High resolution mapping of OCA2 intragenic rearrangements and identification of a founder effect associated with a deletion in Polish albino patients

Oculocutaneous albinism type 2 (OCA2) represents about 30% of OCA worldwide. Using quantitative multiplex fluorescent PCR and very high-resolution array-CGH focussed on the OCA2 gene and surrounding regions in 15q12, we identified new rearrangements. Deletion 1, encompassing exons 3-20, was present in three patients (including one in the homozygous state), and Deletion 2 (exons 1-20) was found in one patient (heterozygous state). The duplication (exons 3-20) was found in one patient in the homozygous state. Using 14 microsatellite markers we determined haplotypes associated with these rearrangements. Deletion 1 was associated with the same haplotype in three patients who were all of Polish origin, which is strongly in favour of a founder effect. Deletion 2 was associated with a distinct haplotype. The homozygous duplication was inherited from the two unrelated parents of the patients on two different haplotypes. Analysis of the sequences around the breakpoints of these rearrangements showed that all occurred within complex arrays of repetitive sequences. The combined use of very high-resolution array-CGH and of microsatellites (including new intragenic ones described here) constitutes a powerful approach for the precise characterization of OCA2 rearrangements, which have been found in more than 20% of OCA2 patients.     

Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport

Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol® do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol®, prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®. Microscopic examination of transfected cells pre-treated with Lutrol® confirmed that more plasmid DNA copies were internalized. Absence of cationic lipid did not impair Lutrol®-mediated DNA internalization, but critically impaired endosomal escape. Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways.     

De novo discovery and multiplexed amplification of microsatellite markers for black alder (Alnus glutinosa) and related species using SSR-enriched shotgun pyrosequencing

Recent developments in sequencing technologies and bioinformatics analyses provide an unprecedented opportunity for cost and time effective high quality microsatellite marker discovery in nonmodel organisms for which no genomic information is available. Here, we use shotgun pyrosequencing of a microsatellite-enriched library to develop, for the first time, microsatellite markers for Alnus glutinosa, a keystone tree species of European riparian woodland communities. From a total of 17?855 short sequences, we identified 590 perfect microsatellites from which 392 had designed primers. A subset of 48 loci were tested for amplification, 12 of which were polymorphic in A. glutinosa. These 12 loci were successfully coamplified in a single multiplex polymerase chain reaction experiment and validated for population genetics applications. In addition, 10 and 8 of these microsatellites were found to be transferable to the related A. incana and A. cordata species. The developed multiplex of 12 microsatellite markers therefore provides new opportunities for experimental evolutionary and forest genetics research in Alnus.     

How important are Rho GTPases in neurosecretion?

Rho GTPases are small GTP binding proteins belonging to the Ras superfamily which act as molecular switches that regulate many cellular function including cell morphology, cell to cell interaction, cell migration and adhesion. In neuronal cells, Rho GTPases have been proposed to regulate neuronal development and synaptic plasticity. However, the role of Rho GTPases in neurosecretion is poorly documented. In this review, we discuss data that highlight the importance of Rho GTPases and their regulators into the control of neurotransmitter and hormone release in neurons and neuroendocrine cells, respectively.     

Microbial processes in a high-latitude fjord (Kongsfjorden, Svalbard): II. Ciliates and dinoflagellates

