Role of the locus and of the resistance gene on gene amplification frequency in methotrexate resistant Leishmania tarentolae

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Abstract
The protozoan parasite Leishmania resists the anti-folate 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 LAarentolae 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 ptt1 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.

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