Differential strain-specific diagnosis of the heartwater agent: Ehrlichia ruminantium.

Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa introduced in the Caribbean and threatening to emerge and spread in the American mainland. Complete genome sequencing was done for two isolates of E. ruminantium of differing phenotype, isolates Gardel (Erga) from Guadeloupe Island and Welgevonden (Erwe) originating from South Africa and maintained in Guadeloupe. The type strain of E. ruminantium (Erwo), previously isolated and sequenced in South Africa; is identical to Erwe with respect to target genes. They make the Erwe/Erwo complex. Comparative analysis of the genomes shows the presence of 49 unique CDS and 28 truncated CDS differentiating Erga from Erwe/Erwo. Three regions of accumulated differences (RAD) acting as mutational hot spots were identified in E. ruminantium. Ten CDS, six unique CDS and four truncated CDS corresponding to major genomic changes (deletions or extensive mutations) were considered as targets for differential diagnosis on four isolates of E. ruminantium: Erga, Erwe/Erwo, Senegal and Umpala. Pairs of PCR primers were developed for each target gene. PCR analysis of the target genes generated strain-specific patterns on Erga and Erwe/Erwo as predicted by comparative genomics, but also for isolates Senegal and Umpala. The target genes identified by bacterial comparative genomics are shown to be highly efficient for strain-specific PCR diagnosis of E. ruminantium and further vaccine management tools.

Comparative genomics of three strains of Ehrlichia ruminantium: a review.

The tick-borne Rickettsiale Ehrlichia ruminantium (E. ruminantium) is the causative agent of heartwater in Africa and the Caribbean. Heartwater, responsible for major losses on livestock in Africa represents also a threat for the American mainland. Three complete genomes corresponding to two different groups of differing phenotypes, Gardel and Welgevonden, have been recently described. One genome (Erga) represents the Gardel group from Guadeloupe Island and two genomes (Erwo and Erwe) belong to the Welgevonden group. Erwo, isolated in South Africa, is the parental strain of Erwe, which was maintained for 18 years in Guadeloupe under different culture conditions than Erwo. The three strains display genomes of differing sizes with 1,499,920 bp, 1,512,977 bp, and 1,516,355 bp for Erga, Erwe, and Erwo, respectively. Gene sequences and order are highly conserved between the three strains, although several gene truncations could be pinpointed, most of them occurring within three regions of accumulated differences (RAD). E. ruminantium displays a strong leading/lagging compositional bias inducing a strand-specific codon usage. Finally, a striking feature of E. ruminantium is the presence of long intergenic regions containing tandem repeats. These repeats are at the origin of an active process, specific to E. ruminantium, of genome expansion/contraction based on the addition or removal of tandem units.

Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features.

The green lineage is reportedly 1,500 million years old, evolving shortly after the endosymbiosis event that gave rise to early photosynthetic eukaryotes. In this study, we unveil the complete genome sequence of an ancient member of this lineage, the unicellular green alga Ostreococcus tauri (Prasinophyceae). This cosmopolitan marine primary producer is the world's smallest free-living eukaryote known to date. Features likely reflecting optimization of environmentally relevant pathways, including resource acquisition, unusual photosynthesis apparatus, and genes potentially involved in C(4) photosynthesis, were observed, as was downsizing of many gene families. Overall, the 12.56-Mb nuclear genome has an extremely high gene density, in part because of extensive reduction of intergenic regions and other forms of compaction such as gene fusion. However, the genome is structurally complex. It exhibits previously unobserved levels of heterogeneity for a eukaryote. Two chromosomes differ structurally from the other eighteen. Both have a significantly biased G+C content, and, remarkably, they contain the majority of transposable elements. Many chromosome 2 genes also have unique codon usage and splicing, but phylogenetic analysis and composition do not support alien gene origin. In contrast, most chromosome 19 genes show no similarity to green lineage genes and a large number of them are specialized in cell surface processes. Taken together, the complete genome sequence, unusual features, and downsized gene families, make O. tauri an ideal model system for research on eukaryotic genome evolution, including chromosome specialization and green lineage ancestry.

