CREATION OF HOMOZYGOUS MUTANTS OF LEISHMANIA-DONOVANI WITH SINGLE TARGETING CONSTRUCTS

Homozygous null mutants of the hypoxanthine-guanine phosphoribosyltran sferase (hgprt) and adenine phosphoribosyltransferase (aprt) loci were created in Leishmania donovani in which both alleles were elimi nated using only a single targeting construct, Functional heterozygotes mer e first generated by homologous recombination after transfection with vectors containing 5'- and 3'-flanking regions of either the hgprt or the aprt gene circumscribing drug resistance markers. Homozygous null mutants were then isolated from the heterozygotes by negative selectio n in media containing subversive substrates of the encoded proteins, i .e. allopurinol for HGPRT and 4-aminopyrazolopyrimidine for APRT, The novel alleles created by homologous recombination were verified by Sou thern blotting, and the effects of gene replacement upon gene expressi on in intact parasites were evaluated by direct enzymatic assay and by immunoblotting, All mutant strains were viable under the selection co nditions and exhibited appropriate drug resistance phenotypes. The abi lity to generate homozygous knockouts with single targeting constructs greatly facilitates the genetic dissection and subsequent biochemical investigations of the purine pathway in Leishmania and has important general implications for the genetic manipulation and analysis of the leishmanial genome.