CELLULAR PROTEINS INVOLVED IN ADENOVIRUS E1A REPRESSION

腺病毒E1A癌基因编码两种目前尚不清楚的生化 在ElA蛋白结构域中映射对细胞重要的活性 转化-转录抑制和细胞DNA合成 诱导 很可能是细胞调节的相互作用 具有特异性EIA蛋白质序列的蛋白质在 这些EIA癌基因的功能。 为了进一步了解EIA抑制和 为了开发鉴定和纯化细胞蛋白因子的方法，我们 将研究两种截然不同的独特增强剂作为模型- 依赖的，ElA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 是细胞类型特异性的，而用于neu的那些不是。 保护EIA 据报道，蛋白质结构域2是neu癌基因抑制所必需的 但对胰岛素抑制是必要的。 我们建议确定 EIA抑制是否需要细胞蛋白质合成 用细胞显微注射法测定胰岛素和neu，并研究ElA结构域 也需要ElA抑制胰岛素和neu抑制， 对于EIA DNA合成诱导。 结果与 用ElA抑制性SV40增强子进行的类似研究将（i）有助于 制定EIA抑制模型，（二）帮助确定是否有两个 EIA抑制现象涉及不同的机制-一个 代表"直接转录抑制"，另一种可能是 反映EIA诱导的细胞DNA途径中的次级事件 （iii）指导研究机制和发展 分离细胞蛋白因子的策略。 我们的主要努力 然后将是（i）开发体外和体内互补测定法 对于参与EIA阻遏的细胞蛋白，（ii）通过 几种方法，特别是通过EIA肽亲和层析， 与EIA结构域相关和/或在EIA中起作用的细胞蛋白 抑制，和（iii）纯化在EIA中起作用的细胞因子 并克隆它们的基因进行详细研究。

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.