TRANSPOSON MUTAGENESIS OF NUCLEAR POLYHEDROSIS VIRUSES

运输是基因重排的一种形式，在 包括原核生物和真核生物。分子基础 动员非逆转录病毒样真核转座子的方法是 不完全理解，但可能涉及重组 类似于在其他类型基因组中工作的机制 重排事件包括免疫球蛋白和T-Sell受体 基因排列，基因扩增，基因破坏，以及 癌基因激活。转座子动员的后果 对于高等生物和/或病毒的进化来说 相当可观。核型多角体病毒的FP突变 (NPV)为分子提供了一个独特的实验系统 转座子运动的几个方面的分析。许多. 这些自发突变是通过宿主的插入而产生的。 在4.95kb的500bp区域内的转座元件 加州金龟子或梅氏山羊草中的HindIII片段 MNPV称为FP基因座。突变的表型导致一种 独特的斑块形态和显性扩增 细胞培养中繁殖过程中的突变体。这些属性 可用于开发体外转座试验 使用克隆的转座子和靶向病毒DNA。建议数 研究重点将放在分析毛霉转座子上 显示靶点特异性和插入的IFP2和TFP3 NPV的FP基因座内的位置偏好。的影响 病毒靶点或转座子末端的突变 这些元素的插入和删除将被研究。 分析转座子的位点特异性插入的能力 分子水平是这一体系的一个显著特征。 标记了LacZ融合基因的转座子将被 在病毒DNA存在的情况下被引入昆虫细胞。这个 独特的斑块形态和/或颜色将有助于 插入和切除事件的检测。我们还将使用 重组核型多角体病毒载体的研究 通过病毒在物种间转座子。重组NPV将成为 携带自主可选择的P-元素构造体的构建 在上一个授权期内产生的。这些 重组核糖核酸将被用于分析潜在的载体 转座子进入黑腹果蝇细胞或胚胎。建议数 研究将使我们更全面地了解 转座子诱变在细菌进化中的意义 病毒及其宿主，并将推动一种 独特的体外分子分析实验系统 高等真核生物中的转座和遗传重组。

Transportation is a form of genetic rearrangement common among both procaryotic and eucaryotic organisms. The molecular basis of mobilization for non-retrovirus-like eucaryotic transposons is incompletely understood, but likely involves recombinational mechanisms similar to those operating in other types of genome rearrangement event including immunoglobin and T-sell receptor gene arrangements, gene amplification, gene disruption, and oncogene activation. The consequences of transposon mobilization for the evolution of higher organisms and/or viruses could be substantial. The FP mutation of nuclear polyhedrosis viruses (NPVs) provides a unique experimental system for the molecular analysis of several aspects of transposon movement. Many of these spontaneous mutants arise through the insertion of host transposable elements within a 500 bp region of the 4.95 kb HindIII fragment in Autographa californica or Galleria mellonella MNPVs called the FP locus. The mutant phenotype results in a distinctive plaque morphology and dominant amplification of the mutants during propagation in cell cultures. These properties can be exploited in developing an in vitro transposition assay using cloned transposons and target viral DNAs. The proposed research will focus on analysis of Trichoplusia ni transposons IFP2 and TFP3 that exhibit target site specificity and insertion site preference within the FP locus of NPVs. The effect of mutations at viral target sites or within transposon termini on the insertion and excision of these elements will be studied. The ability to analyze site-specific insertion of transposons at the molecular level is a distinguishing feature of this system. Transposons that are tagged with a lacZ fusion gene will be introduced into insect cells in the presence of viral DNA. The distinctive plaque morphologies and/or coloration will facilitate detection of insertion and excision events. We will also use recombinant NPV to analyze the potential for vectoring of transposons between species by viruses. Recombinant NPV will be constructed carrying autonomous selectable P-element constructs that have been generated under the previous award period. These recombinant NPVs will be used to analyze potential vectoring of transposons into D. melanogaster cells or embryos. The proposed research will provide a more complete understanding of the significance of transposon mutagenesis in the evolution of viruses and their hosts, and will advance the development of a unique in vitro experimental system for molecular analysis of transposition and genetic recombination in higher eucaryotes.