Chromosome aneuploidy in L. major selected for MTX resistance. The relative expression ratio of each individual gene of chromosomes (a) 22, (b) 28, (c) 11 and (d) 12 of L. major MTX60.4 was contrasted with the expression levels of the same genes in L. major wild-type cells, which were arbitrarily set at 1. Quantitative Southern blots were performed; two distant probes per chromosome were hybridized to HpaII digested DNA from L. major wild-type (lane 1), and L. major MTX60.4 (lane 2) (only one hybridization is shown for chromosomes 11 and 12). The hybridization signals of an α-tubulin (α-tub) probe, whose related gene is unchanged in the resistant strain, were used to standardize all the hybridization signals. HpaII digested total DNA from revertant L. major MTX60.4 parasites after 5, 12, 25, and 30 passages without MTX (lanes 3, 4, 5, and 6, respectively) were added, showing the progressive loss of aneuploid chromosomes in revertants.