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(Received for publication, July 31, 1996)
From the Department of Biochemistry and Molecular Biology, Oregon
Health Sciences University, Portland, Oregon 97201-3098
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
Volume 271, Number 48,
Issue of November 29, 1996
pp. 30840-30846
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
- 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 has important general implications for the
genetic manipulation and analysis of the leishmanial genome.
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