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.

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The DNA-activated serine/threonine protein kinase (DNA-PK) is composed of a large catalytic polypeptide (DNA-PK,,) and Ku, a heterodimeric DNA-binding component (p7O/p8O) that targets DNA-PKc, to DNA. A 41-kbp segment of the DNA-PK¢, 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-PK& gene was mapped to band qll 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.

The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines that carry ''activator'' or ''effector'' constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function 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.

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.

Summary: 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. RESULTS AND DISCUSSIONAn essential feature of any morpholino strategy is the rapidity of the dispersal of the oligonucleotide after injection into the early embryo. To assess the ability of MOs to spread from a single site of injection and be segregated upon subsequent cell divisions, we injected an FITC labelled control MO into the animal pole of one cell at the two-cell stage and examined the embryos throughout development. As shown in Figure 1b, the FITC MO was present throughout the injected half of the neurula stage embryo. This fluorescence was maintained evenly until at least stage 43 (data not shown). Therefore, MOs rapidly spread throughout the injected cell and are faithfully inherited by the progeny of that cell.To test their ability to target an integrated gene we designed a MO to inhibit the translation of GFP (gfp-MO) expressed in a number of transgenic Xenopus lines. One of the lines we used contains a single integration site of a CMV-GFP transgene (line 16 from Marsh-Armstrong, et al., 1999). The major advantage of using a transgenic line harbouring a single integration is that the expression level of all the progeny are equivalent in the progeny inheriting the transgene. We mated F1 transgenic males (heterozygous for the transgene) with wild-type females, giving rise to 50% of the F2 progeny inheriting the transgene and expressing GFP. In this work we aimed to specifically address the efficacy MO based translational inhibition of zygotic transcripts. For this reason it was not possible to use transgenic F1 females from this line as the CMV promoter is maternally expressed, resulting in a large maternal pool of GFP protein (data not shown). gfp-MO was injected into a single blastomere at the two-cell stage and the fluorescence of the embryos was assessed throughout development. In those embryos inheriting the CMV-GFP transgene (50%) gfp-MO completely inhibited the fluorescence on the injected side at doses between 1-20 ng (Fig. 1c). To more quantitatively examine the efficacy of gfp-MO in inhibiting GFP expression, we injected the morpholino at a broad concentration range into both blastomeres at the two-cell stage. Embryos were then examined throughout development for nonspecific phenotypes, GFP fluorescence, and protein levels. As GFP has no function during development, any abnormal phenotype observed in injec...

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