Intrachromosomal tandem duplication and repeat expansion during attempts to inactivate the subtelomeric essential gene GSH1 in Leishmania

Abstract

Gamma-glutamylcysteine synthetase encoded by GSH1 is the rate-limiting enzyme in the biosynthesis of glutathione and trypanothione in Leishmania. Attempts to generate GSH1 null mutants by gene disruption failed in Leishmania infantum. Removal of even a single allele invariably led to the generation of an extra copy of GSH1, maintaining two intact wild-type alleles. In the second and even third round of inactivation, the markers integrated at the homologous locus but always preserved two intact copies of GSH1. We probed into the mechanism of GSH1 duplication. GSH1 is subtelomeric on chromosome 18 and Southern blot analysis indicated that a 10-kb fragment flanked by 466-bp direct repeated sequences was duplicated in tandem on the same chromosomal allele each time GSH1 was targeted. Polymerase chain reaction analysis and sequencing confirmed the generation of novel junctions created at the level of the 466-bp repeats consequent to locus duplication. In loss of heterozygosity attempts, the same repeated sequences were utilized for generating extrachromosomal circular amplicons. Our results are consistent with break-induced replication as a mechanism for the generation of this regional polyploidy to compensate for the inactivation of an essential gene. This chromosomal repeat expansion through repeated sequences could be implicated in locus duplication in Leishmania.