Keith James H, Schaeper Cheryl A, Fraser Tresa S, Fraser Malcolm J
University of Notre Dame, Notre Dame, Indiana, USA.
BMC Mol Biol. 2008 Aug 11;9:73. doi: 10.1186/1471-2199-9-73.
The piggyBac mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of piggyBac, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for piggyBac at amino acid positions D268, D346, and D447.
This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the piggyBac transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the piggyBac transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features.
We found all the designated DDD aspartates reside in clusters of amino acids that conserved among piggyBac family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.
作为一种用于许多真核生物转基因的工具，piggyBac移动元件正迅速受到欢迎。通过研究催化piggyBac移动的转座酶，我们或许能够对该载体系统进行改造，使其成为一种更有效的转基因工具。在之前的一篇论文中，萨卡尔A、西姆C、洪YS、霍根JR、弗雷泽MJ、罗伯逊HM和柯林斯FH提出，在氨基酸位置D268、D346和D447处存在piggyBac广泛存在的“DDE/DDD”基序。
本研究利用定向诱变和基于质粒的迁移率测定来评估这些残基作为piggyBac转座酶催化核心的重要性。我们对“DDD”基序所有三个提议残基以及D450处另一个附近高度保守的天冬氨酸的电荷和物理大小方面的单个点突变进行了功能分析。我们所有的突变对S2细胞培养物中的切除频率都有显著影响。我们还将piggyBac转座酶与其他功能和非功能的近亲成员进行了比对，试图确定最保守的区域并定位一些有趣的特征。
我们发现所有指定的DDD天冬氨酸都位于piggyBac家族转座酶成员中保守的氨基酸簇中。我们的结果表明，所有四个天冬氨酸在细胞环境中发生切除时都或多或少是必需的，但D450似乎对谷氨酸替代具有耐受性。与野生型转座酶相比，所有测试的突变体在细胞培养物中均显著降低了切除频率。
