Creation of null/+ mutants of the α-tubulin gene in Leishmania enriettii by gene cluster deletion

Tubulins are abundant structural proteins in the Leishmania parasite, and tubulin genes are examples of highly expressed genes, which are present in multiple copies arranged in tandem repeats. Functional analysis of such multicopy genes using genetic manipulation has not yet been possible. Here we describe a method for creating deletions of α-tubulin gene clusters by targeted gene replacement and report the isolation of null/+ mutants deleted for either of the two allelic tubulin clusters. We also report null/null mutants in which both clusters have been deleted from their chromosomal loci and in which α-tubulin genes are present as episomal elements. Characterization of tubulin mRNA expression in these mutants indicated a post-transcriptional up-regulation of α-tubulin mRNA stability in null/+ mutants which contained only one-third the normal number of α-tubulin genes. A parallel increase in β-tubulin mRNA levels indicated coordinate regulation of tubulin mRNA in Leishmania.