TRANSPOSON MEDIATED MUTAGENESIS OF NP VIRUSES

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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.