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

玉米中的活化/解离转座因子（Activator/Dissociation, Ac/Ds）被广泛用作许多植物物种的诱变和基因分离工具。然而，转位频率变化很大，在一些物种中，它们对于大规模的基因标记方法来说太低了。本项目旨在阐明尚不清楚的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)我们之前已经证明，浓度依赖性转座酶（TPase）组装成活性低聚物或非生产性聚集体在调节Ac/Ds转座中起关键作用。为了更好地理解这些过程，我们想要确定TPase的催化位点和蛋白质相互作用域。(2)在酵母Ac的初步实验中，我们发现了TPase活性增加的突变体。我们希望研究高活性的生化机制，并产生具有更高活性的TPase突变体。(3)在早期的实验中，TPase的活性是通过植物原生质体的瞬时切除测定来估计的。为了更可靠地确定转基因植物的切除和正向诱变活性，将建立新的转基因植物检测方法。(4)我们将研究Ac/Ds在人类细胞插入诱变中的适用性。

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.