CELLULAR PROTEINS INVOLVED IN ADENOVIRUS E1A REPRESSION

腺病毒E1a癌基因编码两种已知的生化 映射到对细胞重要的ELA蛋白结构域的活动 转化-转录抑制与细胞DNA合成 归纳法。很可能细胞调控的相互作用 具有特定EIA蛋白序列的蛋白质(S)在 这些EIA癌基因的功能。为了进一步了解环评抑制和 为了开发鉴定和纯化细胞蛋白因子的方法，我们 将作为模型研究两种截然不同和独特的增强剂- 依赖的ELA可抑制基因--大鼠胰岛素II基因和大鼠 Neu癌基因。ELA抑制胰岛素所需的细胞因子(S) 是细胞类型特异的，而neu不是。节约的环境影响评估 据报道，蛋白结构域2是抑制neu癌基因所必需的。 而是为了治疗胰岛素抑制。我们建议确定 是否需要细胞蛋白质合成来抑制EIA 细胞微量注射法检测胰岛素和neu及研究ELA结构域 对ELA抑制胰岛素和Neu抑制的要求也是如此 至于EIA DNA合成的诱导。测试结果与实验结果的比较 对ELA可抑制的SV40增强子的类似研究将(I)有所帮助 制定环境影响评估抑制模型，(Ii)帮助确定两个 EIA抑制现象涉及不同的机制--一 代表“直接转录抑制”，另一个可能是 反映EIA诱导的细胞DNA途径中的一个次要事件 综合，以及(Iii)指导机理研究和发展 细胞蛋白因子的分离策略。我们的主要努力 然后将(I)开发体外和体内互补分析方法 对于参与EIA抑制的细胞蛋白，(Ii)通过 几种方法，特别是通过EIA多肽亲和层析， EIA中与ELA结构域和/或功能相关的细胞蛋白 抑制，以及(Iii)纯化在EIA中起作用的细胞因子 并克隆它们的基因以进行详细研究。

The adenovirus E1A oncogenes encodes two porly understood biochemical activities that map in ElA protein domains important for cell transformation - transcriptional repression and cellular DNA-synthesis induction. It is likely that interaction of cellular regulatory protein(s) with specific EIA protein sequences play critical roles in these EIA oncogene functions. To further understand EIA repression and to develop assays to identify and purify cellular protein factors, we will investigate as models two contrasting and unique enhancer- dependent, ElA repressible genes - the rat insulin II gene and the rat neu oncogene. Cellular factor(s) required for ElA repression of insulin are cell-type specific whereas those for neu are not. Conserved EIA protein domain 2 is reported to be required for neu oncogene repression but to be dispensible for insulin repression. We propose to determine whether cellular protein synthesis is required for EIA repression of insulin and neu by a cell microinjection assay and to study ElA domain requirements for ElA repression of insulin and neu represssion, as well as for EIA DNA-synthesis induction. Comparison of results with those of similiar studies with the ElA repressible SV40 enhancer will (i) help formulate models of EIA repression, (ii) help determine whether two different mechanisms are involved in the EIA repression phenomena - one representing "direct transcriptional repression" and the other possibly reflecting a secondary event in an EIA induced pathway of cellular DNA synthesis, and (iii) guide studies on mechanism and the development of strategies for isolation of cellular protein factors. Our major effort will then be (i) to develop in vitro and in vivo complementation assays for cellular proteins involved in EIA repression, (ii) to identify by several approaches, particularly by EIA peptide-affinity chromatography, cellular proteins that associate with ElA domains and/or function in EIA repression, and (iii) to purify cellular factors that function in EIA repression and to clone their genes for detailed studies.