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

The protozoan parasite Leishmania resists the antifolate methotrexate (MTX) by amplifying the R locus dihydrofolate reductase-thymidylate synthase (dh fr-ts) gene, the H locus ptr1 pterin reductase gene, and finally by mutatio n in a common folate/MTX transporter. Amplification of dhfr-ts has never be en observed in Leishmania tarentolae MTX resistant mutants while ptr1 ampli fication is common. We have selected a L.tarentolae ptr1 null mutant for MT X resistance and observed dhfr-ts amplification in this mutant demonstratin g that once a preferred resistance mechanism is unavailable, a second one w ill 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 resi stance gene and the locus on the rate of gene amplification, Transfection s tudies indicated that ptr1 gave higher levels of MTX resistance than dhfr-t s. Consistent with this, when ptr1 was present as part of either the H locu s or the R locus it was invariably amplified, while dhfr-fs was only amplif ied when ptr1 was inactivated, When dhfr-fs was present in a ptr1 null back ground 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 determini ng the frequency of gene amplification in Leishmania.