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Nucleic Acids Research 2005 33(5):1699-1709; doi:10.1093/nar/gki304
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Published online 21 March 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Formation of linear inverted repeat amplicons following targeting of an essential gene in Leishmania

Paul-André Genest, Bas ter Riet, Carole Dumas1, Barbara Papadopoulou1, Henri G. A. M. van Luenen and Piet Borst*

Division of Molecular Biology and Centre of Biomedical Genetics, The Netherlands Cancer Institute Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 1Faculty of Medicine, Department of Medical Biology, Laval University, Infectious Disease Research Center, RC709, CHUL Research Center (CHUQ) 2705 blvd Laurier, Quebec, Canada G1V 4G2

*To whom correspondence should be addressed. Tel: +31 020 512 2880; Fax: +31 020 669 1383; Email: p.borst{at}nki.nl

Received January 19, 2005. Revised February 25, 2005. Accepted February 25, 2005.

Attempts to inactivate an essential gene in the protozoan parasite Leishmania have often led to the generation of extra copies of the wild-type alleles of the gene. In experiments with Leishmania tarentolae set up to disrupt the gene encoding the J-binding protein 1 (JBP1), a protein binding to the unusual base ß-D-glucosyl-hydroxymethyluracil (J) of Leishmania, we obtained JBP1 mutants containing linear DNA elements (amplicons) of ~100 kb. These amplicons consist of a long inverted repeat with telomeric repeats at both ends and contain either the two different targeting cassettes used to inactivate JBP1, or one cassette and one JBP1 gene. Each long repeat within the linear amplicons corresponds to sequences covering the JBP1 locus, starting at the telomeres upstream of JBP1 and ending in a ~220 bp sequence repeated in an inverted (palindromic) orientation downstream of the JBP1 locus. We propose that these amplicons have arisen by a template switch inside a DNA replication fork involving the inverted DNA repeats and helped by the gene targeting.

GenBank/EMBL/DDBJ accession nos+

+AY842844–AY842849


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