On the cellular site of two-pore channel TPC1 action in the Poaceae.

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.

Genetic structure of a linear population of Beta vulgaris ssp maritima (sea beet) revealed by isozyme and RFLP analysis

Knowledge of the genetic structure of plant populations is necessary for the understanding of the dynamics of major ecological processes. It also has applications in conservation biology and risk assessment for genetically modified crops. This paper reports the genetic structure of a linear population of sea beet, Beta vulgaris ssp. maritima (the wild relative of sugar beet), on Furzey Island, Poole Harbour. The relative spatial positions of the plants were accurately mapped and the plants were scored for variation at isozyme and RFLP loci. Structure was analysed by repeated subdivision of the population to find the average size of a randomly mating group. Estimates of F-ST between randomly mating units were then made, and gave patterns consistent with the structure of the population being determined largely by founder effects. The implications of these results for the monitoring of transgene spread in wild sea beet populations are discussed.

Constraints on the permeability structure of alluvial aquifers from P-wave sonic logs

Modern sonic logging tools designed for shallow environmental and engineering applications allow for P-wave phase velocity measurements over a wide frequency band. Methodological considerations indicate that, for saturated unconsolidated sediments in the silt to sand range and source frequencies ranging from approximately 1 to 30 kHz, the observable poro-elastic P-wave velocity dispersion is sufficiently pronounced to allow for reliable first-order estimations of the underlying permeability structure. These predictions have been tested on and verified for a surficial alluvial aquifer. Our results indicate that, even without any further calibration, the thus obtained permeability estimates as well as their variabilities within the pertinent lithological units are remarkably close to those expected based on the corresponding granulometric characteristics.

Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation.

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a (45)Ca(2+) uptake assay (7.11 +/- 1.93 versus 3.61 +/- 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuria.

Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit.

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

Impact of social structure variation on population genetics, social conflicts and life histories in ants

