A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture

Abstract

Chromosome structure and function are influenced by transposable elements, which are mobile DNA segments that can move from place to place. hAT elements are a superfamily of DNA cut and paste elements that move by excision and integration. We have characterized two hAT elements, TcBuster and Space Invaders (SPIN), that are members of a recently described subfamily of hAT elements called Buster elements. We show that TcBuster, from the red flour beetle Tribolium castaneum, is highly active in human cells. SPIN elements are currently inactive elements that were recently highly active in multiple vertebrate genomes, and the high level of sequence similarity across widely diverged species and patchy phylogenetic distribution suggest that they may have moved between genomes by horizontal transfer. We have generated an intact version of this element, SPIN ON , which is highly active in human cells. In vitro analysis of TcBuster and SPIN ON shows that no proteins other than transposase are essential for recombination, a property that may contribute to the ability of SPIN to successfully invade multiple organisms. We also analyze the target site preferences of de novo insertions in the human genome of TcBuster and SPIN ON and compare them with the preferences of Sleeping Beauty and piggyBac, showing that each superfamily has a distinctive pattern of insertion. The high-frequency transposition of both TcBuster and SPIN ON suggests that these transposon systems offer powerful tools for genome engineering. Finally, we describe a Saccharomyces cerevisiae assay for TcBuster that will provide a means for isolation of hyperactive and other interesting classes of transposase mutants.hAT element | target site selection | gene therapy | insertional mutagenesis | transgenesis

Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon

“…We observed a comparable degree of target specificity for insect piggyBac in an analysis of about 13,500 de novo insertions in the human genome (17). One of the non-TTAA insertions in yeast, CTAA, is the same as the most common non-TTAA insertions observed for the insect piggyBac.…”

“…We found that 92.8% of the bat piggyBat sites had a flanking TTAA, which is slightly lower than the 96% observed with insect piggyBac in the human colorectal cancer line HCT116 cells (18) and the 98% observed with insect piggyBac in HeLa cells (17). The distribution of piggyBat and insect piggyBac insertions with respect to particular human chromosomal features was also similar (P = 0.11, χ 2 test; Fig.…”

“…The insect piggyBac helper plasmid contained the piggyBac ORF cloned into the EcoRI/NotI site of pcDNA3.1 myc His A-His (Invitrogen). These DNAs have been previously described (17).…”

Sleeping Beauty Transposition

“…9), which are molecular hallmarks of SB transposition. At very low frequencies, non-TA target sites were also found at insertions generated by SB100X (115,119). About 75% of SB transposon excision events are coupled to chromosomal integration (93) and no extrachromosomal, excised molecules are readily detectable.…”

“…9). SB transposition almost exclusively occurs at TA dinucleotides (5,114,115,116,117,118), and SB integrants therefore are flanked by TA TSDs (Fig. 9), which are molecular hallmarks of SB transposition.…”