Comparative genomic analysis of three strains of Ehrlichia ruminantium reveals an active process of genome size plasticity.

Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa that has been introduced in the Caribbean and is threatening to emerge and spread on the American mainland. We sequenced the complete genomes of two strains of E. ruminantium of differing phenotypes, strains Gardel (Erga; 1,499,920 bp), from the island of Guadeloupe, and Welgevonden (Erwe; 1,512,977 bp), originating in South Africa and maintained in Guadeloupe in a different cell environment. Comparative genomic analysis of these two strains was performed with the recently published parent strain of Erwe (Erwo) and other Rickettsiales (Anaplasma, Wolbachia, and Rickettsia spp.). Gene order is highly conserved between the E. ruminantium strains and with A. marginale. In contrast, there is very little conservation of gene order with members of the Rickettsiaceae. However, gene order may be locally conserved, as illustrated by the tuf operons. Eighteen truncated protein-encoding sequences (CDSs) differentiate Erga from Erwe/Erwo, whereas four other truncated CDSs differentiate Erwe from Erwo. Moreover, E. ruminantium displays the lowest coding ratio observed among bacteria due to unusually long intergenic regions. This is related to an active process of genome expansion/contraction targeted at tandem repeats in noncoding regions and based on the addition or removal of ca. 150-bp tandem units. This process seems to be specific to E. ruminantium and is not observed in the other Rickettsiales.

Comprehensive analysis of the renal transcriptional response to acute uranyl nitrate exposure.

BACKGROUND: Chemical and radiological toxicities related to uranium acute exposure have been widely studied in nuclear fuel workers and military personnel. It is well known that uranyl nitrate induces acute renal failure (ARF). However, the mechanisms of this metal-induced injury are not well defined at the molecular level. RESULTS: Renal function and histology were assessed in mice receiving uranyl nitrate (UN(+)) and controls (UN(-)). To identify the genomic response to uranium exposure, serial analysis gene expression (SAGE) of the kidney was performed in both groups. Over 43,000 mRNA SAGE tags were sequenced. A selection of the differentially expressed transcripts was confirmed by real-time quantitative PCR and Western blotting. UN(+) animals developed renal failure and displayed the characteristic histological lesions of UN nephropathy. Of the >14,500 unique tags identified in both libraries, 224 had a modified expression level; they are known to participate in inflammation, ion transport, signal transduction, oxidative stress, apoptosis, metabolism, and catabolism. Several genes that were identified had not previously been evaluated within the context of toxic ARF such as translationally controlled tumor protein, insulin like growth factor binding protein 7 and ribosomal protein S29, all apoptosis related genes. CONCLUSION: We report a comprehensive description of the UN induced modifications in gene expression levels, including the identification of genes previously unrelated to ARF. The study of these genes and the metabolisms they control should improve our understanding of toxic ARF and enlighten on the molecular targets for potential therapeutic interventions.

Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway.

CFTR expression is tightly controlled by a complex network of ubiquitous and tissue-specific cis-elements and trans-factors. To better understand mechanisms that regulate transcription of CFTR, we examined transcription factors that specifically bind a CFTR CArG-like motif we have previously shown to modulate CFTR expression. Gel mobility shift assays and chromatin immunoprecipitation analyses demonstrated the CFTR CArG-like motif binds serum response factor both in vitro and in vivo. Transient co-transfections with various SRF expression vector, including dominant-negative forms and small interfering RNA, demonstrated that SRF significantly increases CFTR transcriptional activity in bronchial epithelial cells. Mutagenesis studies suggested that in addition to SRF other co-factors, such as Yin Yang 1 (YY1) previously shown to bind the CFTR promoter, are potentially involved in the CFTR regulation. Here, we show that functional interplay between SRF and YY1 might provide interesting perspectives to further characterize the underlying molecular mechanism of the basal CFTR transcriptional activity. Furthermore, the identification of multiple CArG binding sites in highly conserved CFTR untranslated regions, which form specific SRF complexes, provides direct evidence for a considerable role of SRF in the CFTR transcriptional regulation into specialized epithelial lung cells.

