Selectable genetic markers for nematode transgenesis

The nematode Caenorhabditis elegans has been used to study genetics and development since the mid-1970s. Over the years, the arsenal of techniques employed in this field has grown steadily in parallel with the number of researchers using this model. Since the introduction of C. elegans transgenesis, nearly 20 years ago, this system has been extensively used in areas such as rescue experiments, gene expression studies, and protein localization. The completion of the C. elegans genome sequence paved the way for genome-wide studies requiring higher throughput and improved scalability than provided by traditional genetic markers. The development of antibiotic selection systems for nematode transgenesis addresses these requirements and opens the possibility to apply transgenesis to investigate biological functions in other nematode species for which no genetic markers had been developed to date.     

Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system

Transposons have provided important genetic tools for functional genomic screens in lower eukaryotes but have proven less useful in higher eukaryotes because of their low transposition frequency. Here we show that Sleeping Beauty (SB), a member of the Tc1/mariner class of transposons, can be mobilized in mouse somatic cells at frequencies high enough to induce embryonic death and cancer in wild-type mice. Tumours are aggressive, with some animals developing two or even three different types of cancer within a few months of birth. The tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of genes and pathways that induce disease. SB transposition can easily be controlled to mutagenize any target tissue and can therefore, in principle, be used to induce many of the cancers affecting humans, including those for which little is known about the aetiology. The uses of SB are also not restricted to the mouse and could potentially be used for forward genetic screens in any higher eukaryote in which transgenesis is possible.     

Leukaemia disease genes: large-scale cloning and pathway predictions

Retroviral insertional mutagenesis in BXH2 and AKXD recombinant inbred mice induces a high incidence of myeloid or B- and T-cell leukaemia and the proviral integration sites in the leukaemias provide powerful genetic tags for disease gene identification. Some of the disease genes identified by proviral tagging are also associated with human disease, validating this approach for human disease gene identification. Although many leukaemia disease genes have been identified over the years, many more remain to be cloned. Here we describe an inverse PCR (IPCR) method for proviral tagging that makes use of automated DNA sequencing and the genetic tools provided by the Mouse Genome Project, which increases the throughput for disease gene identification. We also use this IPCR method to clone and analyse more than 400 proviral integration sites from AKXD and BXH2 leukaemias and, in the process, identify more than 90 candidate disease genes. Some of these genes function in pathways already implicated in leukaemia, whereas others are likely to define new disease pathways. Our studies underscore the power of the mouse as a tool for gene discovery and functional genomics.     

The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome

We determined the complete genome sequence of Clostridium difficile strain 630, a virulent and multidrug-resistant strain. Our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. These mobile elements are putatively responsible for the acquisition by C. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. The metabolic capabilities encoded in the genome show multiple adaptations for survival and growth within the gut environment. The extreme genome variability was confirmed by whole-genome microarray analysis; it may reflect the organism's niche in the gut and should provide information on the evolution of virulence in this organism.     

Functional impact of PTP1B-mediated Src regulation on oxidative phosphorylation in rat brain mitochondria

Given the presence of Src and PTP1B within rat brain mitochondria, we have investigated whether PTP1B regulates Src activity in mitochondria as in the cytosol. Results showed that Src was stimulated by in vitro addition of ATP to mitochondria, and this stimulation was reversed by a membrane-permeable allosteric inhibitor of PTP1B and by a potent selective Src inhibitor. They also indicated a direct action of PTP1B on phosphorylated tyrosine 527 residue of Src, thus implicating a role for PTP1B in the modulation of Src activity in mitochondria. Putative Src and PTP1B substrates were identified by liquid chromatography tandem mass spectrometry and two-dimensional blue native/SDS-PAGE. Both inhibitors inhibited ADP-stimulated respirations concurrently with Src activation and complex IV activation by ATP, while having no effect or increasing the activity of the other complexes. Our analysis emphasizes the regulatory function of Src and its modulation by PTP1B on oxidative phosphorylation in mitochondria.     

Autoradiographic study of the penetration of non-histone chromatin proteins into differentiating cells

Summary Attention has previously been drawn to a specific effect of NHCP on embryonicPleurodeles cell differentiation. With a modified NHCP labelling technique, autoradiography has revealed a cytoplasmic concentration of labelled NHCP and has not revealed any difference between homospecific and heterospecific NHCP penetration.This work has been supported financially by research grants from the C.N.R.S. (ERA No. 327 and ATP No. 4310 on the cellular differentiation), and INSERM (No. 711019).The authors wish to extend their thands to Dr.Mazarguil who kindly initiated us in the techniques of enzyme binding. Many thanks are also due to Mr.Hawks for most valuable help in correcting the English.