Transcriptional Regulation of pdf in Drosophila

果蝇中pdf的转录调控

• 批准号: 6944365
• 负责人: JAE H PARK
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-02 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6944365
• 关键词: DNA binding protein; Drosophilidae; biological clocks; circadian rhythms; gene mutation; genetic regulation; genetic regulatory element; genetic transcription; molecular cloning; mutant; neuropeptides; polymerase chain reaction; protein protein interaction; regulatory gene; site directed mutagenesis; transposon /insertion element; yeasts

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.

描述（由申请人提供）：大多数生命形式的生理和行为的每日节律由内部昼夜节律起搏器驱动，其使用物理波动刺激来测量时间的流逝。生物钟的紊乱在我们的社会中引起严重的健康问题，包括时差，失眠和轮班相关的疾病。哺乳动物和果蝇（Drosophila melanogaster）中枢起搏功能分子机制的相似性表明，果蝇是研究生物钟的一个很好的模型系统。然而，中央振荡器调节明显的生理和行为节律（输出途径）的机制在很大程度上是未知的。我们最近的研究确定了第一个昼夜信使;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的pdf表达。这些研究表明，任何影响正常PDF生产的因素都可能导致异常节律。考虑到PDF在调节时钟输出中的重要性，本申请的目的是通过执行以下具体目标来检查果蝇中pdf基因的转录调节机制： 1.剖析pdf启动子。这将通过pdf增强子的定点诱变和随后的体内功能测定来实现。将采用酵母单杂交和EMS诱变筛选来寻找pdf转录调控因子。 2.描述一种新的节律突变体。被P元件插入破坏的基因将被分离并进行分子表征。 3.在D. virilis（Dv-pdf）.利用RT-PCR或文库筛选的方法，我们将分离Dv-pdf基因及其调控元件。将在D.黑腹菌