A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13.

Genomic islands (GEIs) are large DNA segments, present in most bacterial genomes, that are most likely acquired via horizontal gene transfer. Here, we study the self-transfer system of the integrative and conjugative element ICEclc of Pseudomonas knackmussii B13, which stands model for a larger group of ICE/GEI with syntenic core gene organization. Functional screening revealed that unlike conjugative plasmids and other ICEs ICEclc carries two separate origins of transfer, with different sequence context but containing a similar repeat motif. Conjugation experiments with GFP-labelled ICEclc variants showed that both oriTs are used for transfer and with indistinguishable efficiencies, but that having two oriTs results in an estimated fourfold increase of ICEclc transfer rates in a population compared with having a single oriT. A gene for a relaxase essential for ICEclc transfer was also identified, but in vivo strand exchange assays suggested that the relaxase processes both oriTs in a different manner. This unique dual origin of transfer system might have provided an evolutionary advantage for distribution of ICE, a hypothesis that is supported by the fact that both oriT regions are conserved in several GEIs related to ICEclc.

A cryptic heterogametic transition revealed by sex-linked DNA markers in Palearctic green toads.

In sharp contrast to birds and mammals, most cold-blooded vertebrates have homomorphic (morphologically undifferentiated) sex chromosomes. This might result either from recurrent X-Y recombination (occurring e.g. during occasional events of sex reversal) or from frequent turnovers (during which sex-determining genes are overthrown by new autosomal mutations). Evidence for turnovers is indeed mounting in fish, but very few have so far been documented in amphibians, possibly because of practical difficulties in identifying sex chromosomes. Female heterogamety (ZW) has long been established in Bufo bufo, based on sex reversal and crossing experiments. Here, we investigate a sex-linked marker identified from a laboratory cross between Palearctic green toads (Bufo viridis subgroup). The F(1) offspring produced by a female Bufo balearicus and a male Bufo siculus were phenotypically sexed, displaying an even sex ratio. A sex-specific marker detected in highly reproducible AFLP genotypes was cloned. Sequencing revealed a noncoding, microsatellite-containing fragment. Reamplification and genotyping of families of this and a reciprocal cross showed B. siculus to be male heterogametic (XY) and suggested the same system for B. balearicus. Our results thus reveal a cryptic heterogametic transition within bufonid frogs and help explain patterns of hybrid fitness within the B. viridis subgroup. Turnovers of genetic sex-determination systems may be more frequent in amphibians than previously thought and thus contribute to the prevalence of homomorphic sex chromosomes in this group.

Islands of euchromatin-like sequence and expressed polymorphic sequences within the short arm of human chromosome 21.

The goals of the human genome project did not include sequencing of the heterochromatic regions. We describe here an initial sequence of 1.1 Mb of the short arm of human chromosome 21 (HSA21p), estimated to be 10% of 21p. This region contains extensive euchromatic-like sequence and includes on average one transcript every 100 kb. These transcripts show multiple inter- and intrachromosomal copies, and extensive copy number and sequence variability. The sequencing of the "heterochromatic" regions of the human genome is likely to reveal many additional functional elements and provide important evolutionary information.

A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.

First detection of Waddlia chondrophila in Africa using SYBR Green real-time PCR on veterinary samples.

Waddlia chondrophila is a strict intracellular microorganism belonging to the order Chlamydiales that has been isolated twice from aborted bovine fetuses, once in USA and once in Germany. This bacterium is now considered as an abortigenic agent in cattle. However, no information is available regarding the presence of this bacterium in Africa. Given the low sensitivity of cell culture to recover such an obligate intracellular bacterium, molecular-based diagnostic approaches are warranted. This report describes the development of a quantitative SYBR Green real-time PCR assay targeting the recA gene of W. chondrophila. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing pathogens that can cause abortion in cattle. The PCR exhibited a good intra-run and inter-run reproducibility. This real-time PCR was then applied to 150 vaginal swabs taken from Tunisian cows that have aborted. Twelve samples revealed to be Waddlia positive, suggesting a possible role of this bacterium in this setting. This new real-time PCR assay represents a diagnostic tool that may be used to further study the prevalence of Waddlia infection.

