BRAIN RNA-MODIFYING ENZYME--CLONING AND CHARACTERIZATION

脑 RNA 修饰酶——克隆和表征

• 批准号: 3418373
• 负责人: TIMOTHY C. WONG
• 金额: $ 15.3万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3418373
• 关键词: clone cells; enzyme activity; enzymes; gene mutation; genetic transcription; host organism interaction; measles virus; molecular cloning; neuroblastoma; nucleic acid probes; posttranscriptional RNA processing; virus genetics; virus infection mechanism

The broad, long-term objective is to understand the Molecular mechanisms of chronic virus infections in the human central nervous system (CNS). Measles virus (MV) causes millions of death annually in infants and children on a global scale. MV can cause acute and subacute CNS infections. Recent research suggests that a neural cell RNA-modifying activity may introduce genetic changes called biased hypermutation in MV and may alter its mode of replication in a CNS infection. The present project investigates this unique virus-host interaction. The specific aims of this project are (1) to test the model that biased hypermutation occurs in the MV transcription complex, (2) to purify the RNA-modifying enzyme implicated in hypermutation and clone the corresponding cellular gene, and (3) to characterize the RNA-modifying enzyme and prove its role in hypermutation by use of the cloned gene. The first goal will be accomplished by studying MV transcription in neuroblastoma cells which contain high RNA-modifying activity or in vitro in neuroblastoma cell lysates. The second goal will be achieved by purifying the RNA-modifying enzyme from bovine brain, which contains the RNA-modifying activity. The purified protein will be sequenced to generate oligonucleotide probes for cloning the cDNA encoding the enzyme. The final goal will be accomplished by generating antibodies and gene probes to study the expression of the RNA-modifying protein in different cell types. The RNA-modifying enzyme will be overexpressed in mammalian cells with low intrinsic RNA-modifying activity. MV will be passaged in those cells to see whether hypermutation occurs with increased frequency.

广泛的长期目标是了解分子机制 人类中枢神经系统（CNS）的慢性病毒感染。 麻疹病毒（MV）每年导致数百万婴儿死亡， 儿童在全球范围内。 MV可引起急性和亚急性CNS 感染. 最近的研究表明，神经细胞RNA修饰 活动可能会在MV中引入称为偏性超突变的遗传变化 并可能改变其在CNS感染中的复制模式。 本 该项目研究了这种独特的病毒-宿主相互作用。 本课题的具体目的是：（1）对模型进行检验， 在MV转录复合物中发生超突变，（2）纯化 与超突变有关的RNA修饰酶，并克隆 相应的细胞基因，和（3）表征RNA修饰的 酶，并通过使用克隆的基因证明其在超突变中的作用。 第一个目标将通过研究MV转录来实现， 含有高RNA修饰活性的神经母细胞瘤细胞或体外 在神经母细胞瘤细胞裂解物中。 第二个目标将通过 从牛脑中纯化RNA修饰酶，其含有 RNA修饰活性。 将对纯化的蛋白质进行测序， 产生用于克隆编码所述酶的cDNA的寡核苷酸探针。 最终的目标将通过产生抗体和基因来实现。 探针来研究RNA修饰蛋白在不同细胞中的表达， 细胞类型。 RNA修饰酶将在哺乳动物中过表达 具有低内在RNA修饰活性的细胞。 MV将在 这些细胞来观察超突变是否以增加的频率发生。