The composition and ecological role of ciliates and dinoflagellates were investigated at one station in Kongsfjorden, Svalbard, during six consecutive field campaigns between March and December 2006. Total ciliate and dinoflagellate abundance mirrored the seasonal progression of phytoplankton, peaking with 5.8 × 10⁴ cells l⁻¹ in April at an average chlorophyll a concentration of 10 μg l⁻¹. Dinoflagellates were more abundant than ciliates, dominated by small athecates. Among ciliates, aloricate oligotrichs dominated the assemblage. A large fraction (>60%) of ciliates and dinoflagellates contained chloroplasts in spring and summer. The biomass of the purely heterotrophic fraction of the ciliate and dinoflagellate community (protozooplankton) was with 14 μg C l⁻¹ highest in conjunction with the phytoplankton spring bloom in April. Growth experiments revealed similar specific growth rates for heterotrophic ciliates and dinoflagellates (<0–0.8 d⁻¹). Food availability may have controlled the protozooplankton assemblage in winter, while copepods may have exerted a strong control during the post-bloom period. Calculations of the potential grazing rates of the protozooplankton indicated its ability to control or heavily impact the phytoplankton stocks at most times. The results show that ciliates and dinoflagellates were an important component of the pelagic food web in Kongsfjorden and need to be taken into account when discussing the fate of phytoplankton and biogeochemical cycling in Arctic marine ecosystems.     

A WASp homolog powers actin polymerization-dependent motility of endosomes in vivo

BACKGROUND: WASp/SCAR proteins activate the Arp2/3 complex to nucleate actin filament assembly and are thought to have important roles in endocytosis. WASp is required for efficient endocytosis of antigen receptors, N-WASp promotes actin polymerization-dependent movement of endomembrane vesicles, and Las17 (a yeast WASp homolog) is required for endocytic internalization. However, it is unknown whether movement of endosomes or other organelles requires activation of the Arp2/3 complex by members of the WASp/SCAR family. RESULTS: Fluorescence video microscopy of yeast cells expressing a GFP-tagged G protein-coupled receptor (Ste2-GFP) as an endocytic marker revealed that endosomes and the lysosome-like vacuole are highly motile. Endosome/vacuole motility required actin polymerization, as indicated by sensitivity to latrunculin A, whereas microtubules were uninvolved. Endosome/vacuole motility did not require actin cables or myosin V (a MYO2 gene product), which moves secretory vesicles and the Golgi apparatus and mediates vacuole segregation. However, endosome motility required Las17, a WASp homolog. In contrast to other processes involving Las17, endosome/vacuole motility required the WCA domain of Las17, which is necessary and sufficient to activate the Arp2/3 complex. CONCLUSIONS: Endosome/vacuole motility in vivo requires actin polymerization stimulated by the WASp homolog Las17. WASp/SCAR family members in mammalian cells may have similar functions. Defects in endosome/lysosome motility may contribute to deficits in lymphocyte or macrophage function observed in human patients lacking WASp or developmental defects in N-WASp-deficient mice.     

Detection of QTL controlling digestive efficiency and anatomy of the digestive tract in chicken fed a wheat-based diet

Background

Improving digestive efficiency is a major goal in poultry production, to reduce production costs, make possible the use of alternative feedstuffs and decrease the volume of manure produced. Since measuring digestive efficiency is difficult, identifying molecular markers associated with genes controlling this trait would be a valuable tool for selection. Detection of QTL (quantitative trait loci) was undertaken on 820 meat-type chickens in a F2 cross between D- and D+ lines divergently selected on low or high AMEn (apparent metabolizable energy value of diet corrected to 0 nitrogen balance) measured at three weeks in animals fed a low-quality diet. Birds were measured for 13 traits characterizing digestive efficiency (AMEn, coefficients of digestive utilization of starch, lipids, proteins and dry matter (CDUS, CDUL, CDUP, CDUDM)), anatomy of the digestive tract (relative weights of the proventriculus, gizzard and intestine and proventriculus plus gizzard (RPW, RGW, RIW, RPGW), relative length and density of the intestine (RIL, ID), ratio of proventriculus and gizzard to intestine weight (PG/I); and body weight at 23 days of age. Animals were genotyped for 6000 SNPs (single nucleotide polymorphisms) distributed on 28 autosomes, the Z chromosome and one unassigned linkage group.

