Manipulating the Xenopus genome with transposable elements

The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities

Evolution of Salmonella excretion by sows during gestation in link with the faecal microbiome

Pork meat is estimated to be responsible of 10 to 20% of human salmonellosis cases. Control strategies at the farm could reduce contamination at the slaughterhouse. One of the targeted sector of the production is the maternity, where sows could be Salmonella reservoirs. The aim of this study was to characterize the faecal microbiome of sows excreting or not Salmonella during gestation phases. A total of 76 sows were selected and fecal matters were analysed at the beginning or the end of gestation period. Salmonella detection was conducted using a method including two selective enrichment media (MSRV and TBG). Nine (9) isolates per positive samples were collected. Among the 76 sows tested, 31 were shedding Salmonella. The sows in the first third of their gestation shed significantly more frequently Salmonella (22/29) than those in the last third (9/47) (χ² P \u3c 0.05). The shedding status of 19 of the sows that were previously sampled in the first third of their gestation was followed, this time in the last third, confirming reduction of the shedding. Association between changes in the intestinal microbiome and this evolution of Salmonella shedding will be explored. MiSeq sequencing is currently being conducted on the feces to identify shifts in the composition or diversity in the microbial community that could be associated to these variations. A large number of Salmonella isolates that were collected were genotyped by a high resolution melt (HRM) technique. These results showed the presence of a major HRM profile (136 isolates / 169) and two minor profiles (24 and 9 /169)

Remobilization of Tol2 transposons in Xenopus tropicalis

<p>Abstract</p> <p>Background</p> <p>The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring <it>Tol2 </it>element from <it>Oryzias latipes </it>efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, <it>Xenopus tropicalis. Tol2 </it>transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.</p> <p>Results</p> <p>To test whether <it>Tol2 </it>transposons integrated in the <it>Xenopus tropicalis </it>genome are substrates for remobilization, we injected <it>in vitro </it>transcribed <it>Tol2 </it>mRNA into one-cell embryos harbouring a single copy of a <it>Tol2 </it>transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single <it>Tol2 </it>transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the <it>Tol2 </it>transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis.</p> <p>Conclusions</p> <p>In this study, we demonstrate that single copy <it>Tol2 </it>transposons integrated into the <it>Xenopus tropicalis </it>genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.</p

Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis

<p>Abstract</p> <p>Background</p> <p>The <it>Sleeping Beauty </it>(<it>SB</it>) transposon system has been used for germline transgenesis of the diploid frog, <it>Xenopus tropicalis</it>. Injecting one-cell embryos with plasmid DNA harboring an <it>SB </it>transposon substrate together with mRNA encoding the <it>SB </it>transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that <it>SB </it>transposons stably integrated into the frog genome are effective substrates for remobilization.</p> <p>Results</p> <p>Transgenic frogs that express the <it>SB</it>10 transposase were bred with <it>SB </it>transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the <it>SB </it>transposons. The remobilized <it>SB </it>transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'.</p> <p>Conclusions</p> <p>In this study, we demonstrate that <it>SB </it>transposons integrated into the <it>X. tropicalis </it>genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.</p

Organic matter composition and heterotrophic bacterial activity at declining summer sea ice in the central Arctic Ocean

The Arctic Ocean is highly susceptible to climate change as evidenced by rapid warming and the drastic loss of sea ice during summer. The consequences of these environmental changes for the microbial cycling of organic matter are largely unexplored. Here, we investigated the distribution and composition of dissolved organic matter (DOM) along with heterotrophic bacterial activity in seawater and sea ice of the Eurasian Basin at the time of the record ice minimum in 2012. Bacteria in seawater were highly responsive to fresh organic matter and remineralized on average 55% of primary production in the upper mixed layer. Correlation analysis showed that the accumulation of dissolved combined carbohydrates (DCCHO) and dissolved amino acids (DAA), two major components of fresh organic matter, was related to the drawdown of nitrate. Nitrate‐depleted surface waters at stations adjacent to the Laptev Sea showed about 25% higher concentrations of DAA than stations adjacent to the Barents Sea and in the central Arctic basin. Carbohydrate concentration was the best predictor of heterotrophic bacterial activity in sea ice. In contrast, variability in sea‐ice bacterial biomass was largely driven by differences in ice thickness. This decoupling of bacterial biomass and activity may mitigate the negative effects of biomass loss due to ice melting on heterotrophic bacterial functions. Overall, our results reveal that changes in DOM production and inventories induced by sea‐ice loss have a high potential to enhance the bacterial remineralization of organic matter in seawater and sea ice of the Arctic Ocean

