PubMed Health⌕ Search

Biomedical subjects

C Blaineau

Publications and source records attributed to C Blaineau.

At least 19 recordsLinked to original sources
PubMed2001

Effect of large targeted deletions on the mitotic stability of an extra chromosome mediating drug resistance in Leishmania.

A mitotically stable linear extra chromosome obtained in a Leishmania donovani strain rendered mycophenolic acid-resistant has been physically mapped. This 290-kb chromosome has an inverted duplicated structure around a central inversion region, and is derived from a conservative amplification event of a approximately 140-kb subtelomeric end of chromosome 19. Large-sized targeted deletions of the central region were performed through homologous recombination using three specific transfection vectors. The size of the extra chromosome was thus successfully reduced from 290 to 260, 200 and 120 kb respectively. The mitotic stability of these chromosomes was then analysed in drug-free cultures over >140 days. Results differed according to the deletion created. By contrast with the smallest deletion the two largest deletions altered mitotic stability, leading to progressive loss of the size-reduced chromosomes with similar kinetics in both mutants. The 30-kb region common to both deletions may therefore be considered as involved in mitotic stability. A 44-kb contig covering this region could be assembled and sequenced. The analysis of this sequence did not reveal any sequence elements typical of centromeric DNA. By contrast, its enrichment in homopolymer tracts suggests that this region might contain an origin of replication.

Animals↗
PubMed1999

High conservation of the fine-scale organisation of chromosome 5 between two pathogenic Leishmania species.

In a previous work we showed a remarkable conservation of the general structure of the genome (chromo-some number and synteny) among different pathogenic species of Old World Leishmania, indicating the absence of major interchromosomal rearrangements during evolution. In the present study, we have compared the fine structure of chromo-some 5 among two of these divergent species (Leishmania major and Leishmania infantum) by means of physical mapping. Remarkably, the 42 markers jointly mapped on these two chromosomes were found in an identical order along the chromosome. This perfect colinearity of the markers is in striking contrast to the large genetic distance that separates these species and suggests that conservation of the fine-scale organisation of chromosomes may be critical in Leishmania. If this colinearity is confirmed on the whole of the chromosome set, the current systematic sequencing programme of the genome of L.major should greatly help in the development of comparative genetics between different species of Leishmania.

Animals↗
PubMed1998

Conserved linkage groups associated with large-scale chromosomal rearrangements between Old World and New World Leishmania genomes.

The genus Leishmania can be taxonomically separated into three main groups: the Old World subgenus L. (Leishmania), the New World subgenus L. (Leishmania) and the New World subgenus L. (Viannia). The haploid genome of Old World Leishmania species has been shown to contain 36 chromosomes defined as physical linkage groups; the latter were found entirely conserved across species. In the present study, we tried to verify whether this conservation of the genome structure extends to the New World species of Leishmania. 300 loci were explored by hybridization on optimized pulsed field gel electrophoresis separations of the chromosomes of polymorphic strains of the six main pathogenic Leishmania species of the New World. When comparing these New World karyotypes with their Old World counterparts, 32 out of 36 linkage groups were found conserved among all species. Four chromosomal rearrangements were found. All species belonging to the L. (Viannia) subgenus were characterized by the presence (i) of a short sequence exchange between chromosomes 26 and 35, and (ii) more importantly, of a fused version of chromosomes 20 and 34 which are separated in all Old World species. 69 additional markers were isolated from a plasmid library specifically constructed from the rearranged chromosomes 20+34 in an attempt to detect mechanisms other than a fusion or breakage: only two markers out of 40 did not belong to the linkage groups 20 and 34. On the other hand, all strains belonging to the New World subgenus L. (Leishmania) were characterized by two different chromosomal rearrangements of the same type (fusion/breakage) as above as compared with Old World species: chromosomes 8+29 and 20+36. Consequently, these two groups of species have 35 and 34 heterologous chromosomes, respectively. Overall, these results show that large-scale chromosomal rearrangements occurred during the evolution of the genus Leishmania, and that the three main groups of pathogenic species are characterized by different chromosome numbers. Nevertheless, translocations seem particularly rare, and the conservation of the major linkage groups should be an essential feature for the compared genetics between species of this parasite.

