The Activator (Ac) element from maize: transposition mechanism and regulation and advancement as a mutagenesis tool

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The Activator/Dissociation (Ac/Ds) transposable elements from maize are extensively used as mutagenesis and gene isolation tools in many plant species. However, the transposition frequencies vary widely and in some species they are too low for large scale gene tagging approaches. This project aims to elucidate the still poorly understood regulation and transposition mechanism of Ac/Ds, to improve its controllability and to extend the applicability of Ac/Ds as mutagenesis tool in vertebrate cells. (1) We previously demonstrated that the concentration-dependent transposase (TPase) assembly into either active oligomers or non-productive aggregates plays a critical role in regulating Ac/Ds transpositon. To better understand these processes we want to identify the TPase catalytic site and protein interaction domains. (2) In preliminary experiments with Ac in yeast we identified TPase mutants with increased activity. We want to investigate the biochemical mechanism of hyperactivity and generate TPase mutants with even higher activity. (3) In earlier experiments TPase activity was estimated by using transient excision assays in plant protoplasts. To more reliably determine the excision and forward mutagenesis activity, novel assays in transgenic plants will be established. (4) We will investigate the suitability of Ac/Ds for insertion mutagenesis in human cells.