Plant molecular biology is currently limited by supporting genetic technology, particularly in the model species Arabidopsis thaliana. This project aims to explore the feasibility of using two site- specific DNA enzyme systems from the yeast Saccharomyces cerevisiase to develop genetic methods in Arabidopsis based on site-specific recombination. The two systems are the FLP recombinase, derived from the yeast 2-micron plasmid, and the HO recombinase, which promotes yeast mating type switching. FLP promotes reciprocal recombination between two asymmetric FRT target sites, resulting in deletion or inversion of the intervening DNA depending on whether the two sites are in direct or inverted respectively; HO promotes gap repair by homologous recombination after cutting at its Y/Z target. Demonstration of either or both activities will allow the subsequent design of sophisticated schemes for targeted genetic events in plants. The goals for the present period are to demonstrate FLP and/or HO activity in plant systems. Available cloned genes encoding each enzyme will be placed downstream of the available strong constitutive CaMV 35S promoter, and activity of the transcribed and translated enzymes will be assayed. For FLP, assays will score deletion or inversion of a reporter GUS (escherichia coli B- glucuronidase) gene located between pair of direct or inverted FRT sites; for HO, assays will score recombination between two copies of GUS reporter gene promoted by gap repair of a Y/Z site in one of them. Assays will be carried out in both transient (electroporated tobacco and Arabidopsis protoplasts) and stable (transgenic Arabidopsis plants) configurations. *** //