Renal toxicogenomic response to chronic uranyl nitrate insult in mice.

Although the nephrotoxicity of uranium has been established through numerous animal studies, relatively little is known about the effects of long-term environmental uranium exposure. Using a combination of conventional biochemical studies and serial analysis of gene expression (SAGE), we examined the renal responses to uranyl nitrate (UN) chronic exposure. Renal uranium levels were significantly increased 4 months after ingestion of uranium in drinking water. Creatinine levels in serum were slightly but significantly increased compared with those in controls. Although no further significant differences in other parameters were noted, substantial molecular changes were observed in toxicogenomic profiles. UN induced dramatic alterations in expression levels of more than 200 genes, mainly up-regulated, including oxidative-response-related genes, genes encoding for cellular metabolism, ribosomal proteins, signal transduction, and solute transporters. Seven differentially expressed transcripts were confirmed by real-time quantitative polymerase chain reaction. In addition, significantly increased peroxide levels support the implication of oxidative stress in UN toxicant response. This report highlights the potential of SAGE for the discovery of novel toxicant-induced gene expression alterations. Here, we present, for the first time, a comprehensive view of renal molecular events after uranium long-term exposure.

Fluorescence in situ hybridization analysis of human oocytes: advantages of a double-labeling procedure.

OBJECTIVE: To improve the fluorescence in situ hybridization (FISH) chromosomal analysis of human oocytes and first polar bodies. DESIGN: In situ chromosomal identification on isolated cells, with combinations of centromeric (or locus-specific) probes and whole-chromosome painting probes for chromosomes 9, 13, 16, 18, 21, and X. SETTING: Montpellier University Hospital. PATIENT(S): Women participating in an IVF program. INTERVENTION(S): Fifty-four in vitro unfertilized oocytes were fixed on slides, and simple or double FISH labeling procedures were performed on preparations. MAIN OUTCOME MEASURE(S): Simultaneous in situ visualization of specific domains and chromosome arms of each targeted chromosome. RESULT(S): Eight chromosomal abnormalities were identified, including two hyperhaploidies, three cases of extra single chromatid, and three cases of balanced separation of sister chromatids. Also, the double-labeling procedure allowed the avoidance of five interpretation errors, owing to additional artefactual signals. CONCLUSION(S): By ensuring precise identification of both chromosomes and single chromatids, the FISH double-labeling procedure limits the risk of erroneous interpretation and allows a more accurate cytogenetic analysis of human oocytes.

Infevers: an evolving mutation database for auto-inflammatory syndromes.

The Infevers database (http://fmf.igh.cnrs.fr/infevers/) was established in 2002 to provide investigators with access to a central source of information about all sequence variants associated with periodic fevers: Familial Mediterranean fever (FMF), TNF Receptor Associated Periodic Syndrome (TRAPS), Hyper IgD Syndrome (HIDS), Familial Cold Autoinflammatory Syndrome/Muckle-Wells Syndrome/Chronic Infantile Neurological Cutaneous and Articular Syndrome (FCAS/MWS/CINCA). The prototype of this group of disorders is FMF, a recessive disease characterized by recurrent bouts of unexplained inflammation. FMF is the pivotal member of an expanding family of autoinflammatory disorders, a new term coined to describe illnesses resulting from a defect of the innate immune response. Therefore, we decided to extend the Infevers database to genes connected with autoinflammatory diseases. We present here the biological content of the Infevers database, including the introduction of two new entries: Crohn/Blau and Pyogenic sterile arthritis, pyoderma gangrenosum and acne (PAPA syndrome). Infevers has a range of query capabilities, allowing for simple or complex interrogation of the database. Currently, the database contains 291 sequence variants in related genes (MEFV, TNFRSF1A, MVK, CARD15, PSTPIP1, and CIAS1), consisting of published data and personal communications, which has revealed or refined the preferential mutational sites for each gene. This database will continue to evolve in its content and to improve in its presentation.

