mammals and fruit flies (Drosophila melanogaster) suggest that Drosophila is an excellent model system to study the biological clock. However, the mechanisms by which the central oscillators regulate the overt physiological and behavioral rhythms (output pathways) are largely unknown. Our recent studies identified the first circadian messenger; it is a neuropeptide, pigment-dispersing factor (PDF). Interestingly, the core-clock transcription factors activate pdf expression in the pacemaker cells. These studies indicate that any factors affecting normal PDF production can cause aberrant rhythms. Given the importance of the PDF in the regulation of clock output, the objectives of this application are to examine transcriptional regulatory mechanisms of the pdf gene in Drosophila by performing the following specific aims: 1. Dissect pdf promoter. This will be accomplished by site-directed mutagenesis of the pdf enhancer and subsequent functional in vivo assay. Yeast one-hybrid and EMS-mutagenesis screen will be employed to find pdf transcriptional regulators. 2. Characterize a new rhythm mutant. The gene disrupted by a P-element insertion will be isolated and characterized molecularly. 3. Clone the pdf gene in D. virilis (Dv-pdf). Using RT-PCR or library screening, we will isolate the Dv-pdf gene and its regulatory elements. The Dv-pdf promoter will be assayed heterologously in D. melanogaster.