From the 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
Molecular Pharmacology, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105
Received for publication, April 24, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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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.
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.
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 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 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
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 Plasmid Constructions
Plasmids were constructed using conventional cloning techniques
(15) and propagated using the E. coli strain TOP10F'
[mcrA 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
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).
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).
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
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 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.
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).
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ura3-52 his3
200 leu2
1
trp1
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).
-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).
-32P-labeled DNA probes. All post-hybridization washes
were performed to a final stringency of 0.1× SSC, 0.1% SDS at
42 °C.
70 °C until use. In
vitro transcription was performed for 10 min at 26 °C in the
presence of 100 µCi of [
-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.
(mrr-hsdRMS-mcrBC)
80
lacZ
M15
lacX74 recA1
deoR araD139
(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.
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
50% homology with the
carboxyl-terminal domain of other eukaryotic DNA topoisomerases I
(Fig 1).
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.
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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.
Quantification of relative transcription rates for various genes in
L. donovani promastigotes
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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
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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The abbreviation used is: PFGE, pulsed field gel electrophoresis.
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