Size-polymorphism of mini-exon gene-bearing chromosomes among natural populations of Leishmania, subgenus Viannia

https://doi.org/10.1016/S0020-7519(99)00010-7Get rights and content

Abstract

In order to explore genomic plasticity at the level of the mini-exon gene-bearing chromosome in natural populations of Leishmania, the molecular karyotype of 84 Leishmania stocks belonging to subgenus Viannia, originating mostly from Peru and Bolivia, and differing according to eco-geographical and clinical parameters, was resolved and hybridised with a mini-exon probe. The results suggest that size variation of the mini-exon gene-bearing chromosome is frequent and important (up to 245-kb size-difference), and partially involves variation (up to 50%) in copy number of mini-exon genes. There is no significant size-difference between mini-exon-bearing chromosomes of Peruvian and Bolivian populations of cutaneous and mucosal isolates of Leishmania (Viannia) braziliensis, but there is between eco-geographical populations of Leishmania (Viannia) peruviana. Leishmania (V.) peruviana presented a significantly smaller mini-exon-bearing chromosome than the other species of subgenus Viannia. The contrast between the general chromosome size heterogeneity and the homogeneity observed in some Peruvian Andean areas is discussed in terms of selective pressure.

Introduction

Chromosomal size-polymorphism is very frequent among Leishmania and other Protozoa, and is being studied for basic purposes1, 2, 3, 4, and less for its contribution to molecular epidemiology5, 6. During an extensive survey of Neotropical Leishmania belonging to subgenus Viannia, we found two interesting chromosomes. The first one, bearing gp63 genes, showed a significantly smaller size in Leishmania (Viannia) guyanensis and Leishmania (Viannia) peruviana than in Leishmania (Viannia) braziliensis7, 8. The second one, bearing rDNA genes, permitted identification of eco-geographical populations among L. (V.) peruviana; size was significantly smaller in populations isolated from southern Peru than northern ones7, 9. In both cases, chromosome size-variation was shown to be due partially to variation in copy number of tandemly repeated genes, respectively gp63 and rDNA8, 9, 10.

Amplification/deletion of repeated sequences appears to be an important and general mechanism leading to protozoan genome poly morphism[11]. In addition, if the sequences involved in these phenomena correspond to important genes, their rearrangement might have phenotypic consequences (due to gene dosage[12], deletion of unique interspersed genes[4], or modification at the level of intergenic sequences known to play a role in transcription regulation[13]), and thus be selected by environmental factors. Accordingly, analysing the genomic organisation of important repeated genes might reveal new markers for eco-epidemiology.

Mini-exon genes are tandemly repeated sequences encoding a factor essential for the functionality of mRNA in trypanosomatids[14]. Previous studies on a few laboratory-maintained lines of Leishmania major showed that amplification/deletion of the mini-exon gene array was partly responsible for size variation of the bearing chromosome[15]. The aim of the work reported here was to explore this phenomenon in natural populations of Leishmania, differing according to eco-geographical and clinical parameters. Therefore, the molecular karyotype of 84 Leishmania stocks representing several species of subgenus Viannia was resolved and hybridised with a mini-exon probe. Size of hybridising chromosomes was evaluated and compared among the different groups of organisms considered. In addition, the relationship between chromosomal size and hybridisation intensity of the mini-exon probe was studied in order to verify the involvement of mini-exon gene in chromosome plasticity.

Section snippets

Stocks

Eighty-four stocks previously characterised by isoenzyme analysis (Bañuls, PhD thesis, 1998) were considered in the present study (Table 1). Most stocks were obtained from an allopatric sampling in Peru and Bolivia. With respect to L. (V.) peruviana, four bio-geographical units (BGUs, classification of Lamas[16]), constituting a north–south transect along the Peruvian Andes, were considered: Huancabamba (HB), Surco-North, Surco-Centre and Surco-South (SUN, SUC and SUS, respectively). Procedures

Chromosome hybridisation patterns of mini-exon genes

The karyotype of 84 stocks belonging to subgenus Viannia was hybridised with a mini-exon gene probe. As expected, patterns were highly polymorphic, with chromosomal size ranging between 360 and 605 kb. In addition, in several stocks, double hybridising bands were encountered (Fig. 1A). Identical patterns were observed when hybridising with pLb-149G (Fig. 1B), a random genomic probe not cross-hybridising with mini-exon genes (not shown). This suggests homology between the different hybridising

Discussion

In previous work, Iovannisci and Beverley[15]showed that size variation of the mini-exon gene-bearing chromosome was partly due to expansion/contraction of the mini-exon gene array for a few laboratory-maintained strains. In the present study, we explored this phenomenon in natural populations of Neotropical Leishmania (subgenus Viannia). Our results show that size variation of the mini-exon gene-bearing chromosome is frequent and important (up to 245 kb size difference), chromosomal plasticity

Acknowledgements

This investigation received financial support from the EC (contract IC18-CT96-0123) and from the Belgian Agency for Cooperation to Development (training fellowship to A.K.).

References (33)

  • C.R Davies et al.

    Lutzomyia verrucarum can transmit Leishmania peruviana, the aetiological agent of Andean cutaneous leishmaniasis

    Trans R Soc Trop Med Hyg

    (1993)
  • E Cupolillo et al.

    Intergenic region typing (IRT): a rapic molecular approach to the characterization and evolution of Leishmania

    Mol Biochem Parasitol

    (1995)
  • P Wincker et al.

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

    Nucleic Acids Res

    (1996)
  • R.D Adam

    Chromosome-size variation in Giardia lamblia: the role of rDNA repeats

    Nucleic Acids Res

    (1992)
  • J.C Dujardin et al.

    From pulsed field to field: contribution of molecular karyotyping for epidemiological studies of New World leishmaniasis

    Arch Inst Pasteur Tunis

    (1993)
  • J.C Dujardin et al.

    Molecular karyotype variation of Leishmania (Viannia) peruviana evidences geographical populations in Peru along a North–South cline

    Ann Trop Med Parasitol

    (1993)
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