Fast multicolor primed in situ protocol for chromosome identification in isolated cells may be used for human oocytes and polar bodies.

OBJECTIVE: To present and evaluate the use of a new ultra-fast multicolor primed in situ (PRINS) procedure for karyotyping human oocytes and first polar bodies. DESIGN: In situ chromosomal identification on isolated cells, using combinations of specific primers for chromosomes 1, 7, 9, 16, and 18 and fluorescent nucleotides. SETTING: Sixteen unfertilized oocytes were obtained from women participating in an IVF program. PATIENT(S): Five patients undergoing an IVF-ET. INTERVENTION(S): In vitro unfertilized oocytes were fixed on slides, and sequential PRINS reactions were performed on each preparation. MAIN OUTCOME MEASURE(S): Ultrarapid in situ identification of three or four chromosomes on oocyte and polar body chromosome spreads. RESULT(S): On the basis of the direct in situ mixing of the colors of fluorochromes (FITC, TRITC, Cascade Blue) that were incorporated in sequential PRINS reactions, this method allows rapid and efficient labeling of three or four individual chromosomes. Each PRINS reaction consists of a unique 4- to 6-minute step for both in situ annealing and elongation. The procedure can be combined with fluorescence in situ hybridization (FISH) reactions. CONCLUSION(S): By simplifying the multicolor PRINS procedure, this new protocol should facilitate the use and adaptation of PRINS to chromosome screening. This approach could be used in parallel or in combination with FISH for efficient aneuploidy assessment on isolated cells.

Stable dicentric duplication-deficiency chromosome 14 resulting from crossing-over within a maternal paracentric inversion.

We report on the extremely rare occurrence of a stable dicentric duplication-deletion chromosome 14 in a viable offspring with multiple malformations and developmental delay. This abnormality was derived from a maternal paracentric inversion in the long arm of chromosome 14. Both classical and molecular cytogenetic techniques were used to perform the chromosomal investigation of this structural abnormality. The immunofluorescent labeling of centromeric proteins shows only one functional centromere on the rearranged chromosome 14. The present observation confirms that paracentric inversions may lead to stable recombinant chromosomes.

Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes.

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. We validated this approach by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Finally, we present strong evidence showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo.

INFEVERS: the Registry for FMF and hereditary inflammatory disorders mutations.

We have established the INFEVERS--INternet periodic FEVERS--website (which is freely accessible at http://fmf.igh.cnrs.fr/infevers/). Our objectives were to develop a specialist site to gather updated information on mutations responsible for hereditary inflammatory disorders: i.e. Familial Mediterranean Fever (FMF), TRAPS (TNF Receptor 1A Associated Syndrome), HIDS (HyperIgD Syndrome), MWS (Muckle-Wells Syndrome)/FCU (Familial Cold Urticaria)/CINCA (Chronic Infantile Neurological Cutaneous and Articular Syndrome). Contributors submit their novel mutations through a 3 step form. Depending on the disease concerned, a member of the editorial board is automatically solicited to overview and validate new submissions, via a special secured web interface. If accepted, the new mutation is available on the INFEVERS web site and the discoverer, who is informed by email, is credited by having his/her name and date of the discovery on the site. The INFEVERS gateway provides researchers and clinicians with a common access location for information on similar diseases, allowing a rapid overview of the corresponding genetic defects at a glance. Furthermore, it is interactive and extendable according to the latest genes discovered.

The MetaFMF website: a high quality tool for meta-analysis of FMF.

We present here the MetaFMF database (freely accessible at http://fmf.igh.cnrs.fr/metaFMF/index_us.html) that attempts to gather and unify, in a common resource, data on phenotype-genotype correlation in familial Mediterranean fever (FMF). A single accession form, including a large number of quality controls, has been implemented such that data, collected worldwide, are included in an homogeneous manner. The inclusion criterion has the objective to avoid interpretational bias: patients will be included only if they bear at least two mutations. The clinical form has been set up by an International editorial board (12 FMF expert centres), which guarantees the validity of the data. Data are anonymous and submitted by a secure interface, in which the researcher is logged in with a specific ID and password. A pilot study on 211 patients has shown the feasibility and relevance of this project. We anticipate that the use of MetaFMF will enable reliable assessment of phenotype-genotype correlations in FMF, and define a set of severe versus mild mutations/genotypes. It should also highlight reasons for previous inconsistencies in such correlations.

Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes.

The effect of maternal age on the incidence of chromosomal abnormalities was investigated on a large sample of 3,042 in vitro unfertilized human oocytes II obtained from 792 women aged 19-46 years and participating in an in vitro fertilization program for various indications. The chromosomal analysis combined a gradual fixation of oocytes and an adapted R-banding technique. A total of 1,397 interpretable karyotypes were obtained. Various types of numerical aberration were observed, involving conventional chromosome nondisjunction (3.5%), single-chromatid nondisjunction (5.9%), complex (0.8%) or extreme aneuploidy (0.5%), diploidy (5.4%), and set of single chromatids (3.8%). No significant difference was found in the mean age of women according to the various types of chromosomal abnormalities. A positive relationship was found between maternal age and the global rate of aneuploidy, in agreement with the findings of epidemiological studies. The incidence of both whole-chromosome nondisjunction and precocious chromatid separation were correlated to maternal aging but the most significant correlation was found between maternal aging and single-chromatid nondisjunction. The rate of diploidy was also correlated to a slight extent to maternal aging, whereas no correlation was found between maternal age and the rate of single-chromatid sets. These data reveal that single-chromatid malsegregation is an essential factor in the age-dependent occurrence of nondisjunction in human oocytes. Disturbance in sister-chromatid cohesion might be a causal mechanism predisposing to premature chromatid separation and subsequently to nondisjunction in female meiosis.

Stable dicentric duplication-deficiency chromosome 14 resulting from crossing-over within a maternal paracentric inversion.

We report on the extremely rare occurrence of a stable dicentric duplication-deletion chromosome 14 in a viable offspring with multiple malformations and developmental delay. This abnormality was derived from a maternal paracentric inversion in the long arm of chromosome 14. Both classical and molecular cytogenetic techniques were used to perform the chromosomal investigation of this structural abnormality. The immunofluorescent labeling of centromeric proteins shows only one functional centromere on the rearranged chromosome 14. The present observation confirms that paracentric inversions may lead to stable recombinant chromosomes.

Reduced MEFV messenger RNA expression in patients with familial Mediterranean fever.

OBJECTIVE: Familial Mediterranean fever (FMF) is the most common inherited periodic syndrome. The disease phenotype and the almost exclusive expression of the causative gene, MEFV, in leukocytes suggest that this gene plays an important role in the inflammatory cascade. Since most of the known mutations are conservative, we sought to determine how minor DNA defects can give rise to the dramatic phenotypic features seen in FMF. METHODS: To address whether the molecular basis of the phenotype-genotype correlation could be related to altered MEFV messenger RNA (mRNA) expression, we quantified the relative abundance of MEFV transcripts in peripheral blood leukocytes from patients with FMF, healthy carriers of a single MEFV mutation, and healthy control subjects. RESULTS: We found significantly lower expression of MEFV mRNA in genetically ascertained FMF patients than in healthy controls (0.7 versus 1.1; P = 0.00001). In healthy carriers, the mRNA levels were intermediate, suggesting a true dose-response relationship between the number of mutations and the abundance of MEFV transcripts. The difference between healthy controls and healthy carriers was significant (1.1 versus 0.8; P = 0.008), demonstrating that the decrease in mRNA expression is related to a molecular defect independent of FMF symptoms. MEFV mRNA expression was also found to be a function of the type of mutations. The lowest MEFV levels were found in healthy carriers and patients with M694V. Moreover, we observed an inverse correlation with the clinical severity score (r = -0.6, P = 0.04 and r = -0.6, P = 0.004 in patients with 1 and 2 M694V mutations, respectively). CONCLUSION: Our results demonstrate that MEFV message levels are related to both the genotype and the phenotype, and suggest that the pathophysiology of FMF relies on a quantitative defect of MEFV mRNA expression.

