GENE EXPRESSION AND SPLICING OF ADENOVIRUS MESSENGER RNA

这项建议的目标是描述处理机制的特征 2型腺病毒在生产过程中合成的信使RNA 感染。这些研究将集中在病毒和细胞mRNA的剪接上。 我们已经开发了一种体外剪接系统，它利用作为底物 腺病毒2Pre-RNAs体外合成 沙门氏菌噬菌体6启动子。几个动力学参数以及 在此定义了体外剪接反应的要求 系统。最近，我们已经获得了108倍的剪接纯化 粗核提取液的活性。 在此应用程序中，我们建议进一步提纯和识别 信使核糖核酸剪接的个别成分。我们会尝试找出 不同纯化产物的潜在RNA剪接中间体 分数。同时，我们将尝试识别剪接 体内产生AD2感染细胞的中间体。研究将会是 目的是确定U1单链RNPs的确切作用和机制 体外信使核糖核酸剪接。我们将分析核苷酸序列 体外剪接对E2前-RNA的要求，以及 核糖核蛋白(RNPs)在存在的前RNAs的形成 提纯的馏分。此外，几种病毒基因的mRNA剪接将 在体外进行研究。我们将首先尝试在体外复制 观察了几种病毒基因的剪接模式，并进行了二次纯化和 确定控制病毒mRNAs加工的因素(S)。

The objective of this proposal is to characterize the processing mechanisms of messenger RNA synthesized by adenovirus type 2 during productive infection. These studies will focus on viral and cellular mRNA splicing. We have developed an in vitro splicing system that utilizes as substrates adenovirus 2 pre-RNAs synthesized in vitro from plasmid DNAs containing the Salmonella Phage 6 promoter. Several Kinetic parameters as well as the requirements for the in vitro splicing reaction were defined in this system. Recently, we have obtained a 108-fold purification of the splicing activity from crude nuclear extracts. In this application we propose to further purify and identify the individual components of mRNA splicing. We will attempt to identify potential RNA splicing intermediates with the different purified fractions. At the same time we will attempt to identify splicing intermediates in in vivo productively Ad2 infected cells. Studies will be carried out to determine the precise role and mechanism of the U1 snRNPs in in vitro mRNA splicing. We will analyze the nucleotide sequence requirement for E2 pre-RNA in vitro splicing, and also the ribonucleoprotein (RNPs) formation of the pre-RNAs in the presence of purified fractions. In addition, mRNA splicing of several viral genes will be studied in vitro. We will attempt first to reproduce in vitro the observed splicing patterns of several viral genes and second to purify and identify the factor(s) involved in the control of processing of viral mRNAs.