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P Leblond

Publications and source records attributed to P Leblond.

36 records · Page 2Linked to original sources
PubMed1993

Analysis of genome instability in Streptomyces ambofaciens.

Genetic instability in Streptomyces ambofaciens DSM 40697 is correlated with genomic instability characterized by multiple rearrangements (deletions and/or amplifications) occurring in a large unstable region. We have focused on one of the two amplifiable DNA loci which were mapped in this region: the amplifiable unit of DNA locus 6 (AUD6). The nucleotide sequence of one AUD6 fragment of 1.9 kb reveals the presence of two open reading frames (ORF1 and ORF2) on the basis of the typical Streptomyces base composition at each of the three positions within codons. ORF1 shows some similarity with a gene encoding a regulatory protein. The presence of potential genes in this unstable locus was unexpected because deletions occurred with high frequency within this region in the genetic instability-derived mutant strains. However, transcription analyses by S1 nuclease protection experiments on the wild-type strain showed transcription of both ORF1 and ORF2. Moreover, the amplified strain reveals increased transcription of ORF1 but no transcription of ORF2. The amplification therefore results in a switch in transcription. The unstable region of S. ambofaciens DSM 40697 therefore is not a 'silent' region because at least some loci are transcribed.

Amino Acid Sequence↗
PubMed1993

Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3(2).

A physical map of the chromosome of Streptomyces lividans 66 ZX7 was constructed by ordering the macrorestriction fragments generated from the genomic DNA with the restriction enzymes AseI and DraI. AseI and DraI linking cosmids (i.e., recombinant cosmids including either AseI or DraI sites) were isolated from a gene bank and used as hybridization probes against Southern transfers of pulsed-field gel electrophoresis (PFGE) restriction patterns. The DraI sites were precisely mapped by PFGE analyses of AseI-DraI double digests and hybridization with the AseI junctions. The 16 AseI and 7 DraI fragments were aligned as a single chromosome of about 8,000 kb. The data supported the interpretation that the chromosome is a linear structure. The related strain Streptomyces coelicolor A3(2) M145, recently mapped by H. Kieser, T. Kieser, and D. A. Hopwood (J. Bacteriol. 174:5496-5507, 1992), was compared with S. lividans at the level of the genomic structure by hybridizing the linking cosmids to Southern transfers of PFGE patterns. In spite of little apparent similarity in their restriction patterns, the comparison of the physical maps revealed a common structure with an identical ordering of the cosmid sequences. This conservation of the map order was further confirmed by assigning genetic markers (i.e., cloned genes and DNA elements relevant to the unstable region) to the AseI fragments.

DNA, Bacterial↗
PubMed1992

Target antigens of anti-platelet antibodies in patients with steroid-resistant ITP of recent onset. Canadian Apheresis Study Group.

Recent application of Western blotting procedures in the detection of anti-platelet antibodies has permitted more refined definition of reactivity. We report on the results of anti-platelet antibody assays in a series of 19 patients with recently diagnosed (less than 6 months duration) steroid-resistant, idiopathic thrombocytopenia purpura (ITP). At presentation, six of the 19 patients had a positive test for platelet-associated IgG (PAIgG) as measured by the direct radial-immunodiffusion (RID) assay, whereas three of the 19 were positive with the direct-platelet-suspension-immunofluorescence test (DPSIFT). The indirect-platelet-suspension-immunofluorescence test (IPSIFT) demonstrated antibodies in seven of the sera. Following Western blot (WB) analysis, the serum of 7/19 patients (only three of which were positive in the IPSIFT) could be shown to react with platelet antigens. Two patterns were seen: in four cases there were bands of apparent molecular weights of 60,000, 55,000 and 50,000; in the other three samples, a single band near 90,000 was demonstrated. Unlike the situation reported for chronic ITP, no reactivity was seen against higher molecular weight antigens other than the 'non-specific' binding at apparent molecular weight 200,000 which is also seen with normal sera. The data suggest that antibodies reacting against specific platelet antigens are present in the serum of some patients with recent onset ITP.

Adult↗
PubMed1991

Genetic instability and associated genome plasticity in Streptomyces ambofaciens: pulsed-field gel electrophoresis evidence for large DNA alterations in a limited genomic region.

Using pulsed-field gel electrophoresis (PFGE) analysis, the amplifiable units of DNA (AUD) loci AUD6 and AUD90 of Streptomyces ambofaciens DSM40697 could be mapped in the wild-type genome within two adjacent AseI restriction fragments estimated to be about 75 and 850 kb. In addition, the genetic instability and formation of very large deletions were strictly correlated. Their sizes were estimated to range from 250 to more than 2,000 kb. These deletions affected the DNA region overlapping both amplifiable loci. PFGE also allowed us to localize the amplified DNA sequences and to establish their structure: amplification takes place at the AUD locus as a tandem array of the wild-type AUD sequence.

DNA, Bacterial↗
PubMed1990

Pulsed-field gel electrophoresis analysis of the genome of Streptomyces ambofaciens strains.

