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

Feed presentation options in Swine early fattening mitigates Salmonella shedding and specifically modulates the faecal microbiota

Aims: The object of this study was to determine the impact of only modifying the processing and/or particle size of pig feed on Salmonella shedding and faecal microbiota. Methods and Results: Pigs were fed a diet that varied only by their processing (pellet or mash) and their particle size (500, 750 or 1250 mu m) for 21 days. Salmonella detection in faeces and seroconversion were determined. Faecal microbiota was assessed by Ion Torrent amplicon sequencing and real-time PCR. Significantly fewer pigs (P &#x227A; 0.05) shed Salmonella in the groups fed mash 500 (1) and mash or pellet 1250 (5 each) compared to the commercial reference group (15) fed pellet 500. Both mash processing and large particle size raised the proportion and number of bacteria from the Bifidobacterium genus in the faecal microbiota of the pigs. Thirteen other taxa significantly varied (P &#x227A; 0.0005) with feed presentation. Conclusion: Mash processing and/or large particle size in pig feed reduces Salmonella shedding prevalence and promotes beneficial populations of digestive microbiota. Significance and Impact of the Study: This study is the first to demonstrate a difference in Salmonella shedding through only modifying pig feed presentation and is the first to extensively describe modifications of faecal microbiota

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

Rule-based knowledge aggregation for large-scale protein sequence analysis of influenza A viruses

Background: The explosive growth of biological data provides opportunities for new statistical and comparative analyses of large information sets, such as alignments comprising tens of thousands of sequences. In such studies, sequence annotations frequently play an essential role, and reliable results depend on metadata quality. However, the semantic heterogeneity and annotation inconsistencies in biological databases greatly increase the complexity of aggregating and cleaning metadata. Manual curation of datasets, traditionally favoured by life scientists, is impractical for studies involving thousands of records. In this study, we investigate quality issues that affect major public databases, and quantify the effectiveness of an automated metadata extraction approach that combines structural and semantic rules. We applied this approach to more than 90,000 influenza A records, to annotate sequences with protein name, virus subtype, isolate, host, geographic origin, and year of isolation. Results: Over 40,000 annotated Influenza A protein sequences were collected by combining information from more than 90,000 documents from NCBI public databases. Metadata values were automatically extracted, aggregated and reconciled from several document fields by applying user-defined structural rules. For each property, values were recovered from ≥88.8% of records, with accuracy exceeding 96% in most cases. Because of semantic heterogeneity, each property required up to six different structural rules to be combined. Significant quality differences between databases were found: GenBank documents yield values more reliably than documents extracted from GenPept. Using a simple set of semantic rules and a reasoner, we reconstructed relationships between sequences from the same isolate, thus identifying 7640 isolates. Validation of isolate metadata against a simple ontology highlighted more than 400 inconsistencies, leading to over 3,000 property value corrections. Conclusion: To overcome the quality issues inherent in public databases, automated knowledge aggregation with embedded intelligence is needed for large-scale analyses. Our results show that user-controlled intuitive approaches, based on combination of simple rules, can reliably automate various curation tasks, reducing the need for manual corrections to approximately 5% of the records. Emerging semantic technologies possess desirable features to support today's knowledge aggregation tasks, with a potential to bring immediate benefits to this field

AML1/ETO Oncoprotein Is Directed to AML1 Binding Regions and Co-Localizes with AML1 and HEB on Its Targets

A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein

Induction of apoptosis in myeloid leukaemic cells by ribozymes targeted against AML1/MTG8

The translocation (8;21)(q22;q22) is a karyotypic abnormality detected in acute myeloid leukaemia (AML) M2 and results in the formation of the chimeric fusion gene AML1/MTG8. We previously reported that two hammerhead ribozymes against AML1/MTG8 cleave this fusion transcript and also inhibit the proliferation of myeloid leukaemia cell line Kasumi-1 which possesses t(8;21)(q22;q22). In this study, we investigated the mechanisms of inhibition of proliferation in myeloid leukaemic cells with t(8;21)(q22;q22) by ribozymes. These ribozymes specifically inhibited the growth of Kasumi-1 cells, but did not affect the leukaemic cells without t(8;21)(q22;q22). We observed the morphological changes including chromatin condensation, fragmentation and the formation of apoptotic bodies in Kasumi-1 cells incubated with ribozymes for 7 days. In addition, DNA ladder formation was also detected after incubation with ribozymes which suggested the induction of apoptosis in Kasumi-1 cells by the AML1/MTG8 ribozymes. However, the ribozymes did not induce the expression of CD11b and CD14 antigens in Kasumi-1 cells. The above data suggest that these ribozymes therefore inhibit the growth of myeloid leukaemic cells with t(8;21)(q22;q22) by the induction of apoptosis, but not differentiation. We conclude therefore that the ribozymes targeted against AML1/MTG8 may have therapeutic potential for patients with AML carrying t(8;21)(q22;q22) while, in addition, the product of the chimeric gene is responsible for the pathogenesis of myeloid leukaemia. © 1999 Cancer Research Campaig

Environmental metabarcoding reveals contrasting belowground and aboveground fungal communities from poplar at a Hg phytomanagement site

Characterization of microbial communities in stressful conditions at a field level is rather scarce, especially when considering fungal communities from aboveground habitats. We aimed at characterizing fungal communities from different poplar habitats at a Hg-contaminated phytomanagement site by using Illumina-based sequencing, network analysis approach, and direct isolation of Hg-resistant fungal strains. The highest diversity estimated by the Shannon index was found for soil communities, which was negatively affected by soil Hg concentration. Among the significant correlations between soil operational taxonomic units (OTUs) in the co-occurrence network, 80% were negatively correlated revealing dominance of a pattern of mutual exclusion. The fungal communities associated with Populus roots mostly consisted of OTUs from the symbiotic guild, such as members of the Thelephoraceae, thus explaining the lowest diversity found for root communities. Additionally, root communities showed the highest network connectivity index, while rarely detected OTUs from the Glomeromycetes may have a central role in the root network. Unexpectedly high richness and diversity were found for aboveground habitats, compared to the root habitat. The aboveground habitats were dominated by yeasts from the Lalaria, Davidiella, and Bensingtonia genera, not detected in belowground habitats. Leaf and stem habitats were characterized by few dominant OTUs such as those from the Dothideomycete class producing mutual exclusion with other OTUs. Aureobasidium pullulans, one of the dominating OTUs, was further isolated from the leaf habitat, in addition to Nakazawaea populi species, which were found to be Hg resistant. Altogether, these findings will provide an improved point of reference for microbial research on inoculation-based programs of tailings dumps

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