A Novel Active DNA Topoisomerase I in Leishmania donovani*

Héctor VillaDagger §, Ana R. Otero MarcosDagger §, Rosa M. RegueraDagger , Rafael Balaña-FouceDagger , Carlos García-EstradaDagger , Yolanda Pérez-PertejoDagger , Babu L. Tekwani||, Peter J. Myler**, Kenneth D. Stuart**, Mary-Ann BjornstiDagger Dagger , and David OrdóñezDagger §§

From the Dagger  Departamento de Farmacología y Toxicología (INTOXCAL), Universidad de León, Campus de Vegazana s/n, 24071 León, Spain, the || National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, Mississippi 38677, the ** Seattle Biomedical Research Institute, Seattle, Washington 98195, and the Dagger Dagger  Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Received for publication, April 24, 2002, and in revised form, October 22, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA topoisomerases are ubiquitous enzymes that catalyze changes in DNA topology by altering the linkage of DNA strands, solving topological problems caused by cellular processes such as DNA replication, transcription, or recombination (1, 2). These enzymes are classified on the basis of the number of DNA strands that they cleave and the covalent bond formed in the enzyme-DNA intermediate. Unlike type II DNA topoisomerases, type I enzymes are ATP-independent, which transiently break a single strand of DNA. Type I DNA topoisomerases are classified into two subfamilies: type IA and type IB. The enzymes of type IA subfamily, including bacterial DNA topoisomerase I and III, eukaryotic DNA topoisomerase III, and reverse gyrase (3, 4), form a tyrosyl linkage with a 5'-phosphate group of one of the DNA strands generated due to the enzyme action (2), whereas the enzymes of type IB subfamily, including eukaryotic and vaccinia virus DNA topoisomerases I (5) and DNA topoisomerase V, establish the tyrosyl bond with the 3'-phosphate group (2). Type 1A topoisomerases relax only negatively supercoiled DNA with Mg2+ requirement, whereas type IB topoisomerases relax both negatively and positively supercoiled DNA even in the absence of a metallic cofactor, although Mg2+ and Ca2+ stimulate the relaxation activity (6, 7).

Type IB DNA topoisomerases are monomeric enzymes, constituted by four domains (8, 9). The nonconserved amino-terminal domain contains putative signals for nuclear localization of the enzyme. The largest domain, the core, is essential for enzyme activity and shows high phylogenetic conservation, particularly in the residues closely interacting with DNA. The third domain is known as the linker, which is poorly conserved and highly variable in length and is not essential for the enzyme activity. Finally, the carboxyl-terminal domain is highly conserved and crucial for the catalytic activity. This domain contains a tyrosine residue (Tyr723 in the human topoisomerase I), which interacts with one of the DNA strands, creating a transient covalent phosphodiester bond between the enzyme and the DNA.

A type I DNA topoisomerase has been purified and characterized from Leishmania donovani promastigotes, the causative agent for visceral leishmaniasis (10). Topoisomerases have been shown as the promising targets for new drug development against leishmaniasis (11). A DNA topoisomerase IB-like gene (LdTOP1A), which encodes for a protein lacking the conventional active site "SKXXY," motif has been characterized in L. donovani. However, heterologous expression of LdTOP1A gene in Escherichia coli produced an inactive protein (12).

The present paper describes the molecular cloning and functional expression of a novel DNA topoisomerase I from L. donovani. Unlike type I DNA topoisomerases from several other organisms, the leishmanial enzyme is encoded by two different genes (LdTOP1A and LdTOP1B) located at two different chromosomes, and the polypeptide encoded by LdTOP1B gene contains the conserved SKXXY motif required for activity. This, to our knowledge, is the first report in which two different genes code for an active DNA topoisomerase I.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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Materials

Media and reagents were purchased from Sigma. Primers were purchased from Amersham Biosciences.

