Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules.

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.

A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system.

BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs). RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.

The DNA-binding domain (DBD) is essential for NR2E3 dimerization and interaction with Crx

Purpose:NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts in concert with the transcription factors Crx and Nrl to repress cone-specific genes and activate rod-specific genes. NR2E3 and Crx have been shown to physically interact by their DNA-binding domain (DBD), which may also be implicated in the dimerization process of the nuclear receptor. However, neither NR2E3 homodimerization nor NR2E3/Crx complex formation has been investigated in detail. Methods:In this present work, we analyzed the dimerization of the NR2E3 protein and its interaction with Crx by bioluminescence resonance energy transfer (BRET2) which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. We investigated, on whole intact cells, the role of NR2E3 DBD-mutations in dimerization and association with Crx. Results:We clearly showed that NR2E3 formed homodimers in HEK-293T cells. Moreover, all causative NR2E3 mutations present in the DBD of the protein showed an alteration in dimerization, except for the R76Q and the R104W mutants. Interestingly, the adRP-linked G56R mutant was the only DBD-NR2E3 mutant that showed a correct interaction with Crx. Finally, we observed a decrease in rhodospin gene transactivation for all DBD-NR2E3 mutants tested and no potentiation for the adRP-linked G56R mutant. In addition, the p.G56R mutant enhanced the transrepression of M-opsin promoter, while all other DBD-NR2E3 mutants did not repress M-opsin transactivation. Conclusions:A defect, either in the dimer formation or in the interaction of NR2E3 with Crx, leads to abnormal transcriptional activity on rhodopsin and M-opsin promoter and to an atypical retinal development; while the titration of Crx by p.G56R-NR2E3 leads to low levels of rhodopsin and M-opsin expression and may be responsible for the strong adRP phenotype.

Satisfaction survey with DNA cards method to collect genetic samples for pharmacogenetics studies

Background: Pharmacogenetic studies are essential in understanding the interindividual variability of drug responses. DNA sample collection for genotyping is a critical step in genetic studies. A method using dried blood samples from finger-puncture, collected on DNA-cards, has been described as an alternative to the usual venepuncture technique. The purpose of this study is to evaluate the implementation of the DNA cards method in a multicentre clinical trial, and to assess the degree of investigators' satisfaction and the acceptance of the patients perceived by the investigators.Methods: Blood samples were collected on DNA-cards. The quality and quantity of DNA recovered were analyzed. Investigators were questioned regarding their general interest, previous experience, safety issues, preferences and perceived patient satisfaction. Results: 151 patients' blood samples were collected. Genotyping of GST polymorphisms was achieved in all samples (100%). 28 investigators completed the survey. Investigators perceived patient satisfaction as very good (60.7%) or good (39.3%), without reluctance to finger puncture. Investigators preferred this method, which was considered safer and better than the usual methods. All investigators would recommend using it in future genetic studies. Conclusion: Within the clinical trial setting, the DNA-cards method was very well accepted by investigators and patients (in perception of investigators), and was preferred to conventional methods due to its ease of use and safety.

Large-scale DNA sequencing in taxonomy and conservation : a case study with the mayfly family Heptageniidae (Ephemeroptera) in the Alps and Madagascar

