Characterization of a Xenopus tropicalis endogenous retrovirus with developmental and stress-dependent expression.

We report on the identification and characterization of XTERV1, a full-length endogenous retrovirus (ERV) within the genome of the western clawed frog (Xenopus tropicalis). XTERV1 contains all the basic genetic elements common to ERVs, including the classical 5'-long terminal repeat (LTR)-gag-pol-env-3'-LTR architecture, as well as conserved functional motifs inherent to each retroviral protein. Using phylogenetic analysis, we show that XTERV1 is related to the Epsilonretrovirus genus. The X. tropicalis genome harbors a single full-length copy with intact gag and pol open reading frames that localizes to the centromeric region of chromosome 5. About 10 full-length defective copies of XTERV1 are found interspersed in the genome, and 2 of them could be assigned to chromosomes 1 and 3. We find that XTERV1 genes are zygotically transcribed in a regulated spatiotemporal manner during frog development, including metamorphosis. Moreover, XTERV1 transcription is upregulated under certain cellular stress conditions, including cytotoxic and metabolic stresses. Interestingly, XTERV1 Env is found to be homologous to FR47, a protein upregulated following cold exposure in the freeze-tolerant wood frog (Rana sylvatica). In addition, we find that R. sylvatica FR47 mRNA originated from a retroviral element. We discuss the potential role(s) of ERVs in physiological processes in vertebrates.

The olig family: phylogenetic analysis and early gene expression in Xenopus tropicalis.

The olig genes form a small subfamily of basic helix-loop-helix transcription factors. They were discovered in 2000 as genes required for oligodendrocyte lineage specification. Since then, olig genes have been identified in various vertebrate species and corresponding sequences accumulated within genomic databases. Until now, three groups of olig genes have been characterized. Our phylogenetic analysis demonstrates the existence of a fourth group, which we named olig4. Genes of the four olig groups are present in actinopterygians and amphibians, whereas mammals only possess olig1, 2, and 3. We also found one olig gene in hemichordates, urochordates, and cephalochordates. Our expression study during Xenopus tropicalis embryogenesis shows that the four olig genes have very distinct expression patterns. Olig1 is very faintly expressed before the tadpole stage, whereas olig2, 3, and 4 are expressed from the gastrula stage onward. The olig3 expression during neurulation suggests a role in early anteroposterior patterning of the brain. All these results indicate that olig genes are involved in several developmental processes during early development.

Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis.

Morpholino (MO) based inhibition of translational initiation represents an attractive methodology to eliminate gene function during Xenopus development (Heasman et al., 2000). However, the degree to which a given target protein can be eliminated and the longevity of this effect during embryogenesis has not been documented. To examine the efficacy of MOs, we have used transgenic Xenopus lines that harbour known numbers of integrations of a GFP reporter under the control of the ubiquitous and highly expressed CMV promoter (Fig. 1a). In addition we have investigated the longevity of the inhibitory effect by using transgenic lines expressing GFP specifically in the lens of tadpoles. These transgenic lines represent the ideal control for the technique as the promoters are highly expressed and GFP can be easily detected by fluorescence and immunoblotting. Moreover, as GFP has no function in development, the levels of inhibition can be tested in an otherwise normal individual. Here we report that MOs are able to efficiently and specifically inhibit the translation of GFP in transgenic lines from Xenopus laevis and Xenopus tropicalis and the inhibitory effect is long-lived, lasting into the tadpole stages. genesis 30:110--113, 2001.

A gene trap approach in Xenopus.

The frog transgenesis technique ultimately promises to make mutagenesis possible through random insertion of plasmid DNA into the genome. This study was undertaken to evaluate whether a gene trap approach combined with transgenesis would be appropriate for performing insertional mutagenesis in Xenopus embryos. Firstly, we confirmed that the transgenic technique results in stable integration into the genome and that transmission through the germline occurs in the expected Mendelian fashion. Secondly, we developed several gene trap vectors, using the green fluorescent protein (GFP) as a marker. Using these vectors, we trapped several genes in Xenopus laevis that are expressed in a spatially restricted manner, including expression in the epiphysis, the olfactory bulb and placodes, the eyes, ear, brain, muscles, tail and intestine. Finally, we cloned one of the trapped genes using 5' rapid amplification of cDNA ends polymerase chain reaction (RACE PCR). These results suggest that the transgenic technique combined with a gene trap approach might provide a powerful method for generating mutations in endogenous genes in Xenopus.