SV40 TUMOR ANTIGEN

DNA肿瘤病毒SV 40是一种非常宝贵的工具， 研究真核生物基因调控的分子方面 复制和病毒肿瘤发生。 一个主要的重点是 SV 40大T的结构、功能及表达调控 抗原已经揭示了异常大量的 关于病毒和细胞过程的重要发现。 因此 到目前为止，我们的研究主要是为了了解 这个有趣的多功能的结构和功能 调节蛋白 例如，仔细而系统的研究 T抗原的研究为深入了解 阻遏和自动调节，启动病毒DNA合成， 病毒编码的酶活性，序列特异性蛋白-DNA 相互作用和病毒诱导的肿瘤发生。 在这份赠款中， 更新，我们把注意力转向协调一致和系统的 研究T抗原表达是如何被 病毒和细胞反式激活因子的相互作用。 在 此外，我们将继续以SV 40为模型进行研究 细胞中发生的复杂调节过程，例如 转录。 同时，我们希望比较 介导其他几种病毒和 与SV 40 T抗原相关的细胞基因， 共同监管网络。 一个主要的努力将致力于识别，净化和 表征各种反式激活细胞因子， 转录因子Sp1，TATA结合因子，CAAT 转录因子（CTF）和增强子结合蛋白（EBP）， 已知与真核生物中的转录有关 细胞 这些转录因子中的许多已经通过 生物化学分级分离和体外转录研究 SV 40，表明它们是DNA结合蛋白。 因此，在本发明中， 我们已经开发的T抗原DNA的方法 结合实验现在可以应用于解剖性质， 蛋白质-DNA相互作用对Sp1，TBF，CTF， 还有EBP 我们还将培养针对这些病毒的抗体 并尝试克隆编码这些因子的基因。 抗体和分子克隆将被用来帮助我们 阐明作用机制并定义潜在的蛋白质- 介导转录特异性的蛋白质相互作用。 此外，我们计划继续研究SV 40大T 通过设计大规模分离程序， 试图结晶并进行X射线衍射研究 这种病毒编码的蛋白质。 最后，我们将利用 体外DNA复制反应，以研究T 抗原在触发SV 40-DNA复制和作为调节剂 转录。

The DNA tumor virus, SV40, has been an invaluable tool for studying molecular aspects of eukaryotic gene regulation, DNA replication, and viral oncogenesis. A major focus on the structure, function and regulated expression of the SV40 large T antigen has already revealed an unusually large number of important findings concerning viral and cellular processes. Thus far, our studies have been largely directed at understanding the structure and function of this interesting multi-functional regulatory protein. For example, a careful and systematic study of T antigen has provided insights into the mechanism of repression and autoregulation, initiation of viral DNA synthesis, viral coded enzymatic activities, sequence-specific protein-DNA interactions, and virally-induced oncogenesis. In this grant renewal, we turn our attention to a concerted and systematic effort at investigating how T antigen expression is regulated by the interplay of viral and cellular trans-activating factors. In addition, we will continue to use SV40 as a model to study complex regulatory processes occurring in the cells, such as transcription. At the same time, we hope to compare the mechanisms mediating expression of several other viral and cellular genes related to SV40 T antigen by the occurrence of common regulatory networks. A major effort will be devoted to identifying, purifying and characterizing various trans-activating cellular factors such as transcription factor Sp1, TATA binding factor (TBF), CAAT transcription factor (CTF), and enhancer binding protein (EBP), that are known to be involved with transcription in eukaryotic cells. Many of these transcription factors have been identified by biochemical fractionation and in vitro transcription studies of SV40, which revealed that they are DNA binding proteins. Thus, the methods that we have already developed for T antigen DNA binding experiments can now be applied to dissect the nature and specificity of the protein-DNA interactions for Sp1, TBF, CTF, and EBP. We will also raise antibodies directed against these proteins and attempt cloning of the genes encoding these factors. The antibodies and molecular clones will be used to help us unravel the mechanism of action and define potential protein- protein interactions that mediate transcriptional specificity. In addition, we plan to continue our studies of the SV40 large T antigen protein by devising large-scale isolation procedures, and attempting to crystallize and carry out X-ray diffraction studies of this virally-encoded protein. Finally, we will make use of in vitro DNA replication reactions to investigate the role of T antigen in triggering SV40-DNA replication and as a regulator of transcription.