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

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...

Full description

Saved in:
Bibliographic Details
Published in:Nucleic acids research 2005-01, Vol.33 (5), p.1699-1709
Main Authors: Genest, Paul-André, Riet, Bas ter, Dumas, Carole, Papadopoulou, Barbara, van Luenen, Henri G. A. M., Borst, Piet
Format: Article
Language:English
Subjects:
Alleles
Animals
Base Sequence
Cell Line
Cinnamates - pharmacology
DNA, Protozoan - chemistry
DNA-Binding Proteins - genetics
Gene Targeting
Genes, Protozoan
Hygromycin B - analogs & derivatives
Hygromycin B - pharmacology
Leishmania - drug effects
Leishmania - genetics
Models, Genetic
Molecular Sequence Data
Mutation
Plasmids - chemistry
Protozoan Proteins - genetics
Repetitive Sequences, Nucleic Acid
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 β-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.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gki304