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

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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
Pages (from-to)1-14
Number of pages14
JournalMolecular and Biochemical Parasitology
Volume103
Issue number1
DOIs
Publication statusPublished - 1999 Sep 20

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Adenosine Kinase
Vidarabine
Toxoplasma
Organism Cloning
Complementary DNA
Clone Cells
Insertional Mutagenesis
Poisons
Expressed Sequence Tags
Nucleic Acid Databases
Nucleosides

All Science Journal Classification (ASJC) codes

  • Parasitology
  • Molecular Biology

Cite this

Sullivan, William J. ; Chiang, Chi-Wu ; Wilson, Craig M. ; Naguib, Fardos N.M. ; El Kouni, Mahmoud H. ; Donald, Robert G.K. ; Roos, David S. / Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus. In: Molecular and Biochemical Parasitology. 1999 ; Vol. 103, No. 1. pp. 1-14.
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Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus. / Sullivan, William J.; Chiang, Chi-Wu; Wilson, Craig M.; Naguib, Fardos N.M.; El Kouni, Mahmoud H.; Donald, Robert G.K.; Roos, David S.

In: Molecular and Biochemical Parasitology, Vol. 103, No. 1, 20.09.1999, p. 1-14.

Research output: Contribution to journalArticle

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