Extrachromosomal circular amplification of a genomic region of Leishmania chromosome 6 that includes the DHFR-TS locus. (a) Genomic organization of the DHFR-TS locus in both L. infantum MTX20.5 and L. major MTX60.4. Relative gene expression data (RNA) were determined using DNA microarrays and relative hybridization data were obtained by comparative genomic hybridization (DNA). Asterisks indicate that the microarray data of these genes were not found to be reliable. Direct repeats are shown with thick arrows and the approximate position of primers 6a and 6b are indicated with half arrows. (b) Chromosome size blot of Leishmania cells hybridized to a DHFR-TS probe. Sizes were determined using a yeast molecular weight marker (Biorad. Hercules, CA, USA). (c) Model for the formation of the extrachromosomal DHFR-TS circular DNA generated through homologous recombination between direct repeats (Figure S1 in Additional data file 2). (d) PCR with primers 6a and 6b to support the model shown in (c). Lane 1, L. infantum wild-type cells; lane 2, L. infantum MTX 20.5; lane 3, L. major wild-type cells; lane 4, L. major MTX60.4.