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The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.

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.

Resume Les leishmanioses sont des affections parasitaires de plus en plus associees a l'immunodepression et plus particulierement la leishmaniose viscerale au cours de l'infection a VIH dans ie sud de l'Europe. D'apres une etude retrospective de l'Organisation mondiale de la sante, 692 cas ont ete recenses jusqu'en 1995, interessant surtout les adultes de 20 a 40 ans. Les manifestations biocliniques sont souvent atypiques avec des localisations inhabituelles en particulier digestives et pulmonaires. Le diagnostic biologique repose essentiellement sur la mise en evidence du parasite par examen direct et culture, plus recemment par polymerase chain reaction, technique d'auenir. Le diagnostic serologique, inconstamment positif, est toutefois utilise en complement. Les therapeutiques antileishmaniennes classiques sont insuffisantes pour une guerison durable.