Les larves aquatiques d'éphémères (Ephemeroptera) colonisent toutes les eaux douces du monde et sont couramment utilisées comme bio-indicateurs de la qualité de l'eau. Le genre Rhithrogena (Heptageniidae) est le deuxième plus diversifié chez les éphémères, et plusieurs espèces européennes ont une distribution restreinte dans des environnements alpins sensibles. Les espèces de Rhithrogena ont été classées en "groupes d'espèces" faciles à identifier. Cependant, malgré leur importance écologique et en terme de conservation, beaucoup d'espèces présentent des différences morphologiques ambiguës, suggérant que lataxonomie actuelle ne refléterait pas correctement leur diversité évolutive. De plus, aucune information sur leurs relations, leur origine, le taux de spéciation ou les mécanismes ayant provoqué leur remarquable diversification dans les Alpes n'est disponible. Nous avons d'abord examiné le statut spécifique d'environ 50% des espèces européennes de Rhithrogena en utilisant un large échantillonnage de populations alpines incluant 22 localités typiques, ainsi qu'une analyse basée sur le modèle général mixte de Yule et de coalescence (GMYC) appliqué à un gène mitochondrial standard (coxl) et à un gène nucléaire développé spécifiquement pour cette étude. Nous avons observé un regroupement significatif des séquences coxl en 31 espèces potentielles, et nos résultats ont fortement suggéré la présence d'espèces cryptiques et de fractionnements taxonomiques excessifs chez les Rhithrogena. Nos analyses phylogénétiques ont démontré la monophylie de quatre des six groupes d'espèces reconnus présents dans notre échantillonnage. La taxonomie ADN développée dans cette étude pose les bases d'une future révision de ce genre important mais cryptique en Europe. Puis nous avons mené une étude phylogénétique multi-gènes entre les espèces européennes de Rhithrogena. Les données provenant de trois gènes nucléaires et de deux gènes mitochondriaux ont été largement concordantes, et les relations entre les espèces bien résolues au sein de la plupart des groupes d'espèces dans une analyse combinant tous les gènes. En l'absence de points de calibration extérieurs tels que des fossiles, nous avons appliqué à nos données mitochondriales une horloge moléculaire standard pour les insectes, suggérant une origine des Rhithrogena alpins à la limite Oligocène / Miocène. Nos résultats ont montré le rôle prépondérant qu'ont joué les glaciations du quaternaire dans leur diversification, favorisant la spéciation d'au moins la moitié des espèces actuelle dans les Alpes. La biodiversité et le taux d'endémisme à Madagascar, notamment au niveau de la faune des eaux douces, sont parmi les plus extraordinaires et les plus menacés au monde. On pense que beaucoup d'espèces d'éphémères sont restreintes à un seul bassin versant (microendémisme) dans les zones forestières, ce qui les rendrait particulièrement sensibles à la réduction et à la dégradation de leur habitat. Mis à part deux espèces décrites, Afronurus matitensis et Compsoneuria josettae, les Heptageniidae sont pratiquement inconnus à Madagascar. Les deux genres ont une distribution discontinue en Afrique, à Madagascar et en Asie du Sud-Est, et leur taxonomie complexe est régulièrement révisée. L'approche standard pour comprendre leur diversité, leur endémisme et leur origine requerrait un échantillonnage étendu sur plusieurs continents et des années de travaux taxonomiques. Pour accélérer le processus, nous avons utilisé des collections de musées ainsi que des individus fraîchement collectés, et appliqué une approche combinant taxonomie ADN et phylogénie. L'analyses GMYC du gène coxl a délimité 14 espèces potentielles à Madagascar, dont 70% vraisemblablement microendémiques. Une analyse phylogénique incluant des espèces africaines et asiatiques portant sur deux gènes mitochondriaux et quatre gènes nucléaires a montré que les Heptageniidae malgaches sont monophylétiques et groupe frère des Compsoneuria africains. L'existence de cette lignée unique, ainsi qu'un taux élevé de microendémisme, mettent en évidence leur importance en terme de conservation. Nos résultats soulignent également le rôle important que peuvent jouer les collections de musées dans les études moléculaires et en conservation. - Aquatic nymphs of mayflies (Ephemeroptera) colonize all types of freshwaters throughout the world and are extensively used as bio-indicators of water quality. Rhithrogena (Heptageniidae) is the second most species-rich genus of mayflies, and several European species have restricted distributions in sensitive Alpine environments and therefore are of conservation interest. The European Rhithrogena species are arranged into "species groups" that are easily identifiable. However, despite their ecological and conservation importance, ambiguous morphological differences among many species suggest that the current taxonomy may not accurately reflect their evolutionary diversity. Moreover, no information about their relationships, origin, timing of speciation and mechanisms promoting their successful diversification in the Alps is available. We first examined the species status of ca. 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule- coalescent (GMYC) model analysis of one standard mitochondrial (coxl) and one newly developed nuclear marker. We observed significant clustering of coxl into 31 GMYC species, and our results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Phylogenetic analyses recovered four of the six recognized species groups in our samples as monophyletic. The DNA taxonomy developed here lays the groundwork for a future revision of this important but cryptic genus in Europe. Then we conducted a species-level, multiple-gene phylogenetic study of European Rhithrogena. Data from three nuclear and two mitochondrial loci were broadly congruent, and species-level relationships were well resolved within most species groups in a combined analysis. In the absence of external calibration points like fossils, we applied a standard insect molecular clock hypothesis to our mitochondrial data, suggesting an origin of Alpine Rhithrogena in the Oligocene / Miocene boundary. Our results highlighted the preponderant role that quaternary glaciations played in their diversification, promoting speciation of at least half of the current diversity in the Alps. Madagascar's biodiversity and endemism are among the most extraordinary and endangered in the world. This includes the island's freshwater biodiversity, although detailed knowledge of the diversity, endemism, and biogeographic origin of freshwater invertebrates is lacking. Many mayfly species are thought to be restricted to single river basins (microendemic species) in forested areas, making them particularly sensitive to habitat reduction and degradation. The Heptageniidae are practically unknown in Madagascar except for two described species, Afronurus matitensis and Compsoneuria josettae. Both genera have a disjunct distribution in Africa, Madagascar and Southeast Asia, and a complex taxonomic status still in flux. The standard approach to understanding their diversity, endemism, and origin would require extensive field sampling on several continents and years of taxonomic work. Here we circumvent this using museum collections and freshly collected individuals in a combined approach of DNA taxonomy and phylogeny. The cox/-based GMYC analysis revealed 14 putative species on Madagascar, 70% of which potentially microendemics. A phylogenetic analysis that included African and Asian species and data from two mitochondrial and four nuclear loci indicated the Malagasy Heptageniidae are monophyletic and sister to African Compsoneuria. The observed monophyly and high microendemism highlight their conservation importance. Our results also underline the important role that museum collections can play in molecular studies, especially in critically endangered biodiversity hotspots like Madagascar.

