FORMATION OF EXTRACHROMOSOMAL CIRCULAR AMPLICONS WITH DIRECT OR INVERTED DUPLICATIONS IN DRUG-RESISTANT LEISHMANIA-TARENTOLAE

Selection for methotrexate resistance in Leishmania spp. is often asso ciated with amplification of the H locus short-chain dehydrogenase-red uctase gene ptr1 as part of extrachromosomal elements. Extensive seque nces are always coamplified and often contain inverted duplications, m ost likely formed by the annealing of inverted repeats present at the H locus. By gene targeting mediated by homologous recombination, sever al repeated sequences were introduced in the vicinity of ptr1. Selecti on for methotrexate resistance in these transfectants led to ptr1 ampl ification as part of small circles with direct or inverted duplication s whether the integrated sequences consisted of direct or inverted rep eats. Hence, for a region to be amplified in L. tarentolae during drug selection, a drug resistance gene is required and must be flanked by (any) homologous repeated sequences. The distance between these repeat s and their orientation will determine the length of the amplicon and whether it contains direct or inverted duplications.