PiggyBac-ing on a primate genome: novel elements, recent activity and horizontal transfer

Abstract

To better understand the extent of Class II transposable element activity in mammals, we investigated the mouse lemur, Microcebus murinus, whole genome shotgun (2X) draft assembly. Analysis of this strepsirrhine primate extended previous research that targeted anthropoid primates and found no activity within the last 37 Myr. We tested the hypothesis that members of the piggyBac Class II superfamily have been inactive in the strepsirrhine lineage of primates during the same period. Evidence against this hypothesis was discovered in the form of three nonautonomous piggyBac elements with activity periods within the past 40 Myr and possibly into the very recent past. In addition, a novel family of piggyBac transposons was identified, suggesting introduction via horizontal transfer. A second autonomous element was also found with high similarity to an element recently described from the little brown bat, Myotis lucifugus, further implicating horizontal transfer in the evolution of this genome. These findings indicate a more complex history of transposon activity in mammals rather than a uniform shutdown of Class II transposition, which had been suggested by analyses of more common model organisms.

FIG. 1.—

Search strategy to identify piggyBac elements in the Microcebus murinus draft assembly. Initial search programs are shown in rectangles, and methods used to process all output are shown in ovals. For BlastN analyses, up to 40 hits were extracted with flanking sequence and used with MUSCLE to generate a consensus; the process was repeated to extend flanks until TIRs, and nonhomologous flanking sequences were observed.

FIG. 2.—

Summary of comparative analyses to determine lineage specificity of selected elements. Individual piggyBac insertion loci recovered from Microcebus murinus were used as probes to query the Otolemur garnettii WGS and also to design primers for PCR-based analyses of Lemur catta, Cheirogaleus medius, and M. murinus (fig. 7). Additionally, multiple primer combinations were designed to amplify the piggyBac1_Mm ORF as per figure 8.

FIG. 3.—

Schematic of piggyBac2_ML from Myotis lucifugus (top) and three similar piggyBac elements from Microcebus murinus. Deletions and duplications relative to M. lucifugus are indicated for any difference greater than 3 bp. The 1,752-bp ORF is shown for M. lucifugus in lighter shading.

FIG. 4.—

Portion of an amino acid alignment of piggyBac1_Mm and other representative piggyBac elements. The alignment includes the Trichoplusia ni element that has been shown to catalyze transposition. Conserved motifs among the transposase sequences are shaded. Numbers and arrows indicate amino acid residue positions in the presumed piggyBac1_Mm ORF that is described in the text. The complete alignment is available as Supplementary Material online.

FIG. 5.—

Results of ORF phylogenetic analysis. Terminal nodes for all known piggyBac transposases are consensus sequences from RepBase (element name followed by genus in which it was identified) or GenBank (accession number followed by genus in which it was identified). Consensus sequences for piggyBac1_Mm and piggyBac2_Mm (boxed) were generated as described in the text.

FIG. 6.—

Histogram showing element frequency over estimated age distributions for the nonautonomous piggyBac TEs. The presumed dates of the Microcebus/Cheirogaleus, Microcebus/Lemur, and Microcebus/Otolemur divergences are indicated by white, gray, and black arrows, respectively.

FIG. 7.—

Example alignment of a mouse lemur-specific Class II insertion. The WGS contig sequence is at the top with comparisons with experimentally derived sequences from Microcebus murinus, Cheirogaleus medius, and Lemur catta below. The bottom sequence is the consensus of npiggy1_Mm. TIRs are underlined, and TSDs are shaded.

FIG. 8.—

PCR amplification of piggyBac1_Mm ORF fragments from lemuriform primates. At the bottom of the figure, relative primer locations are provided on a simplified map of piggyBac1_Mm.