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C Pourcel

Publications and source records attributed to C Pourcel.

At least 19 recordsLinked to original sources
PubMed2005

CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.

The remarkable repetitive elements called CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repeats interspaced with non-repetitive elements or 'spacers'. CRISPRs are present in both archaea and bacteria, in association with genes involved in DNA recombination and repair. In the Yersinia pestis genome, three such elements are found at three distinct loci, one of them being highly polymorphic. The authors have sequenced a total of 109 alleles of the three Y. pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate. In nine strains of Yersinia pseudotuberculosis, 132 spacers were found, of which only three are common to Y. pestis isolates. In Y. pestis of the Orientalis biovar investigated in detail here, deletion of motifs is observed but it appears that addition of new motifs to a common ancestral element is the most frequent event. This takes place at the three different loci, although at a higher rate in one of the loci, and the addition of new motifs is polarized. Interestingly, the most recently acquired spacers were found to have a homologue at another locus in the genome, the majority of these inside an inactive prophage. This is believed to be the first time that the origin of the spacers in CRISPR elements has been explained. The CRISPR structure provides a new and robust identification tool.

Bacteriophages↗
PubMed2005

Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto.

Leptospira interrogans sensu stricto is responsible for the most frequent and severe cases of human leptospirosis. The epidemiology and clinical features of leptospirosis are usually associated with the serovars and serogroups of Leptospira. Because of the difficulties associated with serological identification of Leptospira strains, we evaluated a novel PCR-based method for typing L. interrogans serovars. Based upon the genome sequence of L. interrogans serovar Lai type strain 5660, 44 loci were analyzed by PCR for their variability in size due to the presence of variable-number tandem repeats (VNTR). Seven VNTR loci were found to be powerful markers for serovar identification, epidemiology, and phylogenetic studies of L. interrogans. This rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.

Bacterial Typing Techniques↗
PubMed2004

Tandem repeats analysis for the high resolution phylogenetic analysis of Yersinia pestis.

BACKGROUND: Yersinia pestis, the agent of plague, is a young and highly monomorphic species. Three biovars, each one thought to be associated with the last three Y. pestis pandemics, have been defined based on biochemical assays. More recently, DNA based assays, including DNA sequencing, IS typing, DNA arrays, have significantly improved current knowledge on the origin and phylogenetic evolution of Y. pestis. However, these methods suffer either from a lack of resolution or from the difficulty to compare data. Variable number of tandem repeats (VNTRs) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses in a growing number of pathogens and have given promising results for Y. pestis as well. RESULTS: In this study we have genotyped 180 Y. pestis isolates by multiple locus VNTR analysis (MLVA) using 25 markers. Sixty-one different genotypes were observed. The three biovars were distributed into three main branches, with some exceptions. In particular, the Medievalis phenotype is clearly heterogeneous, resulting from different mutation events in the napA gene. Antiqua strains from Asia appear to hold a central position compared to Antiqua strains from Africa. A subset of 7 markers is proposed for the quick comparison of a new strain with the collection typed here. This can be easily achieved using a Web-based facility, specifically set-up for running such identifications. CONCLUSION: Tandem-repeat typing may prove to be a powerful complement to the existing phylogenetic tools for Y. pestis. Typing can be achieved quickly at a low cost in terms of consumables, technical expertise and equipment. The resulting data can be easily compared between different laboratories. The number and selection of markers will eventually depend upon the type and aim of investigations.

Africa↗
PubMed2003

Characterization of a tandem repeat polymorphism in Legionella pneumophila and its use for genotyping.

We have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats [VNTRs]) in the genome of Legionella pneumophila. Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains. For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain. Most of these markers did not amplify the DNA of other Legionella species or other bacteria used as controls. A polymorphism was observed for seven markers among the L. pneumophila strains tested. To check whether these markers could be used to compare strains of L. pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods. The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains. Sequencing of one minisatellite from 14 reference strains was performed. Comparison of the sequences allowed a classification and confirmed the existence of subspecies of L. pneumophila. We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples. This allowed the rapid identification of the L. pneumophila colonies and gave a first hint as to the presence of several strains in a single sample.

Amino Acid Sequence↗
PubMed2001

A dominant modifier of transgene methylation is mapped by QTL analysis to mouse chromosome 13.

