Polynucleotide High Performance Affinity Chromatography

描述(申请人提供)：目标是改进转录因子的DNA亲和层析的性能，并增加对遗传调节的理解。许多转录因子(如c-jun、c-myc等)最初是作为癌基因被发现的，基因调控对于理解遗传病是很重要的。这与人类健康和福祉的相关性是显而易见的。我们将：1.开发一种新颖和创新的启动子表征方法。现在研究启动子结构的方法是鉴定其序列中包含的每个反应元件，使用凝胶迁移率改变分析来检测分离的元件是否确实与核蛋白结合，然后使用DNA亲和层析和其他方法来纯化蛋白质，一次一个。对于复杂的启动子来说，这种线性方法既繁琐又漫长。启动子捕获是在上一个资助阶段发展起来的，它有可能提供一种全新的方法来表征启动子；使用凝胶印迹技术在几个步骤中定位和鉴定所有结合的转录因子。我们最近将2DGE与西南印迹和On-印迹消化相结合，以实现对TFS的灵敏检测，并允许直接从印迹进行质谱学表征。结合启动子捕获，这些启动子复合体很可能从几个凝胶印迹中分析出来。我们将确定这种新方法是否可以加快研究进展。2.将开发新的三维凝胶电泳法(3DGE)来解决难治性TFS。将开发三种新的3DGE方法来进一步分离系统寡核苷酸捕获(SOT)和启动子捕获(PT)获得的蛋白质。每种情况下的第一维依赖于特定的DNA结合电泳，然后是第二和第三维的PI和SDS-PAGE。这些新技术将解决任何棘手的转录因子鉴定问题。3.替代诱捕方法将解决目前诱捕技术存在的问题。SOT已被证明是现有的最有效的转录因子纯化技术，但它并不完美。用于捕获的寡核苷酸的末端是与DNA修复蛋白结合的污染物，这可能会使识别变得模糊。我们将开发使用硫醇-二硫化物化学或固定化金属亲和层析的替代方法来解决这个问题。拟议的研究结果将是研究基因调控的真正创新和变革性的方法。这些实验将需要四年时间。 公共卫生相关性：许多癌基因是转录因子，表明转录因子与癌症的相关性，以及转录因子对细胞分裂和其他生物过程的重要性。要了解遗传病和遗传调控，还必须了解转录因子的功能。在这里，我们改进了这些重要蛋白质的纯化和性质。

DESCRIPTION (provided by applicant): The goal is to improve the performance of the DNA-affinity chromatography of transcription factors and to increase understanding of genetic regulation. Many of the transcription factors (TFs; e.g., c- jun, c-myc) were originally discovered as oncogenes and genetic regulation is important to the understanding of genetic disease. The relevance to human health and well-being is clear. We will: 1. Develop a novel and innovative method for characterizing promoters. The way promoter structure is now studied is to identify each response element contained within its sequence, use the electrophoretic mobility shift assay to detect whether the isolated element is in fact bound by a nuclear protein, and then use DNA-affinity chromatography and other methods to purify the proteins, one-at-a-time. For complex promoters, this linear approach is tedious and prolonged. Promoter Trapping was developed in the last funding period and has the potential to provide a completely novel way to characterize a promoter; mapping and identification of all transcription factors that bind in a few steps using gel blots. We have recently combined 2DGE with southwestern blotting and on- blot digestion to allow the sensitive detection of TFS and allow mass spectrometry characterization directly from the blot. Combined with promoter trapping, these promoter complexes can likely be analyzed from a few gel blots. We will determine if this new approach can speed research progress. 2. Novel 3-dimensional gel electrophoresis (3DGE) methods will be developed to solve intractable TFs. Three new 3DGE methods will be developed to further separate the proteins obtained from systematic oligonucleotide trapping (SOT) and promoter trapping (PT). The first dimension in each case relies on specific DNA binding electrophoresis, then a second and third dimension of pI and SDS-PAGE. These new techniques will resolve any intractable transcription factor identifications. 3. Alternative trapping methods will solve problems with current trapping technology. SOT has been proven to be the most powerful techniques available for purifying transcription factors but it is not perfect. The ends of the oligonuleotide used for trapping are binding DNA repair proteins as contaminants which can obscure identification. We will develop alternatives which use either thiol-disulfide chemistry or alternatively, immobilized metal affinity chromatography, to solve this problem. The result of the proposed studies will be truly innovative and transformative ways to study genetic regulation. These experiments will require four years. PUBLIC HEALTH RELEVANCE: Many oncogenes are transcription factors, showing the relevance of transcription factors to cancer and also the importance of transcription factors to cell division and other biological processes. To understand genetic disease and genetic regulation, the function of transcription factors must also be understood. Here, we improve the purification and characterization of these important proteins.