Spectrometric Nucleic Acid-Associated Protein ID

光谱法核酸相关蛋白 ID

• 批准号: 6657388
• 负责人: James G Wetmur
• 金额: $ 25.43万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-10 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6657388
• 关键词: DNA; Escherichia coli; SDS polyacrylamide gel electrophoresis; crosslink; gene expression; genetic promoter element; genetic regulatory element; gonadotropin releasing factor; lac operon; mass spectrometry; nucleic acid purification; nucleic acid sequence; polymerase chain reaction; protein binding; protein protein interaction; restriction endonucleases; technology /technique development; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant) The advent of microarray technology has allowed, for the first time, the ability to assess the genome-wide changes in transcription that occur in response to specific stress, drugs or toxicants. The waves of gene changes that occur in response to a specific stimulus are transmitted by the assembly of transcription factors at specific promoters. Thus a key component of the code which couples environmental stimuli to changes in gene expression is the synthesis, modification and aggregation of activating and inactivating transcription factors at the promoters which are induced or suppressed. Studies of this crucial component of information flow in genetic networks have been limited by the difficulty to identify and characterize the proteins that interact with intact regulatory regions of genes whose expression is modulated. While great advances have been made in identifying which transcription factors are capable of activating particular gene constructs, direct knowledge is limited about which factors activate the actual gene in an intact chromosome and the secondary modification of those factors when interacting with the intact gene. In order to facilitate studies into these questions, the investigators propose to develop a new technique that will allow mass spectrometric identification of the proteins associated with specific gene promoters. If successful, this new technique has the potential to vastly improve the researchers' capacity to identify and characterize the protein assembly that interacts with specific promoters following an external stimulus and thereby significantly contribute to unraveling the structure and organization of genetic networks. The investigators propose to develop and validate this approach with a prokaryotic model, and then to apply the technique to the modulation of a well characterized eukaryotic promoter that forms a central part of the environmentally sensitive reproductive system. Specifically, the technique will be applied to the identification and characterization of proteins that interact with the mouse LhB promoter when induced by pulsatile hormone administration. This gene has a well characterized tertiary gene promoter that requires induction and synthesis of other transcription factors for its induction. The researchers will utilize this newly developed technique to identify the transcription factors that bind the intact LhB promoter during pulsatile GnRH induction.

描述（由申请人提供） 微阵列技术的出现首次允许评估响应特定压力，药物或毒物而发生的全基因组变化的能力。响应特定刺激发生的基因变化波是通过特定启动子的转录因子的组装来传递的。因此，将环境刺激与基因表达的变化结合的代码的关键组成部分是在启动子处的激活和灭活转录因子的合成，修改和聚集，这些转录因子被诱导或抑制。遗传网络中信息流的这种关键成分的研究受到识别和表征与表达调节的基因完整调节区域相互作用的蛋白质的困难的限制。尽管在识别哪些转录因子能够激活特定基因构建体方面取得了巨大进步，但直接知识限制了哪些因素激活完整染色体中的实际基因以及与完整基因相互作用时这些因素的次要修饰。 为了促进对这些问题的研究，研究人员建议开发一种新技术，该技术将允许对与特定基因启动子相关的蛋白质进行质谱鉴定。 如果成功的话，这项新技术有可能大大提高研究人员的能力，以识别和表征与外部刺激后与特定启动子相互作用的蛋白质组件的能力，从而显着促进了遗传网络的结构和组织。研究人员建议通过实质性模型开发和验证这种方法，然后将技术应用于构成环境敏感的生殖系统中心的良好表征的真核启动子的调节。 具体而言，该技术将应用于通过脉冲激素给药诱导时与小鼠LHB启动子相互作用的蛋白质的鉴定和表征。该基因具有良好的三级基因启动子，需要诱导和合成其他转录因子才能诱导。研究人员将利用这种新开发的技术来识别在脉冲GNRH诱导过程中结合完整LHB启动子的转录因子。