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Endogenous Tc1/mariner transposons in Xenopus

Tc1-like elements (TLEs) belong to the Tc1-mariner superfamily of transposable elements that uses a cut and paste mechanism involving a DNA molecule intermediate to move. TLEs mobility properties make them potential tools for transgenesis and mutagenesis in various animal models. Native elements such as Minos or artificial elements reconstructed by molecular engineering such as Sleeping Beauty and Frog Prince are transpositionally active.

Because of this possibility to use them as transgenesis tools, we wanted to have a better overview of the endogenous TC1-Mariner of the two Xenopus species we use as developmental model organisms: Xenopus laevis and Xenopus tropicalis. The other reason of such a study resides in the fact that an engineered transposon used in a living organism could be submitted to regulatory interactions from endogenous active transposons.

A number of distinct TLE lineages were shown to co-exist in zebrafish, salmon and amphibian. In Xenopus laevis, two lineages, TXr and TXz, have been described and characterized. TXr is closely related to the teleost fish transposons Tzf/Tdr and TXz is not related to any TLEs described so far. In X. tropicalis, three types of transposase ORF sequences were found using degenerated PCR. However, these sequences were not representative of full-length TLE in this species.

In this study, we report the first identification of several diverse TLE lineages within the genome of X. tropicalis.

We have used genomic sequencing data extracted from the first assembly of the Xenopus tropicalis genome combined with a degenerated PCR approach to identify multiple lineages of Tc1 related transposable elements. Full-length elements were isolated in each lineage and characterized. Most of them exhibit the typical characteristics of Tc1-like elements (TLEs). An open reading frame (ORF) encoding a 340-350 aa transposase containing a [D, D(34)E] signature was found as well as conserved inverted terminal repeats (ITRs) at each extremities. These ITRs could vary in length, depending on the TLE lineage.

These new TLEs were named Eagle, Froggy, Jumpy, Maya, Xeminos, XtTXr and XtTXz. Phylogenetic analyses indicate that their closest relatives are present in the genomes of actinopterygian and amphibian. Interestingly, Maya and Xeminos share remarkable characteristics. Maya contains a [D,D(36)E] motif but is not related to any described TLE so far. Xeminos is the first vertebrate TLE strongly related to an invertebrate lineage. Finally, we have identified for most of these TLEs, copies containing an intact transposase ORF suggesting that these elements may still be active.

Mariner-like elements (MLEs) belong to the Tc1-mariner superfamily of DNA transposons, which is very widespread in animal genomes. We report here the first complete description of a MLE, Xtmar1, within the genome of a poikilotherm vertebrate, the amphibian Xenopus tropicalis. A close relative, XMLE, is also characterized within the genome of a sibling species, Xenopus laevis. The phylogenetic analysis of the relationships between MLE transposases reveals that Xtmar1 is closely related to Hsmar2 and Bytmar1 and that, together they form a second distinct lineage of the irritans subfamily. All members of this lineage are also characterized by the 36-43 bp size of their imperfectly conserved ITRs, and by the-8 bp motif located at their outer extremity.

Taking into account the fact that Xtmar1, Xlmar1 and Hsmar2 are present in species located at both extremities of the vertebrate evolutionary tree, MLE relatives belonging to the same subfamily were searched for in the available sequencing projects using the amino acid consensus sequence of the Hsmar2 transposase as an in-silico probe. We found that irritans MLEs are present in chordate genomes including most craniates. This therefore suggests that these elements have been present within chordate genomes for 750 Myr and that their main mode of maintenance in these species has been via vertical transmission. The very small number of stochastic losses observed in the data available suggests that their inactivation during evolution has been very slow.

Transposition events of endogenous TLEs such as Tzf transposons have been described in zebrafish genome. This transposition occurs at a frequency detectable in a single generation. The analysis of genomic sequences with intact ORFs raises the question of their activity in X. tropicalis genome. Future work will address the possibility of current active transposition of TLEs used to develop transformation tools for the genetic manipulation of X. tropicalis.

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André MAZABRAUD, (Group Leader)
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