M. Navarro, G. Ortiz and M. Segovia
The protozoan parasite Leishmania is the causative
agent of visceral/cutaneous leishmaniosis, an important
public health problem in the Mediterranean countries,
Asia, Africa and Latin America (1). Moreover
Leishmania has emerged as an opportunistic pathogen of
HIV-infected humans. Since vaccines are not yet in sight,
the main line of defense now available against
Leishmania is chemotherapy. However, this parasite is
able to acquire drug resistance. Although different
mechanisms of drug resistance have been found (2), DNA
amplification is the most important of them (3). Thus
almost all the amplification events described so far in
Leishmania are accompanied by drug resistance (4). By
these means Leishmania obtains several copies of one or
more genes which contribute to the resistance to
methotrexate, arsenite, tunicamycin, vinblastine, etc. (3).
Table 1. Promastigote cultures with different drugs.
Mini
Day 1
Day 2
Day 3
Day 4
Day 7
Control
M_
75*
90
90
125
130
M+
50
75
75
80
90
Arsenite
M_
100
65
75
70
65
M+
70
75
90
100
55
Methotrexate
M_
90
70
75
60
65
M+
75
70
85
55
50
Tunicamycin
M_
75
100
110
100
110
M+
80
75
100
105
85
*Number indicate 105 cells/ml.
We can then conclude that the LD1 minichromosomes
of Leishmania appear in very short periods of time and
they are not implicated in resistance to drugs such as
methotrexate, arsenite and tunicamycin.
ACKNOWLEDGEMENTS
BIBLIOGRAPHY
Servicio de Microbiología, Hospital General Universitario, Murcia.
In our laboratory, differently cloned lines from the same
stock of L. major have been obtained (5). Some of them
showed minichromosomes of different sizes which were
absent in the original stock. These linear DNAs have been
shown to be DNA amplifications of the LD1 region
situated in the 1.9 megabase chromosome (6).
To test if these amplifications were implicated in drug
resistance, as most amplifications are in Leishmania, lines
either with or without minichromosomes were grown with
different drugs which usually kill Leishmania. Thus
promastigotes of a cloned line either containing the 230
kb minichromosome (CO1 M+) or having no
minichromosome (CO1 M_), were grown with: arsenite,
methotrexate, tunicamycin or in the absence of any drug.
At higher concentrations of the antibiotics (more than 100
mM) none of the lines was able to grow. At lower
concentrations (1 µM), the growth of the CO1 M_ line
(M_ in Table 1) was similar to that of CO1 M+ (M+ in
Table 1). Hence no differences between the lines with the
minichromosome and those without it were found. In
addition, the minichromosomes could not be forced to
appear in promastigotes lacking them by stepwise
increasing of methotrexate, arsenite or tunicamycin (data
not shown).
The apparent spontaneous appearance of these elements
in different lines of Leishmania led us to analyze the dy-
namics of the minichromosomes. For this purpose two
tubes previously cryopreserved from the CO1 M_ cloned
line were thawed, grown in RPMI medium and analyzed
by Pulsed-Field Gel Electrophoresis (5). While one of the
cultures had no minichromosome as the parental line did,
the other one showed a minichromosome of 210 kb only
forty days after thawing. These results reveal that the
minichromosomes develop in a very short period of time:
less than forty days. No previous DNA amplification in
Leishmania could emerge in such a short period of time.
For instance, amplification in drug resistance can be
forced to occur by a stepwise selection process of one year,
using increasing drug concentrations (7).
Part of this work was supported by the FIS grant 94/0728 from the
Spanish Government.
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chromo
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Overproduction of a bifunctional thymidilate synthetase-dihydrofolate reductase
and DNA amplification in methotrexate-resistant Leishmania tropica.
Proc Natl Acad Sci USA 1983; 80: 2132-2136.
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