BACKGROUND The piggyBac transposable element is a popular tool for germ-line transgenesis of eukaryotes. Despite this, little is known about the mechanism of transposition or the transposase (TPase) itself. A thorough understanding of just how piggyBac works may lead to more effective use of this important mobile element. A PSORTII analysis of the TPase amino acid sequence predicts a bipartite nuclear localization signal (NLS) near the c-terminus, just upstream of a putative ZnF (ZnF). RESULTS We fused the piggyBac TPase upstream of and in-frame with the enhanced yellow fluorescent protein (EYFP) in the Drosophila melanogaster inducible metallothionein protein. Using Drosophila Schneider 2 (S2) cells and the deep red fluorescent nuclear stain Draq5, we were able to track the pattern of piggyBac localization with a scanning confocal microscope 48 hours after induction with copper sulphate. CONCLUSION Through n and c-terminal truncations, targeted internal deletions, and specific amino acid mutations of the piggyBac TPase open reading frame, we found that not only is the PSORTII-predicted NLS required for the TPase to enter the nucleus of S2 cells, but there are additional requirements for negatively charged amino acids a short length upstream of this region for nuclear localization.

中文翻译：

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piggyBac 转座酶的分析揭示了 94 个 c 端残基中的功能性核靶向信号。

背景 PiggyBac 转座因子是用于真核生物种系转基因的流行工具。尽管如此，对转座机制或转座酶 (TPase) 本身知之甚少。彻底了解 piggyBac 的工作原理可能会导致更有效地使用这一重要的移动元素。TPase 氨基酸序列的 PSORTII 分析预测在 c 端附近有一个二分核定位信号 (NLS)，就在推定的 ZnF (ZnF) 的上游。结果 我们在黑腹果蝇诱导型金属硫蛋白中将 piggyBac TPase 与增强型黄色荧光蛋白 (EYFP) 的上游和框内融合。使用果蝇施耐德 2 (S2) 细胞和深红色荧光核染色剂 Draq5，在用硫酸铜诱导 48 小时后，我们能够用扫描共聚焦显微镜跟踪 PiggyBac 定位的模式。结论通过piggyBac TPase开放阅读框的n端和c端截断、靶向内部缺失和特定氨基酸突变，我们发现TPase进入S2细胞核不仅需要PSORTII预测的NLS，而且对于核定位，该区域上游较短的带负电荷的氨基酸有额外的要求。