Publications

Plasmid instability is a significant concern in the industrial utilization of microorganisms for protein or DNA production. Here we report on the development of a new and highly effective stabilization system based on the use of the ccd antidote/poison genes. For the first time, we separated the antidote gene from the poison gene: localizing the former in the plasmid and integrating the latter in the bacterial chromosome. We show that this separate-component-stabilization (SCS) strategy: (i) allows for perfect stabilization without the use of antibiotics; (ii) increases three to five times the recombinant protein production levels; and (iii) does not require any specific modification of the protein production process or culture medium. We illustrate that point by using the classical T7 promotor (i.e., used in most expression systems). Finally, we demonstrate that the SCS system increases by five the yield in DNA production, a result especially important for the design and production of gene therapy constructs void of any antibiotic resistance gene.

Mammalian Hox genes encode transcription factors that are crucial for proper morphogenesis along the various body axes. Despite their extensive structural and functional characterization, the nature of their target genes remains elusive. We have addressed this question by using DNA microarrays to screen for genes whose expression in developing distal forelimbs and genital eminences was significantly modified in the absence of the full Hoxd gene complement. This comparative approach not only identified specific candidate genes, but also allowed the examination of whether a similar Hox expression pattern in distinct tissues leads to the modulation of the same or different downstream genes. We report here a set of potential target genes, most of which were not previously known to play a role in the early stages of either limb or genital bud development. Interestingly, we find that the majority of these candidate genes are differentially expressed in both structures, although often at different times. This supports the idea that both appendices involve similar genetic controls, both upstream and downstream of the Hox gene family. These results highlight the surprising mechanistic relationship between these rather different body parts and suggest a common developmental strategy to build up the most distal appendicular structures of the body, i.e. the digits and the penis/clitoris.

The Myxogastria are common soil microorganisms with a life cycle comprised of a plasmodial trophic stage and large fruiting bodies generally visible with the unaided eye. Until now, their classification has been based exclusively on a combination of morphological, ultrastructural, and developmental characters. Our study is the first attempt to examine phylogenetic relationships among these taxa using molecular data. Partial small-subunit ribosomal RNA and/or elongation factor 1-alpha gene sequences were obtained from eleven, mostly field-collected species representing the five orders of Myxogastria. Nineteen sequences were obtained and subjected to phylogenetic analysis together with 10 sequences available from GenBank. Separate and combined analyses of the two data sets support the division of Myxogastria into three distinct groups. The most basal clade consists of the Echinosteliales, an order considered to have affinities with Protostelia. The three species examined possess unpigmented or slightly pigmented spores. The second group consists of Liceales and Trichiales, taxa characterized by the presence of clear, but pigmented, spores. The third group consists of the two remaining orders, Physarales and Stemonitales, both possessing dark spores. This suggests that spore pigmentation is an evolutionarily conservative character in myxogastrians, and that the simple morphology of echinostelids is not a derived feature.

We performed extensive and realistic simulations of the colonization process of Europe by Neolithic farmers, as well as their potential admixture and competition with local Palaeolithic hunter-gatherers. We find that minute amounts of gene flow between Palaeolithic and Neolithic populations should lead to a massive Palaeolithic contribution to the current gene pool of Europeans. This large Palaeolithic contribution is not expected under the demic diffusion (DD) model, which postulates that agriculture diffused over Europe by a massive migration of individuals from the Near East. However, genetic evidence in favour of this model mainly consisted in the observation of allele frequency clines over Europe, which are shown here to be equally probable under a pure DD or a pure acculturation model. The examination of the consequence of range expansions on single nucleotide polymorphism (SNP) diversity reveals that an ascertainment bias consisting of selecting SNPs with high frequencies will promote the observation of genetic clines (which are not expected for random SNPs) and will lead to multimodal mismatch distributions. We conclude that the different patterns of molecular diversity observed for Y chromosome and mitochondrial DNA can be at least partly owing to an ascertainment bias when selecting Y chromosome SNPs for studying European populations.

