TRANSPOSON MUTAGENESIS OF NUCLEAR POLYHEDROSIS VIRUSES

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Transposition is a form of genetic rearrangement common among both prokaryotic and eukaryotic organisms. It likely involves recombinational mechanisms that are probably active in many types of genome rearrangements including T-cell receptor and immunoglobulin gene rearrangements, gene amplification, gene disruption, and oncogene activation. The consequences of transposon mobilization for the evolution of higher organisms and/or eukaryotic viruses could be substantial. The FP mutants of nuclear polyhedrosis viruses (NPVs) provide a unique experimental system for molecular analysis of key aspects of transposon movement. Many of these spontaneous mutants arise through the insertion of host transposons within a 500 bp region of the viral genome 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, lacZ fusion genetagged 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 two closely related 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. We will also introduce recombinant NPV carrying various P-element constructs into Drosophila melanogaster cell cultures and embryos to analyze the potential for vectoring of transposons between species by viruses. The PI has made significant contributions in understanding the genetics of FP mutants and is now ready to develop this unique system as an experimental model for eukaryotic transposition phenomena. The PI has completed one three year grant and is beginning a five year continuation grant on this subject, and should not yet be considered an established investigator. This award will allow the PI to direct efforts towards establishing his lab as a focus of research on transposon genetics. The University is committed to developing a vigorous research program in Molecular Genetics. The PI is the first tenured Molecular Geneticist at this institution and is responsible in large part for the further development of the research program. This award will enhance the PIs ability to take a more direct role in training graduate students and participating in the research development of the newer faculty in this program.