GENE EXPRESSION AND SPLICING OF ADENOVIRUS MESSENGER RNA

本提案的目的是描述处理机制的特征 腺病毒2型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的mRNA剪接。 我们已经开发了一种体外剪接系统， 腺病毒2前体RNA体外合成自质粒DNA， 沙门氏菌噬菌体6启动子。 几个动力学参数以及 体外剪接反应的要求在本发明中定义。 系统 最近，我们获得了108倍纯化的剪接 核提取物的活性。 在本申请中，我们提出进一步纯化和鉴定 mRNA剪接的单个组分。 我们会尝试找出 潜在的RNA剪接中间体， 分数。 同时，我们将尝试识别剪接 中间体在体内生产性Ad2感染的细胞中。 研究将 进行了研究，以确定U1 snRNP的确切作用和机制， 体外mRNA剪接。 我们将分析 E2前体RNA体外剪接的要求，以及 核糖核蛋白（RNP）的前体RNA的存在下， 纯化的级分。 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 观察到几种病毒基因的剪接模式，其次是纯化， 鉴定参与控制病毒mRNA加工的因子。

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.