Effect of large targeted deletions on the mitotic stability of an extra chromosome mediating drug resistance in Leishmania

A mitotically stable linear extra chromosome obtained in a Leishmania donov ani strain rendered mycophenolic acid-resistant has been physically mapped. This 290-kb chromosome has an inverted duplicated structure around a centr al inversion region, and is derived from a conservative amplification event of a similar to 140-kb subtelomeric end of chromosome 19. Large-sized targ eted deletions of the central region were performed through homologous reco mbination using three specific transfection vectors. The size of the extra chromosome was thus successfully reduced from 290 to 260, 200 and 120 kb re spectively. The mitotic stability of these chromosomes was then analysed in drug-free cultures over > 140 days. Results differed according to the dele tion created. By contrast with the smallest deletion the two largest deleti ons altered mitotic stability, leading to progressive loss of the size-redu ced chromosomes with similar kinetics in both mutants. The 30-kb region com mon to both deletions may therefore be considered as involved in mitotic st ability. A 44-kb contig covering this region could be assembled and sequenc ed. The analysis of this sequence did not reveal any sequence elements typi cal of centromeric DNA. By contrast, its enrichment in homopolymer tracts s uggests that this region might contain an origin of replication.