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G Vergnaud

Publications and source records attributed to G Vergnaud.

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↗
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 tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis.

BACKGROUND: Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies. RESULTS: This report presents a database (http://minisatellites.u-psud.fr) of tandem repeats from publicly available bacterial genomes which facilitates the identification and selection of tandem repeats. We illustrate the use of this database by the characterization of minisatellites from two important human pathogens, Yersinia pestis and Bacillus anthracis. In order to avoid simple sequence contingency loci which may be of limited value as epidemiological markers, and to provide genotyping tools amenable to ordinary agarose gel electrophoresis, only tandem repeats with repeat units at least 9 bp long were evaluated. Yersinia pestis contains 64 such minisatellites in which the unit is repeated at least 7 times. An additional collection of 12 loci with at least 6 units, and a high internal conservation were also evaluated. Forty-nine are polymorphic among five Yersinia strains (twenty-five among three Y. pestis strains). Bacillus anthracis contains 30 comparable structures in which the unit is repeated at least 10 times. Half of these tandem repeats show polymorphism among the strains tested. CONCLUSIONS: Analysis of the currently available bacterial genome sequences classifies Bacillus anthracis and Yersinia pestis as having an average (approximately 30 per Mb) density of tandem repeat arrays longer than 100 bp when compared to the other bacterial genomes analysed to date. In both cases, testing a fraction of these sequences for polymorphism was sufficient to quickly develop a set of more than fifteen informative markers, some of which show a very high degree of polymorphism. In one instance, the polymorphism information content index reaches 0.82 with allele length covering a wide size range (600-1950 bp), and nine alleles resolved in the small number of independent Bacillus anthracis strains typed here.

Bacillus anthracis↗
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↗
PubMed2001

Children of chernobyl cleanup workers do not show elevated rates of mutations in minisatellite alleles.

The disaster at the Chernobyl Nuclear Power Plant in April 1986 was accompanied by the release of large amounts of radioisotopes, resulting in the contamination of extensive regions of the Ukraine, Byelorus and the Russian Federation. Cleanup workers (liquidators) and people living on land contaminated with radioactive materials were most exposed. To assess the genetic effects of exposure to ionizing radiation after the Chernobyl accident, we have measured the frequency of inherited mutant alleles at seven hypermutable minisatellite loci in 183 children born to Chernobyl cleanup workers (liquidators) and 163 children born to control families living in nonirradiated areas of the Ukraine. There was no significant difference in the frequency of inherited mutant alleles between the exposed and control groups. The exposed group was then divided into two subgroups according to the time at which the children were conceived with respect to the fathers' work at the power plant. Eighty-eight children were conceived either while their fathers were working at the facility or up to 2 months later (Subgroup 1). The other 95 children were conceived at least 4 months after their fathers had stopped working at the Chernobyl site (Subgroup 2). The frequencies of mutant alleles were higher for the majority of loci (i.e. 1.44 times higher for CEB1) in Subgroup 1 than in Subgroup 2. This result, if confirmed, would reconcile the apparently conflicting results obtained in the chronically exposed Byelorus population and the Hiroshima-Nagasaki A-bomb survivors.

Alleles↗
PubMed2000

The SH2D2A gene encoding the T-cell-specific adapter protein (TSAd) is localized centromeric to the CD1 gene cluster on human Chromosome 1.

The SH2D2A gene encoding the T-cell-specific adapter protein (TSAd), was isolated from a human Chromosome (Chr) 1 cosmid library (LLNL, UK HGMP). The gene spans 11 kilobases and contains nine exons and eight introns. Four alternative transcript variants were observed in activated T cells. Three single-nucleotide polymorphisms were identified within intron 2. A variable number of GA repeats was found at position -340 from the first coding ATG. Linkage analysis using this marker in eight CEPH families showed that the SH2D2A gene is located close to the D1S2624 marker on Chr 1q21-1q22. Physical mapping of a PAC and BAC contig containing the CD1 gene cluster telomeric to D1S2624 failed to identify a clone harboring the SH2D2A gene. Thus the SH2D2A gene is located centromeric to the CD1 gene cluster on Chr 1.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Minisatellites: mutability and genome architecture.

Minisatellites have been found in association with important features of human genome biology such as gene regulation, chromosomal fragile sites, and imprinting. Our knowledge of minisatellite biology has greatly increased in the past 10 years owing to the identification and careful analysis of human hypermutable minisatellites, experimental models in yeast, and recent in vitro studies of minisatellite recombination properties. In parallel, minisatellites have been put forward as potential biomarkers for the monitoring of genotoxic agents such as ionizing radiation. We summarize and discuss recent observations on minisatellites. In addition we take advantage of recent whole chromosome sequence data releases to provide a unifying view which may facilitate the annotation of tandem repeat sequences.

Animals↗
PubMed1999

Meiotic instability of human minisatellite CEB1 in yeast requires DNA double-strand breaks.

Minisatellites are tandemly repeated DNA sequences of 10-100-bp units. Some minisatellite loci are highly unstable in the human germ line, and structural analysis of mutant alleles has suggested that repeat instability results from a recombination-based process. To provide insights into the molecular mechanism of human minisatellite instability, we developed Saccharomyces cerevisiae strains carrying alleles of the most unstable human minisatellite locus, CEB1 (ref. 2). We observed that CEB1 is destabilized in meiosis, resulting in a variety of intra- and inter-allelic gains or losses of repeat units, similar to rearrangements described in humans. Using mutations affecting the initiation of recombination (spo11) or mismatch repair (msh2 pms1 ), we demonstrate that meiotic destabilization depends on the initiation of homologous recombination at nearby DNA double-strand break (DSBs) sites and involves a 'rearranged heteroduplex' intermediate. Most of the human and yeast data can be explained and unified in the context of DSB repair models.