Animals↗
PubMed1997

A direct method for the chromosomal assignment of DNA markers in Leishmania.

A simple method for the chromosomal assignment of any DNA marker would be an important tool for the ongoing project to map the genome of the protozoan parasite Leishmania. The Leishmania chromosomes enter pulsed field gel electrophoresis (PFGE) gels under current electrophoretic conditions, but their direct identification in a given strain is hampered by their stacking in a few chromosomal bands, and by the very frequent size variations of the same chromosome among parasite strains. To overcome these problems. we determined the complete karyotypes of 12 Old World Leishmania cloned strains. This enabled us to select three of these strains that display great chromosome size polymorphisms, such that every chromosome can be individualized by a specific pattern after hybridization onto these three karyotypes. The complete resolution of the genomes of these three strains can be carried out with only three electrophoretic conditions. This makes a series of three blots sufficient for the assignment of any new marker on a particular Leishmania chromosome.

Animals↗
PubMed1996

Medium-range restriction maps of five chromosomes of Leishmania infantum and localization of size-variable regions.

The chromosomes of the human protozoan parasite Leishmania exhibit striking size polymorphisms among different strains. To define the structural basis for these variations, we have constructed full-length restriction maps of five chromosomes of 370 to 490 kb in Leishmania infantum clone LEM1317. Rare-cutting sites for the enzymes AseI, DraI, XbaI, SspI, SpeI, and SfiI were mapped by partial and complete digestion of either gel-purified chromosomes or total DNA. Sixty-eight anonymous DNA probes were localized on these maps, as well as the mini-exon and dihydrofolate reductase-thymidilate synthase gene probes. These maps were compared with those from other strains of L. infantum and Leishmania donovani. This showed that the distribution of the restriction sites was conserved in these two close species. Four regions involved in the size variations of three chromosomes were localized; subtelomeric sequences were responsible for size variability in three of four cases. The whole of this study takes a particular significance in the frame of the project of complete physical mapping and sequencing of the Leishmania genome.

Animals↗
PubMed1996

The Leishmania genome comprises 36 chromosomes conserved across widely divergent human pathogenic species.

All the physical linkage groups constituting the genome of Leishmania infantum have been identified for the first time by hybridization of specific DNA probes to pulsed field gradient-separated chromosomes. The numerous co-migrating chromosomes were individualised using the distinctive size polymorphisms which occur among strains of the L. infantum/L. donovani complex as a tool. A total of 244 probes, consisting of 41 known genes, 66 expressed sequence tags (ESTs) and 137 anonymous DNA sequences, were assigned to a specific linkage group. We show that this genome comprises 36 chromosomes ranging in size from 0.35 to -3 Mb. This information enabled us to compare the genome structure of L. infantum with those of the three other main Leishmania species that infect man in the Old World, L. major, L. tropica and L. aethiopica. The linkage groups were consistently conserved in all species examined. This result is in striking contrast to the large genetic distances that separate these species and suggests that conservation of the chromosome structure may be critical for this human pathogen. Finally, the high density of markers obtained during the present study (with a mean of 1 marker/130 kb) will speed up the construction of a detailed physical map that would facilitate the genetic analysis of this parasite, for which no classical genetics is available.

Animals↗
PubMed1995

Conservation among Old World Leishmania species of six physical linkage groups defined in Leishmania infantum small chromosomes.

We have characterised 49 DNA probes specific for each of the six smallest chromosomes in Leishmania infantum and have examined the allocation of these probes in the molecular karyotypes of the other Old World Leishmania species Leishmania donovani, Leishmania major, Leishmania tropica and Leishmania aethiopica. These 49 probes define 6 physical linkage groups in the molecular karyotypes of various strains of L. infantum. 40 of these probes hybridise in the other Old World Leishmania species and show a remarkably conserved linkage pattern. No interchromosomal exchange nor fusion could be detected. Thus, in spite of the chromosomal size polymorphisms, the general structure of the genome seems to be conserved in the six smallest chromosomes among Old World Leishmania species. This structural genomic homogeneity should be helpful for mapping studies of any Old World Leishmania genomes.

Animals↗
PubMed1995

A polymorphic minisatellite sequence in the subtelomeric regions of chromosomes I and V in Leishmania infantum.

A minisatellite DNA sequence is described for the first time in Leishmania infantum. It is borne by four chromosomes and consists of an 81-bp repeat unit organised in several clusters. On chromosomes I and V of L. infantum, the clusters are tightly located in the size-variable subtelomeric regions. The organisation of this sequence may be related to that of the subtelomeric interspersed repeat sequences identified in the human genome. The sequencing of seven repeat units, some subcloned from the same cluster, allowed the definition of a consensus sequence of 81 bp, particularly G/C rich (73%). Two subfamilies were clearly defined: one exhibits a 91-95% homology with the consensus sequence; the second one comprises two monomers sharing a 91% homology but only 77% homology with the consensus sequence. The two types of monomers can be found in the same cluster. These data suggest interactions between monomers and a possible role of this sequence in the instability of these regions. Finally, restriction fragment length polymorphisms were revealed by this sequence among various strains of L. infantum. Besides allowing the detection of recombination events in the unstable regions of the chromosomes, this new marker may become a useful tool in the study of the parasite population dynamics in leishmaniasis foci.

Animals↗
PubMed1994

Structural organisation of microsatellite families in the Leishmania genome and polymorphisms at two (CA)n loci.

In the present study, we have analysed the frequency and distribution of several microsatellite DNAs [(CA)n, (GGT)n and (GCA)n] in the genome of Leishmania. Hybridisation analysis on the molecular karyotypes of different Leishmania strains showed the presence of these three microsatellites on all chromosomes of the parasite. The number of microsatellite clusters appeared grossly similar among strains from different Old World complexes. However, these three microsatellite families showed an uneven distribution among heterologous chromosomes of the same strain. Moreover, restriction analysis of chromosome I in various strains of Leishmania infantum showed a strong clustering of these microsatellites in the same chromosomal region. A partial genomic library was screened with a (CA)n probe, and 21 positive clones were isolated. The sequencing of these clones confirmed the association of various microsatellites such as (CA)n, (CT)n, and (GCA)n. Finally, specific polymerase chain reaction amplification of two cloned (CA)n loci demonstrated allelic size polymorphisms among strains within L. infantum and Leishmania donovani. Most of the 34 strains analysed were found to be monoallelic, while two alleles were found in a small number of strains. The interest of these sequences for studies on ploidy and population genetics of the parasite is discussed.

Animals↗
PubMed1992

Molecular karyotype analysis in Leishmania.

The advent of pulsed field electrophoresis has allowed a direct approach to the karyotype of Leishmania. The molecular karyotype thus obtained is a stable characteristic of a given strain, although minor modifications may occur during in vitro maintenance. Between 20 and 28 chromosomal bands can be resolved depending on the strain, ranging in size from approximately 250 to 2600 kb. The technique has revealed a striking degree of polymorphism in the size and number of the chromosomal bands between different strains, and this seems independent of the category (species, zymodeme, population) to which the strains belong. It appears that only certain strains originating from the same geographic area may share extensive similarities. This polymorphism can largely be accounted for by chromosome size variations, which can involve up to 25% of the chromosome length. As a result, homologous chromosomes can exist in versions of markedly different size within the same strain. When this occurs with several different chromosomes, the interpretation of PFE patterns appears difficult without prior identification of the size-variable chromosomes and of the chromosome homologies. DNA deletions and amplifications have been shown to account for some of these size modifications, but other mechanisms are probably involved; nevertheless, interchromosomal exchange does not seem to play a major role in these polymorphisms. These chromosomal rearrangements, yet in an early stage of characterization, exhibit two relevant features: they seem (1) to affect essentially the subtelomeric regions and (2) to occur in a recurrent nonrandom manner. Chromosomal rearrangements sharing the same characteristics have been identified in yeast and other protozoa such as Trypanosoma and Plasmodium. The significance of this hypervariability for the biology of the parasite remains unknown, but it can be expected that such mechanisms have been maintained for some purpose; genes specifically located near chromosome ends might benefit from rapid sequence change, alternating activation, or polymorphism of expression. The chromosomal plasticity could represent a general mode of mutation in these parasites, in parallel with genetic exchange which may be uncommon in nature. The molecular characterization of these rearrangements, the identification of each chromosome with the help of physical restriction maps and linkage maps, and the collation of such data on a number of strains and species should allow a significant progress in the understanding of the genetics of Leishmania, in particular as regards ploidy, generation of phenotypic diversity, and genome evolution. Finally, like other models, this is susceptible to improve our knowledge of DNA-DNA interactions and of the chromosome functional structure and dynamics.

Animals↗
PubMed1992

Multiple forms of chromosome I, II and V in a restricted population of Leishmania infantum contrasting with monomorphism in individual strains suggest haploidy or automixy.

We have resolved the molecular karyotypes of 22 Leishmania infantum strains isolated between 1980 and 1988 in a restricted geographic area and belonging to zymodemes MON-11, -29 and -33. Three strains were isolated from sandflies and all the others from human cutaneous lesions. A high degree of karyotypic homology is observed among these strains, contrasting with the highly polymorphic MON-1 strains isolated in the same area. We have analysed the time-dependent evolution of size variants of chromosomes I to V, each identified by chromosome-specific DNA probes. More evidence is given for the role of subtelomeric regions in chromosomal size variation in Leishmania for both chromosomes I and II. At the population level, the chromosomes I, II and V are present in respectively 8, 4 and 3 distinct sizes. Furthermore, and despite the small size of the sample, various combinations were observed among these different chromosomal forms. These results could be explained by the occurrence of a high rate of recurrent mutations or of genetic exchange. In contrast, only one chromosomal form was observed in individual karyotypes for the chromosomes I-V. These results could tally with the hypothesis of a haploid organisation for these chromosomes and strains, or, in the frame of a diploid organisation, with the hypothesis of a predominantly automictic sexuality giving rise to 2 identical forms of the homologues in the same strain.

Animals↗
PubMed1992

Leishmania: sex, lies and karyotype.

The exploration of the genome of the tryponosomotid protozoan Leishmania has been difficult until recently owing to a number of obstacles, not least our ignorance of the ploidy and of the number of chromosomes (as in many other protozoa, the latter do not condense during mitosis), the uncertainty of the species concept in these allegedly asexual protozoa and the absence of classical genetic studies. Here, Patrick Bastien, Christine Bloineou and Michel Pages discuss the advances in this field brought about by the advent of molecular biology and its techniques, with on emphasis on ploidy and genetic exchange. In particular, they discuss the data from pulsed field gel electrophoresis (PFGE). When coupled with DNA restriction analysis, PFGE constitutes a powerful tool for the direct examination o f chromosomes of protozoa.

Journal Article↗
PubMed1991

Long-range restriction maps of size-variable homologous chromosomes in Leishmania infantum.

In order to clarify the interpretation of molecular karyotype polymorphisms in Leishmania, the three smallest chromosomes from five cloned strains of Leishmania infantum were identified by chromosome-specific anonymous DNA probes. The presence in the same clone of homologous chromosomes of a different size was demonstrated. The chromosome size polymorphism appeared even more dramatic, with size variations affecting up to 20% of the chromosome, and the two smallest bands in one strain being equivalent to six bands in another strain. Long-range restriction maps of five different-sized homologues of chromosome I showed the size-variation to be located to a terminal fragment in 4 out of 5 cases, and to a central fragment in one. The size-variable sequence was present on at least three other chromosomes as determined by hybridisation analysis. This suggests an instability of the subtelomeric regions such as that in Plasmodium falciparum. Lastly, the finding of several pairs of distinct-sized homologous chromosomes, together with other studies, strongly suggest that Leishmania is diploid in at least part of its chromosomal complement.

Animals↗
PubMed1990

Interclonal variations in molecular karyotype in Leishmania infantum imply a 'mosaic' strain structure.

The molecular karyotypes of 36 clones derived from 8 strains of Leishmania infantum were examined by pulsed-field gel electrophoresis. Although there appeared to be a high degree of genetic relatedness between the clones and the parent strain, a limited degree of polymorphism was noted in 50% of the clones, expressed mainly as the presence of an additional chromosome or as a chromosome size modification. Repeated subcloning in one strain showed that chromosomal rearrangements could occur during the cloning process. Chromosome homologies were examined by Southern analysis with chromosome-specific DNA probes. The results suggest a disomy for some chromosomes, but cannot exclude aneuploidy. The mechanisms possibly leading to such heterogeneity are discussed: they could involve frequent DNA amplification/deletion, and imply a 'mosaic' structure of the cultured strains or clones, with different individuals possessing differently sized versions of the same chromosomes.

Animals↗
PubMed1985

Developmentally regulated cell surface structures on mouse and human embryonal carcinoma cell lines.

Three monoclonal antibodies 5.1.H, 8.7.D and 13.7.A raised against semi-purified Tera 1 membrane fractions recognize distinct onco-foetal antigens which are developmentally regulated on cells such as Tera 2 clone 13 and appear to be restricted in their expression to undifferentiated ectoblastic cells and certain organized cystic structures mimicking the foetal intestine. These antigens, absent from normal adult tissues, differ markedly from glycosidic stage-specific antigens such as 75.12 which, while functioning as embryonal carcinoma differentiation markers, are also expressed on certain adult tissues. No evidence for a role of fucosyltransferases in regulating either 75.12 or SSEA-1 antigen expression on embryonal carcinoma cells or for the presence of lectin-like structures recognizing these antigens on such cells was found.

Adult↗
PubMed1985

Acute myeloblastic leukemia with active tyrosine protein kinase.

High level of tyrosine protein kinase activity was found in a membrane fraction isolated from an acute myeloblastic leukemia, out of 24 leukemias of different origin investigated. The major substrate for tyrosine phosphorylation in vitro was a 58 kDA protein (p58). The phosphorylation proceeded actively at 0 degrees C and was strongly stimulated by Mn2+ ions. Comparison by partial proteolysis of the p58 with similar phosphoproteins from a T-lymphoma line (KE37) and from lectin stimulated lymphocytes showed high similarity. The possible role of the tyrosine kinase activity in this leukemia is discussed.

Gastrins↗
PubMed1984

Stimulation of tyrosine phosphorylation in lectin treated human lymphocytes.

Large increases in tyrosine phosphorylation have been detected in subcellular matrixes isolated from lectin treated human lymphocytes. In lectin stimulated cells proteins of molecular weight 105, 75, 58 and 35 kDa contained phosphotyrosine (P-tyr) whereas non-stimulated cells had no 105 and low levels of P-tyr in proteins of 75, 58 and 35 kDa. In stimulated cells increased tyrosine kinase activity was also shown using gastrin as substrate. In both stimulated and non-stimulated cells the 58 kDa phosphoprotein was the most heavily labelled, after partial proteolysis of the 58 kDa different phosphopeptides were generated. A peptide with a sequence analogous to the autophosphorylated tyrosine site of pp60src inhibited tyrosine phosphorylation in stimulated cells. The lymphocyte system provides a useful tool to study normal tyrosine protein kinases and their role in cellular proliferation.

Detergents↗