INTERACTIONS OF P53 AND HMG-I(Y) IN SPERMATOGENESIS

该计划有两个广泛的目标：（1）研究和阐明p53和人HMG-I（Y）在合成连接DNA上的特异性相互作用以及蛋白质相互作用的可能性;（2）在体外和体内研究p53启动子的染色质结构以及HMG-I（Y）结合对染色质结构的影响。 第一个包括确定这些蛋白质与四向连接点的结合是否是互斥的，并在碱基对分辨率下识别这些蛋白质的结合位点。 第二部分将在体外和体内研究HMG-I（Y）对p53启动子染色质结构的影响。此外，将通过化学交联研究和抗体检测方法鉴定特异性HMG-I（Y）-p53蛋白-蛋白相互作用。 合成的四向连接、p53共有位点和p53启动子区将用作DNA底物。 这些实验将包括羟基自由基足迹，电泳迁移率，报告基因表达，和光交联技术。

The proposal has two broad goals: (1) to study and elucidate the specific interactions of p53 and human HMG-I(Y) on synthetic junction DNA and the possibility that the proteins interact; and (2) to study the chromatin structure of the p53 promoter in vitro and in vivo and the effects of HMG-I(Y) binding on chromatin structure. The first includes determining whether binding of these proteins to four-way junctions is mutually exclusive and identifying the binding sites of these proteins at base-pair resolution. The second will examine the effect of HMG-I(Y) on the p53 promoter chromatin structure in vitro and in vivo. Additionally, specific HMG-I(Y)-p53 protein-protein interactions will be identified by chemical crosslinking studies and antibody detection methods. Synthetic four-way junctions, p53 consensus sites, and the p53 promoter region will be used as DNA substrates. These experiments will include hydroxyl radical footprinting, electrophoretic mobility, reporter gene expression, and photocrosslinking techniques.