Phylogenetic and functional characterization of the hAT transposon superfamily

Abstract

Transposons are found in virtually all organisms and play fundamental roles in genome evolution. They can also acquire new functions in the host organism and some have been developed as incisive genetic tools for transformation and mutagenesis. The hAT transposon superfamily contains members from the plant and animal kingdoms, some of which are active when introduced into new host organisms. We have identified two new active hAT transposons, AeBuster1, from the mosquito Aedes aegypti and TcBuster from the red flour beetle Tribolium castaneum. Activity of both transposons is illustrated by excision and transposition assays performed in Drosophila melanogaster and Ae. aegypti and by in vitro strand transfer assays. These two active insect transposons are more closely related to the Buster sequences identified in humans than they are to the previously identified active hAT transposons, Ac, Tam3, Tol2, hobo, and Hermes. We therefore reexamined the structural and functional relationships of hAT and hAT-like transposase sequences extracted from genome databases and found that the hAT superfamily is divided into at least two families. This division is supported by a difference in target-site selections generated by active transposons of each family. We name these families the Ac and Buster families after the first identified transposon or transposon-like sequence in each. We find that the recently discovered SPIN transposons of mammals are located within the family of Buster elements.

Figure 1.—

Consensus phylogenetic tree showing the relationship of amino acid transposase sequences, between 50 selected full-length hAT elements based on a maximum-likelihood optimality criterion (50% majority rule consensus). Numbers next to most nodes show quartet puzzling reliability based on 10,000 puzzling steps, a measure of nodal support similar to bootstrapping, produced by the program TREE-PUZZLE. Nodal support inside the SPIN, AeBuster3, SPIN_Ml_a clade is not shown for purposes of clarity. The shaded areas indicate the proposed division of the hAT superfamily into the Buster and Ac families. The scale bar represents a phylogenetic distance of 1 amino acid substitution per site.

Figure 2.—

Footprint sequences remaining at empty sites following excision of either AeBuster1 (A) or TcBuster (B) from donor plasmids in Ae. aegypti embryos. TSDs are underlined and the transposon is shown within the block arrows. The sequences of five empty sites arising from the excision of AeBuster1 and six empty sites arising from the excision of TcBuster are shown with additional DNA that is inserted into the empty sites shown between the TSDs. Smaller arrows show how this is related to sequences within the TSDs.

Figure 3.—

WebLogo (http://weblogo.berkeley.edu/logo.cgi) TSDs generated by AeBuster 1 and TcBuster in developing embryos of Ae. aegypti and D. melanogaster.

Figure 4.—

Strand transfer reactions using precleaved left and right AeBuster1 ends.5′-end-labeled 40mer oligonucleotides containing the L and R ends with their 16-bp TIRs were incubated with AeBuster1 transposase and a pUC19 target DNA and then displayed on an agarose gel. In a single end join (SEJ), a single transposon end oligonucleotide is joined to the target plasmid; in a double end join (DEJ), to transposon ends oligonucleides join to the target DNA at the same position, linearizing the plasmid. Lane 1, left end–no transposase; lane 2, left end–plus transposase; lane 3, right end–no transposase; lane 4, right end–plus transposase. All the reactions shown were run on the same gel and are from the same gel image but have been cropped and arranged for easier viewing.