We have studied mitochondrial DNA variation in a local population of the leaf beetle species Gonioctena olivacea, to check whether its apparent low dispersal behaviour affects its pattern of genetic variation at a small geographical scale. We have sampled 10 populations of G. olivacea within a rectangle of 5 x 2 km in the Belgian Ardennes, as well as five populations located approximately along a straight line of 30 km and separated by distances of 3-12 km. For each sampled individual (8-19 per population), a fragment of the mtDNA control region was polymerase chain reaction-amplified and sequenced. Sequence data were analysed to test whether significant genetic differentiation could be detected among populations separated by such relatively short distances. The reconstructed genealogy of the mitochondrial haplotypes was also used to investigate the demographic history of these populations. Computer simulations of the evolution of populations were conducted to assess the minimum amount of gene flow that is necessary to explain the observed pattern of variation in the samples. Results show that migration among populations included in the rectangle of 5 x 2 km is substantial, and probably involves the occurrence of dispersal flights. This appears difficult to reconcile with the results of a previous ecological field study that concluded that most of this species dispersal occurs by walking. While sufficient migration to homogenize genetic diversity occurs among populations separated by distances of a few hundred metres to a few kilometres, distances greater than 5 km results in contrast in strong differentiation among populations, suggesting that migration is drastically reduced on such distances. Finally, the results of coalescent simulations suggest that the star-like genealogy inferred from the mtDNA sequence data is fully compatible with a past demographic expansion. However, a metapopulation structure alone (without the need to invoke a population expansion event) cannot be dismissed as the cause of this star shape.

In mammals, circadian oscillators not only exist in specialized neurons of the suprachiasmatic nucleus, but in almost all peripheral cell types. These oscillators are operative even in established fibroblast cell lines, such as Rat-1 cells or NIH3T3 cells, and in primary fibroblasts from mouse embryos or adult animals. This can be demonstrated by treating such cells for a short time period with high concentrations of serum or chemicals that activate a large number of known signaling pathways. The possibility of studying circadian rhythms in cultured cells should facilitate the biochemical and genetic dissection of the circadian clockwork and should promote the discovery of new clock components.

Sensory neurons expressing members of the seven-transmembrane V1r receptor superfamily allow mice to perceive pheromones. These receptors, which exhibit no sequence homology to any known protein except a weak similarity to taste receptors, have only been found in mammals. In the mouse, the V1r repertoire contains >150 members, which are expressed by neurons of the vomeronasal organ, a structure present exclusively in some tetrapod species. Here, we report the existence of a single V1r gene in multiple species of a non-terrestrial, vomeronasal organ-lacking taxon, the teleosts. In zebrafish, this V1r gene is expressed in chemosensory neurons of the olfactory rosette with a punctate distribution, strongly suggesting a role in chemodetection. This unique receptor gene exhibits a remarkably high degree of sequence variability between fish species. It likely corresponds to the original V1r present in the common ancestor of vertebrates, which led to the large and very diverse expansion of vertebrate pheromone receptor repertoires, and suggests the presence of V1rs in multiple nonmammalian phyla.

Acanthoscelides obtectus Say is a bruchid species of Neotropical origin, and is specialized on beans of the Phaseolus vulgaris L. group. Since the domestication and diffusion of beans, A. obtectus has become cosmopolitan through human-mediated migrations and is now a major pest in bean granaries. Using phylogeographic methods applied to mitochondrial DNA (mtDNA) and nuclear microsatellite molecular markers, we show that the origin of this species is probably further south than Mesoamerica, as commonly thought. Our results also indicate that A. obtectus and its Mesoamerican sister species Acanthoscelides obvelatus, two morphologically close species differing principally in voltinism, speciated in allopatry: A. obtectus (multivoltine) arising in Andean America and A. obvelatus (univoltine) in Mesoamerica. In contrast to Mesoamerica where beans fruit once yearly, wild beans in Andean America fruit year-round, especially in regions showing little or no seasonality. In such habitats where resources are continuously present, multivoltinism is adaptive. According to existing hypotheses, multivoltinism in A. obtectus is a new adaptation that evolved after bean domestication. Our data suggest the alternative hypothesis that multivoltinism is an older trait, adapted to exploit the year-round fruiting of wild beans in relatively aseasonal habitats, and allowed A. obtectus to become a pest in bean granaries. This trait also permitted this species to disperse through human-mediated migrations associated with diffusion of domesticated beans. We also show that diversity of Old World A. obtectus populations can be quite well explained by a single colonization event about 500 bp. Human-mediated migrations appear not to be rare, as our results indicate a second more recent migration event from Andean America to Mexico.