RESUMEN
In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63-RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR-RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni; reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63-RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63-RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63-RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR-RFLP). PCR-RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63-RFLP and PCR-RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.
Asunto(s)
Glicoproteínas/genética , Leishmania/genética , Leishmaniasis/epidemiología , Animales , Southern Blotting , Electroforesis en Gel de Agar , Electroforesis en Acetato de Celulosa , Variación Genética/genética , Glicoproteínas/química , Isoenzimas/análisis , Leishmania/química , Leishmania/clasificación , Leishmaniasis/parasitología , Hibridación de Ácido Nucleico , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo Restrictivo , América del Sur/epidemiologíaRESUMEN
The genomic organization of gp63 genes in 4 and 7 isolates of Leishmania braziliensis and L. peruviana, respectively was studied by RFLP analysis with 3 restriction enzymes (Bgl I, Sal I and Apa I). Our results showed a marked polymorphism among isolates. Some characters were specific to L. braziliensis or to L. peruviana, and others specific to the respective biogeographical populations of L. peruviana. The average minimum copy number of gp63 genes was found to be higher in L. braziliensis (71) than in L. peruviana (46), suggesting that deletion of gp63 genes might be partially involved in the size decrease of the chromosome bearing gp63 genes, observed between those 2 species (from 700 to 610 kb). Our results may suggest the existence of at least 2 arrays of heterologous gp63 repeats, varying in relative copy number between L. braziliensis and L. peruviana, and among isolates of the latter species. Rearrangement of the gp63 genes was observed during long-term in vitro maintenance of a reference strain of L. braziliensis. These observations document the existence of a dynamic gp63 gene organization in Leishmania of the braziliensis complex.
Asunto(s)
Genes Protozoarios , Leishmania braziliensis/genética , Metaloendopeptidasas/genética , Proteínas Protozoarias/genética , Animales , Clonación Molecular , Cósmidos , Amplificación de Genes , Reordenamiento Génico , Leishmania braziliensis/aislamiento & purificación , Hibridación de Ácido Nucleico , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
Molecular karyotype of 45 reference populations of Neotropical leishmanias was analyzed with ethidium bromide staining and with 6 chromosome-derived probes selected from a genomic library of Leishmania (Viannia) braziliensis. Size-conserved patterns were identified and found to be specific to subgenus Viannia and to its constitutive species. An important issue for epidemiology and clinical investigations was the discrimination between L. (V.) peruviana and L. (V.) braziliensis, 2 species found very similar by other genetic techniques, but responsible for totally different clinical patterns. The suggested existence of genetically distinct demes, or karyodemes, within the group-species might also show to be of importance, as these populations might differ in virulence, host-specificity and clinical manifestations.
Asunto(s)
Leishmania braziliensis/genética , Polimorfismo Genético , Animales , Sondas de ADN , Electroforesis en Gel de Campo Pulsado , Cariotipificación , Leishmania braziliensis/clasificación , Leishmania mexicana/genética , Hibridación de Ácido Nucleico , Mapeo RestrictivoRESUMEN
Circular extrachromosomal elements were observed in a variety of Leishmania species. We show here that two lines originating from the same isolate have been found to contain a circular DNA molecule of 26.6 kb and a linear chromosome of about 250 kb, respectively, which share a homology of more than 20 kb. The circular DNA molecule and its related region on the linear chromosome were cloned and their restriction maps compared. This investigation reveals information about chromosome rearrangement in L. mexicana M379. Further examination will enable us to understand the nature of chromosome rearrangement such as circularization or linearization.