Leishmania and Yeast Strains

For protein expression Saccharomyces cerevisiae strain EKY3 [MAT alpha  ura3-52 his3Delta 200 leu2 Delta 1 trp1 Delta 63 top1 D::TRP1], deficient in DNA topoisomerase I activity, was used. L. donovani strain LSB-51.1 (MHOM/S.D./00/Khartoum) was maintained as promastigotes at 26 °C in Schneider's insect medium containing 10% fetal bovine serum (Invitrogen), penicillin (50 units/ml), and streptomycin (50 µg/ml).

Cloning of DNA Topoisomerase I

To generate a DNA probe for isolation of the L. donovani LdTOP1A gene (GenBankTM accession number AF303577), 100 pmol of two degenerated primers based on sequence homology alignments 5'-GAT/CACGATCGTCGGT/CTGCTG-3'(sense), corresponding to amino acid residues DTVGCC, and 5'-GTAG/CGTA/GCGGAACACCTT-3'(antisense), coding to the conserved sequence KVFRTY, were added to a reaction mixture containing 100 ng of Leishmania genomic DNA, 200 µM concentration each of dNTP, and 2.5 units of Taq DNA polymerase (Stratagene). A single 264-bp PCR product was obtained and subcloned into pGEM-T vector (Promega). A random labeled probe (Random Primed DNA labeling kit, Roche Molecular Biochemicals) was prepared using this 264-bp PCR product as template to screen a L. donovani lambda -EMBL3 genomic library (a gift from Dr. Meade, University of Mississippi Medical Center, Jackson, MS) (13). 5 × 104 plaque-forming units were plated and screened by the PCR probe. One positive bacteriophage clone was isolated, which was further purified through tertiary screening, and sequenced on both strands. Sequence analysis was performed by DNAstar, whereas comparisons with other genes of the data base were performed using the search algorithm BLAST (14).

Nucleic Acid Isolation, Pulsed Field Gel Electrophoresis (PFGE),1 and Hybridization Analysis

Genomic DNA was isolated from 2 × 109 L. donovani promastigotes by standard procedures (15). Total RNA was isolated from 109 cells using an RNA isolation kit (Qiagen). Plasmid DNAs were isolated by the alkaline lysis procedure. Chromosomal localization of the genes was conducted by PFGE. Briefly, L. donovani LSB-51.1 (MHOM/S.D./00/Khartoum) promastigotes were harvested by centrifugation, washed twice in phosphate-buffered saline, resuspended in phosphate-buffered saline, and mixed with 2% agarose at the ratio of 1:1 (v/v). Processing of the samples was made at 50 °C for 48 h in 10 ml of 0.5 M EDTA, pH 8.0, 1% Sarkosyl, and 150 µl of 2 mg/ml fresh-made proteinase K. Separation of the chromosomal bands was achieved at 14 °C in 1% agarose gels with 0.5 × TBE running buffer (45 mM Tris borate), using a clamped homogeneous electrical field electrophoresis (CHEF, Bio-Rad) with a 35-120-s ramping pulse at 6 V/cm for 24 h. S. cerevisiae chromosomes were used as molecular weight markers. After staining with ethidium bromide, gels were blotted onto nylon membranes (Sigma) by alkaline transfer. DNA, RNA, and chromosomal blots were hybridized with the randomly primed alpha -32P-labeled DNA probes. All post-hybridization washes were performed to a final stringency of 0.1× SSC, 0.1% SDS at 42 °C.

Nuclear Run-on Assay

Transcription in isolated nuclei was achieved as described previously by Quijada et al. (16). Briefly, logarithmic phase promastigotes were harvested by centrifugation and suspended in ice-cold hypotonic buffer. Cells were lysed by vortexing in the presence of Nonidet P-40 and Triton X-100. Immediately nuclei washing buffer was added, and the nuclei were pelleted (3000 × g), washed, and stored at -70 °C until use. In vitro transcription was performed for 10 min at 26 °C in the presence of 100 µCi of [alpha -32P]UTP (3000 Ci/mmol) (Amersham Biosciences). The reaction was stopped, and the radiolabeled nascent RNA was extracted by phenol:chloroform. Non-incorporated isotopes were removed on ProbeQuant G-50 microcolumns (Amersham Biosciences). 3 µg of each plasmid, to be probed with the nascent RNA, was linearized, denatured, and transferred to a positively charged nylon membrane. The membrane was then subjected to hybridization with the purified labeled RNA.

Plasmid Constructions

Plasmids were constructed using conventional cloning techniques (15) and propagated using the E. coli strain TOP10F' [mcrA Delta (mrr-hsdRMS-mcrBC) phi 80Delta lacZDelta M15 Delta lacX74 recA1 deoR araD139 Delta (ara-leu) 7697 galU galK rpsL endA1 nupG (F': lacYq Tn10 TetR)] (Invitrogen). The sequences were verified by dideoxy sequencing along both the critical junction sequence sites.

YCpGAL1-LdTOP1A-URA Construction-- The 1.9-kb LdTOP1A gene was amplified from L. donovani genomic DNA, using a sense primer with a flanking BamHI site and a RGS(His)6 tag: 5'-GCGGATCCGACATGAGAGGATCGCACCACCACCACCACCACAAGGTGGAGAATAGCAAGATGGGGGTGAAG-3' and an antisense primer with a flanking XbaI site: 5'-CCTCTAGAGGACTCCGACACCTACAGACGAACAGAGTCACTCG-3', which correspond, respectively, to amino-terminal and carboxyl-terminal ends of the LdTOP1A gene. Restriction sites are underlined, and the start codon for DNA topoisomerase I is indicated in bold. The amplified fragment was cloned into the BamHI-SpeI site of the YCpGAL1-URA vector (a gift from Dr. J. C. Wang, Harvard University, Cambridge, MA). The resultant construct, YCpGAL1-LdTOP1A-URA contained the LdTOP1A gene driven by the galactose-inducible GAL1 promoter.

YCpGAL1-LdTOP1B-URA Construction-- A 838-bp BamHI-ClaI fragment from pSK-LdTOP1B was subloned into the YCpGAL1-URA vector. This construct contained the LdTOP1B gene also driven by GAL1 promoter.

pESC LdTOP1A-LdTOP1B-URA Construction-- In a two-fragment ligation reaction, the construct was created by insertion of LdTOP1A previously cut with BamHI-XhoI from YCpGAL1-LdTOP1A-URA and LdTOP1B cut with NotI-SpeI from YCpGAL1-LdTOP1B-URA. The resultant construct encodes the full-length L. donovani DNA topoisomerase I driven by the GAL1 and GAL10 promoters.

YCpGAL1-hTOP-URA described previously (17) was used for expression of human topoisomerase I. pUC18-rDNA, used in the nuclear run on assay, was kindly provided by Dr. Requena (Centro de Biología Molecular, Severo Ochoa UAM, Madrid, Spain).

pGEM3Z-LdTOP1A and pGEM3Z-LdTOP1B were constructed by insertion of LdTOP1A and LdTOP1B genes in the BamHI and HindIII restriction sites of pGEM3Zf(+) vector (Promega).

Protein Expression

S. cerevisiae strain EKY3 was transformed with different constructs, viz. YCpGAL1-LdTOP1A-URA, YCpGAL1-LdTOP1B-URA, or pESC LdTOP1A-LdTOP1B-URA, carrying the URA3 selectable marker, by treatment with lithium acetate (18-20). Transformants were selected on synthetic complete-uracil medium. At least four independent clones were selected from each transformation. After 6-h induction with 2% galactose in synthetic complete ura-raffinose medium, the cells were harvested by centrifugation, washed, and resuspended at the ratio of 2 g of wet cells/2 ml of TEEG buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 10% glycerol) supplemented with or without 0.2 M KCl and a mixture of protease inhibitors (1 × sodium fluoride, 1 × sodium bisulfite, 2 × Complete Mini® (Roche Molecular Biochemicals)). Cell extracts were prepared by disruption with acid-washed glass beads according to a procedure previously described (21, 22). Briefly, cells were subjected to one freeze/thaw cycle at -80 °C, lysed by vortexing with 425-600 µm glass beads, and the extracts cleared by centrifugation at 15,000 × g for 30 min at 4 °C.

In Vitro Relaxation Assay

DNA topoisomerase I activity was assayed by the relaxation of negatively supercoiled plasmid DNA. DNA topoisomerase I proteins were incubated in a 20-µl reaction volume containing 0.3 µg of pHC624 DNA (2015 bp, plasmid substrate), 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol, 10 mM EDTA, 50 mg/ml gelatin, 150 mM KCl. Human (23) and Leishmania enzyme activities were assayed for 30 min at 37 °C. Reactions were terminated by the addition of 1% SDS, and the extent of plasmid DNA relaxation was assessed by electrophoresis in a 1% agarose gel in 0.1 M Tris borate buffer, pH 8.0, at 5 V/cm for 4 h. The gels were visualized under UV illumination after staining with ethidium bromide and photographed (24).

    RESULTS
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INTRODUCTION
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Sequence Analysis and Genomic Organization-- A single open reading frame consisting of 1908 bp was isolated (LdTOP1A gene, GenBankTM accession number AF303577) showing a 50% identity to Homo sapiens DNA topoisomerase I sequence (GenBankTM accession number J03250). The open reading frame encoded for putative polypeptide of 636 amino acids, with a predicted molecular mass of 73 kDa, which is slightly smaller than human (765 amino acids) and S. cerevisiae (769 amino acids) enzymes. The conserved core domain is present, whereas the carboxyl-terminal domain, which contains the active site, is absent in this gene (Fig. 1).


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Fig. 1.   Alignment of LdTOP1B gene product with other eukaryotic type I DNA topoisomerases. The sequence was translated to protein and aligned with other DNA topoisomerase I proteins from different organisms, including the L. donovani topoisomerase I-like gene reported previously (11). GeneBankTM accession numbers are as follows: LdTOP1B gene, AY062908; LdTOP1A gene, AF145121; Plasmodium falciparum, X83758; S. cerevisiae, K03007; H. sapiens, J03250.

The presence of a tyrosine residue has been described at the enzyme catalytic site of all DNA topoisomerases I characterized so far, except in Leishmania. To search for a new gene encoding a DNA topoisomerase I active site, PCR was performed using specific oligonucleotides, whose sequence was based on Leishmania Genome Sequencing Project (GenBankTM accession number AL389894) (which operates with Leishmania major Friendlin strain) (25). The sense primer sequence was 5'-CGTGAAAGGCAAGTCTGAGG-3', and the antisense primer was 5'-AGGCGGCATGTGAATTAAAG-3'. Genomic DNA from L. donovani LSB-51.1 (MHOM/S.D./00/Khartoum) was used as a template. A single 826-bp PCR product was obtained, cloned, and sequenced, revealing a 95% identity with the L. major sequence (LdTOP1B GenBankTM accession number AY062908). This fragment contained the highly conserved SKXXY motif with the tyrosine as the active site residue, and the sequence alignment analysis displayed approx 50% homology with the carboxyl-terminal domain of other eukaryotic DNA topoisomerases I (Fig 1).

To determine the LdTOP1A gene copy number, Southern blot studies were performed as described under "Experimental Procedures" using the 264-bp PCR as a probe. A single band was obtained (Fig. 2A), revealing that it is a single copy gene confirming the earlier results (12). The same experiment was performed for the second gene (LdTOP1B), using the 826-bp PCR fragment described above. Fig. 2B showed a single hybridizing band, thus suggesting that this gene is also present as a single copy in the Leishmania genome. Chromosomal location analysis revealed that LdTOP1B gene is placed at a single chromosomal band of approx 0.4 Mb. These data concur with the Leishmania Genome Sequencing Project findings, according to which the LdTOP1B gene expressed sequence tag has been identified on chromosome 4 (0.46 Mb) in L. major (www.ebi.ac.uk/parasites/LGN/chromo4.html). These results show clearly that LdTOP1A and LdTOP1B genes are located on different chromosomes, since the LdTOP1A gene was located on a chromosomal band of 1.6-1.9 Mb (Fig. 2C).


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Fig. 2.   Genomic organization of LdTOP1A and LdTOP1B genes encoding DNA topoisomerase I in L. donovani. A, Southern analysis of L. donovani LdTOP1A gene (filter-probed with a 264-bp fragment). B, Southern analysis of L. donovani LdTOP1B gene (filter-probed with a 826-bp fragment). C, PFGE analysis of L. donovani indicating localization of LdTOP1A and LdTOP1B genes on different chromosomal bands.

Transcription Analysis-- Northern blot analysis was conducted to explore the possibility that the two genes may undergo post-transcriptional processing, which may lead to sharing of a common mRNA. A single hybridizing band of ~2.3 kb was observed with the LdTOP1B gene probe, which differs from the ~3.6-kb mRNA band observed for LdTOP1A gene (see Fig. 3A). To test whether these two mRNAs are transcribed at a similar rate, nuclear run-on experiments were conducted using nuclei isolated from logarithmic phase promastigotes. The rate of transcription of each gene was determined relative to the rate of rDNA transcription. Genes whose relative rates of transcription were measured include the LdTOP1A, LdTOP1B, and rDNA genes, pGEM-3Zf(+) and pUC-18 plasmids (3 µg of double-stranded, linearized, and denatured plasmids). The results of Fig. 3B show the transcription of LdTOP1A and LdTOP1B genes relative to the rDNA transcription. The results of three independent experiments are shown in Table I. The hybridization signal to the rDNA gene was arbitrary chosen as 1.0, and the other signals were reported relative to that value. Despite the fact that LdTOP1A and LdTOP1B genes are located at different genomic clusters, their transcription rates, quantified in a phosphorimager, were similar ~10-13-fold lower with respect to the rDNA signal.


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Fig. 3.   Expression analysis of LdTOP1A and LdTOP1B genes encoding DNA topoisomerase I in L. donovani. A, Northern analysis of mRNA from L. donovani promastigotes (day 2 culture). Lane MWM, molecular weight markers; lane 1, total RNA; lane 2, filter-probed with a 264-bp fragment; lane 3, filter-probed with a 826-bp fragment. B, levels of nascent LdTOP1A and LdTOP1B transcripts in isolated nuclei of L. donovani by nuclear run-on assay.

                              
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Table I
Quantification of relative transcription rates for various genes in L. donovani promastigotes
Nuclear run-on transcripts from promastigotes were hybridized to plasmid DNAs immobilized on nylon filter, and the hybridization signals were quantified on a phosphorimager. Values are relative signal intensities normalized for the rDNA plasmid value (mean ± S.D. of three independent experiments).

DNA Topoisomerase I Activity-- As shown in Fig 4. DNA topoisomerase I activity was reconstituted using a deficient S. cerevisiae strain (EKY3, see "Experimental Procedures"). LdTOP1A and LdTOP1B genes were co-expressed together (Fig. 4A) in a pESC-URA vector, which contains the GAL1 and GAL10 yeast promoters in opposing orientation. Co-expression of the two genes cloned in this vector guarantees protein-protein interactions after induction in the yeast host strain. Nevertheless, when LdTOP1A (Fig. 4B) and LdTOP1B (Fig. 4C) genes were individually expressed (each one in a different experiment), the resulting proteins did not show topoisomerase activity. Expression of the human TOP 1 gene under similar conditions produced a functional protein, which catalyzed the plasmid relaxation activity in vitro (Fig. 4D).