Nutrigenomics and nutrigenetics: the emerging faces of nutrition.

The recognition that nutrients have the ability to interact and modulate molecular mechanisms underlying an organism's physiological functions has prompted a revolution in the field of nutrition. Performing population-scaled epidemiological studies in the absence of genetic knowledge may result in erroneous scientific conclusions and misinformed nutritional recommendations. To circumvent such issues and more comprehensively probe the relationship between genes and diet, the field of nutrition has begun to capitalize on both the technologies and supporting analytical software brought forth in the post-genomic era. The creation of nutrigenomics and nutrigenetics, two fields with distinct approaches to elucidate the interaction between diet and genes but with a common ultimate goal to optimize health through the personalization of diet, provide powerful approaches to unravel the complex relationship between nutritional molecules, genetic polymorphisms, and the biological system as a whole. Reluctance to embrace these new fields exists primarily due to the fear that producing overwhelming quantities of biological data within the confines of a single study will submerge the original query; however, the current review aims to position nutrigenomics and nutrigenetics as the emerging faces of nutrition that, when considered with more classical approaches, will provide the necessary stepping stones to achieve the ambitious goal of optimizing an individual's health via nutritional intervention.

Mitochondrial DNA of Nellore and European x Nellore crossing cattle of high performance

The objective of this work was to evaluate, through a polymorphism in the ND5 gene of the bovine mitochondrial DNA, the frequency of Bos taurus indicus mtDNA individuals in a sample of Nellore purebred origin animals (n = 69) and crossbred animals originated from crosses of European sires and Nellore purebred origin females (n = 275). Only 2.26% (8/354) of the animals presented Bos taurus indicus mtDNA. The high frequency of Bos taurus taurus mtDNA in these animals can be a consequence of selection, once the animals studied are originated from selected lineages of high performance for meat production.

Electron microscopic visualization of protein-DNA interactions at the estrogen responsive element and in the first intron of the Xenopus laevis vitellogenin gene.

Stable protein-DNA complexes can be assembled in vitro at the 5' end of Xenopus laevis vitellogenin genes using extracts of nuclei from estrogen-induced frog liver and visualized by electron microscopy. Complexes at the three following sites can be identified on the gene B2: the transcription initiation site, the estrogen responsive element (ERE) and in the first intron. The complex at the transcription initiation site is stabilized by dinucleotides and thus represents a ternary transcription complex. The formation of the complexes at the two other sites is enhanced by estrogen and is reduced by tamoxifen, an antagonist of estrogen, while this latter effect is reversed by adding an excess of hormone. No sequence homology is apparent between the site containing the ERE and the binding site in intron I and functional tests in MCF-7 cells suggest that these two sites are not equivalent. Finally, we made use of previously characterized deletion mutants of the 5' flanking region of the gene B1, a close relative of the gene B2, to demonstrate that the 13-bp palindromic core element of the ERE is involved in the formation of the complexes observed upstream of the transcription initiation site.

Patient with Fanconi Syndrome (FS) and retinitis pigmentosa (RP) caused by a deletion and duplication of mitochondrial DNA (mtDNA).

