The Zymovars of Vibrio cholerae: Multilocus Enzyme Electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several geneticaly diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae) from Western and Northeastern Colombia

Random amplified polymorphic DNA (RAPD) markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8) but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8). According to molecular variance analysis, the genetic distance between populations was significant (phiST 0.1131, P < 0.001). The variation among individuals within populations (phiST 0.8869, P < 0.001)was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.

Exogenous selection shapes germination behaviour and seedling traits of populations at different altitudes in a Senecio hybrid zone.

BACKGROUND AND AIMS: The Senecio hybrid zone on Mt Etna, Sicily, is characterized by steep altitudinal clines in quantitative traits and genetic variation. Such clines are thought to be maintained by a combination of 'endogenous' selection arising from genetic incompatibilities and environment-dependent 'exogenous' selection leading to local adaptation. Here, the hypothesis was tested that local adaptation to the altitudinal temperature gradient contributes to maintaining divergence between the parental species, S. chrysanthemifolius and S. aethnensis. METHODS: Intra- and inter-population crosses were performed between five populations from across the hybrid zone and the germination and early seedling growth of the progeny were assessed. KEY RESULTS: Seedlings from higher-altitude populations germinated better under low temperatures (9-13 °C) than those from lower altitude populations. Seedlings from higher-altitude populations had lower survival rates under warm conditions (25/15 °C) than those from lower altitude populations, but also attained greater biomass. There was no altitudinal variation in growth or survival under cold conditions (15/5 °C). Population-level plasticity increased with altitude. Germination, growth and survival of natural hybrids and experimentally generated F(1)s generally exceeded the worse-performing parent. CONCLUSIONS: Limited evidence was found for endogenous selection against hybrids but relatively clear evidence was found for divergence in seed and seedling traits, which is probably adaptive. The combination of low-temperature germination and faster growth in warm conditions might enable high-altitude S. aethnensis to maximize its growth during a shorter growing season, while the slower growth of S. chrysanthemifolius may be an adaptation to drought stress at low altitudes. This study indicates that temperature gradients are likely to be an important environmental factor generating and maintaining adaptive divergence across the Senecio hybrid zone on Mt Etna.

Communally breeding Bechstein's bats have a stable social system that is independent from the postglacial history and location of the populations.

Investigating macro-geographical genetic structures of animal populations is crucial to reconstruct population histories and to identify significant units for conservation. This approach may also provide information about the intraspecific flexibility of social systems. We investigated the history and current structure of a large number of populations in the communally breeding Bechstein's bat (Myotis bechsteinii). Our aim was to understand which factors shape the species' social system over a large ecological and geographical range. Using sequence data from one coding and one noncoding mitochondrial DNA region, we identified the Balkan Peninsula as the main and probably only glacial refugium of the species in Europe. Sequence data also suggest the presence of a cryptic taxon in the Caucasus and Anatolia. In a second step, we used seven autosomal and two mitochondrial microsatellite loci to compare population structures inside and outside of the Balkan glacial refugium. Central European and Balkan populations both were more strongly differentiated for mitochondrial DNA than for nuclear DNA, had higher genetic diversities and lower levels of relatedness at swarming (mating) sites than in maternity (breeding) colonies, and showed more differentiation between colonies than between swarming sites. All these suggest that populations are shaped by strong female philopatry, male dispersal, and outbreeding throughout their European range. We conclude that Bechstein's bats have a stable social system that is independent from the postglacial history and location of the populations. Our findings have implications for the understanding of the benefits of sociality in female Bechstein's bats and for the conservation of this endangered species.

The fine-scale architecture of structural variants in 17 mouse genomes.

BACKGROUND: Accurate catalogs of structural variants (SVs) in mammalian genomes are necessary to elucidate the potential mechanisms that drive SV formation and to assess their functional impact. Next generation sequencing methods for SV detection are an advance on array-based methods, but are almost exclusively limited to four basic types: deletions, insertions, inversions and copy number gains. RESULTS: By visual inspection of 100 Mbp of genome to which next generation sequence data from 17 inbred mouse strains had been aligned, we identify and interpret 21 paired-end mapping patterns, which we validate by PCR. These paired-end mapping patterns reveal a greater diversity and complexity in SVs than previously recognized. In addition, Sanger-based sequence analysis of 4,176 breakpoints at 261 SV sites reveal additional complexity at approximately a quarter of structural variants analyzed. We find micro-deletions and micro-insertions at SV breakpoints, ranging from 1 to 107 bp, and SNPs that extend breakpoint micro-homology and may catalyze SV formation. CONCLUSIONS: An integrative approach using experimental analyses to train computational SV calling is essential for the accurate resolution of the architecture of SVs. We find considerable complexity in SV formation; about a quarter of SVs in the mouse are composed of a complex mixture of deletion, insertion, inversion and copy number gain. Computational methods can be adapted to identify most paired-end mapping patterns.

Post-genomic update on a classical candidate gene for coronary artery disease: ESR1.

