title: Formation of linear inverted repeat amplicons following targeting of an essential gene in Leishmania.
abstract: 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 beta-D-glucosyl-hydroxymethyluracil (J) of Leishmania, we obtained JBP1 mutants containing linear DNA elements (amplicons) of approximately 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 approximately 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.
authors.affiliation: Division of Molecular Biology and Centre of Biomedical Genetics, The Netherlands Cancer Institute Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Paul-André
authors.initials: PA
authors.lastname: Genest
authors.forename: Bas
authors.initials: B
authors.lastname: ter Riet
authors.forename: Carole
authors.initials: C
authors.lastname: Dumas
authors.forename: Barbara
authors.initials: B
authors.lastname: Papadopoulou
authors.forename: Henri G A M
authors.initials: HG
authors.lastname: van Luenen
authors.forename: Piet
authors.initials: P
authors.lastname: Borst
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002934:Cinnamates / Q000494:pharmacology; D016054:DNA, Protozoan / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000235:genetics*; D018390:Gene Targeting; D017125:Genes, Protozoan; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D007891:Leishmania / Q000187:drug effects / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000737:chemistry; D015800:Protozoan Proteins / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D002934:Cinnamates; D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; C402790:J-specific DNA-binding protein, protozoa; D015800:Protozoan Proteins; D006921:Hygromycin B; C026273:hygromycin A