TRANSPOSON MEDIATED MUTAGENESIS OF NP VIRUSES

核型多角体病毒（NuclearPolyhedrosisVirus，NPV）是一种重要的生物防治剂 用于农业和医疗害虫。 它们也被设计成 作为原核和真核基因的高效表达载体 感染的昆虫细胞培养物中。 NPV的自发FP突变体是 在连续传代时扩增，并代表了大规模接种的主要障碍 在细胞培养物中培养这些病毒， 杀虫剂或用作表达载体。 许多FP突变体似乎 在500 bp区域插入重复宿主序列后产生 苜蓿银纹夜蛾MNPV的4.95kb Hind III片段，以及在一些 在某些情况下，这些插入是转座因子。 这似乎是一个 研究真核转座的理想系统。 它有 易于识别的斑块形态表型的优点 随着插入事件的发生，快速隔离和评估 一次产生许多突变体，并且有可能产生一种 使用克隆的转座子和靶病毒DNA的体外系统。 这个项目 将更充分地表征最感兴趣的插入宿主序列， 转座子TFP3和本文中称为IFP2的重复序列， 通过双脱氧测序。 这些插入物中的区域 将通过插入或删除来识别对它们的移动性重要的 诱变 这些插入在FP突变体或 将在几次细胞培养传代后分离的野生型病毒， 确定，病毒基因组中的替代插入位点将被 映射。 计算机分析插入物周围的病毒序列 现场将确定是否有任何可能的二级结构， 可能会促进这个区域的重组。 携带TFP3转座子的 将构建选择标记基因并将其转移到病毒中， 基因组，这种病毒载体将被用来感染细胞培养物的一个 不太敏感的物种，以分析NPV是否可以作为媒介物， 转座因子在物种间的转移。 这些实验将 更全面地理解了 转座子介导的NPV突变，并将奠定重要的基础 为了将该系统开发为真核生物的实验模型， 换位

Nuclear polyhedrosis viruses (NPV) are candidate biological control agents for agricultural and medical insect pests. They have also been engineered as highly efficient expression vectors for procaryotic and eucaryotic genes in infected insect cell cultures. Spontaneous FP mutants of NPV are amplified upon serial passage and represent a major obstacle to mass cultivation of these viruses in cell cultures for either production as insecticides or use as expression vectors. Many FP mutants appear to be generated upon insertion of repetitive host sequences at a 500 bp region of the 4.95 kb Hind III fragment of Autographa californica MNPV and in some cases these insertions are transposable elements. This appears to be an ideal system for the study of eucaryotic transposition. It has the advantages of an easily identified plaque morphology phenotype associated with the insertion event, capability for rapid isolation and evaluation of many mutants at once, and the possibility for the development of an in vitro system using cloned transposons and target viral DNA. This project will more fully characterize the most interesting inserted host sequences, the transposon TFP3 and the repetitive sequence herein designated IFP2, through dideoxy sequencing. Regions in these insertions which are important for their mobility will be identified by insertion or deletion mutagensis. The relative frequency of these insertions in FP mutant or wild type virus isolated after several cell culture passages will be determined, and alternate insertion sites in the viral genome will be mapped. Computer analysis of the viral sequences surrounding the insertion sites will determine if there are any probable secondary structures which might facilitate recombination in this region. A TFP3 transposon carrying a selectable marker gene will be constructed and transferred to the viral genome, and this viral vector will be used to infect cell cultures of a less susceptible species to analyze if NPV may act as vehicles for the transfer of transposable elements between species. These experiments will yield a more complete understanding of the evolutionary significance of transposon mediated mutagenesis of NPV, and will lay important groundwork for the development of this system as an experimental model of eucaryotic transposition.