Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells
Key findings
A functional Tc1-like transposon (Sleeping Beauty, SB) was reconstructed from inactive salmonid fish transposable elements through iterative PCR-based mutagenesis. The reconstruction involved correcting mutations in transposase coding sequences from Tss1.1, Tss1.2, and Tsg1 elements across 10 iterative steps (SB1-SB10), creating a consensus open reading frame with restored catalytic activity.
SB10 transposase demonstrated functional transposition activity in human HeLa cells, generating G418-resistant colonies when co-transfected with a neomycin resistance-marked transposon donor construct (pT/neo). Integration assays showed genuine transposition events with characteristic target site duplications (TA dinucleotides) at insertion junctions, confirming cut-and-paste transposition mechanism.
Recombinant N-terminal SB transposase fragment (N123, first 123 amino acids) exhibited specific DNA-binding activity to transposon inverted repeat sequences in gel shift and DNaseI footprinting assays. The transposase recognized 12 bp direct repeat (DR) core motifs within 30 bp binding sites at transposon termini, with binding specificity determined by sequences flanking the DR motifs.