Nucleic Acids Research, Vol 27, Issue 18 3653-3659, Copyright © 1999 by Oxford University Press
C Kundig, E Leblanc, B Papadopoulou and M Ouellette
The protozoan parasite Leishmania resists the antifolate methotrexate (MTX)
by amplifying the R locus dihydrofolate reductase-thymidylate synthase (
dhfr-ts ) gene, the H locus ptr1 pterin reductase gene, and finally by
mutation in a common folate/MTX transporter. Amplification of dhfr-ts has
never been observed in Leishmania tarentolae MTX resistant mutants while
ptr1 amplification is common. We have selected a L.tarentolae ptr1 null
mutant for MTX resistance and observed dhfr-ts amplification in this mutant
demonstrating that once a preferred resistance mechanism is unavailable, a
second one will take over. By introducing the ptr1 gene at the R locus and
the dhfr-ts gene at the H locus by gene targeting, we investigated the role
of the resistance gene and the locus on the rate of gene amplification.
Transfection studies indicated that ptr1 gave higher levels of MTX
resistance than dhfr-ts. Consistent with this, when ptr1 was present as
part of either the H locus or the R locus it was invariably amplified,
while dhfr-ts was only amplified when ptr1 was inactivated. When dhfr-ts
was present in a ptr1 null background on both the H locus and the R locus,
amplification from the H locus was more frequent suggesting that both the
gene and the locus are determining the frequency of gene amplification in
Leishmania.
ARTICLES
Role of the locus and of the resistance gene on gene amplification frequency in methotrexate resistant Leishmania tarentolae
Centre de Recherche en Infectiologie du CHUL and Departement de Biologie Medicale, Division de Microbiologie, Faculte de Medecine, Universite Laval, Quebec, Canada.
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