Biomedical subjects

M Blot

Publications and source records attributed to M Blot.

At least 19 recordsLinked to original sources

PubMed2004

Genomic changes arising in long-term stab cultures of Escherichia coli.

Genomic scans of clones isolated from long-term stab cultures of Escherichia coli K-12 showed the loss of two large segments of the genome, with each lost segment being approximately 20 kb long. A detailed analysis of one of the deletions, located between 5.4 and 5.9 min, revealed that similar deletions had arisen in several other stab cultures. All deletions of this type exhibited a right terminus ending precisely at an IS5A element and a left terminus that varied over an approximately 5-kb range but was bordered in all but two cases by sequences belonging to the preferred consensus target sequence for IS5, YTAR. The ubiquity of such deletions in independent stab cultures and the increase in their frequency over time argue that they have a selective advantage. It is speculated that the loss of the crl locus is responsible for the selective advantage of the deletions.

DNA Transposable Elements↗

PubMed2001

Detection and activity of insertion sequences in environmental strains of Burkholderia.

The presence of two insertion sequences, IS406 and IS407, was tested by polymerase chain reaction (PCR) amplification in 25 strains representing 15 Burkholderia species and the close relative Ralstonia pickettii. A total of 50% of the 25 strains contained at least one of the two insertion sequences (ISs) and a statistically significant correlation was found between the occurrences of IS406 and IS407. Moreover, PCR-RFLP studies of the amplified fragments showed that IS406 is largely conserved among all the strains tested, whereas IS407 is rather polymorphic. Transposition activity was studied in Burkholderia vietnamiensis TVV75, using the pGBG1 target plasmid. This entrapping plasmid permitted the isolation and characterization of three active IS, able to activate the plasmid-borne tetA gene after transposition. Sequencing permitted the identification of these mobile genetic elements as isoforms of IS402, IS407 and IS1416. PCR amplification products provided IS probes, which were used to determine the copy-numbers of IS402, IS407 and IS1416 in the genome of B. vietnamiensis TVV75, by Southern blotting. Copy numbers are 12, 3 and 11 respectively. To our knowledge, this is the first description of active insertion sequences in B. vietnamiensis.

Blotting, Southern↗

PubMed2001

Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B.

Twelve populations of Escherichia coli B all lost D-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs(-) mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs(+) progenitor. These Rbs(-) mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 x 10(-5) per cell generation, of mutation from Rbs(+) to Rbs(-), which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbs genes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150 element located immediately upstream of the rbs operon. The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS150 copy and the one upstream of the rbs operon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functional rbs operon to two Rbs(-) mutants, and we constructed another Rbs(-) strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs(-) mutants have a competitive advantage relative to their Rbs(+) counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs(-) mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.

Adaptation, Physiological↗

PubMed2000

A broad-host-range plasmid for isolating mobile genetic elements in gram-negative bacteria.

Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.

Agrobacterium tumefaciens↗

PubMed2000

Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements.

As part of a long-term evolution experiment, two populations of Escherichia coli B adapted to a glucose minimal medium for 10,000 generations. In both populations, multiple IS-associated mutations arose that then went to fixation. We identify the affected genetic loci and characterize the molecular events that produced nine of these mutations. All nine were IS-mediated events, including simple insertions as well as recombination between homologous elements that generated inversions and deletions. Sequencing DNA adjacent to the insertions indicates that the affected genes are involved in central metabolism (knockouts of pykF and nadR), cell wall synthesis (adjacent to the promoter of pbpA-rodA), and ill-defined functions (knockouts of hokB-sokB and yfcU). These genes are candidates for manipulation and competition experiments to determine whether the mutations were beneficial or merely hitchhiked to fixation.

Biological Evolution↗

PubMed1999

Genomic evolution during a 10,000-generation experiment with bacteria.

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.

Biological Evolution↗

PubMed1998

Cytochrome c biogenesis is involved in the transposon Tn5-mediated bleomycin resistance and the associated fitness effect in Escherichia coli.

The transposon Tn5 ble gene and the Escherichia coli alkylation-inducible aidC locus are co-operatively involved in the resistance to the anti-cancer drug and DNA-cleaving agent bleomycin and enhance fitness of bacteria in the absence of the drug. In this report, we demonstrate that the aidC locus is identical to nrfG, the last gene of the nrf operon involved in the periplasmic formate-dependent nitrite reduction. In the presence of Ble, NrfG expression is specifically induced and restores both bleomycin resistance and its associated beneficial growth effect in an aidC- strain. In vitro DNA protection assays reveal that purified Ble prevents bleomycin-mediated DNA breakage, as do bleomycin-binding proteins. Similarities between haems of the cytochrome c biogenesis nrf pathway and iron bleomycin suggest a DNA repair-independent molecular mechanism for both bleomycin resistance and increased viability. The Ble protein binds bleomycin and prevents DNA breakage. It also induces the nrf locus that may assimilate bleomycin into haem for extracellular transport or inactivate bleomycin. Inactivation of potent DNA oxidants confers a better fitness to the bacterium carrying the transposon, suggesting a symbiotic relationship between host and transposon.

