Distribution of Polymorphic and Non-Polymorphic Microsatellite Repeats in Xenopus tropicalis

The results of our bioinformatics analysis have found over 91,000 di-, tri-, and tetranucleotide microsatellites in our survey of 25% of the X. tropicalis genome, suggesting there may be over 360,000 within the entire genome. Within the X. tropicalis genome, dinucleotide (78.7%) microsatellites vastly out numbered tri- and tetranucleotide microsatellites. Similarly, AT-rich repeats are overwhelmingly dominant. The four AT-only motifs (AT, AAT, AAAT, and AATT) account for 51,858 out of 91,304 microsatellites found. Individually, AT microsatellites were the most common repeat found, representing over half of all di-, tri-, and tetranucleotide microsatellites. This contrasts with data from other studies, which show that AC is the most frequent microsatellite in vertebrate genomes (Toth et al. 2000). In addition, we have determined the rate of polymorphism for 5,128 non-redundant microsatellites, embedded in unique sequences. Interestingly, this subgroup of microsatellites was determined to have significantly longer repeats than genomic microsatellites as a whole. In addition, microsatellite loci with tandem repeat lengths more than 30 bp exhibited a significantly higher degree of polymorphism than other loci. Pairwise comparisons show that tetranucleotide microsatellites have the highest polymorphic rates. In addition, AAT and ATC showed significant higher polymorphism than other trinucleotide microsatellites, while AGAT and AAAG were significantly more polymorphic than other tetranucleotide microsatellites

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

Disruption of Gastrulation and Heparan Sulfate Biosynthesis in EXT1-Deficient Mice

AbstractMutations in the EXT1 gene are responsible for human hereditary multiple exostosis type 1. The Drosophila EXT1 homologue, tout-velu, regulates Hedgehog diffusion and signaling, which play an important role in tissue patterning during both invertebrate and vertebrate development. The EXT1 protein is also required for the biosynthesis of heparan sulfate glycosaminoglycans that bind Hedgehog. In this study, we generated EXT1-deficient mice by gene targeting. EXT1 homozygous mutants fail to gastrulate and generally lack organized mesoderm and extraembryonic tissues, resulting in smaller embryos compared to normal littermates. RT-PCR analysis of markers for visceral endoderm and mesoderm development indicates the delayed and abnormal development of both of these tissues. Immunohistochemical staining revealed a visceral endoderm pattern of Indian hedgehog (Ihh) in wild-type E6.5 embryos. However, in both EXT1-deficient embryos and wild-type embryos treated with heparitinase I, Ihh failed to associate with the cells. The effect of the EXT1 deletion on heparan sulfate formation was tested by HPLC and cellular glycosyltransferase activity assays. Heparan sulfate synthesis was abolished in EXT1 −/− ES cells and decreased to less than 50% in +/− cell lines. These results indicate that EXT1 is essential for both gastrulation and heparan sulfate biosynthesis in early embryonic development

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

Dynamic relationship of the epithelium and mesenchyme during salivary gland initiation: the role of Fgf10.

Peer reviewe

Lab-on-a-Chip: From Astrobiology to the International Space Station

The continual and long-term habitation of enclosed environments, such as Antarctic stations, nuclear submarines and space stations, raises unique engineering, medical and operational challenges. There is no easy way out and no easy way to get supplies in. This situation elevates the importance of monitoring technology that can rapidly detect events within the habitat that affect crew safety such as fire, release of toxic chemicals and hazardous microorganisms. Traditional methods to monitor microorganisms on the International Space Station (ISS) have consisted of culturing samples for 3-5 days and eventual sample return to Earth. To augment these culture methods with new, rapid molecular techniques, we developed the Lab-on-a-Chip Application Development - Portable Test System (LOCAD-PTS). The system consists of a hand-held spectrophotometer, a series of interchangeable cartridges and a surface sampling/dilution kit that enables crew to collect samples and detect a range of biological molecules, all within 15 minutes. LOCAD-PTS was launched to the ISS aboard Space Shuttle Discovery in December 2006, where it was operated for the first time during March-May 2007. The surfaces of five separate sites in the US Lab and Node 1 of ISS were analyzed for endotoxin, using cartridges that employ the Limulus Amebocyte Lysate (LAL) assay; results of these tests will be presented. LOCAD-PTS will remain permanently onboard ISS with new cartridges scheduled for launch in February and October of 2008 for the detection of fungi (Beta-glucan) and Gram-positive bacteria (lipoteichoic acid), respectively

Harnessing case isolation and ring vaccination to control Ebola.

As a devastating Ebola outbreak in West Africa continues, non-pharmaceutical control measures including contact tracing, quarantine, and case isolation are being implemented. In addition, public health agencies are scaling up efforts to test and deploy candidate vaccines. Given the experimental nature and limited initial supplies of vaccines, a mass vaccination campaign might not be feasible. However, ring vaccination of likely case contacts could provide an effective alternative in distributing the vaccine. To evaluate ring vaccination as a strategy for eliminating Ebola, we developed a pair approximation model of Ebola transmission, parameterized by confirmed incidence data from June 2014 to January 2015 in Liberia and Sierra Leone. Our results suggest that if a combined intervention of case isolation and ring vaccination had been initiated in the early fall of 2014, up to an additional 126 cases in Liberia and 560 cases in Sierra Leone could have been averted beyond case isolation alone. The marginal benefit of ring vaccination is predicted to be greatest in settings where there are more contacts per individual, greater clustering among individuals, when contact tracing has low efficacy or vaccination confers post-exposure protection. In such settings, ring vaccination can avert up to an additional 8% of Ebola cases. Accordingly, ring vaccination is predicted to offer a moderately beneficial supplement to ongoing non-pharmaceutical Ebola control efforts

Light Induction of a Vertebrate Clock Gene Involves Signaling through Blue-Light Receptors and MAP Kinases

AbstractThe signaling pathways that couple light photoreception to entrainment of the circadian clock have yet to be deciphered. Two prominent groups of candidates for the circadian photoreceptors are opsins (e.g., melanopsin) and blue-light photoreceptors (e.g., cryptochromes). We have previously showed that the zebrafish is an ideal model organism in which to study circadian regulation and light response in peripheral tissues. Here, we used the light-responsive zebrafish cell line Z3 to dissect the response of the clock gene zPer2 to light. We show that the MAPK (mitogen-activated protein kinase) pathway is essential for this response, although other signaling pathways may also play a role. Moreover, action spectrum analyses of zPer2 transcriptional response to monochromatic light demonstrate the involvement of a blue-light photoreceptor. The Cry1b and Cry3 cryptochromes constitute attractive candidates as photoreceptors in this setting. Our results establish a link between blue-light photoreceptors, probably cryptochromes, and the MAPK pathway to elicit light-induced transcriptional activation of clock genes

Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H)1 CD4(+) and CD8(+) T cells and a systemic LACK-specific T(H)1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H)1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H)1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania