International Journal for Parasitology 36 (2006) 211–217 www.elsevier.com/locate/ijpara Characterization of LST-R533: Uncovering a novel repetitive element in Leishmania* André L. Pedrosa a, Andrea M. Silva b, Jeronimo C. Ruiz b, Angela K. Cruz b,* b a Departamento de Ciências Biológicas, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil Received 26 June 2005; received in revised form 7 October 2005; accepted 14 October 2005 Abstract We have previously isolated and sequenced a novel repetitive element, now named LST-R533, which is present in four different regions of one extremity of Leishmania major chromosome 20. The repeats are polymorphic in size, ranging from 367 to 533 bp and contain an internal 81 bp sequence with highly conserved segments (14–81 bp long) dispersed throughout the parasite’s genome. These sequences were not found in coding regions of any predicted gene in L. major Friedlin genome, but are part of untranslated regions of some Leishmania transcripts. Analysis of the 81 bp sequence revealed significant degrees of identity with retrotransposons described in several other organisms. The presence of the sequence in other species from genus Leishmania was determined by Southern hybridisation and DNA sequencing. This analysis indicated the conservation of the 81-nucleotide element in all the Leishmania species evaluated. No sequences corresponding to LST-R533 or the 81 bp element were found on either Trypanosoma brucei or Trypanosoma cruzi databanks. q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Leishmania; Genome analysis; Repetitive sequences; LST-R533; Retrotransposons 1. Introduction Parasites from the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that affects an estimated three million people worldwide. Approximately 30 species of the parasite compose the genus, which is divided in subgenera Leishmania and Viannia, and 22 of them are associated with human infections (WHO, 1990). Different species of the parasite are responsible for causing a wide range of clinical manifestations, which vary from self-limiting cutaneous ulcers to the visceral form, a fatal disease if not treated. The parasite plays a major role in determining the form and progression of leishmaniasis (Spath et al., 2003; Naderer et al., 2004). Although the molecular mechanisms related to parasite * Nucleotide sequence data reported in this study are available in the GenBank under the accession numbers AF339905, AF339906, AF339907, DQ092335, DQ092336, DQ092337and DQ092338. * Corresponding author. Address: Avenida Bandeirantes, 3900, Bairro Monte Alegre, Ribeirão Preto, São Paulo, CEP 14049-900, Brazil. Tel.: C55 163 602 3318; fax: C55 163 633 1786. E-mail address: akcruz@fmrp.usp.br (A.K. Cruz). pathogenicity are not well understood, the genetic plasticity of the organism may be a crucial feature. In fact, this parasite does not seem to have full ploidy control and several strains of Leishmania have been shown to be aneuploid or suffer dramatic alterations in their ploidy (Cruz et al., 1993; Myler et al., 1999; Ghedin et al., 2004; Martinez-Calvillo et al., 2005). In addition, Leishmania strains reveal a high degree of intra- and interspecific chromosomal size polymorphism probably as a result of recombination processes involving repetitive elements, frequently observed at chromosomal extremities. They also show variation of tandemly repeated sequences (Iovannisci and Beverley, 1989; Ravel et al., 1996; Sunkin et al., 2000; Ghedin et al., 2004). In trypanosomatids, repetitive sequences are involved in central aspects of parasitism such as antigenic variation, documented in Trypanosoma brucei (Borst and Rudenko, 1994) and gene amplification (reviewed in Beverley, 1991). Repetitive elements in Leishmania are important components of circular amplified regions of the genome, which are frequently associated with development of resistance to drugs (Singh et al., 2001; Beverley et al., 1984). They may also be of particular interest for species identification and taxonomy, 0020-7519/$30.00 q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2005.10.002 212 A.L. Pedrosa et al. / International Journal for Parasitology 36 (2006) 211–217 since several classes of repetitive elements characterised in protozoan parasites are species-specific (reviewed in FloeterWinter and Shaw, 2001; Wickstead et al., 2003). In Leishmania, the majority of repetitive sequences described are associated with chromosomal extremities (Ravel et al., 1995; Fu and Barker, 1998; Sunkin et al., 2000; Pedrosa et al., 2001). We have previously described the isolation and sequencing of a recombinant cosmid, 117E08 (E8), which represents one extremity of chromosome 20 of Leishmania major LV39 (Pedrosa et al., 2001; Tosi et al., 1997). The presence of a repetitive element distributed in three different regions within the 35 kb of the insert was shown by sequencing, annotation and hybridisation experiments (Pedrosa et al., 2001). Here we describe the characterisation of an interspersed element, LST-R533, which is present as four copies in one of the extremities of L. major chromosome 20. It bears an internal 81 bp fragment, which is dispersed in the genome of the different Leishmania species tested and showed significant sequence identity with sections of retrotransposons characterised in other organisms. 2. Materials and methods 2.1. Leishmania culture and manipulation Promastigotes of Leishmania (Leishmania) major LV39 (Rho/SU/59/P), L. (L.) major Friedlin (MHOM/IL/80/Friedlin), L. (L.) major LT252 clonal line CC1 (MHOM/IR/83/IR), Leishmania (Leishmania) amazonensis (MPRO/BR/1972/M1841-LV-79), Leishmania (Leishmania) mexicana (MNYC/BZ/1962/M379), Leishmania (Leishmania) donovani (MHOM/ET/67/HU3), Leishmania (Sauroleishmania) hoogstraali (RHEM/SD/1963/NG26) and Leishmania (Sauroleishmania) tarentolae (ATCC 30267) were grown at 26 8C in M199 medium (Gibco BRL) supplemented as described (Kapler et al., 1990). Leishmania (Viannia) braziliensis 2904 (MHOM/BR/75/M2904) and L. (V.) braziliensis CE3227 (MHOM/BR/94/H-3227) were grown under similar conditions in a medium further supplemented with 2% human urine. Cells at density of 1.0–2.0!107 promastigotes/ ml were pelleted at 2000!g for 10 min at 4 8C for genomic DNA preparation. L. major strains were provided by S.M. Beverley (Department of Molecular Microbiology, Washington University, St Louis, USA) and P. Bastien (Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Montpellier, France). New World and reptile species were provided by J. Shaw (Departamento de Parasitologia, Universidade de São Paulo, Brazil). 2.2. Cosmid isolation, sequencing and sequence analysis Cosmid E8 was isolated from a L. major LV39 genomic library with the hexameric repeats from the telomeres of T. brucei as a probe (Tosi et al., 1997) and sequenced using a transposon-based strategy (Pedrosa et al., 2001). Sequence searches and multiple sequence alignments were performed by BLAST (Altschul et al., 1997) and ClustalW (Thompson et al., 1994), respectively. Sequences described in this work are available at GenBank under accession numbers AF339905, AF339906, AF339907, DQ092335, DQ092336, DQ092337 and DQ092338. 2.3. Manipulation of nucleic acids Episomal DNA was purified using a Plasmid Maxi Kit (QIAGEN Incorporated, Valencia, CA, USA) and digested with endonucleases Bam HI, Hind III, Xba I and Xho I as recommended (New England Biolabs, Beverley, MA). Agarose blocks containing the intact chromosomes of Leishmania promastigotes were prepared and digested as previously described (Coburn et al., 1991). Transfer of DNA from agarose gels to nylon membranes (Gene Screen Plus, NEN Life Science Products, Boston, MA) was carried out as described (Sambrook et al., 1989). Promastigote forms of L. major LV39 clonal line were harvested in the logarithmic and stationary phases of growth and total RNA was extracted using TRIzol (Gibco BRL, Grand Island, NY). Amastigotes were obtained from footpad lesions of infected BALB/c mice 30 days after an intradermal injection of 1.0!107 stationary phase promastigotes of L. major LV39 and total RNA was extracted using TRIzol (Antoniazi et al., 2000). Aliquots containing 10 mg of total RNA from both amastigote and promastigote forms of the parasite were equilibrated in formamide, formaldehyde and MOPS buffer (MOPS 0.02 M, sodium acetate 8.0 mM, EDTA 1.0 mM), denatured by heating the sample for 3 min at 95 8C and fractionated in a 1% agarose formaldehyde/MOPS gel. 2.4. Hybridisation experiments Gel-purified restriction fragments and PCR products were radiolabelled by random priming (Feinberg and Vogelstein, 1983) and used as probes in hybridisation experiments. Southern and northern blotting were conducted as described (Sambrook et al., 1989). Hybridisations were carried out overnight at 67 8C, following standard procedures (Sambrook et al., 1989). Membranes were washed twice in 2!SSPE (NaCl 0.3 mM, NaH2PO4 20 mM, EDTA 2 mM, pH 7.4), 0.5% SDS at 67 8C and once in 0.1!SSPE, 0.1% SDS at room temperature, each time for 15 min (Cruz and Beverley, 1990) and exposed to a Kodak Diagnostic Film. 2.5. Pulsed field gel electrophoresis Agarose-embedded chromosomes were separated in 1.0% agarose gels using a Bio-Rad CHEF-DR II apparatus. Chromosomes smaller than 1 Mb were resolved under the following conditions: 4.5 V/cm, ramping from 50 to 120 s for 48 h at 14 8C. Southern blotting, hybridisation and washing under high stringency conditions were conducted as previously described (Cruz and Beverley, 1990). A.L. Pedrosa et al. / International Journal for Parasitology 36 (2006) 211–217 213 Fig. 1. Isolation of LST-R533. (A) Hind III fragments of cosmid E8 were fractionated in a 0.8% agarose gel and stained with ethidium bromide (1) and the corresponding blot was probed with the E8-HH1.1 restriction fragment (2). (B) Southern hybridisation of Hind III (1) and Hind III/Bam HI (2) digested Leishmania major LV39 genomic DNA using the fragment E8-HH1.1 as a probe. The molecular marker used was Hind III-digested lambda DNA. Fragment lengths are shown on the left side of panels A and B (in kb). Arrowheads indicate the E8-HH1.1 restriction fragment. (C) Northern hybridisation of total RNA from LV39 promastigotes in the initial and middle logarithmic phases (1 and 2, respectively), in the stationary phase (3) and from lesion amastigotes (4) hybridised with the LST-R533 amplicon (see Fig. 2). (D) Control hybridisation of 0.8 kb Xho I fragment of DHFRTS from L. major. (E) Ethidium bromide stained gel showing ribosomal RNA bands. The molecular marker is the 0.1–2.0 kb RNA ladder (Invitrogen). (E) Schematic representation of one extremity (50 kb) of chromosome 20 from L. major. Open boxes represent the four copies of LST-R533. The filled box stands for the chromosomal extremity. The magnified Hind III sites represent restriction fragment E8-HH1.1. Horizontal lines represent the fragments recognised by the E8-HH1.1 probe in A (numbers indicate the fragment sizes, in kb). H, Hind III; B, Bam HI; Tel, telomeric repeat. 2.6. PCR amplification Leishmania DNA embedded in agarose blocks was prepared for PCR after three successive washes in TE (Tris–HCl 10.0 mM, pH 7.4 and EDTA 1.0 mM), followed by a heating step in TE (440 mL) at 80 8C for 5 min to melt the agarose. Aliquots of 5 mL, containing approximately 100 pg of genomic DNA, were used as templates in a 50 ml-reaction containing 1!PCR buffer (Perkin–Elmer, Foster City, CA), 200 mM dNTPs, 1.0 mM of each primer and 0.5 units of Taq DNA polymerase (Perkin–Elmer). Reactions were performed using a first 3-min step of denaturation at 95 8C, followed by 30 cycles of 45 s at 95 8C, 40 s at 55 8C, 55 s at 72 8C and a final extension step of 10 min at 72 8C. The primers used were LSTR1 (5 0 -GCCCCGCTATCCCTCTGCTGACG-3 0 ) and LSTR2 (5 0 -GCCTCGCAGACGCTCCCATTGT-3 0 ). PCR products were visualised in a 1.2% agarose gel with ethidium bromide staining. 2.7. Cloning Cosmid E8 was digested with Hind III and the 1.1 kb fragment was cloned in pUC19. PCR products were purified using the PEG method (Zhen and Swank, 1993) and cloned in pGEM using the pGEMR-T Easy Vector kit (Promega Biosciences Inc.). Ligation products were transformed by electroporation in Escherichia coli DH10B and selected in 1.5% agar-LB medium containing 50 mg/ml ampicillin. III-digested cosmid E8 and L. major digested genomic DNA revealed the expected fragments of 1.1, 4.3 and 5.4 kb (Fig. 1A and B). Strong hybridisation signals were observed at the high molecular weight range of Hind III-digested genomic DNA (Fig. 1B). The shorter Hind III band (w20 kb) originates the w4.7 kb band seen the Hind III/Bam HI digestion. The higher molecular weight signal observed in the Hind III digested genomic DNA is probably due to the presence of several long restriction fragments with low sequence identity to the probe, since a corresponding strong signal is not observed in the Hind III/Bam HI digestion (Fig. 1B, lane 2). Additionally, in the genomic DNA fainter signals were observed in several fragments of variable sizes. The available genomic sequence of L. major Friedlin was searched with the consensus sequence of the three copies of the element (Ivens et al., 2005). The fourth copy of the element was identified in the same extremity of chromosome 20. The restriction map obtained from the available sequence is consistent with the Southern blot results (Fig. 1B and E). Sequences obtained from L. major lineages LV39 and Friedlin were, on average, 99% identical (Fig. 5 and data not shown). The sequences were previously named LSTR378 (Leishmania Sub-Telomeric Reiterated 378 bp element (Pedrosa et al., 2001). We have renamed them LST-R533 (the number corresponds to the size of the longest repeat) and numbers 1–4 have been added to indicate their position in the chromosomal end (Fig. 1E). 3.2. Sequence analysis and annotation of LST-R533 3. Results 3.1. Isolation of LST-R533 The hybridisation of the 1.1 kb Hind III restriction fragment bearing one copy of LST-R533 to blots containing the Hind Sequence analysis of LST-R533-1, -2, -3 and -4 elements demonstrated that they are direct repeats in the L. major genome and polymorphic in size (486, 533, 506 and 367 bp long, respectively). The core consensus sequence is 322 nucleotides long (Fig. 2) and the mean GC content of 214 A.L. Pedrosa et al. / International Journal for Parasitology 36 (2006) 211–217 Fig. 2. Multiple sequence alignment of the four copies of LST-R533 found in one extremity of chromosome 20 of Leishmania major Friedlin. Nucleotides used as the annealing sites for primers LSTR1 (reverse) and LSTR2 (forward) are underlined. Nucleotides in bold represent the 81-nt conserved element. the repeat is 68.5% (range 68–70%). Annotation of LST-R533 in the complete genomic sequence of L. major Friedlin confirmed that LST-R533-1 is found juxtaposed to the typical telomeric and subtelomeric repeats of Leishmania described previously (Fu and Barker, 1998). LST-R533K2 to -4 are found at approximate distances of 10.6, 25.6 and 37.4 kb from the chromosomal extremity (Fig. 1E). Moreover, LST-R533-2, -3 and -4 are located in intergenic regions of 1.4, 1.8 and 2.4 kb, respectively. All annotated open reading frames (ORFs) flanking the four copies of LST-R533 are present in L. major Friedlin genome as single copy sequences. 3.3. Genomic distribution of LST-R533 A BLAST search of LST-R533-2 against the L. major Friedlin genome revealed an internal region of 81 bp from LSTR533 particularly abundant in the parasite’s genome (Fig. 2, bold-faced nucleotides). A subsequent BLAST search using the 81 bp element uncovered the presence of sequences ranging from 14 to 81 bp and with a minimum of 95% identity in 308 different intergenic regions of all chromosomes of the parasite. Short segments of sequences within the 81-nt element presented significant identity with retrotransposons described in several organisms (Table 1) (Jurka and Kapitonov, 1999; Kapitonov and Jurka, 1999, 2001). These segments coincide with the regions of the highest sequence conservation within the L. major genome. The LSTR-533 PCR-amplified fragment (Fig. 2, region encompassing the underlined nucleotides) was used as a probe in a northern blot containing total RNA from promastigotes and amastigotes of L. major LV39 and the presence of two transcripts was detected (Fig. 1C). The hybridisation of a probe representing the dihydrofolate reductase thymidylate synthase gene (DHFRTS) and the ethidium bromide stained bands of ribosomal RNA were used as controls for the detection of a transcript from a single copy gene and for RNA loading and integrity, respectively (Fig. 1D and E). No portions of LSTR533 were found within annotated coding regions of the L. major genome. Moreover, we have not found any sequences with significant identity to both LST-R533 and the 81bp element in T. brucei or T. cruzi genome databases. 3.4. Distribution of LST-R533 in Leishmania species A labelled amplicon representing LST-R533 recognised major chromosomal bands compatible with the approximate Table 1 Transposable elements presenting sequence identity with the 81 bp-element from Leishmania major Transposable element (length in bp) Organism Identity (%) Coordinates (within the 81 bp element) GenBank accession number/ Reference Dr000883 (842) Retrosor1_I (702) Sz-17 (3386) L1M2B_5 (3252) Helitronya1A_CE (3084) Paltra3_CE (360) Vandal1 (15,093) Zebrafish Sorghum bicolour Oryza sativa Homo sapiens Caenorhabiditis elegans Caenorhabiditis elegans Arabidopsis thaliana 100 100 100 100 100 100 100 4–17 8–21 13–26 7–19 67–79 68–80 38–96 AL596027 AF098806 AF111709 Jurka and Kapitonov, 1999 Kapitonov and Jurka, 2001 Kapitonov and Jurka, 1999 Kapitonov and Jurka, 1999 A.L. Pedrosa et al. / International Journal for Parasitology 36 (2006) 211–217 215 Fig. 3. Distribution of LST-R533 in the genome of Leishmania. (A) PFGE-separated chromosomes of different species of Leishmania: 1, Leishmania major CC1; 2, L. major Friedlin; 3, Leishmania braziliensis 2904; 4, L. braziliensis CE3227; 5, Leishmania amazonensis; 6, Leishmania mexicana; 7, Leishmania hoogstraali; 8, Leishmania tarentolae; 9, Leishmania donovani; 10, Molecular marker: lambda DNA concatamers. (B) Hybridisation of the probe LST-R533 to the Southern of the gel shown in A. C: compression zone. length of chromosome 20 (760 kb) in all species analysed and a faint signal for species from the subgenus Sauroleishmania (Fig. 3A and B). The probe also recognised fainter signals in other chromosomal bands in Leishmania strains from subgenera Leishmania, Viannia and Sauroleishmania (Fig. 3B). The same amplicon was probed to genomic DNA from L. major digested with Xho I and Xba I. Two to four major restriction fragments and several other fainter signals were recognised in all lineages tested were observed (Fig. 4A). In agreement with pulse field gel electrophoresis (PFGE) results, only faint signals were detected in lanes corresponding to species from subgenus Sauroleishmania Fig. 4A and C. We cloned and sequenced PCR products obtained with primers LSTR1 and LSTR2 from different species of Leishmania spp. in order to investigate cross species conservation of LST-R533. Amplified fragments were polymorphic in size and multiple sequence alignment (ClustalW) of the Leishmania sequences obtained indicated the conservation of the region among the different species tested (Fig. 5). Furthermore, BLAST search revealed that the 81 bp sequence presents 83% identity with a sequence in the locus containing a P-type ATPase of L. donovani, which includes the LdH1A and LdH1B genes. The LdH1A sequence was used in order to localise the homologous gene in the L. major Friedlin genome, which bears two copies of the homologous genes, both annotated as H1A and located at the 53 kb extreme of chromosome 18. Two copies of internal regions of LSTR533 were found downstream of the second H1A gene in this chromosome, the first at 2.9 kb (C strand) from the stop codon and the second at 14.8 kb (K strand). 