Linear amplicons as precursors of amplified circles in methotrexate-resistant Leishmania tarentolae.

摘要

A study was carried out to follow the formation of amplicons in Leishmania at different steps of methotrexate selection by tagging individual alleles with different dominant markers. Both alleles of the H locus of L. tarentolae [Sauroleishmania tarentolae] (TarIIWT) were tagged using gene targeting with the neomycin and hygromycin phosphotransferase genes (neo and hyg). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags revealed that both alleles could be amplified in the same cell and that chromosomal deletions were frequent. When the methotrexate concentration was increased in subsequent selection steps, circles were observed inseveral mutants. A hyg marker was introduced into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. It is concluded that the tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.