Biology and utilization of Hermes, a class II transposable element from the genome of the house fly, Musca domestica

The availability of germ line transformation for insects, which are important medical and agricultural pest species, offers the chance to develop new methods to disease and pest control. The focus of this dissertation is the application and biology of the Hermes transposable element, one of the four transformation systems used for genetic manipulation of non-drosophilid insects. The research presented here was conducted to (i) improve the application of Hermes for germline transformation of insects and (ii) to improve our understanding on how this element transposes in its host genome.; To improve Hermes application, a general Hermes transformation vector, pH[act5c:EGFP], was developed, that contained an autonomous marker gene under control of a highly conserved promoter. This vector was used in several insect species in which no genetic mutants are available. Additionally, Hermes was established as a gene vector for a new host, the Mediterranean fruit fly, C. capitata, an important agricultural pest.; To clarify the mechanism by which Hermes transposes the ORF of its transposase was mapped for domains facilitating the crucial functions of nuclear import, transposase dimerization, and DNA transposition. To reach its substrate, the Hermes element, the transposase must move to the host cell nucleus. Using Hermes-GFP fusion proteins expressed transiently in Schneider 2 cells, the N-terminus was found to be the only transposase region necessary for nuclear import. Based on data from a few transposable elements, binding of transposase molecules to each other is a crucial step in element mobility. Using the yeast Two-Hybrid system, a protein-interaction domain responsible for dimerization and/or oligomerization was identified in the Hermes transposase carboxyl-terminus. Its sequence and overall structure are conserved in closely related transposable elements. To test the hypothesis that Hermes has an active center referred to as a DDE signature and known for a wide variety of prokaryotic and eukaryotic transposases, inter-plasmid transposition assays using various Hermes mutants were performed. Only two of the three proposed DDE sites within the Hermes transposase appeared necessary for transposition.