LINEAR AMPLICONS AS PRECURSORS OF AMPLIFIED CIRCLES IN METHOTREXATE-RESISTANT LEISHMANIA-TARENTOLAE

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes (neo and hyg), Selection of these recombinan t parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in th e same cell and that chromosomal deletions are frequent. When methotre xate concentration was increased in subsequent selection steps, circle s were observed in several mutants. We have introduced a hyg marker in to linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, ci rcles with the hyg marker were observed, showing that circles can inde ed be formed from linear amplicons, The tagging of H locus alleles per mits appreciation of the extent of genetic rearrangements leading to a mplicon formation in Leishmania cells selected for drug resistance.