Alleles↗
PubMed1999

Finding new human minisatellite sequences in the vicinity of long CA-rich sequences.

Microsatellites and minisatellites are two classes of tandem repeat sequences differing in their size, mutation processes, and chromosomal distribution. The boundary between the two classes is not defined. We have developed a convenient, hybridization-based human library screening procedure able to detect long CA-rich sequences. Analysis of cosmid clones derived from a chromosome 1 library show that cross-hybridizing sequences tested are imperfect CA-rich sequences, some of them showing a minisatellite organization. All but one of the 13 positive chromosome 1 clones studied are localized in chromosomal bands to which minisatellites have previously been assigned, such as the 1pter cluster. To test the applicability of the procedure to minisatellite detection on a larger scale, we then used a large-insert whole-genome PAC library. Altogether, 22 new minisatellites have been identified in positive PAC and cosmid clones and 20 of them are telomeric. Among the 42 positive PAC clones localized within the human genome by FISH and/or linkage analysis, 25 (60%) are assigned to a terminal band of the karyotype, 4 (9%) are juxtacentromeric, and 13 (31%) are interstitial. The localization of at least two of the interstitial PAC clones corresponds to previously characterized minisatellite-containing regions and/or ancestrally telomeric bands, in agreement with this minisatellite-like distribution. The data obtained are in close agreement with the parallel investigation of human genome sequence data and suggest that long human (CA)s are imperfect CA repeats belonging to the minisatellite class of sequences. This approach provides a new tool to efficiently target genomic clones originating from subtelomeric domains, from which minisatellite sequences can readily be obtained. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ000377-AJ000383.]

Bacteriophage P1↗
PubMed1999

[Structure and evolution of human sub-telomeric regions].

Recent progress in the field of human genome analysis has led to the development of new concepts in the definition of subtelomeric domains. Analysis of DNA sequences from human and yeast chromosome ends have shown that short stretches of degenerate TTAGGG are found at a distance from the telomeric repeats. These stretches define a boundary between two structurally different regions. The distal domain is characterised by numerous, short segments of interrupted homology to many other human telomeric regions and to a number of ESTs. The proximal domain shows much longer uninterrupted homology to a few chromosome ends. This domain evolved quickly within primates at least, as demonstrated by the detailed study of locus DNF92 which spread very recently in humans from 17 qter to at least ten other chromosome ends. At the different sites, presence-absence polymorphisms are observed within humans. The region remained single locus at the paralogous site in higher primates. Conversely, a human and orangutan single locus telomeric domain occupies multiple chromosome ends in chimpanzee. Balanced translocation is the likely mechanism through which the spreading occurred. Some members of the olfactory receptor gene family show a similar behaviour: multiple telomeric locations, and presence-absence polymorphism. Strikingly, the set of chromosome ends occupied by the two regions is identical, except for the two ancestral sites. Moreover, the relative frequency of detection of the region at the different sites indicates some kind of competition between the two regions. Consequently, these two regions represent major new tools to investigate recent human genome evolution and human genome diversity in different populations.

Animals↗
PubMed1998

Analysis of distribution in the human, pig, and rat genomes points toward a general subtelomeric origin of minisatellite structures.

We have developed approaches for the cloning of minisatellites from total genomic libraries and applied these approaches to the human, rat, and pig genomes. The chromosomal distribution of minisatellites in the three genomes is strikingly different, with clustering at chromosome ends in human, a seemingly almost even distribution in rat, and an intermediate situation in pig. A closer analysis, however, reveals that interstitial sites in pig and rat often correspond to terminal cytogenetic bands in human. This observation suggests that minisatellites are created toward chromosome ends and their internalization represents secondary events resulting from rearrangements involving chromosome ends.

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↗
PubMed1997

Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident.

Analysis of germline mutation rate at human minisatellites among children born in areas of the Mogilev district of Belarus heavily polluted after the Chernobyl accident has been extended, both by recruiting more families from the affected region and by using five additional minisatellite probes, including multi-locus probe 33.6 and four hypervariable single-locus probes. These additional data confirmed a twofold higher mutation rate in exposed families compared with non-irradiated families from the United Kingdom. An elevated rate was seen at all three independent sets of minisatellites (detected separately by multi-locus probes 33.15, 33.6 and six single-locus probes), indicating a generalised increase in minisatellite germline mutation rate in the Belarus families. Within the Belarus cohort, mutation rate was significantly greater in families with higher parental radiation dose estimated for chronic external and internal exposure to caesium-137, consistent with radiation induction of germline mutation. The spectra of mutation seen in the unexposed and exposed families were indistinguishable, suggesting that increased mutation observed over multiple loci arises indirectly by some mechanism that enhances spontaneous minisatellite mutation.

Adult↗
PubMed1997

Molecular-cytogenetic detection of a deletion of 1p36.3.

We report a deletion of 1p36.3 in a child with microcephaly, mental retardation, broad forehead, deep set eyes, depressed nasal bridge, flat midface, relative prognathism, and abnormal ears. The phenotype is consistent with that described for partial monosomy for 1p36.3. Reverse chromosome painting and microsatellite and Southern blot analyses were used to map the extent of the deletion. Fluorescence in situ hybridisation (FISH) analysis using probes from every telomere indicates that the rearrangement is likely to be a chromosomal truncation or rearrangement involving subtelomeric repetitive DNA. The deletion was identified by screening a sample of children and adults with idiopathic mental retardation. In conjunction with previous work on this sample, we estimate that 7.4% of the group have subtelomeric rearrangements.

Adolescent↗