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.

Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation.

In vertebrates, BMP signaling before gastrulation suppresses neural development. Later in development, BMP signaling specifies a dorsal and ventral fate in the forebrain and dorsal fate in the spinal cord. It is therefore possible that a change in the competence of the ectoderm to respond to BMP signaling occurs at some point in development. We report that exposure of the anterior neural plate to BMP4 before gastrulation causes suppression of all neural markers tested. To determine the effects of BMP4 after gastrulation, we misexpressed BMP4 using a Pax-6 promoter fragment in transgenic frog embryos and implanted beads soaked in BMP4 in the anterior neural plate. Suppression of most anterior neural markers was observed. We conclude that most neural genes continue to require suppression of BMP signaling into the neurula stages. Additionally, we report that BMP4 and BMP7 are abundantly expressed in the prechordal mesoderm of the neurula stage embryo. This poses the paradox of how the expression of most neural genes is maintained if they can be inhibited by BMP signaling. We show that at least one gene in the anterior neural plate suppresses the response of the ectoderm to BMP signaling. We propose that the suppressive effect of BMP signaling on the expression of neural genes coupled with localized suppressors of BMP signaling result in the fine-tuning of gene expression in the anterior neural plate.

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.

DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product.

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-stranded break repair and V(D)J recombination, comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit, DNA-PKcs. Here, we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays, however, indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly, DNA-PKcs is most similar to PI kinase family members involved in cell cycle control, DNA repair, and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p, Tor2p, and FRAP, S. pombe rad3, and the product of the ataxia telangiectasia gene, mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action.

Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8.

The DNA-activated serine/threonine protein kinase (DNA-PK) is composed of a large (approximately 460 kDa) catalytic polypeptide (DNA-PKcs) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PKcs to DNA. A 41-kbp segment of the DNA-PKcs gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PKcs gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells.