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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.

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 ∼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.

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.

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.

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.

Background Trypanosomatids exhibit a unique gene organization into large directional gene clusters (DGCs) in opposite directions. The transcription "strand switch region" (SSR) separating the two large DGCs that constitute chromosome 1 of Leishmania major has been the subject of several studies and speculations. Thus, it has been suspected of being the single replication origin of the chromosome, the transcription initiation site for both DGCs or even a centromere. Here, we have used an inter-species compared genomics approach on this locus in order to try to identify conserved features or motifs indicative of a putative function. Results We isolated, and compared the structure and nucleotide sequence of, this SSR in 15 widely divergent species of Leishmania and Sauroleishmania. As regards its intrachromosomal position, size and AT content, the general structure of this SSR appears extremely stable among species, which is another demonstration of the remarkable structural stability of these genomes at the evolutionary level. Sequence alignments showed several interesting features. Overall, only 30% of nucleotide positions were conserved in the SSR among the 15 species, versus 74% and 62% in the 5' parts of the adjacent XPP and PAXP genes, respectively. However, nucleotide divergences were not distributed homogeneously along this sequence. Thus, a central fragment of approximately 440 bp exhibited 54% of identity among the 15 species. This fragment actually represents a new Leishmania-specific CDS of unknown function which had been overlooked since the annotation of this chromosome. The encoded protein comprises two trans-membrane domains and is classified in the "structural protein" GO category. We cloned this novel gene and expressed it as a recombinant green fluorescent protein-fused version, which showed its localisation to the endoplasmic reticulum. The whole of these data shorten the actual SSR to an 887-bp segment as compared with the original 1.6 kb. In the rest of the SSR, the percentage of identity was much lower, around 22%. Interestingly, the 72-bp fragment where the putatively single transcription initiation site of chromosome 1 was identified is located in a low-conservation portion of the SSR and is itself highly polymorphic amongst species. Nevertheless, it is highly C-rich and presents a unique poly(C) tract in the same position in all species. Conclusion This inter-specific comparative study, the first of its kind, (a) allowed to reveal a novel genus-specific gene and (b) identified a conserved poly(C) tract in the otherwise highly polymorphic region containing the putative transcription initiation site. This allows hypothesising an intervention of poly(C)-binding proteins known elsewhere to be involved in transcriptional control.

Chromosome fragmentation (CF) constitutes one means of manipulating eukaryotic genomes and provides a powerful tool for examining both the structure and function of chromosomes. During the past 15 yr, CF, which is based on the use of transfection, has been widely used in yeast and mammals to elucidate the functional elements required for normal chromosome maintenance. However, in view of the relatively late development of parasite genome projects, this strategy has only been used recently in parasites. Here, we describe basic methods for CF (except telomere-mediated fragmentation) experiments and analysis in Leishmania. Current limitations of this methodology are precisely the lack of knowledge of the nature of centromeres and autonomously replicating sequences in this and other protozoa, the poor understanding of precise recombination mechanisms, as well as the fact that the deletion of unknown genes essential to parasite survival may interfere with recombination events and chromosomal rearrangements. Still, this powerful method has enriched our basic knowledge of chromosomal structure and maintenance.

The Leishmania genome project reference strain, Leishmania major Friedlin, is trisomic for chromosome 1. The complete sequence of this chromosome has revealed that the genes are grouped into two large clusters of the polycistronic type, each borne by one DNA strand and located on each side of a 1.6-kb sequence often termed the switch region. Several hypotheses concerning the role of this switch region have been put forward (region of initiation of transcription for both gene clusters, origin of replication or centromeric sequence). In the present study, we have deleted this region on the three copies of chromosome 1 by sequential targeted replacements. The absence of the switch region did not alter the mitotic stability of the three deleted chromosomes. This region therefore does not appear necessary for chromosomal replication or segregation. However, during the third targeting round, which aimed at knocking out the last switch region, a fourth copy of chromosome 1 that retained this region appeared in all clones analysed. This suggests that the persistence of this switch region is necessary for parasite survival. We then showed that the presence/absence of the switch region did not act upon the expression of a resistance marker gene inserted beforehand into the left gene cluster of the same chromosomal molecule. This result suggests that the presence of this 1.6-kb sequence is not necessary for the expression of all genes on chromosome 1.

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 Ase I, Dra I, Xba I, Ssp I, Spe I, and Sfi I 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.

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.

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.

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.