Anti-Bacterial Agents↗

PubMed1995

Somatic intrachromosomal homologous recombination events in populations of plant siblings.

Intrachromosomal homologous recombination in whole tobacco plants was analyzed using beta-glucuronidase as non-selectable marker. We found that recombination frequencies were additive for transgenes in allelic positions and could be enhanced by treatment of plants with DNA-damaging agents. We compared the patterns of distribution of recombination events of different transgenic lines of tobacco and Arabidopsis with the respective Poisson distributions. Some lines showed Poisson-like distributions, indicating that recombination at the transgene locus was occurring in a random fashion in the plant population. In other cases, however, the distributions deviated significantly from Poisson distributions indicating that for specific transgene loci and/or configurations recombination events are not randomly distributed in the population. This was due to overrepresentation of plants with especially many as well as especially few recombination events. Analysis of one tobacco line indicated furthermore that the distribution of recombination events could be influenced by treating the seedlings with external factors. Our results suggest that different plant individuals, or parts of them, might exhibit different transient 'states' of recombination competence. A possible model relating 'recombination silencing' and transcription silencing to heterochromatization of the transgene locus is discussed.

Arabidopsis↗

PubMed1995

Dynamics of IS-related genetic rearrangements in resting Escherichia coli K-12.

An analysis of restriction fragment length polymorphism (RFLP) using eight residential insertion sequence (IS) elements as hybridization probes reveals that the genome of resting bacteria is more dynamic than it was long believed. Escherichia coli strains stored in agar stabs for up to 30 yr accumulate a genetic variation which is correlated to time of storage. This spontaneous mutagenesis is often IS-specific, with particularly high activity for IS5, and thus suggests that transpositional DNA rearrangements are a major cause for the observed genetic polymorphism. The RFLP patterns indicate a burst of IS30 transposition to occur occasionally. Mutation rate is estimated by two different methods to roughly 10(-5) IS-related DNA rearrangements per bacterial chromosome per hour of storage for the eight IS elements studied. A pedigree derived from the RFLP data reveals that populations had evolved independently in each stab and showed no signs of convergence. Relics of an assumed ancestral population were still present in the stab cultures, but the elder stabs provided mostly mutants. These results indicate that cells placed under nutritional deprivation might have a highly plastic genome and suggest that such plasticity might play an adaptive role.

DNA Probes↗

PubMed1994

The Tn5 bleomycin resistance gene confers improved survival and growth advantage on Escherichia coli.

The bleomycin resistance gene (ble) of transposon Tn5 is known to decrease the death rate of Escherichia coli during stationary phase. Bleomycin is a DNA-damaging agent and bleomycin resistance is produced by improved DNA repair which also requires the host genes aidC and polA coding, respectively, for an alkylation-inducible gene product and DNA polymerase I. In the absence of the drug, this DNA repair system is believed to cause the slower death rate of bleomycin-resistant bacteria. In this study, the effect of ble and aidC genes on the viability of bacteria and their growth rate in chemostat competitions was studied. The results indicate, that bleomycin-resistant bacteria display greater fitness under these conditions. Another beneficial effect of transposon Tn5 had been previously attributed to the insertion sequence IS 50 R. We were not able to reproduce this result with IS 50 R, however, the complete transposon was beneficial under similar conditions. Moreover, we showed the Tn5 fitness effect to be aidC-dependent. The ble gene was discovered after the fitness effect of IS 50 R had been established; it has not previously been considered to mediate the beneficial effect of Tn5. This possibility is discussed based on the molecular mechanism of bleomycin resistance.

Bleomycin↗

PubMed1994

Transposable elements and adaptation of host bacteria.

A transposable element (TE) is a mobile sequence present in the genome of an organism. TEs can cause lethal mutations by inserting into essential genes, promoting deletions or leaving short sequences upon excision. They therefore may be gradually eliminated from mixed populations of haploid micro-organisms such as Escherichia coli if they cannot balance this mutation load. Horizontal transmission between cells is known to occur and promote the transfer of TEs, but at rates often too low to compensate for the burden to their hosts. Therefore, alternative mechanisms should be found by these elements to earn their keep in the cells. Several theories have been suggested to explain their long-term maintenance in prokaryotic genomes, but little molecular evidence has been experimentally obtained. In this paper, the permanence of transposable elements in bacterial populations is discussed in terms of costs or benefits for the element and for the host. It is observed that, in all studies yet reported, the elements do not behave in their host as selfish DNA but as a co-operative component for the evolution of the couple.

Bacteria↗

PubMed1994

Insertion sequence-related genetic variation in resting Escherichia coli K-12.

Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage. This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS). Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition. IS5 was particularly active in the generation of variation. All subclones in which IS30 had been active testify to a burst of IS30 transposition. This was correlated with a loss of prototrophy and a reduced growth on rich media. A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones. Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared. A few patterns were each represented by several subclones displaying improved fitness. These results offer insights into the role of IS elements in the plasticity of the E. coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures.

Biological Evolution↗

PubMed1993

Genetic diversity among Borrelia burgdorferi isolates: more than three genospecies?

In order to assess the genetic relationships among isolates of Borrelia burgdorferi, we have determined chromosomal DNA restriction fragment length polymorphisms (RFLP) of 27 isolates. Using five restriction enzymes and seven DNA probes, we recorded 246 different hybridizing bands. The resulting matrix was subjected to factorial correspondence analysis and to phylogenetic analysis using a parsimony program. These analyses demonstrate two closely related genospecies and three individual strains. RFLP variations among these three strains are much more pronounced than among the members of the other two genospecies. This may indicate the existence of more than three genospecies. Plasmid analysis did not contribute significantly to the genotypic differentiation.

Borrelia burgdorferi Group↗

PubMed1993

Tn5-mediated bleomycin resistance in Escherichia coli requires the expression of host genes.

The transposon Tn5 expresses a gene, ble, whose product increases the viability of Escherichia coli and also confers resistance to the DNA-cleaving antibiotic bleomycin and the DNA-alkylating agent ethylmethanesulphonate. We find that the Ble protein induces expression of an alkylation inducible gene, aidC, and that both the AidC gene product and DNA polymerase I are required for Ble to confer bleomycin resistance. These findings support models in which Ble enhances DNA repair and suggest that Tn5 confers a fitness advantage to the host bacterium by increasing the repair of spontaneous DNA lesions. Such co-operation between a transposon and its host suggests that Tn5 is a symbiotic rather than a selfish DNA element.

Bacterial Proteins↗

PubMed1992

Statistical evidence for a random commitment of pluripotent cephalic neural crest cells.

The neural crest (NC) of vertebrate embryos yields cell types belonging to the neural, melanocytic and mesectodermal lineages. To test the possibility that the precursors of these lineages segregate from pluripotent cells by a process involving stochastic determinants, we have analyzed with statistical methods the associations between six differentiated cell types in 201 clones obtained in vitro from migrating cephalic NC cells. Our analysis suggests that neuronal, adrenergic and Schwann cells are not randomly associated, whereas these neural cell types differentiate in the clones independently of both melanocytes and cartilage. These results raise the possibility that pluripotent NC progenitors give rise to the precursors of the major NC-derived lineages (neural, melanocytic and mesectodermal) by a process involving stochastic restrictions of their developmental potentialities.

Animals↗

PubMed1991

Bleomycin-resistance gene derived from the transposon Tn5 confers selective advantage to Escherichia coli K-12.

The plasmid pRAB2 contains a silent operon derived from the transposon Tn5 and carrying the gene neo for neomycin-kanamycin resistance and a truncated ble gene (ble333) for bleomycin resistance. Spontaneous mutants that express the two resistances provide Escherichia coli cells an improved fitness during the phase of decline in the absence of the antibiotics. It is shown that the ble333 gene product is responsible for this better fitness. These results can explain a previously described selective advantage attributed to the presence of Tn5. The improved fitness of bleomycin-resistant bacteria is proposed to relate to DNA repair by the ble gene product. The consequences of the presence of an accessory gene improving fitness are discussed in terms of evolutionary stable strategy of a transposon in populations of E. coli.

Bleomycin↗

PubMed1984

Prolonged total parenteral nutrition in a pregnant woman with acute pancreatitis.

During the 22nd week of her first pregnancy, a 28-year-old woman developed an attack of acute pancreatitis 10 months after pancreatoduodenectomy for chronic pancreatitis of alcoholic origin. She received total parenteral nutrition without complications for 83 days until the cesarean delivery of a child weighing 2,120 g. During that time, clinical, biological, and echographic signs of pancreatitis progressively disappeared. The favorable outcome for both mother and fetus suggests that prolonged TPN may be a useful measure in the management of severe digestive disease occurring during pregnancy.

Acute Disease↗

PubMed1980

[Rare hemoglobins in Guadeloupe and in the Lesser Antilles (author's transl)].

Screening of abnormal hemoglobins on 25 136 samples allowed us to detect from 1974-1979 rare or uncommon hemoglobins as N Baltimore, Korle Bu, Lepore, K Woolwich, J Broussais and J Baltimore. Hemoglobins N Baltimore, J Broussais, and J Baltimore are detected for the first time in Guadedoupe. The identification of these asymptomatic hemoglobins in this part of the Carribbean region provides information concerning the exact origin of the black population of these islands; e.g., hemoglobin K Woolwich and N Baltimore are found only in specific regions of West Africa.

Fetal Hemoglobin↗