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.