The single-copy hepatitis B virus transgene in the E36 transgenic mouse strain undergoes methylation changes in a parent-of-origin, tissue, and strain-specific fashion. In a C57BL/6 background, the paternally transmitted transgene is methylated in 30% of cells, whereas it is methylated in more than 80% of cells in (BALB/c x C57BL/6) F1 mice. We established previously that several genetic factors were likely to contribute to the transgene methylation profile, some with demethylating and some with de novo methylating activities. Using quantitative trait loci (QTL) mapping, we have now localized one major modifier locus on chromosome 13 (Mod13), which explains a 30% increase in the methylation level of this transgene with no effect on the flanking endogenous sequences. No other QTL could be identified, except for a demethylating activity of low significance located on chromosome 12. Recombinant inbred mice containing a BALB/c allele of Mod13 were then used to show that the presence of Mod13 is sufficient to induce de novo methylation. A segregation between de novo methylation and repression of transgene expression was uncovered, suggesting that this genetic system is also useful for the identification of factors that interpret methylation patterns in the genome.

Animals↗
PubMed2000

A new family of genes and pseudogenes potentially expressing testis- and brain-specific leucine zipper proteins in man and mouse.

We have characterized a new mouse gene highly transcribed in the testis, and a derived intronless gene expressed in the embryo. The latter gene is present in Mus musculus domesticus and in Mus musculus castaneus but is absent in Mus spretus. The sequencing of different clones from a testis cDNA library reveals a complex transcriptional regulation for the intron-containing gene. The use of several promoters, alternative splicing and trans-splicing, and of two different polyadenylation sites account for the diversity. The different cDNAs encode proteins with features of basic helix-loop-helix leucine zipper (bHLH-ZIP) DNA-binding factors with homology to a new brain-specific factor. The presence of multiple CK2 and PKC phosphorylation sites suggests that their activity may be regulated by phosphorylation. In man, a pseudogene, apparently derived from the same transcript as in mouse and showing 90% homology in the coding region, is present within an intron of another gene. Interestingly, although the human pseudogene is highly mutated in human, in the mouse it has only four nucleotide changes compared with the cDNA of origin, and is still capable of encoding a protein.

Alternative Splicing↗
PubMed2000

Association of glucocorticoids and cyclosporin A or rapamycin prevents E-selectin and IL-8 expression during LPS- and TNFalpha-mediated endothelial cell activation.

BACKGROUND: Endothelial cell (EC) activation plays an important role in inflammation, hemostasis, and organ rejection of allogeneic and xenogeneic transplantation. These processes leads to rapid and transient up-regulation of proinflammatory molecules, such as the adhesion molecule E-selectin and the chemotactic cytokine IL-8. The purpose of this study was to investigate the specific effects of several major and potentially synergistic immunosuppressive drugs-cyclosporin A (CsA), rapamycin (Rap), and glucocorticoids (GC)-on lipopolysaccharide (LPS)- or tumor necrosis factor (TNF)alpha-induced EC activation METHODS: The ability of immunosuppressive drugs, used alone or in combination, to prevent in vitro TNFalpha- and LPS-induced expression of E-selectin and interleukin 8 on porcine ECs, as well as their effect on leukocyte-EC interaction, were investigated. In addition, we studied the in vivo effect of these drugs after i.v. administration of recombinant TNFalpha to rats. RESULTS: At high concentrations, which correspond to the acceptable experimental levels in primate xenograft recipients, CsA, Rap, and GC individually inhibited E-selectin protein induction in a dose-dependent manner in cultured porcine ECs treated with LPS with an additive effect when the drugs were associated. The pattern of drug-mediated inhibition was related to the stimulus used to activate ECs (i.e., LPS vs. TNFalpha). Reduced expression of E-selectin on ECs activated in the presence of the tested immunosuppressive drugs correlated with a weaker adhesion of human U937 cells to ECs. Messenger RNA analysis demonstrated that the presence of CsA, Rap, and GC during EC activation inhibited E-selectin and interleukin 8 at the gene expression level. LPS-mediated induction of IbetaBalpha expression was not observed in ECs treated with CsA, whereas GC reduced its transcripts by approximately 50%. It is interesting that in vivo studies confirmed that CsA and GC inhibited EC activation at therapeutic doses (1 mg/kg and 10 mg/kg for GC and CsA, respectively) and showed that the combination of CsA and GC efficiently prevents TNFalpha-mediated induction of E-selectin on cardiac ECs. CONCLUSION: Our data show that, besides their specific immunosuppressive effects on T cells, CsA, Rap, and GC can efficiently contribute to the attenuation of EC activation in vivo and the resulting inhibition is enhanced by the association of CsA with GC.

Animals↗
PubMed2000

Negative regulation of autoreactive B cells in transgenic mice expressing a human pathogenic cold agglutinin.

