A potentially functional mariner transposable element in the protist Trichomonas vaginalis

Abstract

Mariner transposable elements encoding a D,D34D motif-bearing transposase are characterized by their pervasiveness among, and exclusivity to, animal phyla. To date, several hundred sequences have been obtained from taxa ranging from cnidarians to humans, only two of which are known to be functional. Related transposons have been identified in plants and fungi, but their absence among protists is noticeable. Here, we identify and characterize Tvmar1, the first representative of the mariner family to be found in a species of protist, the human parasite Trichomonas vaginalis. This is the first D,D34D element to be found outside the animal kingdom, and its inclusion in the mariner family is supported by both structural and phylogenetic analyses. Remarkably, Tvmar1 has all the hallmarks of a functional element and has recently expanded to several hundred copies in the genome of T. vaginalis. Our results show that a new potentially active mariner has been found that belongs to a distinct mariner lineage and has successfully invaded a nonanimal, single-celled organism. The considerable genetic distance between Tvmar1 and other mariners may have valuable implications for the design of new, high-efficiency vectors to be used in transfection studies in protists.

Figure 1.

Tvmar1 structure and sequence. a) Schematic comparison of Mos1, Himar1 and Tvmar1 elements. ITRs (arrow heads), ORF (open box), helix-turn-helix motif (hatched box), aspartic acid residues (D) that form the D,D34D catalytic motif, putative NLS (dotted boxes), and first seven methionine residues in the Tvmar1-encoded ORF (vertical lines) are shown. b) Sequence of the Tvmar1 element isolated from strain G3, with translation of the longest ORF. The Tvmar1 ORF is aligned to the two active mariner elements, Mos1 from Drosophila mauritiana (gi 84871) and Himar1 from Haematobia irritans (Lampe, Churchill, and Robertson 1996), and to two mariner elements from Homo sapiens, the host of Trichomonas vaginalis, Hsmar1 (gi 1263080) and Hsmar2 (gi 1698454). Inverted terminal repeats (ITRs) are underlined. Positions at which the Tvmar1 ORF sequence is identical to a consensus of the five transposases are shown in bold and highlighted. Positions of the aspartic acid residues in the D,D34D active site of mariner transposases are marked with ‘+’ below the alignment.

Figure 1.

Tvmar1 structure and sequence. a) Schematic comparison of Mos1, Himar1 and Tvmar1 elements. ITRs (arrow heads), ORF (open box), helix-turn-helix motif (hatched box), aspartic acid residues (D) that form the D,D34D catalytic motif, putative NLS (dotted boxes), and first seven methionine residues in the Tvmar1-encoded ORF (vertical lines) are shown. b) Sequence of the Tvmar1 element isolated from strain G3, with translation of the longest ORF. The Tvmar1 ORF is aligned to the two active mariner elements, Mos1 from Drosophila mauritiana (gi 84871) and Himar1 from Haematobia irritans (Lampe, Churchill, and Robertson 1996), and to two mariner elements from Homo sapiens, the host of Trichomonas vaginalis, Hsmar1 (gi 1263080) and Hsmar2 (gi 1698454). Inverted terminal repeats (ITRs) are underlined. Positions at which the Tvmar1 ORF sequence is identical to a consensus of the five transposases are shown in bold and highlighted. Positions of the aspartic acid residues in the D,D34D active site of mariner transposases are marked with ‘+’ below the alignment.

Figure 2.

Alignment of 5' and 3' inverted terminal repeats (ITRs) of mariner elements. The internal segment of the transposable elements is abbreviated with two forward slashes (//). Completely conserved positions (black box) and those conserved in all but one sequence (grey box) are highlighted. The elements represented are Tvmar1, from T. vaginalis, Mos1, from D. mauritiana, Himar1, from H. irritans, Hsmar1 and Hsmar2, both from H. sapiens, AhMLE, from the moth Adoxophyes honmai (gi 15982565), Cpmar1 from the green lacewing Chrysoperla plorabunda (gi 156617), Ammar1 from the bee Apis mellifera (gi 27465074), and Cemar1 from Caenorhabditis elegans (Robertson and Asplund 1996)

Figure 3.

Phylogenetic position of Tvmar1 (GenBank Accession No. AY282463) in the (IS630/Tc1/mariner superfamily. mariner subfamilies and related transposons (Tc1, ItmD37E and plant mariner-like elements) are shown. Elements are identified by host name and GenInfo Identifier (gi). One of two most parsimonious trees is presented, which differ only in the resolution of the D. mauritiana-D. simulans-D. teissieri triad. Percent bootstrap support for transposon families, mariner subfamilies and deeper branches is shown (parsimony and neighbor-joining values above and below the branches, respectively). Resolution of deep branches agrees with previous analyses (Shao and Tu 2001; Robertson 2002).

Figure 4.

Sequence logo of the regions flanking Tvmar1 insertions. The 25 nucleotides that precede (a) or follow (b) each Tvmar1 copy are represented in each logo. The vertical axis is a measure of sequence information, has a maximum value of 2, and is proportional to the level of sequence conservation at each position.

Figure 5.

Tvmar1 activity and distribution. a) Northern blot autoradiograph showing a single ∼1Kb transcript identified after hybridization of Tvmar1 to polyA mRNA of T. vaginalis strain G3. b) Autoradiograph of a Southern blot of total genomic DNA extracted from T. vaginalis strains B7RC2, T1, C1, with a control lane of G3, and hybridized with Tvmar1 from G3.

Figure 6.

Distribution of Tvmar1 among species of Trichomonad, as detected by Southern blot hybridization. TV: Trichomonas vaginalis G3 strain (Kent, UK); TT: Trichomonas tenax (ATCC30207); PH: Pentatrichomonas hominis (Hs-3:NIH); TF: Tritrichomonas foetus (ATCC30924); DH: Ditrichomonas honibergii (ATCC50322); PK: Pseudotrichomonas keilini Bishop (ATCC50321). Genomic DNA was digested with DraI; TT and PH were additionally digested with BamHI. a) The absence of signal after 1 hour exposure in all lanes except T. vaginalis suggest that close relatives of Tvmar1 are absent from the other Trichomonad species surveyed. b) The results of a prolonged exposure at low stringency levels suggest that if relatives of Tvmar1 exist in other Trichomonads, they are only very distant homologs to the T. vaginalis element.