Polynucleotide High Performance Affinity Chromatography

描述（由申请人提供）：目的是提高转录因子的DNA亲和层析的性能，并增加对遗传调控的理解。许多转录因子（TF;例如，c-jun、c-myc）最初被发现为致癌基因，并且遗传调节对于理解遗传疾病是重要的。与人类健康和福祉的相关性是显而易见的。我们将：1.开发一种新的和创新的方法来表征启动子。现在研究启动子结构的方法是识别其序列中包含的每个响应元件，使用电泳迁移率变动分析来检测分离的元件是否实际上与核蛋白结合，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，这种线性方法是冗长乏味的。启动子捕获是在上一个资助期开发的，有可能提供一种全新的方法来表征启动子;使用凝胶印迹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与西南印迹和印迹上消化相结合，以允许灵敏地检测TFS并允许直接从印迹进行质谱表征。与启动子捕获相结合，这些启动子复合物可以从几个凝胶印迹分析。我们将确定这种新方法是否能加速研究进展。2.新的三维凝胶电泳（3DGE）方法将被开发来解决棘手的TF。三个新的3DGE方法将进一步分离从系统寡核苷酸捕获（SOT）和启动子捕获（PT）获得的蛋白质。在每种情况下，第一个维度依赖于特异性DNA结合电泳，然后是pI和SDS-PAGE的第二和第三维。这些新技术将解决任何棘手的转录因子鉴定。3.替代捕获方法将解决当前捕获技术的问题。SOT已被证明是纯化转录因子最有效的技术，但它并不完美。用于捕获的寡核苷酸的末端结合DNA修复蛋白作为污染物，这可能使鉴定变得模糊。我们将开发替代品，使用硫醇-二硫化物化学或固定化金属亲和色谱法来解决这个问题。拟议研究的结果将是研究遗传调控的真正创新和变革性方法。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，显示了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。为了了解遗传疾病和遗传调控，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表征。

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.