The goal of this project is to develop mariner transposable elements as genetic tools for arthropods, primarily insects, of medical importance. The impact of arthropod vectors of disease around the world is enormous, and novel approaches to their control are needed. These mariner transposons are potentially useful for gene tagging for cloning, for enhancer trapping, and most importantly as transformation vectors. There are currently no efficient transformation vectors available for any non- drosophilid arthropod, so ideas of developing genetic sexing or sterile male techniques are untestable. Imaginative ideas for developing vector- incompetent strains of mosquitoes are untenable without transformation systems, and mariners might even spread such desirable traits. Finally, basic research on the molecular genetics of insects is handicapped by the lack of these tools. Distantly related mariner elements were previously known from the fruit fly Drosophila mauritiana and the cecropia moth Hyalophora cecropia, and are of the short inverted terminal repeat type. The D. mauritiana element is capable of transforming D. melanogaster. Related mariner elements were discovered in other insects using PCR with degenerate primers designed to regions of amino acid conservation between the transposase genes of the fruit fly and cecropia moth elements. They are present in representative of most insect orders, including Anopheles gambiae, plus a centipede and a mite. From sequences of these PCR fragments there is evidence for recent horizontal transfer of two particular elements across orders of insects. The consensus amino acid sequence representing each of these horizontally transferred mariners is almost certain to be an active transposase that has already demonstrated functionality in the cellular environments of diverse insects. These consensus-encoding elements will be identified in species or generated by in vitro mutagenesis using PCR. In either case,""""""""marooned"""""""" constructs that have the consensus transposase gene flanked by its natural regulatory regions, but without inverted terminal repeats, will be created by PCR. These will be tested for transposase activity in Drosophila melanogaster first, by assaying ability to mobilize in trans a dominant marker gene flanked by appropriate mariner termini. Active constructs will then be tested for activity in target species of medical importance, such as various mosquitoes. In these species other dominant markers being identified by workers on particular species will be needed, and methods for mechanical introduction of DNA into embryos will have to be developed. Constructs that are active in any particular species are then immediately available as transformation vectors. In addition, if two or more distinct and potentially independent mariner transformation systems can be developed, they might be employed for gene tagging and enhancer trapping systems.