A Search for Possible Long Range Spin Dependent Interactions of the Neutron from Exotic Vector Boson Exchange

We present a search for possible spin dependent interactions of the neutron with matter through exchange of spin 1 bosons with axial vector couplings as envisioned in possible extensions of the Standard Model. This was sought using a slow neutron polarimeter that passed transversely polarized slow neutrons by unpolarized slabs of material arranged so that interactions would tilt the plane of polarization and develop a component along the neutron momentum. The result for the rotation angle, ϕ′=[2.8±4.6(stat.)±4.0(sys.)]×10−5 rad/m is consistent with zero. This result improves the upper bounds on the neutron-matter coupling g2A by about three orders of magnitude for force ranges in the mm– μm regime

Hypolithic Microbial Community of Quartz Pavement in the High-Altitude Tundra of Central Tibet

The hypolithic microbial community associated with quartz pavement at a high-altitude tundra location in central Tibet is described. A small-scale ecological survey indicated that 36% of quartz rocks were colonized. Community profiling using terminal restriction fragment length polymorphism revealed no significant difference in community structure among a number of colonized rocks. Real-time quantitative PCR and phylogenetic analysis of environmental phylotypes obtained from clone libraries were used to elucidate community structure across all domains. The hypolithon was dominated by cyanobacterial phylotypes (73%) with relatively low frequencies of other bacterial phylotypes, largely represented by the chloroflexi, actinobacteria, and bacteriodetes. Unidentified crenarchaeal phylotypes accounted for 4% of recoverable phylotypes, while algae, fungi, and mosses were indicated by a small fraction of recoverable phylotypes

Immunogenicity of Fractional Doses of Tetravalent A/C/Y/W135 Meningococcal Polysaccharide Vaccine: Results from a Randomized Non-Inferiority Controlled Trial in Uganda

Meningitis are infections of the lining of the brain and spinal cord and can cause high fever, blood poisoning, and brain damage, as well as result in death in up to 10% of cases. Epidemics of meningitis occur almost every year in parts of sub-Saharan Africa, throughout a high-burden area spanning Senegal to Ethiopia dubbed the “Meningitis Belt.” Most epidemics in Africa are caused by Neisseria meningitidis (mostly serogroup A and W135). Mass vaccination campaigns attempt to control epidemics by administering meningococcal vaccines targeted against these serogroups, among others. However, global shortages of these vaccines are currently seen. We studied the use of fractional (1/5 and 1/10) doses of a licensed vaccine to assess its non-inferiority compared with the normal full dose. In a randomized trial in Uganda, we found that immune response and safety using a 1/5 dose were comparable to full dose for three serogroups (A, Y, W135), though not a fourth (C). In light of current shortages of meningococcal vaccines and their importance in fighting meningitis epidemics around the world, we suggest fractional doses be taken under consideration in mass vaccination campaigns

Quantitative Proteome Profiling of C. burnetii under Tetracycline Stress Conditions

The recommended antibiotic regimen against Coxiella burnetii, the etiological agent of Q fever, is based on a semi-synthetic, second-generation tetracycline, doxycycline. Here, we report on the comparison of the proteomes of a C. burnetii reference strain either cultured under control conditions or under tetracycline stress conditions. Using the MS-driven combined fractional diagonal chromatography proteomics technique, out of the 531 proteins identified, 5 and 19 proteins were found significantly up- and down-regulated respectively, under tetracycline stress. Although the predicted cellular functions of these regulated proteins did not point to known tetracycline resistance mechanisms, our data clearly reveal the plasticity of the proteome of C. burnetii to battle tetracycline stress. Finally, we raise several plausible hypotheses that could further lead to more focused experiments on studying tetracycline resistance in C. burnetii and thus reduced treatment failures of Q fever