Summary The evolution of social structures and breeding systems in animals is a complex process that combines ecological, genetical and social factors. This thesis sheds light on important changes in population genetics, life-history and social behavior that are associated with variation in social structure in ants. The socially polymorphic ant Formica selysi was chosen as the model organism because single- and multiple-queen colonies occur in close proximity within a single large population. The shift from single- to multiple-queen colonies is generally associated with profound changes in dispersal behavior and mode of colony founding. In chapter 1, we examine the genetic consequences of variation in social structure at both the colony and population levels. A detailed microsatellite analysis reveals that both colony types have similar mating systems, with few or no queen turnover. Furthermore, the complete lack of genetic differentiation observed between single- and multiple-queen colonies provides no support to the hypothesis that change in queen number leads to restricted gene flow between social forms. Besides changes in the genetic composition of the colony, the variation in the number of queens per colony is associated with changes in a network of behavioral and life-history traits that have been described as forming a "polygyny syndrome". In chapter 2, we demonstrate that multiple-queen colonies profoundly differ from single-queen ones in terms of size, nest density and lifespan of colonies, in weight of queens produced, as well as in allocation to reproductive individuals relative to workers. These multifaceted changes in life-history traits can provide various fitness benefits to members of multiple-queen colonies. Increasing the number of queens in a colony usually results in a decreased level of aggression towards non-nestmates. The phenotype matching hypothesis predicts that, compared to single-queen colonies, multiple-queen colonies have more diverse genetically-derived cues used for recognition, resulting in a lower ability to discriminate non-nestmates. In sharp contrast to this hypothesis, we show in chapter 3 that single- and multiple-queen colonies exhibit on average similar levels of aggression. Moreover, stronger aggression is recorded between colonies of different social structure than between colonies of the same social structure. Several hypotheses propose that the evolution of multiple-queen colonies is at least partly due to benefits resulting from an increase in colony genetic diversity. The task-efficiency hypothesis holds that genetic variation improves task performance due to a more complete or more sensitive expression of the genetically-based division of labor. In .chapter 4, we evaluate if higher colony genetic diversity increases worker size polymorphism and thus may improve division of labor. We show that despite the fact that worker size has a heritable component, higher levels of genetic diversity do not result in more polymorphic workers. The smaller size and lower polymorphism levels of workers of multiple-queen colonies compared to single-queen ones further indicate that an increase in colony genetic diversity does not increase worker size polymorphism but might improve colony homeostasis. In chapter 5, we provide clear evidence for an ongoing conflict between queens and workers on sex allocation, as predicted by kin selection theory. Our data show that queens of F. selysi strongly influence colony sex allocation by biasing the sex ratio of their eggs. However, there is also evidence that workers eliminated some male brood, resulting in a population sex-investment ratio that is between the queens' and workers' equilibria. Résumé L'évolution des structures sociales et systèmes d'accouplement chez les animaux est un processus complexe combinant à la fois des facteurs écologiques, génétiques et sociaux. Cette thèse met en lumière des changements importants dans la génétique des populations, les traits d'histoire de vie et les comportements sociaux qui sont associés à des variations de structure sociale chez les fourmis. Durant ce travail, nous avons étudié une population de Formica selysi composée à la fois de colonies à une reine et de colonies à plusieurs reines. La transition de colonie à une reine à colonie à plusieurs reines est généralement associée à des changements profonds dans le comportement de dispersion ainsi que le mode de fondation des sociétés. Dans le chapitre 1, nous examinons les conséquences génétiques de la variation de structure sociale tant au niveau de la colonie qu'au niveau de la population. Une analyse détaillée à l'aide de marqueurs microsatellites nous révèle que les deux types de colonies ont des systèmes d'accouplements similaires avec peu ou pas de renouvellement de reines. L'absence totale de différenciation génétique entre les colonies à une et à plusieurs reines n'apporte aucun support à l'hypothèse selon laquelle un changement dans le nombre de reines conduit à un flux de gènes restreint entre les deux formes sociales. A côté de changements dans la composition génétique de la colonie, la variation du nombre de reines dans une colonie est associée à une multitude de changements comportementaux et de traits d'histoire de vie qui ont été décrits comme formant un "syndrome polygyne". Dans le chapitre 2, nous démontrons que les colonies à plusieurs reines diffèrent profondément des colonies à une reine en terme de taille, densité de nids, longévité des colonies, poids des nouvelles reines produites ainsi que dans l'allocation entre les individus reproducteurs et les ouvrières. Ces changements multiples dans les traits d'histoire de vie peuvent apporter des bénéfices variés en terme de fitness aux colonies à plusieurs reines. L'augmentation du nombre de reines dans une colonie est généralement associée à une baisse du degré d'agressivité envers les fourmis étrangères au nid. L'hypothèse "phénotype matching" prédit que les colonies à plusieurs reines ont une plus grande diversité dans les facteurs d'origine génétique utilisés pour la reconnaissance, résultant en une capacité diminuée à discriminer une fourmi étrangère au nid. Contrairement à cette hypothèse, nous montrons dans le chapitre 3 que les colonies à une et à plusieurs reines ont des niveaux d'agressivité similaires. De plus, une agressivité accrue est observée entre colonies de structures sociales différentes comparée à des colonies de même structure sociale. Plusieurs hypothèses ont proposé que l'évolution de colonies ä plusieurs reines soit en partie due aux bénéfices résultant d'une augmentation de la diversité génétique dans la colonie. L'hypothèse "task efficiency" prédit que la diversité génétique améliore l'efficacité à effectuer certaines tâches grâce à une expression plus complète et plus souple d'une division du travail génétiquement déterminée. Nous évaluons dans le chapitre 4 si un accroissement de la diversité génétique augmente le polymorphisme de taille des ouvrières, d'où peut ainsi découler une meilleure division du travail. Nous montrons qu'en dépit du fait que la taille des ouvrières soit un caractère héritable, une forte diversité génétique ne se traduit pas par un plus fort polymorphisme chez les ouvrières. Les ouvrières de colonies à plusieurs reines sont plus petites et moins polymorphes que celles des colonies à une seule reine. Dans le chapitre 5, nous démontrons l'existence d'un conflit ouvert entre reines et ouvrières à propos de l'allocation dans les sexes, comme le prédit la théorie de la sélection de parentèle. Nos données révèlent que les reines de F. selysi influencent fortement l'allocation dans les sexes en biaisant la sexe ratio des oeufs. Cependant, certains indices indiquent que les ouvrières éliminent une partie du couvain mâle, ce qui a pour effet d'avoir un investissement dans les sexes au niveau de la population intermédiaire entre les intérêts des reines et des ouvrières.

Genetic structure of the genus Leptospira by multilocus enzyme electrophoresis

Thirty strains from the 11 species of the genus Leptospira were studied by multilocus enzyme electrophoresis at 12 enzyme loci, all of which were polymorphic. The mean number of alleles per locus was 6.5. Twenty-five electrophoretic types were distinguished. Grouping of the strains by cluster analysis was in general agreement with species delineation as determined by DNA-DNA hybridization, except for the strains of Leptospira meyeri and Leptospira inadai, which were scattered throughout the genus, reflecting previously recognized taxonomic uncertainties. Analysis of the clonality within Leptospira interrogans sensu stricto indicated that this population was relatively heterogeneous and a lack of gene linkage disequilibrium could not be excluded. There was a genetic discrimination between the pathogenic species and the saprophytic ones. The phenotypically intermediate species (L. inadai and Leptospira fainei) were also genetically separated and were probably closer to the saprophytes than to the pathogens.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

Crystal structure of NLRC4 reveals its autoinhibition mechanism.

Nucleotide-binding and oligomerization domain-like receptor (NLR) proteins oligomerize into multiprotein complexes termed inflammasomes when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of mouse NLRC4 in a closed form. The adenosine diphosphate-mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain HD2 repressively contacted a conserved and functionally important α-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.