Identification of the pre-T-cell receptor alpha chain in nonmammalian vertebrates challenges the structure-function of the molecule.

In humans and mice, the early development of αß T cells is controlled by the pre-T-cell receptor α chain (pTα) that is covalently associated with the T-cell receptor ß (TCRß) chain to form the pre-T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand-independent manner through self-oligomerization mediated by pTα. Using in silico and gene synteny-based approaches, we identified the pTα gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is remarkably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pTα functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pTα cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pTα was exclusively to enable expression of the TCRß chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pTα signaling.

Characterization of a novel Xenopus tropicalis cell line as a model for in vitro studies.

Cell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was ~24 h. Its karyotype was genetically stable with a chromosome number of 2n = 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized "Speedy" should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model.

Transposition of a reconstructed Harbinger element in human cells and functional homology with two transposon-derived cellular genes.

Ancient, inactive copies of transposable elements of the PIF/Harbinger superfamily have been described in vertebrates. We reconstructed components of the Harbinger3_DR transposon in zebrafish, including a transposase and a second, transposon-encoded protein that has a Myb-like trihelix domain. The reconstructed Harbinger transposon shows efficient cut-and-paste transposition in human cells and preferentially inserts into a 15-bp consensus target sequence. The Myb-like protein is required for transposition and physically interacts with the N-terminal region of the transposase via its C-terminal domain. The Myb-like protein enables transposition in part by promoting nuclear import of the transposase, by directly binding to subterminal regions of the transposon, and by recruiting the transposase to the transposon ends. We investigated the functions of two transposon-derived human proteins: HARBI1, a domesticated transposase-derived protein, and NAIF1, which contains a trihelix motif similar to that described in the Myb-like protein. Physical interaction, subcellular localization, and DNA-binding activities of HARBI1 and NAIF1 suggest strong functional homologies between the Harbinger3_DR system and their related, host-encoded counterparts. The Harbinger transposon will serve as a useful experimental system for transposon biology and for investigating the enzymatic functions of domesticated, transposon-derived cellular genes.

The ancient mariner sails again: transposition of the human Hsmar1 element by a reconstructed transposase and activities of the SETMAR protein on transposon ends.

Hsmar1, one of the two subfamilies of mariner transposons in humans, is an ancient element that entered the primate genome lineage approximately 50 million years ago. Although Hsmar1 elements are inactive due to mutational damage, one particular copy of the transposase gene has apparently been under selection. This transposase coding region is part of the SETMAR gene, in which a histone methylatransferase SET domain is fused to an Hsmar1 transposase domain. A phylogenetic approach was taken to reconstruct the ancestral Hsmar1 transposase gene, which we named Hsmar1-Ra. The Hsmar1-Ra transposase efficiently mobilizes Hsmar1 transposons by a cut-and-paste mechanism in human cells and zebra fish embryos. Hsmar1-Ra can also mobilize short inverted-repeat transposable elements (MITEs) related to Hsmar1 (MiHsmar1), thereby establishing a functional relationship between an Hsmar1 transposase source and these MITEs. MiHsmar1 excision is 2 orders of magnitude more efficient than that of long elements, thus providing an explanation for their high copy numbers. We show that the SETMAR protein binds and introduces single-strand nicks into Hsmar1 inverted-repeat sequences in vitro. Pathway choices for DNA break repair were found to be characteristically different in response to transposon cleavage mediated by Hsmar1-Ra and SETMAR in vivo. Whereas nonhomologous end joining plays a dominant role in repairing excision sites generated by the Hsmar1-Ra transposase, DNA repair following cleavage by SETMAR predominantly follows a homology-dependent pathway. The novel transposon system can be a useful tool for genome manipulations in vertebrates and for investigations into the transpositional dynamics and the contributions of these elements to primate genome evolution.

Factors acting on Mos1 transposition efficiency.