Cold agglutinins (CA) are autoantibodies that bind to erythrocyte carbohydrates at low temperatures and induce complement-mediated cell lysis, thus causing hemolytic anemia. Tolerance mechanisms towards CA-expressing B cells and the factors inducing pathogenic CA production are unknown. In order to develop an animal model for CA disease, we have produced transgenic mice expressing the heavy or the light chain of a human CA, previously shown to be pathogenic to the mouse. Expression of the human H chain alone resulted in a B cell maturation block at the pro-B stage, and did not induce allelic exclusion. In double-transgenic mice, co-expression of the human H and L chains restored B cell development but the majority of bone marrow cells expressing the human IgM were eliminated by deletion. In the periphery, B cells were depleted, and a large proportion of the remaining cells co-expressed a human and a murine H chain, secreting "mixed" IgM. A few autoreactive cells, predominating in the peritoneal cavity, escaped tolerance mechanisms and secreted transgenic IgM. The autoreactive B cells are amenable to polyclonal stimulation, making these transgenic mice a suitable model for a human autoimmune disease.

Agglutinins↗
PubMed1999

Mutated cytochrome b as a determinant of a new monoclonal antibody (H8.98) on renal carcinoma cell lines recognized by a Vgamma3Vdelta1(+) T-cell clone.

We generated a monoclonal antibody (MAb), H8.98, that recognizes an antigen shared by 50% of examined renal carcinoma cell (RCC) lines and is susceptible to lysis by a Vgamma3Vdelta1(+) T-cell clone derived from RCC tumor-infiltrating lymphocytes. H8.98 inhibited Vgamma3Vdelta1(+ )T-cell clone-mediated lysis of RCC lines. It did not stain normal kidney lines, melanomas, fibroblasts, Burkitt's lymphoma or Epstein-Barr virus-transformed B-cell lines but it did stain 2 of 4 tested breast cancer lines. Through screening of a renal carcinoma cDNA library using H8.98, we isolated a cDNA clone which, upon sequencing, was found to be cytochrome b with 2 point mutations.

Amino Acid Sequence↗
PubMed1998

Intracellular expression in pig cells of anti-alpha1,3galactosyltransferase single-chain FV antibodies reduces Gal alpha1,3Gal expression and inhibits cytotoxicity mediated by anti-Gal xenoantibodies.

BACKGROUND: The carbohydrate structure Gal alpha1,3Gal expressed on pig cells is the major antigen recognized by xenoreactive natural antibodies in the higher primates. In xenotransplantation, natural antibodies binding to that structure initiate hyperacute rejection, and the anti-Gal alpha1,3Gal antibodies that are elicited probably take part in later phases of vascularized graft rejection. This epitope also appears to be involved in innate cellular responses. Inactivation of alpha1,3 galactosyltransferase in transgenic pigs would certainly lead to the success of xenotransplantation, but gene knockout in pigs is not feasible yet. METHODS: As a novel strategy to inhibit alpha1,3 galactosylation, we generated recombinant single-chain Fv (ScFv) antibodies directed against pig alpha1,3-galactosyltransferase and evaluated the effect of their intracellular expression on enzyme activity and Gal alpha1,3Gal expression. RESULTS: After in vitro transfection in pig cells, the scFv antibody anti-pig alpha1,3-galactosyltransferase reduced the amount or function of enzyme by up to 70% as evidenced by immunofluorescence and measurement of cell-associated activity. Consequently, Gal alpha1,3Gal on cell membranes was reduced to the same extent. This led to a profound (more than 90%) reduction in the cytotoxicity involving anti-Gal antibodies and complement. CONCLUSION: Although not sufficient to knock out the overall human anti-pig natural xenoreactivity, intracellular expression of the scFv antibody anti-alpha1,3-galactosyltransferase in pig cells significantly decreases the amount of Gal alpha1,3Gal and could be important to protect cells from elicited antibodies as well as from innate effectors.

Animals↗
PubMed1998

Recent human-specific spreading of a subtelomeric domain.

The recent spreading of a subtelomeric region at nine different human chromosome ends was characterized by a combination of segregation analyses, physical mapping, junction cloning, and FISH investigations. The events occurred very recently in human genome evolution as demonstrated by sequence analysis of different alleles and the single location of the ancestral site at chromosome 17qter in chimpanzee and orangutan. The domain successfully colonized most 1p, 5q, and 6q chromosome ends and is also present at a significant frequency of 6p, 7p, 8p, 11p, 15q, and 19p ends. On 6qter, the transposed domain is immediately distal to the highly conserved, single-copy gene PDCD2.