BACKGROUND: We report a patient with a highly unusual presentation of a mitochondrial disorder. HISTORY AND SIGNS: An 8-year old girl presented with muscular cramps as well as height and weight deceleration. Investigations revealed lactic acidosis, electrolytic imbalance and urinary loss of glucose and electrolytes secondary to proximal renal tubulopathy consistent with Fanconi syndrome (FS). Ophthalmic examination revealed asymptomatic retinitis pigmentosa (RP) with no other ocular manifestations. A mitochondriopathy was suspected and genetic analysis performed. THERAPY AND OUTCOME: Southern blotting documented a heteroplasmic mutation of mtDNA with deletion/duplication. Three discrete mitochondrial genomes were detected: normal; deletion of 6.7 kb and a deletion/duplication consisting of 1 normal and 1 deleted genome. The relative proportions varied considerably between tissues. CONCLUSIONS: The association of FS and RP combines features of Kearns-Sayre syndrome and Pearson marrow-pancreas syndrome, without being typical of either. This highly unusual clinical presentation emphasises the need for systemic investigation of patients with FS and further underlines the importance of mtDNA analysis in patients with unexpected associations of affected tissues.

The homologous regulators ANR of Pseudomonas aeruginosa and FNR of Escherichia coli have overlapping but distinct specificities for anaerobically inducible promoters.

The anaerobic transcriptional regulator ANR induces the arginine deiminase and denitrification pathways in Pseudomonas aeruginosa during oxygen limitation. The homologous activator FNR of Escherichia coli, when introduced into an anr mutant of P. aeruginosa, could functionally replace ANR for anaerobic growth on nitrate but not for anaerobic induction of arginine deiminase. In an FNR-positive E. coli strain, the ANR-dependent promoter of the arcDABC operon, which encodes the enzymes of the arginine deiminase pathway, was not expressed. To analyse systematically these distinct induction patterns, a lacZ promoter-probe, broad-host-range plasmid containing various -40 regions (the ANR/FNR recognition sequences) and -10 promoter sequences was constructed. These constructs were tested in P. aeruginosa and in E. coli expressing either ANR or FNR. In conjunction with the consensus -10 hexamer of E. coli sigma 70 RNA polymerase (TATAAT), the consensus FNR site (TTGAT ..... ATCAA) was recognized efficiently by ANR and FNR in both hosts. By contrast, when promoters contained the Arc box (TTGAC .... ATCAG), which is found in the arcDABC promoter, or a symmetrical mutant FNR site (CTGAT .... ATCAG), ANR was a more effective activator than was FNR. Conversely, an extended 22 bp, fully symmetrical FNR site allowed better activation with FNR than with ANR. Combination of the arc promoter -10 sequence (CCTAAT) with the Arc box or the consensus FNR site resulted in good ANR-dependent expression in P. aeruginosa but gave practically no expression in E. coli, suggesting that RNA polymerase of P. aeruginosa differs from the E. coli enzyme in -10 recognition specificity. In conclusion, ANR and FNR are able to activate the RNA polymerases of P. aeruginosa and E. coli when the -40 and -10 promoter elements ae identical or close to the E. coli consensus sequences.

Spatial visualization of DNA in solution.

An image analysis method is presented which allows for the reconstruction of the three-dimensional path of filamentous objects from two of their projections. Starting with stereo pairs, this method is used to trace the trajectory of DNA molecules embedded in vitreous ice and leads to a faithful representation of their three-dimensional shape in solution. This computer-aided reconstruction is superior to the subjective three-dimensional impression generated by observation of stereo pairs of micrographs because it enables one to look at the reconstructed molecules from any chosen direction and distance and allows quantitative analysis such as determination of distances, curvature, persistence length, and writhe of DNA molecules in solution.

DNA barcodes for soil animal taxonomy

The biodiversity of soil communities remains very poorly known and understood. Soil biological sciences are strongly affected by the taxonomic crisis, and most groups of animals in that biota suffer from a strong taxonomic impediment. The objective of this work was to investigate how DNA barcoding - a novel method using a microgenomic tag for species identification and discrimination - permits better evaluation of the taxonomy of soil biota. A total of 1,152 barcode sequences were analyzed for two major groups of animals, collembolans and earthworms, which presented broad taxonomic and geographic sampling. Besides strongly reflecting the taxonomic impediment for both groups, with a large number of species-level divergent lineages remaining unnamed so far, the results also highlight a high level (15%) of cryptic diversity within known species of both earthworms and collembolans. These results are supportive of recent local studies using a similar approach. Within an impeded taxonomic system for soil animals, DNA-assisted identification tools can facilitate and improve biodiversity exploration and description. DNA-barcoding campaigns are rapidly developing in soil animals and the community of soil biologists is urged to embrace these methods.