BACKGROUND: After age, sex is the most important risk factor for coronary artery disease (CAD). The mechanism through which women are protected from CAD is still largely unknown, but the observed sex difference suggests the involvement of the reproductive steroid hormone signaling system. Genetic association studies of the gene-encoding Estrogen Receptor α (ESR1) have shown conflicting results, although only a limited range of variation in the gene has been investigated. METHODS AND RESULTS: We exploited information made available by advanced new methods and resources in complex disease genetics to revisit the question of ESR1's role in risk of CAD. We performed a meta-analysis of 14 genome-wide association studies (CARDIoGRAM discovery analysis, N=≈87,000) to search for population-wide and sex-specific associations between CAD risk and common genetic variants throughout the coding, noncoding, and flanking regions of ESR1. In addition to samples from the MIGen (N=≈6000), WTCCC (N=≈7400), and Framingham (N=≈3700) studies, we extended this search to a larger number of common and uncommon variants by imputation into a panel of haplotypes constructed using data from the 1000 Genomes Project. Despite the widespread expression of ERα in vascular tissues, we found no evidence for involvement of common or low-frequency genetic variation throughout the ESR1 gene in modifying risk of CAD, either in the general population or as a function of sex. CONCLUSIONS: We suggest that future research on the genetic basis of sex-related differences in CAD risk should initially prioritize other genes in the reproductive steroid hormone biosynthesis system.

'Good-genes' and 'compatible-genes' effects in an Alpine whitefish and the information content of breeding tubercles over the course of the spawning season.

Some models of sexual selection predict that individuals vary in their genetic quality and reveal some of this variation in their secondary sexual characteristics. Alpine whitefish (Coregonus sp.) develop breeding tubercles shortly before their spawning season. These tubercles are epidermal structures that are distributed regularly along the body sides of both males and females. There is still much unexplained variation in the size of breeding tubercles within both sexes and with much overlap between the sexes. It has been suggested that breeding tubercles function to maintain body contact between the mating partners during spawning, act as weapons for defence of spawning territories, or are sexual signals that reveal aspects of genetic quality. We took two samples of whitefish from their spawning place, one at the beginning and one around the peak of spawning season. We found that females have on average smaller breeding tubercles than males, and that tubercle size partly reveals the stage of gonad maturation. Two independent full-factorial breeding experiments revealed that embryo mortality was significantly influenced by male and female effects. This finding demonstrates that the males differed in their genetic quality (because offspring get nothing but genes from their fathers). Tubercle size was negatively linked to some aspects of embryo mortality in the first breeding experiment but not significantly so in the second. This lack of consistency adds to inconsistent results that were reported before and suggests that (i) some aspects of genetic quality are not revealed in breeding tubercles while others are, or (ii) individuals vary in their signaling strategies and the information content of breeding tubercles is not always reliable. Moreover, the fact that female whitefish have breeding tubercles of significant size while males seem to have few reasons to be choosy suggests that the tubercles might also serve some functions that are not linked to sexual signaling.

IS6110 restriction fragment length polymorphism typing of Mycobacterium tuberculosis isolates from East Azerbaijan Province of Iran

To investigate the genetic variation among Mycobacterium tuberculosis isolates in the East Azerbaijan Province of Iran and to evaluate the level of and risk factors for recent transmission of tuberculosis (TB), we performed IS6110-based restriction fragment length polymorphism analysis of strains, isolated from 105 patients during the period of September 2002 to March 2003 in TB centers and university hospitals of the province. Among 105 isolates, 81 different IS6110 patterns were found, of which 70 were observed only once and 11 were shared by two to eight isolates. Ninety-six isolates (91.4%) were found to have more than five copies of IS6110 and together with high patterns polymorphism, shows that IS6110-RFLP typing could be useful for studying the epidemiology of TB in Azerbaijan. The minimum estimated rate of recent transmission was 23%, suggesting that the degree of recent transmission in East Azerbaijan Province is relatively low. Clustering was not associated with age, sex or site of infection of TB but drug-resistant isolates were less likely to be clustered than sensitive isolates (p < 0.05).

The amphioxus genome and the evolution of the chordate karyotype.

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

Evolution of life history traits in a natural population of the shrew Crocidura russula

