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Nucleic Acids Research, 2001, Vol. 29, No. 15 3231-3240
© 2001 Oxford University Press

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

Pascal Dubessay, Christophe Ravel, Patrick Bastien, Marie-Françoise Lignon, Buddy Ullman1, Michel Pagès and Christine Blaineau*

CNRS UMR5093 ‘Génome et Biologie Moléculaire des Protozoaires Parasites’, Laboratoire de Parasitologie-Mycologie, Faculté de Médecine, 163 Rue A. Broussonet, F-34090 Montpellier, France and 1Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97201, USA

A mitotically stable linear extra chromosome obtained in a Leishmania donovani strain rendered mycophenolic acid-resistant has been physically mapped. This 290-kb chromosome has an inverted duplicated structure around a central inversion region, and is derived from a conservative amplification event of a ~140-kb subtelomeric end of chromosome 19. Large-sized targeted deletions of the central region were performed through homologous recombination using three specific transfection vectors. The size of the extra chromosome was thus successfully reduced from 290 to 260, 200 and 120 kb respectively. The mitotic stability of these chromosomes was then analysed in drug-free cultures over >140 days. Results differed according to the deletion created. By contrast with the smallest deletion the two largest deletions altered mitotic stability, leading to progressive loss of the size-reduced chromosomes with similar kinetics in both mutants. The 30-kb region common to both deletions may therefore be considered as involved in mitotic stability. A 44-kb contig covering this region could be assembled and sequenced. The analysis of this sequence did not reveal any sequence elements typical of centromeric DNA. By contrast, its enrichment in homopolymer tracts suggests that this region might contain an origin of replication.

* To whom correspondence should be addressed. Tel: +33 4 67 63 55 13; Fax: +33 4 67 63 00 49; Email: genpara@sc.univ-montp1.fr +AF315645, AF176312, AF319040, AY028171, AF393161–AF393182


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