Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues
University of Notre Dame, Notre Dame, Indiana, USA
BMC Molecular Biology 2008, 9:72 doi:10.1186/1471-2199-9-72Published: 11 August 2008
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).
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.
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.