Coordinate Gene Regulation in Animal Cells

协调动物细胞中的基因调控

• 批准号: 7213423
• 负责人: JOHN T LIS
• 金额: $ 50.6万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7213423
• 关键词: Address; Affinity; Alleles; Animals; Architecture; Binding; Biochemical; Biochemical Process; Biological Assay; Biological Models; Cells; Chemicals; Chromatin; Chromosomes; Complement; Coupled; Cultured Cells; Cyclophosphamide/Fluorouracil/Prednisone; DNA-protein crosslink; Disease; Drosophila genus; Eukaryota; Eukaryotic Cell; Event; Fluorescence Resonance Energy Transfer; Gene Activation; Gene Expression Regulation; Gene Structure; Generations; Genes; Genetic Transcription; Heat-Shock Response; Image; Imaging Techniques; Immunofluorescence Immunologic; In Vitro; Individual; Investigation; Kinetics; Laboratories; Life; Mediating; Messenger RNA; Methodology; Methods; Microscopy; Modeling; Molecular; Movement; Nuclear; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Polymerase; Polymerase Chain Reaction; Process; Processed Genes; Production; Proteins; RNA; RNA Interference; RNA Polymerase II; RNA Processing; Recruitment Activity; Regulation; Regulator Genes; Relative (related person); Resolution; Reverse Transcription; Role; Running; Series; Specificity; Staining method; Stains; Structure; Surface; System; Temperature; Testing; Time; Transcript; Transcriptional Activation; Transgenes; Transgenic Organisms; Whole Organism; Work; aptamer; fly; in vivo; inhibitor/antagonist; molecular modeling; mutant; new technology; novel; polypeptide; promoter; protein protein interaction; reconstitution; transcription factor; two-photon

DESCRIPTION (provided by applicant): The heat shock (HS) genes are a highly-regulated set of genes particularly well-suited to investigate fundamental features of inducible mRNA production. Here, both established and new technologies will be used to identify factors that participate in the HS gene mRNA production, and to define with high temporal and spatial resolution when, where, and with whom these factors interact during the process of gene activation. The consequences on the hsp70 promoter and gene caused by depleting or inactivating specific transcription factors or particular surfaces of these factors will be evaluated in vivo. Aim 1 is a series of screens to identify factors involved in HS-induced transcription and RNA processing using Polytene chromosome immunofluorescence, RNAi treatments of cell cultures, and assays of existing fly mutants. Aim 2 examines promoter architecture and function during HS gene activation and the kinetics of factor recruitment by complementary methods of immuno-staining of polytene chromosomes and real-time PCR-quantified ChIP assays. These strategies will be augmented by in vivo chemical footprinting assays, quantification of specific transcripts by real-time, reverse-transcription PCR and Northern assays, and assays of elongationally-engaged RNA Polymerase II (Pol II) by nuclear run-on. Additionally, pairs of CFP- and YFP-tagged transcription factors will be examined for their interactions on polytene chromosomes using two photon and FRET microscopy, providing a unique view of factor interactions at specific loci during gene activation in real time and in living cells. Aim 3 tests the role of specific factors in promoter architecture and function using a battery of complementary and progressively higher resolution strategies to deplete or inactivate particular factors or particular surfaces of these factors. Strategies include RNAi treatments, use of existing Drosophila mutants, generation and use of fast-acting temperature-sensitive alleles, application of highly-specific drugs/inhibitors, and generation and use of high affinity RNA aptamers to different interacting surfaces of key transcription factors. In all cases, the effects on promoter and gene structure and function will be examined by the in vivo strategies described in Aim 2. The resulting information will be key in establishing molecular models for the various steps in the transcriptional activation of genes and the coupled RNA processing events in vivo, and in providing the background necessary for a molecular understanding of normal and disease states.

描述(由申请人提供)： 热休克(HS)基因是一组高度调控的基因，特别适合于研究可诱导的mRNA产生的基本特征。在这里，将使用现有的和新的技术来识别参与HS基因mRNA生产的因素，并以高时间和空间分辨率定义这些因素在基因激活过程中何时、何地和谁相互作用。耗尽或失活特定转录因子或这些因子的特定表面对HSP70启动子和基因造成的后果将在体内进行评估。目的1是一系列筛选，利用多线染色体免疫荧光、细胞培养的RNAi处理和现有苍蝇突变体的分析来确定参与HS诱导转录和RNA加工的因素。目的研究HS基因激活过程中启动子的结构和功能，并结合多线染色体免疫染色和实时荧光定量芯片技术，研究HS基因的因子募集动力学。这些策略将通过体内化学足迹分析、通过实时、反转录PCR和Northern分析对特定转录物的定量以及通过核运行对延伸参与的RNA聚合酶II(POL II)的分析来加强。此外，CFP和YFP标记的转录因子对将使用双光子和FRET显微镜检查它们在多线染色体上的相互作用，从而提供实时和活细胞中特定位置的因子相互作用的独特视角。AIM 3利用一系列互补且分辨率逐渐提高的策略来耗尽或灭活特定因子或这些因子的特定表面，以测试特定因子在启动子结构和功能中的作用。策略包括RNAi治疗，利用现有的果蝇突变体，产生和使用快速作用的温度敏感等位基因，应用高度特异的药物/抑制剂，以及产生和使用高亲和力的RNA适体到关键转录因子的不同相互作用表面。在所有情况下，对启动子和基因结构和功能的影响将通过目标2中描述的体内策略进行检验。由此产生的信息将是为基因转录激活的各个步骤和体内耦合的RNA加工事件建立分子模型的关键，并为了解正常和疾病状态的分子提供必要的背景。