GENETICS AND BIOCHEMISTRY OF POTATO VIRUS X REPLICATION

描述：(改编自《调查者摘要》)：马铃薯X病毒 (PVX)体系是理解RNA一般作用机制的极好模型 复制和潜在地用于设计新的抗病毒策略。PVX有一个 相对较小的基因组，只产生少量的亚基因组RNA。 烟草原生质体复制系统和依赖于可溶性模板的 摘录将被用来回答四个问题。哪些顺式作用序列和 体外结构会影响PVX RNA的积累吗？序列和结构 对正链和负链RNA的合成都很重要 野生型接种烟草原生质体中的RNA积累分析 和突变的PVX转录本。PVX RNA的模板要求是什么 体外合成？依赖PVX模板的体外转录系统 将被优化并用于定义负-和的模板要求 正链RNA合成。它们的蛋白质成分是什么？ 体内合成PVX RNA所需的蛋白质-蛋白质相互作用？这个 体外合成PVX RNA所需的蛋白质成分将被提纯 并进行了测序。通过序列分析或从以下来源获得相应的cDNA克隆 将使用酵母双杂交文库筛选来评估蛋白质-蛋白质 RNA合成所需的相互作用。宿主和/或病毒蛋白是否相互作用 与PVX RNA中的调控元件有关？蛋白质结合的程度 未感染和感染的植物或原生质体提取物对野生型和突变体 PVX转录本将通过凝胶移位和免疫沉淀分析来确定。 纯化的结合蛋白将通过亲和层析分离出来 测序。对RNA-蛋白质相互作用的进一步分析将利用cDNA 和与测序的蛋白质相对应的蛋白质。从这些网站获得的数据 特定的目标将定义RNA所涉及的组件和机制 综合。

DESCRIPTION: (Adapted from the Investigator's abstract): The potato virus X (PVX) system is an excellent model for understanding general mechanisms of RNA replication and potentially for designing novel antiviral strategies. PVX has a relatively small genome from which only a few subgenomic RNAs are produced. Both a tobacco protoplast replication system and a soluble template-dependent extract will be used to answer four questions. Which cis-acting sequences and structures affect PVX RNA accumulation in vitro? Sequences and structures that are important for synthesis of plus-and minus-strand RNAs will be determined by analysis of RNA accumulation in tobacco protoplasts inoculated with wild-type and mutant PVX transcripts. What are the template requirements for PVX RNA synthesis in vitro? The PVX template-dependent in vitro transcription system will be optimized and utilized to define template requirements for minus-and plus-strand RNA synthesis. What are the protein constituents and protein-protein interactions required for PVX RNA synthesis in vivo? The protein constituents required for PVX RNA synthesis in vitro will be purified and sequenced. Corresponding cDNA clones obtained by sequence analysis or from a yeast two-hybrid library screen will be used to assess protein-protein interactions required for RNA synthesis. Do host and/or viral proteins interact with regulatory elements in PVX RNA? The extent of binding of proteins from uninfected and infected plant or protoplast extracts to wild-type and mutant PVX transcripts will be determined by gel shift and immunoprecipitation assays. Purified binding proteins will be isolated by affinity chromatography for sequencing. Further analyses of RNA-protein interactions will utilize the cDNAs and proteins corresponding to sequenced proteins. Data obtained from these specific aims will define the components and mechanisms involved in RNA synthesis.