TRANSFORMATION OF CELLS BY THE V-REL ONCOGENE

这项建议描述了将加深我们理解的实验 由Rel家族引起的转录调控和肿瘤发生 蛋白质和与REL蛋白相关的蛋白质。尽管 主要的焦点是Vrel逆转录病毒癌蛋白，其他REL(cRel，NF- KappaB p50/p105、p49/p100等)和IkappaB样蛋白(p40、MAD-3、 等)也将被描述为。大多数研究将在以下情况下进行 禽类蛋白质，因为禽类系统仍然是唯一一个 人们已经看到了Rel蛋白对细胞的弗兰克转化。我们的长- Range Goal是对Rel蛋白质 控制细胞的正常和异常生长。因此，有几个 相关的具体目标。 首先，使用以前对我们来说成功的方法，我们 将分离出更多与禽类REL相关的cDNA。例如，我们将 克隆鸡P49/P100基因。使用这个cdna，我们将确定 P100是否是剩余的未确定的Vrel相关蛋白。我们 将在体外和体内对Vrel蛋白复合体进行表征。 使用Gal4融合蛋白、体外诱变和遗传选择 在酵母中，我们将描述对转录很重要的序列 Rel和IkappaB蛋白的激活。此外，我们还将尝试 确定与这些激活结构域相互作用的蛋白质 鸡细胞和酵母菌。 我们将确定磷酸化对鸡cRel的影响， 特别是在亚细胞位置、DNA结合的调节方面 和二聚化。 最后，将进行实验，以解决该机制 由Vrel进行的变形。首先，我们将通过以下方式来描述该机制 哪个Vrel抑制c-rel启动子并激活含有Sp1位点的 推动者。其次，我们将描述VREL中符合以下条件的序列 对其转型活动以及可能发生的变化是必要的 以使CREL变形。在创建了Vrel的条件突变体之后， 我们将尝试识别由Vrel直接控制的基因。 这里提出的实验将与基因控制相关， 肿瘤的发生、发展，也许是对艾滋病毒复制的控制，以及 旨在扩大和扩大实验室正在进行的研究，旨在 了解Rel蛋白的作用机制。

This proposal describes experiments that will further our understanding of transcriptional control and oncogenesis caused by the Rel family of proteins and proteins that associate with Rel proteins. Although the primary focus is on the vRel retroviral oncoprotein, other Rel (cRel, NF- kappaB p50/p105, p49/p100, etc.) and IkappaB-like proteins (p40, MAD-3, etc.) will also be characterized. Most studies will be performed with avian proteins, since the avian system is still the only system in which frank transformation of cells by Rel proteins has been seen. Our long- range goal is an understanding of the mechanism by which Rel proteins control normal and abnormal cell growth. As such, there are several related Specific Aims. First, using approaches that have previously been successful for us, we will isolate additional avian rel-related cDNAs. For example, we will clone a chicken p49/p100 cDNA. Using this cDNA, we will determine whether p100 is the remaining unidentified vRel-associated protein. We will characterize vRel protein complexes in vitro and in vivo. Using Gal4 fusion proteins, in vitro mutagenesis, and genetic selections in yeast, we will characterize sequences important for transcriptional activation in Rel and IkappaB proteins. In addition, we will attempt to identify the proteins with which these activation domains interact in chicken cells and in yeast. We will determine the effect of phosphorylation on chicken cRel, specifically in terms of regulation of subcellular location, DNA binding and dimerization. Finally, experiments will be performed to address the mechanism of transformation by vRel. First, we will characterize the mechanism by which vRel represses the c-rel promoter and activates Sp1 site-containing promoters. Second, we will characterize sequences in vRel that are necessary for its transforming activity, and the changes that can occur to make cRel transforming. After creating conditional mutants of vRel, we will attempt to identify genes controlled directly by vRel. The experiments proposed here will have relevance to gene control, oncogenesis, development, perhaps the control of HIV replication, and are meant to extend and expand ongoing studies in the laboratory aimed at understanding the mechanism of action of Rel proteins.