Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus

William J. Sullivan, Chi Wu Chiang, Craig M. Wilson, Fardos N.M. Naguib, Mahmoud H. El Kouni, Robert G.K. Donald, David S. Roos

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

A genetic approach has been exploited to investivate adenylate salvage pathways in the protozoan parasite Toxoplasma gondii, a purine auxotroph. Using a new insertional mutagenesis vector designed to facilitate the rescue of tagged loci even when multiple plasmids integrate as a tandem array, 15 independent clonal lines resistant to the toxic nucleoside analog adenine arabinoside (AraA) were generated. Approximately two-thirds of these clones lack adenosine kinase (AK) activity. Parallel studies identified an expressed sequence tag (EST) exhibiting a small region of weak similarity to human AK, and this locus was tagged in several AK-deficient insertional mutants. Library screening yielded full-length cDNA and genomic clones. The T. gondii AK gene contains five exons spanning a ~3 kb locus, and the predicted coding sequence was employed to identify additional AK genes and cDNAs in the GenBank and dbEST databases. A genomic construct lacking essential coding sequence was used to create defined genetic knock-outs at the T. gondii AK locus, and AK activity was restored using a cDNA-derived minigene. Hybridization analysis of DNA from 13 AraA-resistant insertional mutants reveals three distinct classes: (i) AK- mutants tagged at the AK locus; (ii) AK- mutants not tagged at the AK locus, suggesting the possibility that another locus may be involved in regulating AK expression; and (iii) mutants with normal AK activity (potential transport mutants). Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)1-14
Number of pages14
JournalMolecular and Biochemical Parasitology
Volume103
Issue number1
DOIs
StatePublished - Sep 20 1999
Externally publishedYes

Keywords

  • Adenosine kinase
  • Adenosine transport
  • Gene targeting
  • Insertional mutagenesis
  • Knock-out mutants
  • Molecular parasitology
  • Purine metabolism

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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