The genome of four Streptomyces ambofaciens strains from different geographical origins (ATCC15154, DSM40697, ETH9247 and ETH 11317) was analysed by pulsed-field gel electrophoresis (PFGE). The PFGE technique has allowed the study of the extrachromosomal content of these strains and the characterization of their genomic DNA by restriction analyses. Electrophoretic migration of undigested DNA allowed us to detect a 80 kb-length linear molecule with concatemeric forms in S. ambofaciens ATCC15154. These extrachromosomal molecules were shown to be homologous to the circular plasmid pSAM1 (80 kb) suggesting that pSAM1 could exist not only in circular form but also in linear form. In the same way a 45 kb-length linear molecule was detected in S. ambofaciens ETH9427 and ETH11317. In contrast, no extrachromosomal DNA could be detected in S. ambofaciens DSM40697. The analysis of the macrorestriction patterns using the rate-cutting enzymes AseI and DraI indicated a close relationship between the DSM- and ETH- strains. Indeed, three types of restriction patterns were distinguished: while S. ambofaciens ETH9427 and ETH11317 were characterized by the same pattern and share more than 75% of comigrating fragments with the strain DSM40697, S. ambofaciens ATCC15154 exhibited a restriction pattern different from the other three. The total genome sizes of S. ambofaciens ATCC15154, DSM40697, ETH9427 and ETH11317 were estimated to be about 6500, 8000, 8200 and 8200 kb, respectively.

Blotting, Southern↗
PubMed1990

Genetic instability and hypervariability in Streptomyces ambofaciens: towards an understanding of a mechanism of genome plasticity.

Many Streptomyces species exhibit a very high degree of genetic instability which is usually manifested as genomic rearrangements such as large deletions. In Streptomyces ambofaciens DSM40697, two levels of genetic instability were previously described: (i) a basic genetic instability similar to that reported for other strains, and (ii) hypervariability, a phenomenon that we believe to be a new aspect of instability closely associated with DNA amplification. A large DNA region undergoes deletions, amplifications and large genomic changes strictly associated with both aspects of genetic instability. The genetic and molecular analyses of the different aspects of genetic instability allow us to propose that they result from a cascade of molecular events and to investigate the relationships between genetic instability phenomena and genome fluidity.

DNA, Bacterial↗
PubMed1989

Hypervariability, a new phenomenon of genetic instability, related to DNA amplification in Streptomyces ambofaciens.

The wild-type strain Streptomyces ambofaciens DSM 40697 exhibits a high degree of genetic instability. Pigment-defective colonies were observed in the progeny of wild-type colonies at a frequency of about 0.01. While only 13% of these pigment-defective colonies gave rise to homogeneous progeny exhibiting the mutant parental phenotype, 87% of the mutant colonies gave rise to hetergeneous progeny without a preponderant phenotype. This new phenomenon of instability was called hypervariability. In addition, 21% of the mutant strains arising in hypervariable progeny contained highly reiterated DNA sequences, while amplified DNA sequences could be detected in neither stable pigment-defective mutant clones nor in wild-type clones. These results indicate a frequent association between genetic instability and hypervariability and a frequent association between hypervariability and amplification of DNA sequences.

DNA, Bacterial↗
PubMed1988

Characterization of two families of spontaneously amplifiable units of DNA in Streptomyces ambofaciens.

Four highly amplified DNA sequences (ADS) ranging from 5.8 to 24.8 kb were found in spontaneous mutant strains of Streptomyces ambofaciens DSM 40697. Restriction patterns of total DNA were hybridized with purified ADS6 (24.8 kb) as a probe to detect the amplifiable regions in the wild-type (WT) genome. The results suggested that the amplifiable unit of DNA (AUD) was present as a single copy in the WT genome. Moreover, similarities suggested by the restriction maps of three of the ADS were confirmed by hybridization experiments. The fourth ADS did not hybridize with the three others. Therefore, two families of DNA sequences are potentially amplifiable in the S. ambofaciens genome.

DNA, Bacterial↗
PubMed1984

[Progressive complications associated with the use of a bovine heterograft for vascular access].

Three bovine carotid heterografts, used for vascular access, were analysed after removal following the development of aneurysms in two cases and thrombosis in one. The first prosthesis was implanted in a 17-year-old man who suffered from myasthenia gravis and was undergoing plasmapheresis. It became notably dilated several months after implantation, and resection was necessary 14 months after insertion. The second graft was placed in a 31-year-old woman being treated for a severe chronic potassium insufficiency. It was removed 16 months after insertion because of thrombosis. The third bovine carotid heterograft was inserted in a 48-year-old patient on long-term hemodialysis and was removed 3.5 months later because of aneurysmal dilatation adjacent to the arterial anastomosis. These evolutionary and degenerative changes associated with the bovine carotid heterograft illustrate the limitations of this material as an interpositional limb for arteriovenous fistulas in angioaccess therapy. Small weaknesses have sometimes been noted in the microstructure of the graft material studied before implantation.

Adolescent↗
PubMed1976

Isolation of variant lymphoma cells with reduced growth requirements for extracellular calcium and magnesium and enhanced oncogenicity.

By exposing an established cell line of malignant mouse lymphocytic cells (L5178Y) to a culture environment low in calcium and magnesium, we have isolated and maintained in continuous culture a variant population with reduced growth requirements for the cations. The variant cells are larger, have enhanced aggregability, tend to form dispersed colonies in semisolid medium, and have increased oncogenicity. Variants and their progenitors share similar morphology, maximum proliferative rate, and stability of phenotype. The significance of these findings is discussed and an analogy suggested between the selective influence of calcium and magnesium deprivation in vitro and the evolution of thymic dysplasias in divalent cation-deprived rodents.

Animals↗