Animals↗
PubMed1998

Control of expression and methylation of a hepatitis B virus transgene by strain-specific modifiers.

In transgenic animals, genotype-specific modifiers exert a control over transgene methylation and expression that may or may not be position dependent. These factors belong to different classes, some of them possibly related to modifiers of position-effect variegation in Drosophila. The study of hepatitis B virus (HBV) gene expression in transgenic mice has revealed the existence of many factors influencing transcription, including hormones and tissue-specific transcription factors. We now report the effect of genotype-specific modifiers on HBV surface antigen (HBsAg) expression and transgene methylation. Compared with the C57BL/6 background, the DBA/2 and 129sv backgrounds cause enhancement of HBsAg expression, with little or not effect on transgene methylation or transcription. In contrast, a single cross with a BALB/c mouse is responsible for de novo methylation and silencing of the transgene in all offspring. Several modifiers appear to segregate in the progeny of a transgenic E36 male mouse crossed with (C57BL/6 x BALB/c) F1 females, with the emergence of a high-expressor group. Our observations suggest that different modifiers act cooperatively, at both the transcriptional and post-transcriptional levels, as part of a complex system regulating transgene expression. This transgenic model provides a system to genetically map new mouse strain-specific modifiers, some of them involved in epigenetic modification and transcription control.

Animals↗
PubMed1997

Porcine alpha1,3-galactosyltransferase: tissue-specific and regulated expression of splicing isoforms.

Expression of the Gal alpha1,3 Gal epitope on membrane glycolipids and glycoproteins is known to vary widely from one tissue to another. In the course of studying the mechanisms underlying this variability, we have isolated from pig cDNA four sequences corresponding to four isoforms of alpha1,3-galactosyltransferase (alpha1,3GT), the Golgi enzyme that links galactose in alpha1,3 on the galactose residue of N-acetyllactosamine. The isoforms differ from each other in the alternative presence of two nucleotide stretches of 36 and 63 base pairs in a segment encoding the stem region of the protein. Stable expression experiments show that all four isoenzymes can confer alpha-galactosyltransferase activity to HeLa cells, and that they are all located within the Golgi compartment, indicating that variations in length in the stem region do not affect enzyme activity or cellular localization. Analysis of RNA from different pig organs and cells shows quantitative differences between tissues in levels of alpha1,3GT, as well as qualitative differences, the four isoforms being unequally represented in different tissues.

Animals↗
PubMed1996

Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing the major epitope for human xenoreactive antibodies (Gal alpha 1-3Gal), blood group H determinant and N-glycolylneuraminic acid.

Glycosphingolipids were isolated from primary cultures of porcine endothelial cells labelled with 14C-galactose or 14C-glucosamine. They were characterized by their mobility on thin layer chromatogram, their sensitivity to exoglycosidases, and their labelling with antibodies. In addition to the major glycosphingolipids, globotetra- and globotriaosylceramide, minor ones were identified as penta- and heptaglycosylceramide of the neolactoseries terminated by either Gal alpha 1-3Gal- (xenoreactive epitope) or Fuc alpha 1-2Gal- (H determinant). Two gangliosides were found, GM3 and GD3, and N-glycolylneuraminic acid was their major sialic acid. Therefore, porcine endothelial cells differ from human endothelial cells by expression of glycosphingolipids that are absent in man: two Gal alpha 1-3Gal-terminated glycolipids recognized by human natural antibodies, and two N-glycolylneuraminic acid-terminated gangliosides which are potent immunogens.

ABO Blood-Group System↗
PubMed1996

Transgenesis in rats: technical aspects and models.

The production of transgenic rats by DNA-microinjection into fertilized ova has now become an established procedure, although fewer than 20 lines have been described during the last 5 years. Overall, transgenic rats remain more difficult to produce than transgenic mice, but satisfactory yields have been obtained by several laboratories. A review of the methods used to generate transgenic rats shows considerable variation between different laboratories, particularly in choice of strain, superovulation protocols and the use of embryo culture before reimplantation. In some instances, the production of transgenic rats has provided data that are new and relevant, compared to data obtained in mice bearing the same transgene. Models have been developed for human diseases such as hypertension and autoimmunity, and applications have been found in the study of carcinogenesis and in pharmacological research. Transgenic rat technology also opens up interesting perspectives for transplantation research, in which microsurgery is an essential procedure. Intensive research is in progress in several laboratories to produce rat embryonic stem (ES) cell lines, but existing lines have not participated in germ line formation a prerequisite for their use in gene knock out experiments.

Animals↗