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

玉米中的激活子/解离(Ac/DS)转座元件在许多植物物种中被广泛用作诱变和基因分离工具。然而，转座频率差异很大，在一些物种中，转座频率太低，不适合大规模的基因标记方法。本项目旨在阐明Ac/DS的调控和转位机制，提高其可控性，扩大Ac/DS作为脊椎动物细胞诱变工具的适用性。(1)我们先前的研究表明，浓度依赖的转座酶(TPase)在调节Ac/DS转座子中起着关键作用，无论是活性寡聚体还是非生产性聚合体。为了更好地理解这些过程，我们想要确定TPase的催化位置和蛋白质相互作用区域。(2)在酵母菌Ac的初步实验中，我们筛选到了活性较高的突变株。我们想要研究高活性的生化机制，并产生更高活性的TPase突变体。(3)在早期的实验中，植物原生质体中的TPase活性是通过瞬时切除试验来估计的。为了更可靠地确定切除和正向诱变活性，将在转基因植物中建立新的检测方法。(4)研究Ac/DS在人类细胞中进行插入突变的适宜性。

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.