Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues.
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.


CONCLUSIONS
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.
Keith
James H
JH
University of Notre Dame, Notre Dame, Indiana, USA. jkeith2@nd.edu
Fraser
Tresa S
TS
Fraser
Malcolm J
MJ
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D000880:Anthraquinones; D002478:Cells, Cultured; D004251:DNA Transposable Elements / Q000235:genetics*; D004331:Drosophila melanogaster / Q000166:cytology; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000737:chemistry*; D019895:Transposases / Q000737:chemistry* / Q000235:genetics
C520537:1,5-bis((2-(methylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione; D000880:Anthraquinones; D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D019895:Transposases