A Hyperactive Transposase of the Maize Transposable Element Activator (Ac)
A Hyperactive Transposase of the Maize Transposable Element Activator (Ac)
复制标题
DOI：
10.1534/genetics.112.139642
复制
发表时间：
2012-07-01
期刊：
影响因子：
3.3
通讯作者：
Kunze, Reinhard
中科院分区：
文献类型：
--
作者：
Lazarow, Katina;Du, My-Linh;Kunze, Reinhard
来自玉米的激活子/解离（Ac/Ds）转座元件被广泛用作植物中的插入诱变和基因分离工具，最近还在青鳉和斑马鱼中使用。它们对于具有长世代周期或具有低基因密度的大基因组的植物物种是特别有价值的。然而，Ac/Ds转座频率变化很大，在某些物种中，它们对于大规模诱变来说太低了。我们发现了一种活性极高的Ac转座酶衍生物，AcTlase（4x），它在酵母酿酒酵母中催化比野生型转座酶更频繁100倍的Ds切除，而切除的Ds元件的重新整合频率不变（57%）。与植物中的野生型转座酶相比，AcTPase 4x催化Ds插入优先进入编码区和遗传连锁位点，但突变蛋白显然已经失去了野生型蛋白的插入位点的弱偏倚，具有升高的鸟嘌呤-胞嘧啶含量和非随机蛋白-DNA扭曲。在拟南芥中，AcTdR（4x）也表现出过度活性，其中它相对于野生型AcTdR影响Ds切除增加超过六倍，因此可能有助于促进基于Ac/Ds的插入诱变方法。
Activator/Dissociation (Ac/Ds) transposable elements from maize are widely used as insertional mutagenesis and gene isolation tools in plants and more recently also in medaka and zebrafish. They are particularly valuable for plant species that are transformation-recalcitrant and have long generation cycles or large genomes with low gene densities. Ac/Ds transposition frequencies vary widely, however, and in some species they are too low for large-scale mutagenesis. We discovered a hyperactive Ac transposase derivative, AcTPase(4x), that catalyzes in the yeast Saccharomyces cerevisiae 100-fold more frequent Ds excisions than the wild-type transposase, whereas the reintegration frequency of excised Ds elements is unchanged (57%). Comparable to the wild-type transposase in plants, AcTPase4x catalyzes Ds insertion preferentially into coding regions and to genetically linked sites, but the mutant protein apparently has lost the weak bias of the wild-type protein for insertion sites with elevated guanine-cytosine content and nonrandom protein-DNA twist. AcTPase(4x) exhibits hyperactivity also in Arabidopsis thaliana where it effects a more than sixfold increase in Ds excision relative to wild-type AcTPase and thus may be useful to facilitate Ac/Ds-based insertion mutagenesis approaches.