Ultrastructural changes of the internal limiting membrane removed during indocyanine green assisted peeling versus conventional surgery for an idiopathic epimacular membrane

Purpose:To evaluate the histological features of cellular retinal fragments on the internal limiting membrane (ILM) removed during epiretinal membrane peeling surgery with and without the aid of ICG diluted in 5% glucose Methods:ILM specimens removed from 88 eyes undergoing vitrectomy and membrane peeling surgery for idiopathic epiretinal membrane between 1995 and 2003 were reviewed retrospectively. Surgery was performed in all cases by the same surgeon using the same technique. ICG was diluted with 5% glucose. Histological analysis focused on the presence and characteristics of retinal structures on the retinal surface of the ILM. Statistical analysis compared the results between group I (conventional surgery without ICG) and group II (ICG-assisted peeling) Results:Seventy-one eyes underwent EMM surgery without the aid of ICG (group I) and seventeen underwent EMM ICG-assisted surgery assisted using ICG (group II). The amount of Muller cell debris on the retinal surface of the ILM was more significant in the group I (no ICG) than in the group II (ICG) (40.8 versus 11.8; p = 0.024). Large fragments of Muller cells were more frequently observed in the group I (no ICG) than in the group II (ICG) (63.4% versus 23.5%; p= 0.003).The presence of larger retinal elements such as neural axons and vessels were observed attached to retinal face of the ILM in 5 (7%) cases of the no-ICG group. No such retinal elements were detected in any of the histological ILM specimens of the ICG-assisted group Conclusions:The use of ICG diluted with 5% glucose in the aid of ILM removal during epiretinal membrane surgery was associated with less retinal debris attached to retinal face of the ILM compared to surgery in which ICG was not used. Our findings contradict previous reports in the literature, in which use of ICG diluted with balanced salt solution (BSS) was associated with more retinal fragments attached to the retinal face of the ILM. According to our results, we hypothesize that diluting ICG with 5% glucose may decrease the adhesion of the ILM to the underlying retinal layers such that less retinal debris is removed with peeling of the ILM.

In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8(+) T cell responses.

The present study evaluates the potential of third-generation lentivirus vectors with respect to their use as in vivo-administered T cell vaccines. We demonstrate that lentivector injection into the footpad of mice transduces DCs that appear in the draining lymph node and in the spleen. In addition, a lentivector vaccine bearing a T cell antigen induced very strong systemic antigen-specific cytotoxic T lymphocyte (CTL) responses in mice. Comparative vaccination performed in two different antigen models demonstrated that in vivo administration of lentivector was superior to transfer of transduced DCs or peptide/adjuvant vaccination in terms of both amplitude and longevity of the CTL response. Our data suggest that a decisive factor for efficient T cell priming by lentivector might be the targeting of DCs in situ and their subsequent migration to secondary lymphoid organs. The combination of performance, ease of application, and absence of pre-existing immunity in humans make lentivector-based vaccines an attractive candidate for cancer immunotherapy.

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.

Genomic islands: tools of bacterial horizontal gene transfer and evolution.

Abstract Bacterial genomes evolve through mutations, rearrangements or horizontal gene transfer. Besides the core genes encoding essential metabolic functions, bacterial genomes also harbour a number of accessory genes acquired by horizontal gene transfer that might be beneficial under certain environmental conditions. The horizontal gene transfer contributes to the diversification and adaptation of microorganisms, thus having an impact on the genome plasticity. A significant part of the horizontal gene transfer is or has been facilitated by genomic islands (GEIs). GEIs are discrete DNA segments, some of which are mobile and others which are not, or are no longer mobile, which differ among closely related strains. A number of GEIs are capable of integration into the chromosome of the host, excision, and transfer to a new host by transformation, conjugation or transduction. GEIs play a crucial role in the evolution of a broad spectrum of bacteria as they are involved in the dissemination of variable genes, including antibiotic resistance and virulence genes leading to generation of hospital 'superbugs', as well as catabolic genes leading to formation of new metabolic pathways. Depending on the composition of gene modules, the same type of GEIs can promote survival of pathogenic as well as environmental bacteria.