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Fig. 4.   In vitro plasmid DNA relaxation assay. A, yeast extracts co-expressing LdTOP1A-LdTOP1B genes. B, yeast extracts overexpressing only LdTOP1A gene. C, yeast extracts overexpressing only LdTOP1B gene. D, yeast extracts overexpressing hTOP1 gene. Lanes a, b, c, and d are, respectively, serial dilutions of the yeast extracts (1/1, 1/10, 1/50, and 1/100), incubated with negatively supercoiled plasmid DNA in reaction buffer for 30 min at 37 °C as detailed under "Experimental Procedures." Reactions were terminated by the addition of SDS, and the products were resolved in agarose gels, followed by ethidium bromide staining. Supercoiled (Sc) and relaxed (R) plasmid DNA topoisomers are as indicated.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This paper describes the molecular cloning and characterization of a new gene (LdTOP1B) encoding the carboxyl-terminal domain of DNA topoisomerase I in L. donovani. The results suggest that two different proteins, codified by two different genes located on different chromosomes, were required to reconstitute a catalytically active DNA topoisomerase I in Leishmania. Similar intensities of the hybridization signals obtained with LdTOP1A and LdTOP1B genes in the nuclear run-on assays indicate that the abundance of nascent RNA transcripts derived from both genes was similar, and an interaction, probably at post-translational level, should occur to reconstitute an active DNA topoisomerase I.

A type I DNA topoisomerase of 67 kDa has been purified from L. donovani promastigotes nuclear extracts by Chakraborty et al. (10). Das et al. (25) have recently described that this enzyme harbors a serine in place of the usual catalytic tyrosine. In addition a theoretical protein model for L. donovani topoisomerase was presented, suggesting that the serine 553 acts as the reactive nucleophile for enzyme catalysis. However, it is difficult to understand how a serine residue can stand-in for phosphodiesterase activity, since some experiments in which active yeast DNA topoisomerase I was mutated at their active site (Tyr727) to Ser or Phe resulted in enzyme inactivation (23). On the other hand, a DNA topoisomerase I-like gene, lacking the sequence corresponding to a conventional active site motif, has been described in L. donovani. Heterologous expression of the LdTOP1A gene in E. coli resulted in production of a catalytically inactive protein (12).

In previous studies with human DNA topoisomerase I, Stewart et al. (26) were able to reconstitute the enzyme activity by mixing a 58-kDa recombinant core domain with a series of different recombinant carboxyl-terminal fragments, which bind tightly to the core domain forming 1:1 complex probably through non-covalent interactions. This model hypothesizes that the core and carboxyl-terminal domains of topoisomerase I are folded independently and then are simply associated with each other to form an active enzyme (26). A similar mechanism may be suggested for independent refolding of LdTOP1A and LdTOP1B gene products, resulting in reconstitution of an active topoisomerase I in L. donovani. The results therefore suggest the presence of a novel type of dimeric topoisomerase I in L. donovani. Understanding of distinct molecular characteristics of the leishmanial topoisomerase I and regulation of expression of the enzyme during parasite growth may be useful for development of selective inhibitors of leishmanial topoisomerase I as promising leishmanicidal agents.

    ACKNOWLEDGEMENTS

We thank William Colley and John Vance (St. Jude Children's Research Hospital, Memphis, TN), Santiago Martinez-Calvillo (Seattle Biomedical Research Institute, Seattle, WA), and José María Requena and his group (Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid, Spain) for their help in molecular techniques. We also thank Francisco Fierro (Universidad de León, León, Spain) and Iris Segura for technical support in PFGE and John Chris Meade (University of Mississippi Medical Center, Jackson, MS) for the Leishmania genomic library.

    FOOTNOTES

* This work was supported by Comisión Interministerial de Ciencia y Tecnología (Grants PM98/0036 and PB96/0159), Junta de Castilla y León (Grants LE05/01 and LE06/02), National Institutes of Health Grant CA 58755, and by ALSAC.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to the work.

Both authors awarded fellowships from the Ministerio de Ciencia y Tecnología, Spain.

§§ To whom correspondence and proofs should be sent: Dept. de Farmacología y Toxicología (INTOXCAL), Universidad de León, Campus de Vegazana s/n, 24071 León, Spain. Tel.: 34-987-291-257; Fax: 34-987-291-590; E-mail: dftrbf@unileon.es.

Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M203991200

    ABBREVIATIONS

The abbreviation used is: PFGE, pulsed field gel electrophoresis.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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