TRANSFORMATION OF CELLS BY THE V-REL ONCOGENE

这个提议描述了一些实验， Rel家族引起的转录控制和肿瘤发生 蛋白质和与Rel蛋白相关的蛋白质。 虽然 主要关注的是vRel逆转录病毒癌蛋白，其他Rel（cRel，NF-κ B）， kappaB p50/p105、p49/p100等）和IkappaB样蛋白（p40，MAD-3， 等等）。也将被定性。 大多数研究将使用 鸟类蛋白质，因为鸟类系统仍然是唯一的系统， 已经看到Rel蛋白质对细胞的直接转化。 我们长久以来- 范围目标是理解Rel蛋白质 控制正常和异常的细胞生长。 因此，有几个 相关的具体目标。 首先，使用以前成功的方法，我们 将分离出更多的鸟类相关cDNA。 比如我们会 克隆鸡p49/p100 cDNA。 利用这个cDNA，我们将确定 p100是否是剩余的未鉴定的vRel相关蛋白。 我们 将在体外和体内表征vRel蛋白复合物。 使用Gal 4融合蛋白、体外诱变和遗传选择 在酵母中，我们将描述转录重要序列， Rel和IkappaB蛋白的激活。 此外，我们将努力 确定与这些激活结构域相互作用的蛋白质， 鸡细胞和酵母中。 我们将确定磷酸化对鸡cRel的影响， 特别是在调节亚细胞定位，DNA结合 和二聚化。 最后，将进行实验以解决 通过vRel进行转换。 首先，我们将描述该机制， vRel抑制c-rel启动子并激活含Sp1位点的 发起人。 其次，我们将描述vRel中的序列， 它的转化活动所必需的，以及可能发生的变化 使cRel转化。 在创建vRel的条件突变体后， 我们将尝试鉴定由vRel直接控制的基因。 这里提出的实验将与基因控制有关， 肿瘤的发生、发展，也许是HIV复制的控制， 旨在扩展和扩大实验室正在进行的研究， 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.