Results

Nine QTL for digestive efficiency traits, 11 QTL for anatomy-related traits and two QTL for body weight at 23 days of age were detected. On chromosome 20, two significant QTL at the genome level co-localized for CDUS and CDUDM, i.e. two traits that are highly correlated genetically. Moreover, on chromosome 16, chromosome-wide QTL for AMEn, CDUS, CDUDM and CDUP, on chromosomes 23 and 26, chromosome-wide QTL for CDUS, on chromosomes 16 and 26, co-localized QTL for digestive efficiency and the ratio of intestine length to body weight and on chromosome 27 a chromosome-wide QTL for CDUDM were identified.

Conclusions

This study identified several regions of the chicken genome involved in the control of digestive efficiency. Further studies are necessary to identify the underlying genes and to validate these in commercial populations and breeding environments.     

Response of the Unicellular Diazotrophic Cyanobacterium Crocosphaera watsonii to Iron Limitation

Iron (Fe) is widely suspected as a key controlling factor of N₂ fixation due to the high Fe content of nitrogenase and photosynthetic enzymes complex, and to its low concentrations in oceanic surface seawaters. The influence of Fe limitation on the recently discovered unicellular diazotrophic cyanobacteria (UCYN) is poorly understood despite their biogeochemical importance in the carbon and nitrogen cycles. To address this knowledge gap, we conducted culture experiments on Crocosphaera watsonii WH8501 growing under a range of dissolved Fe concentrations (from 3.3 to 403 nM). Overall, severe Fe limitation led to significant decreases in growth rate (2.6-fold), C, N and chlorophyll a contents per cell (up to 4.1-fold), N₂ and CO₂ fixation rates per cell (17- and 7-fold) as well as biovolume (2.2-fold). We highlighted a two phased response depending on the degree of limitation: (i) under a moderate Fe limitation, the biovolume of C. watsonii was strongly reduced, allowing the cells to keep sufficient energy to maintain an optimal growth, volume-normalized contents and N₂ and CO₂ fixation rates; (ii) with increasing Fe deprivation, biovolume remained unchanged but the entire cell metabolism was affected, as shown by a strong decrease in the growth rate, volume-normalized contents and N₂ and CO₂ fixation rates. The half-saturation constant for growth of C. watsonii with respect to Fe is twice as low as that of the filamentous Trichodesmium indicating a better adaptation of C. watsonii to poor Fe environments than filamentous diazotrophs. The physiological response of C. watsonii to Fe limitation was different from that previously shown on the UCYN Cyanothece sp, suggesting potential differences in Fe requirements and/or Fe acquisition within the UCYN community. These results contribute to a better understanding of how Fe bioavailability can control the activity of UCYN and explain the biogeography of diverse N₂ fixers in ocean.     

Tetrodotoxin‐resistant voltage‐gated sodium channel Nav1.8 constitutively interacts with ankyrin G

The tetrodotoxin‐resistant (TTX‐R) voltage‐gated sodium channel Na_v1.8 is predominantly expressed in peripheral afferent neurons, but in case of neuronal injury an ectopic and detrimental expression of Na_v1.8 occurs in neurons of the CNS. In CNS neurons, Na_v1.2 and Na_v1.6 channels accumulate at the axon initial segment, the site of the generation of the action potential, through a direct interaction with the scaffolding protein ankyrin G (ankG). This interaction is regulated by protein kinase CK2 phosphorylation. In this study, we quantitatively analyzed the interaction between Na_v1.8 and ankG. GST pull‐down assay and surface plasmon resonance technology revealed that Na_v1.8 strongly and constitutively interacts with ankG, in comparison to what observed for Na_v1.2. An ion channel bearing the ankyrin‐binding motif of Na_v1.8 displaced the endogenous Na_v1 accumulation at the axon initial segment of hippocampal neurons. Finally, Na_v1.8 and ankG co‐localized in skin nerves fibers. Altogether, these results indicate that Na_v1.8 carries all the information required for its localization at ankG micro‐domains. The constitutive binding of Na_v1.8 with ankG could contribute to the pathological aspects of illnesses where Na_v1.8 is ectopically expressed in CNS neurons.