4. Discussion Here we describe the characterisation of LST-R533, a polymorphic repeated element found in inter-ORF regions of one extremity of L. major chromosome 20. LST-R533-2 is the longest copy of the element and LST-R533-1 is truncated at the 5 0 -end, whereas LST-R533-3 and K4 are truncated at the 3 0 end. The expression profiles of genes located at this chromosomal extremity in promastigote forms of the parasite have been previously investigated and differences in the expression of genes along the chromosomal extremity studied were not detected (Pedrosa et al., 2001). Short elements (14–81 nucleotides long) present within the LST-R533 are dispersed Fig. 4. (A) Hybridisation of the probe LST-R533 to the Xho I (X) and Xba I (Xb) digested genomic DNA from the species shown in Fig. 3. (B) Control hybridisation with a 0.8 kb Xho I fragment of DHFRTS from Leishmania major showing complete digestion of DNA samples. (C) Agarose gel of the digested genomic DNA stained by ethidium bromide as a control for DNA loading. Molecular markers used are fragments of Hind III-digested lambda DNA. 216 A.L. Pedrosa et al. / International Journal for Parasitology 36 (2006) 211–217 Fig. 5. Sequence alignment of the LST-R533 amplified from Leishmania spp. genomic DNA. Multiple sequence alignment of the 81-nucleotide element from different species of Leishmania: Lm_Fried, Leishmania major Friedlin; Lm_LV39, L. major LV39; Ld_HU3, Leishmania donovani HU3; La_LV79, Leishmania amazonensis LV79; Lb_2904, Leishmania braziliensis 2904; Lh_NG26, Leishmania hoogstraali. Nucleotides in bold represent the 81-nt conserved element. and conserved in 308 inter-ORF regions of virtually all L. major chromosomes. Such sequence conservation within noncoding regions is compatible with selective pressure and suggests a functional significance of this short segment for the parasite. Sequence analysis revealed the presence of two internal regions of the 81 bp element that presented high sequence identity with retrotransposons isolated from several organisms (Jurka and Kapitonov, 1999; Kapitonov and Jurka, 1999, 2001). However, we have not found an intact copy of such element that could be responsible for its mobilisation in the Leishmania genome. In fact, the expression of a retroposonlike element in a region spanning the putative promoter from two variant surface glycoprotein gene expression sites of T. brucei has already been demonstrated (Lodes et al., 1993). The detection of L. major transcripts recognised by the LSTR533 probe, associated with the fact that portions of the repeat were only found in inter-ORF regions, indicates that the portions of transposable elements described here may be part of untranslated regions (UTRs) of some transcripts of the parasite. Sequence data, PFGE hybridisation and restriction fragment length polymorphism analysis are consistent and allow us to speculate that different Leishmania species whose complete genomic sequences are not yet available have a similar genomic organisation. The observation of strong hybridisation of the LST-R533 probe to a single chromosomal band in the PFGE and the fainter signals in other chromosomal bands of all pathogenic species, associated with L. major genome data, support the hypothesis that short segments within LST-R533 are transposon relics dispersed throughout the genome of these parasites. The faint hybridisation signals in PFGE of the subgenus Sauroleishmania and the conservation of the 81 bp element sequence are consistent with the lower number of copies of this small region in this species. Preliminary comparative analysis between the genomes of different species of Leishmania revealed a low conservation of the non-coding regions (Laurentino et al., 2004). Moreover, a bias of GC content between coding and non-coding regions is a constant (Ivens et al., 2005; Laurentino et al., 2004). On the other hand, LST-R533 sequence analysis revealed a GC content of w68.5%, a considerably high value for an intergenic region in L. major, whose mean GC content is approximately 57% (Ivens et al., 2005). Furthermore, the 81 bp conserved element were found to be distributed throughout the L. major genome and was present in all the human pathogenic strains of Leishmania from New and Old World species tested. The cross-species conservation of the 81 bp element in non-coding regions is intriguing and allows us to speculate that a formerly active transposable element became a sequence of functional relevance for the parasite. This is not the first report indicating the presence of retrotransposon elements in trypanosomatids and it adds to previous data published by Ghedin et al. (2004) demonstrating the occurrence of a retroelement-like sequence in L. major genome. That element, named LmChr1-DIRE, is present in one extremity of L. major Friedlin chromosome 1. Our observation of two conserved blocks of sequence resembling an inactive retrotransposon dispersed in the genome of Leishmania is novel and suggests that the dispersion of this sequence in the genus Leishmania occurred before separation of Leishmania and Viannia subgenera. Acknowledgements We thank Tânia Paula de Aquino Defina and Viviane Ambrósio Trombela for technical assistance. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 99/12403-3 (AKC). AMS and JCR are supported by fellowships from FAPESP. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Antoniazi, S., Lima, H.C., Cruz, A.K., 2000. Overexpression of miniexon gene decreases virulence of Leishmania major in BALB/c mice in vivo. Mol. Biochem. Parasitol. 107, 57–69. Beverley, S.M., 1991. Gene amplification in Leishmania. Annu. Rev. Microbiol. 45, 417–444. A.L. Pedrosa et al. / International Journal for Parasitology 36 (2006) 211–217 Beverley, S.M., Coderre, J.A., Santi, D.V., Schimke, R.T., 1984. Unstable DNA amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization. Cell 38, 431–439. Borst, P., Rudenko, G., 1994. Antigenic variation in African trypanosomes. Science 264, 1872–1873. Coburn, C.M., Otteman, K.M., McNeely, T., Turco, S.J., Beverley, S.M., 1991. Stable DNA transfection of a wide range of trypanosomatids. Mol. Biochem. Parasitol. 46, 169–179. Cruz, A., Beverley, S.M., 1990. Gene replacement in parasitic protozoa. Nature 348, 171–173. Cruz, A.K., Titus, R., Beverley, S.M., 1993. Plasticity in chromosome number and testing of essential genes in Leishmania by targeting. Proc. Natl Acad. Sci. USA 90, 1599–1603. Feinberg, A.P., Vogelstein, B., 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13. Floeter-Winter, L.M., Shaw, J.J., 2001. New horizons in the identification and taxonomy of the Leishmania and the diagnosis of leishmaniasis: the expansion of molecular techniques. Res. Adv. Microbiol. 4, 63–79. Fu, G., Barker, D.C., 1998. Characterisation of Leishmania telomeres reveals unusual telomeric repeats and conserved telomere-associated sequence. Nucleic Acids Res. 26, 2161–2167. Ghedin, E., Bringaud, F., Peterson, J., Myler, P., Berriman, M., Ivens, A., Andersson, B., Bontempi, E., Eisen, J., Angiuoli, S., Wanless, D., Von Arx, A., Murphy, L., Lennard, N., Salzberg, S., Adams, M.D., White, O., Hall, N., Stuart, K., Fraser, C.M., El-Sayed, N.M., 2004. Gene synteny and evolution of genome architecture in trypanosomatids. Mol. Biochem. Parasitol. 134, 183–191. Iovannisci, D.M., Beverley, S.M., 1989. Structural alterations of chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of the mini-exon array. Mol. Biochem. Parasitol. 34, 177– 188. Ivens, A.C., Peacock, C.S., Worthey, E.A., Murphy, L., Aggarwal, G., Berriman, M., Sisk, E., Rajandream, M.A., Adlem, E., Aert, R., Anupama, A., Apostolou, Z., Attipoe, P., Bason, N., Bauser, C., Beck, A., Beverley, S.M., Bianchettin, G., Borzym, K., Bothe, G., Bruschi, C.V., Collins, M., Cadag, E., Ciarloni, L., Clayton, C., Coulson, R.M., Cronin, A., Cruz, A.K., Davies, R.M., De Gaudenzi, J., Dobson, D.E., Duesterhoeft, A., Fazelina, G., Fosker, N., Frasch, A.C., Fraser, A., Fuchs, M., Gabel, C., Goble, A., Goffeau, A., Harris, D., Hertz-Fowler, C., Hilbert, H., Horn, D., Huang, Y., Klages, S., Knights, A., Kube, M., Larke, N., Litvin, L., Lord, A., Louie, T., Marra, M., Masuy, D., Matthews, K., Michaeli, S., Mottram, J.C., MullerAuer, S., Munden, H., Nelson, S., Norbertczak, H., Oliver, K., O’Neil, S., Pentony, M., Pohl, T.M., Price, C., Purnelle, B., Quail, M.A., Rabbinowitsch, E., Reinhardt, R., Rieger, M., Rinta, J., Robben, J., Robertson, L., Ruiz, J.C., Rutter, S., Saunders, D., Schafer, M., Schein, J., Schwartz, D.C., Seeger, K., Seyler, A., Sharp, S., Shin, H., Sivam, D., Squares, R., Squares, S., Tosato, V., Vogt, C., Volckaert, G., Wambutt, R., Warren, T., Wedler, H., Woodward, J., Zhou, S., Zimmermann, W., Smith, D.F., Blackwell, J.M., Stuart, K.D., Barrell, B., 2005. The genome of the kinetoplastid parasite, Leishmania major. Science 309, 436–442. Jurka, J., Kapitonov, V.V., 1999. Sectorial mutagenesis by transposable elements. Genetica 107, 239–248. Kapitonov, V.V., Jurka, J., 1999. Molecular paleontology of transposable elements from Arabidopsis thaliana. Genetica 107, 27–37. Kapitonov, V.V., Jurka, J., 2001. Rolling-circle transposons in eukaryotes. Proc. Natl Acad. Sci. USA 98, 8714–8719. 217 Kapler, G.M., Coburn, C.M., Beverley, S.M., 1990. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol. Cell. Biol. 10, 1084–1094. Laurentino, E.C., Ruiz, J.C., Fazelinia, G., Myler, P.J., Degrave, W., AlvesFerreira, M., Ribeiro, J.M., Cruz, A.K., 2004. A survey of Leishmania braziliensis genome by shotgun sequencing. Mol. Biochem. Parasitol. 137, 81–86. Lodes, M.J., Smiley, B.L., Stadnyk, A.W., Bennett, J.L., Myler, P.J., Stuart, K., 1993. Expression of a retroposon-like sequence upstream of the putative Trypanosoma brucei variant surface glycoprotein gene expression site promoter. Mol. Cell. Biol. 13, 7036–7044. Martinez-Calvillo, S., Stuart, K., Myler, P.J., 2005. Ploidy changes associated with disruption of two adjacent genes on Leishmania major chromosome 1. Int. J. Parasitol. 35, 419–429. Myler, P.J., Audleman, L., deVos, T., Hixson, G., Kiser, P., Lemley, C., Magness, C., Rickel, E., Sisk, E., Sunkin, S., Swartzell, S., Westlake, T., Bastien, P., Fu, G., Ivens, A., Stuart, K., 1999. Leishmania major friedlin chromosome 1 has an unusual distribution of protein-coding genes. Proc. Natl Acad. Sci. USA 96, 2902–2906. Naderer, T., Vince, J.E., McConville, M.J., 2004. Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host. Curr. Mol. Med. 4, 649–665. Pedrosa, A.L., Ruiz, J.C., Tosi, L.R., Cruz, A.K., 2001. Characterisation of three chromosomal ends of Leishmania major reveals transcriptional activity across arrays of reiterated and unique sequences. Mol. Biochem. Parasitol. 114, 71–80. Ravel, C., Wincker, P., Bastien, P., Blaineau, C., Pages, M., 1995. A polymorphic minisatellite sequence in the subtelomeric regions of chromosomes I and V in Leishmania infantum. Mol. Biochem. Parasitol. 74, 31–41. Ravel, C., Wincker, P., Blaineau, C., Britto, C., Bastien, P., Pages, M., 1996. Medium-range restriction maps of five chromosomes of Leishmania infantum and localization of size-variable regions. Genomics 35, 509–516. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Singh, A.K., Papadopoulou, B., Ouellette, M., 2001. Gene amplification in amphotericin B-resistant Leishmania tarentolae. Exp. Parasitol. 99, 141– 147. Spath, G.F., Garraway, L.A., Turco, S.J., Beverley, S.M., 2003. The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts. Proc. Natl Acad. Sci. USA 100, 9536–9541. Sunkin, S.M., Kiser, P., Myler, P.J., Stuart, K., 2000. The size difference between Leishmania major friedlin chromosome one homologues is localized to sub-telomeric repeats at one chromosomal end. Mol. Biochem. Parasitol. 109, 1–15. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Tosi, L.R., Casagrande, L., Beverley, S.M., Cruz, A.K., 1997. Physical mapping across the dihydrofolate reductase-thymidylate synthase chromosome of Leishmania major. Parasitology 114, 521–529. WHO. 1990. World Health Organization Expert Committee: Control of the Leishmaniasis. WHO Technical Report Series, p. 793. Wickstead, B., Ersfeld, K., Gull, K., 2003. Repetitive elements in genomes of parasitic protozoa. Microbiol. Mol. Biol. Rev. 67, 360–375. Zhen, L., Swank, R.T., 1993. A simple and high yield method for recovering DNA from agarose gels. Biotechniques 14, 894–898.