BIGH3 (TGFBI) Arg124 mutations influence the amyloid conversion of related peptides in vitro.

Amyloid deposits with Arg124 mutated TGFBI protein have been identified in autosomal dominant blinding corneal dystrophies. We assessed in vitro the mechanisms determining TGFBI protein amyloid transformation involving mutations of Arg124. Eight peptides synthesized following the TGFBI protein sequence, centered on codon Arg124 holding the previously reported amyloidogenic mutations and the respective controls were studied. Cys124 and His124 mutated peptide preparations contained significantly higher amounts of amyloid than the native peptide. Blocking the SH group of Cys124 and deleting the first four NH2-terminal amino acids including Val112-Val113 resulted in a decrease in amyloid fibril formation while deletion of the nine CONH2-terminal residues increased amyloid fibril concentration. Fourrier transformed-infrared spectroscopy analysis of the different peptide solutions showed an increase in beta-pleated sheet structures in those with enhanced amyloid yielding. We designed a peptide (BB1) likely to counteract the role of Val112-Val113 in amyloid fibril formation. Incubation of Cys124 peptide with BB1 indeed resulted in a 35% inhibition of amyloid fibril formation. Our results are in keeping with the clinical observations of Arg124 mutation-linked amyloidosis and show the importance of Val112-Val113, disulfide and hydrogen bonding in increasing the beta-pleated conformation and amyloid formation. These findings shed new light on the molecular mechanisms of TGFBI protein amyloidogenesis and encourage further research on the use of specifically designed peptides as putative therapeutic agents for these disabling diseases.

Porcine odorant-binding protein selectively binds to a human olfactory receptor.

Odorant-binding proteins (OBPs) represent a highly abundant class of proteins secreted in the nasal mucus by the olfactory neuroepithelium. These proteins display binding affinity for a variety of odorant molecules, thereby assuming the role of carrier during olfactory perception. However, no specific interaction between OBP and olfactory receptors (ORs) has yet been shown and early events in olfaction remain so far poorly understood at a molecular level. Two human ORs, OR 17-209 and OR 17-210, were fused to a Green Fluorescent Protein and stably expressed in COS-7 cell lines. Interaction with OBP was investigated using a highly purified radioiodinated porcine OBP (pOBP) preparation, devoid of any ligand in its binding cavity. No specific binding of the pOBP tracer could be detected with OR 17-209. In contrast, OR 17-210 exhibited specific saturable binding (K(d) = 9.48 nM) corresponding to the presence of a single class of high-affinity binding sites (B(max) = 65.8 fmol/mg prot). Association and dissociation kinetics further confirmed high-affinity interaction between pOBP and OR 17-210. Autoradiographic studies of labeled pOBP to newborn mouse slices revealed the presence of multiple specific binding sites located mainly in olfactory tissue but also in several other peripheral tissues. Our data thus demonstrate a high-affinity interaction between OBP and OR, indicating that under physiological conditions, ORs may be specifically associated with an OBP partner in the absence of odorant. This provides further evidence of a novel role for OBP in the mechanism of olfactory perception.

Transcriptome analysis of monocytic leukemia cell differentiation.

The human leukemia cell line U937 is a well-established model for studying monocytic cell differentiation. We used a modified protocol (SADE) of serial analysis of gene expression (SAGE) and developed a SADE linker-anchored PCR assay to investigate the pattern of expression of known genes and to identify new transcripts in proliferating cells and during cell growth arrest and differentiation. We implemented new informatic tools to compare expression profiles before and after exposure of cells to differentiation inducers. From the analysis of 47,388 tags, we identified 13,806 distinct transcripts, 265 of which showed significant variations (P<0.01). Among 1219 well-identified genes, major changes concerned transcription and translation components, cytoskeleton, and macrophage-specific genes. Nearly half of the tags, some of them expressed at high levels, matched partially characterized genes or ESTs, or revealed yet-unknown transcripts, providing a wealth of new candidate genes that may reveal novel aspects of terminal monocytic differentiation.