Creation of homozygous mutants of Leishmania donovani with single targeting constructs.

The Journal of Biological Chemistry
H Y HwangB Ullman

Abstract

Homozygous null mutants of the hypoxanthine-guanine phosphoribosyltransferase (hgprt) and adenine phosphoribosyltransferase (aprt) loci were created in Leishmania donovani in which both alleles were eliminated using only a single targeting construct. Functional heterozygotes were 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 selection 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 Southern blotting, and the effects of gene replacement upon gene expression in intact parasites were evaluated by direct enzymatic assay and by immunoblotting. All mutant strains were viable under the selection conditions and exhibited appropriate drug resistance phenotypes. The ability to generate homozygous knockouts with single targeting constructs greatly facilitates the genetic dissection and subsequent biochemical investigations of the purine pathway in Leishmania and ha...Continue Reading

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Citations

Jan 30, 2004·Molecular and Biochemical Parasitology·Jan M Zarella-BoitzBuddy Ullman
May 3, 2002·Molecular and Biochemical Parasitology·Huafang ShiElisabetta Ullu
Jan 13, 2012·The Journal of Biological Chemistry·Jan M BoitzBuddy Ullman
Mar 1, 2012·The Journal of Biological Chemistry·Zachary N WilsonPhillip A Yates
Aug 26, 1998·Proceedings of the National Academy of Sciences of the United States of America·G VasudevanS M Landfear
Jun 26, 2012·Trends in Parasitology·Jan M BoitzNicola S Carter
Oct 7, 2010·Molecular Microbiology·Nicola S CarterBuddy Ullman
Mar 7, 2014·International Journal for Parasitology. Drugs and Drug Resistance·Alexandra LüscherPascal Mäser
May 27, 2014·Frontiers in Immunology·Rudra Chhajer, Nahid Ali
Jun 28, 2016·Molecular and Biochemical Parasitology·Jan M BoitzBuddy Ullman
Aug 1, 1997·The Journal of Biological Chemistry·H Y Hwang, B Ullman
Nov 10, 2020·Molecular and Biochemical Parasitology·Gino L TurraMarcel Deponte

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