Mechanism of Recombination Between RNAs Associated With Turnip Crinkle Virus

摘要芜菁皱缩病毒是研究RNA重组机制的理想系统。基因组是最小的病毒RNA基因组之一，与卫星RNA和许多小的亚病毒RNA （Sat-RNA）相关，后者是基因组RNA内或基因组RNA与亚病毒RNA之间重组事件的产物。体外重组和原生质体重组系统的发展使得对该系统中介导重组的要素和因子有了更完整的表征。我们的工作得出了一个重组模型，该模型表明外壳蛋白可能通过将模板定位在邻近位置来帮助介导重组事件。为了测试这个模型并继续描述事件，我们将：1。建立原生质体并进行体外重组实验。2. 使用诱变和化学和酶探针对基序I（重组RNA所有连接处右侧的序列）进行结构研究。3. 使用凝胶转移法和4确定外膜蛋白在sat-RNA D和sat-RNA C之间重组中的作用。依赖RNA聚合酶复制其基因组和亚基因组RNA的病毒受到核苷酸高度固有错误结合的影响，这可能导致相关物种的异质混合物。因此，RNA重组现在被认为是，也许仍然是病毒进化的一个主要因素。该实验室发现芜菁皱缩病毒包含几乎整个卫星RNA序列，该卫星RNA连接到基因组RNA的两个区域，这表明发生了非同源重组。这一事实，以及小的基因组大小和以非随机方式产生的重组分子表明，对重组事件所涉及的序列、结构和机制的详细检查将是富有成效的。在许多方面，更好地了解病毒在进化过程中使用的机制是至关重要的，而这项工作所追求的具体目标所产生的信息将加强这种理解。

Simon MCB-9315948 Abstract Turnip crinkle virus is an ideal system to study the mechanism of RNA recombination. The genome is among the smallest viral RNA genomes and is associated with satellite RNAs and numerous small, subviral RNAs (Sat-RNA) which are the products of recombination events within the genomic RNA or between the genomic and subviral RNAs. The development of an in vitro recombination as well as a protoplast recombination system has allowed a more complete characterization of the elements and factors which mediate recombination in this system. A model of recombination has resulted from our work which suggests that coat protein helps to mediate the recombination event by possibly positioning the templates in proximity. In order to test this model and to continue characterizing the event, we will: 1. Establish protoplast and in vitro assays for recombination. 2. Conduct structural studies on motif I (the sequences found at the right sides of all junctions of recombinant RNA) using both mutagenesis and chemical and enzymatic probes. 3. Determine the role of coat protein in recombination between sat-RNA D and Sat-RNA C using gel-shift assays and 4. Assay for binding of the RNA-dependent RNA polymerase to motif I. %%% Viruses which depend on RNA-dependent RNA polymerases for replication of their genomic and sub-genomic RNAs are subject to high inherent mis-incorporation of nucleotides that can lead to heterogeneous mixtures of related species. Thus, RNA recombination is now thought to have been, and perhaps still is, a major factor in the evolution of viruses. The discovery that the turnip crinkle virus contained nearly the entire sequence of a satellite RNA joined to two regions of genomic RNA by this laboratory suggested that non-homologous recombination occurs. That fact, as well as small genome size and the generation of recombinant molecules in a non-random manner suggested that a detailed examination of the sequences, structures and mechanisms inv olved in the recombinational event would be productive. It is critical in many ways to have a better understanding of the mechanisms used by viruses in the evolutionary process and the information resulting from the specific goals pursued in this work will enhance that understanding.