Abstract Life history traits encompass all the decisions concerning fitness an individual is faced with during his life. The study of these traits is crucial to understand the factors shaping the biology of living organisms. Up until now, most of the information on the evolution of life history traits comes from laboratory studies. While these studies are interesting to test the effect of specific parameters, their conclusions are difficult to extrapolate to natural populations. Investigating the evolution of life history traits in natural populations is of great interest. This may be tricky because it requires information on reproduction, survival and morphology of individuals. Mark-recapture methods allow most of this information to be obtained. However, when direct observations of a species are not possible due to its ecology, indirect methods must be used to infer lifetime reproductive success. In this case, molecular markers are particularly helpful in assessing the genetic relationships between individuals and allow the construction of a pedigree. This thesis focuses on a natural population of a small insectivorous mammal, the greater white-toothed shrew, Crocidura russula. Because of its hidden lifestyle, the two complementary techniques mentioned above were combined to gather information on this population. The data were used to explore diverse aspects of evolutionary biology. We demonstrated that the high genetic variance displayed by the species was not maintained by its mating system because this shrew was less monogamous than previously thought. The large genetic diversity was most likely promoted by gene flow from the neighborhood. Dispersal was thus a central topic in this thesis. We showed that dispersal was not driven by inbreeding avoidance. In addition, we did not find any inbreeding depression in the population. Dispersal was promoted by a high number of vacant territories in the population for both sexes, meaning that territory acquisition played an important role in driving dispersal. Moreover, dispersal propensity was shown to have a genetic basis and, once achieved, to have no effect on individual fitness. Body mass was found to be a life history trait strongly influenced by sexual and viability selection in both sexes. Larger individuals had higher access to reproduction through territory acquisition and defense than lighter ones. By contrast, intermediate size individuals were favored by viability selection presumably because of ecological constraints and metabolic costs. Finally, we demonstrated that the majority of the life history traits in our shrew population has the potential to evolve because they maintained substantial amounts of additive genetic variance. Nonetheless, life history traits had no significant heritability due to their high level of nonadditive or environmental variance. Résumé Les traits d'histoire de vie comprennent toutes les décisions auxquelles un individu est confronté au cours de sa vie et qui concernent sa valeur adaptative. L'étude de ces traits est cruciale pour comprendre les facteurs qui façonnent la biologie des êtres vivants. Jusqu'à ce jour, la majorité des informations sur l'évolution des traits d'histoire de vie provient d'études réalisées en laboratoire. Alors que ces études sont intéressantes pour tester l'effet de paramètres spécifiques, leurs conclusions sont difficilement extrapolables aux populations naturelles. Il est particulièrement intéressant d'étudier l'évolution des traits d'histoire de vie dans des populations naturelles. Toutefois, ces études peuvent se révéler difficiles parce qu'elles requièrent des informations sur la reproduction, la survie et la morphologie des individus. Des méthodes de marquage-recapture permettent d'obtenir ces informations. Cependant, lorsque l'écologie de l'espèce rend les obervations directes impossibles, des méthodes indirectes doivent être utilisées pour obtenir le succès reproducteur des individus. Dans ce cas, les marqueurs moléculaires sont particulièrement utiles pour évaluer les relations génétiques entre individus et permettre la construction d'un pedigree. Cette thèse porte sur une population naturelle d'un petit mammifère insectivore, la musaraigne musette, Crocidura russula. Parce que cette espèce présente un mode de vie souterrain, les deux techniques complémentaires mentionnées ci-dessus ont été combinées pour acquérir les informations nécessaires. Les données ont été utilisées pour explorer divers aspects de biologie evolutive. Nous avons montré que la grande quantité de variance génétique trouvée chez cette espèce n'est pas maintenue par son système d'appariement. Celle-ci s'est en effet avérée être moins monogame que ce qui était admis jusqu'ici. Sa grande diversité génétique est plutôt entretenue par le flux de gènes provenant du voisinage. La dispersion a donc été un sujet phare dans cette thèse. Nous avons montré qu'elle n'est pas provoquée par un évitement de la consanguinité et nous n'avons pas trouvé de dépression de consanguité dans notre population. L'acquisition d'un territoire joue par contre un rôle important dans la dispersion. En outre, la dispersion possède une base génétique chez cette espèce. De plus, une fois qu'ils ont dispersé, les individus n'ont pas une valeur adaptative differente d'individus philopatriques. Le poids s'est avéré être un trait d'histoire de vie fortement influencé par la sélection sexuelle et de viabilité chez les deux sexes. Les gros individus ont accès à la reproduction parce qu'ils acquièrent et défendent un territoire plus facilement que les plus légers. Au contraire, les individus de taille intermédiaire sont favorisés par la sélection de viabilité, certainement à cause de contraintes écologiques et de coûts métaboliques. Finalement, nous avons montré que la majorité des traits d'histoire de vie dans notre population a le potentiel d'évoluer parce qu'elle maintient des quantités considérables de variance génétique additive. Néanmoins, l'héritabilité de ces traits d'histoire de vie n'est pas significative à cause de la grande quantité de variance non-additive ou environmentale associée à ces traits.

Use of phylogeny to resolve the taxonomy of the widespread and highly polymorphic African giant shrews (Crocidura olivieri group, Crocidurinae, Mammalia).

The aim of this study is to provide a better understanding of the genetic relationships within the widespread and highly polymorphic group of African giant shrews (Crocidura olivieri group). We sequenced 769 base pairs (bp) of the mitochondrial cytochrome b gene and 472 bp of the mitochondrial control region over the entire geographic range from South Africa to Morocco. The analyses reveal four main clades associated with different biomes. The largest clade occurs over a range covering Northwest and Central Africa and includes samples of C. fulvastra, C. olivieri, and C. viaria. The second clade is composed of C. goliath from Gabon, while South African C. flavescens, and C. hirta form two additional clades. On the basis of these results, the validity of some taxa in the C. olivieri group should be re-evaluated.