EXPRESSION OF PAPOVAVIRUS TUMOR ANTIGENS

对更方便、更通用的克隆载体的需求 外源基因在真核细胞中的表达促使我们发展 一种病毒载体系统，可以容纳和表达大量的 外源基因序列。我们的系统旨在允许特定的选择 高水平表达外源基因产物的重组病毒 在病毒转录启动子的控制下。在过去的三年里 多年来，我们已经制定了许多策略来表达高水平 在哺乳动物病毒载体系统中获得SV40肿瘤抗原。我们成功了 通过构建SV40A基因获得超量表达 A基因在转录下的重组病毒 控制腺病毒的主要晚期启动子。然而，我们发现， T基因的高效翻译似乎需要特定的引导者 与T抗原基因5‘端连接的序列。这一发现 让我们假设在我们东道主中有效地启动翻译 载体系统需要特定的顺式作用序列元件 常见于腺病毒晚期mRNAs的5‘末端。在此，我们建议 识别最近前导序列中的翻译控制信号 并探讨病毒mRNAs与功能之间的关系 三方前导序列与mRNAs的选择性翻译 病毒感染的宿主细胞。我们打算进一步探索我们的能力 通过同源基因定位外源DNA在腺病毒基因组中 重组。除了构建和分离重组 病毒，我们还将应用一系列技术来表征 表达不同的基因的基因组、转录本和蛋白质结构 腺病毒重组体。最后，我们建议将该表达式推广到 通过构建包含SV40辅助分子的克隆载体来构建 功能基因与其他外源基因的融合以选择 其他基因的表达。特别是，我们计划建造一系列 表达多发性肿瘤抗原的重组载体的设计 病毒。我们对多发性肿瘤T抗原生化特性的兴趣 源于研究表明，尽管多瘤A基因编码 一种在结构和功能上与SV40T相似的多肽 抗原的排列方式有很大差异 影响T抗原介导转录的调控序列 在多发性瘤和SV40中的病毒DNA合成。

The need for more convenient and versatile cloning vectors for the expression of foreign genes in eukaryotic cells has prompted us to develop a viral vector system that can accomodate and express a large range of foreign gene sequences. Our system is designed to allow specific selection of recombinant viruses that express high levels of foreign gene products under the control of viral transcription promoters. In the past three years, we have developed a number of strategies for expressing high levels of the SV40 tumor antigen in a mammalian viral vector system. We succeeded in obtaining overproduction of the SV40 A gene mRNA by constructing recombinant viruses in which the A gene was under the transcriptional control of the adenovirus major late promoter. However, we discovered that efficient translation of T mRNA appeared to require specific leader sequences to be attached to the 5' end of the T antigen mRNA. This finding let us to hypothesize that efficient initiation of translation in our host vector system requires specific cis-acting sequence elements that are commonly found at the 5' ends of adenovirus late mRNAs. Here we propose to identify the translational control signals in the leader sequences of late viral mRNAs and to explore the relationship between the function of the tripartite leader sequence and the selective translation of mRNAs in virus-infected host cells. We intend to further explore our ability to position foreign DNA within the adnovirus genome by homologous recombination. In addition to constructing and isolating recombinant viruses, we will also apply a battery of techniques to characterize the genome, transcript and protein structures of the genes expressed various adenovirus recombinants. Finally, we propose to generalize the expression system by constructing cloning vectors that contain the SV40 helper function gene fused to other foreign genes in order to select for expression of other genes. In particular, we plan to construct a series of recombinant vectors designed to express the tumor antigen of polyoma virus. Our interest in the biochemical properties of polyoma T antigen stems from studies which indicate that although the polyoma A gene encodes a polypeptide that is analogous in structure and function to the SV40 T antigen, there are significant differences between the arrangement of regulatory sequences that influence the T antigen mediated transcription and viral DNA synthesis in polyoma and SV40.