BACKGROUND: Mariner-like elements (MLEs) are widespread DNA transposons in animal genomes. Although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the MLEs transposition in vivo. RESULTS: The transposition of Mos1, first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first in vitro experimental demonstration that MITEs of MLE origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates Mos1 transposition, yielding excision products unable to re-integrate. Finally, the super-helicity of the DNA transposon donor has a dramatic impact on the transposition efficiency. CONCLUSION: The study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. In vitro, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. We propose a model for small transposons transposition that bypasses DNA bending constraints.

Characterization of multiple lineages of Tc1-like elements within the genome of the amphibian Xenopus 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 were 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.

Dual and Opposite Effects of hRAD51 Chemical Modulation on HIV-1 Integration.

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication.

Nuclear importation of Mariner transposases among eukaryotes: motif requirements and homo-protein interactions.

Mariner-like elements (MLEs) are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS) in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174), and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm.

Database of queryable gene expression patterns for Xenopus.

The precise localization of gene expression within the developing embryo, and how it changes over time, is one of the most important sources of information for elucidating gene function. As a searchable resource, this information has up until now been largely inaccessible to the Xenopus community. Here, we present a new database of Xenopus gene expression patterns, queryable by specific location or region in the embryo. Pattern matching can be driven either from an existing in situ image, or from a user-defined pattern based on development stage schematic diagrams. The data are derived from the work of a group of 21 Xenopus researchers over a period of 4 days. We used a novel, rapid manual annotation tool, XenMARK, which exploits the ability of the human brain to make the necessary distortions in transferring data from the in situ images to the standard schematic geometry. Developmental Dynamics 238:1379-1388, 2009. (c) 2009 Wiley-Liss, Inc.

Rapid gynogenetic mapping of Xenopus tropicalis mutations to chromosomes.

Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations. Here we present a simple method for mapping mutations to chromosomes using gynogenesis and centromeric markers. When coupled with available genomic resources, gross mapping facilitates evaluation of candidate genes as well as higher resolution linkage studies. Using gynogenesis, we have mapped the genetic locations of the 10 X. tropicalis centromeres, and performed fluorescence in situ hybridization to validate these locations cytologically. We demonstrate the use of this very small set of centromeric markers to map mutations efficiently to specific chromosomes. Developmental Dynamics 238:1398-1406, 2009. (c) 2009 Wiley-Liss, Inc.

The mariner transposons belonging to the irritans subfamily were maintained in chordate genomes by vertical transmission.

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, XlMLE, 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- to 43-bp size of their imperfectly conserved inverted terminal repeats and by the -8-bp motif located at their outer extremity. Since XlMLE, Xlmar1, and Hsmar2 are present in species located at both extremities of the vertebrate evolutionary tree, we looked for MLE relatives belonging to the same subfamily 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 the main way they have been maintained 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.

Survivin increased vascular development during Xenopus ontogenesis.

Survivin is a member of the inhibitor of apoptosis proteins (IAP) family. These proteins contain one to three zinc-binding motifs termed bacculoviral IAP-binding repeats (BIRs). Survivin contains a single BIR motif. Contrary to other members that directly interact with caspases and inhibit apoptosis, Survivin is believed to have both antiapoptotic and proliferative functions. In mammals, Survivin is not detected in most adult tissues except in endothelial cells of newly formed capillaries and large blood vessels. Importantly, Survivin is highly expressed in all common human cancers. To gain a better view of Survivin expression and function during development, we used the amphibian Xenopus developmental model. We show that the genomes of X. laevis, X. tropicalis, Zebrafish, fugu pufferfish, and rainbow trout encode two different Survivin genes (Su1 and Su2), contrary to mammalian genomes, which encode a single one. In X. laevis, these two genes have a differential spatiotemporal transcription pattern. Transgenic expression of Su1 leads to an enlargement of tadpole's blood vessels with an increase in the number of endothelial cells. This effect requires a functional BIR domain and the p34/cdc2 phosphorylation site. It does not seem to rely on the antiapoptotic activity of Su1 as it is not observed in tadpoles overexpressing other antiapoptotic factors such as XIAP or BclXL. We conclude that Su1 ubiquitous gain of function leads directly or indirectly to an increase in blood vessels size via the proliferation of endothelial cells.