BACKGROUND 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. RESULTS 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. CONCLUSION 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.

中文翻译：

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对 piggyBac 转座酶催化核心必不可少的高度保守的天冬氨酸残基的突变分析。

背景 PiggyBac 移动元件作为用于许多真核生物转基因的工具正在迅速普及。通过研究催化 piggyBac 运动的转座酶，我们可能能够修改该载体系统，使其成为更有效的转基因工具。在之前的出版物中，Sarkar A、Sim C、Hong YS、Hogan JR、Fraser MJ、Robertson HM 和 Collins FH 提出了在 PiggyBac 的氨基酸位置 D268、D346 和D447。结果 本研究利用定向诱变和基于质粒的迁移测定来评估这些残基作为 piggyBac 转座酶催化核心的重要性。我们已经在功能上分析了“DDD”的所有三个建议残基中关于电荷和物理大小的单个点突变 基序以及附近的另一个高度保守的天冬氨酸在 D450。我们所有的突变对 S2 细胞培养物中的切除频率都有显着影响。我们还将 piggyBac 转座酶与其他功能性和非功能性的近亲家族成员进行了比对，以尝试识别最高度保守的区域并定位一些有趣的特征。结论 我们发现所有指定的 DDD 天冬氨酸都存在于 PiggyBac 家族转座酶成员之间保守的氨基酸簇中。我们的结果表明，所有四种天冬氨酸在某种程度上都是细胞环境中发生切除所必需的，但 D450 似乎对谷氨酸替代具有耐受性。与野生型转座酶相比，所有测试的突变体都显着降低了细胞培养物中的切除频率。