Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene: Use as a selectable marker for stable transformation

Robert G.K. Donald, Darrick Carter, Buddy Ullman, David S. Roos

Research output: Contribution to journalArticle

319 Scopus citations

Abstract

A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis. Parasite mutants resistant to 6-thioxanthine arose at a frequency of ∼3 × 10-7. Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species. Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed. Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli. Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms. Internally deleted clones spanning the genomic locus were used to create "knock-out" parasites, which lack all detectable HXGPRT activity. Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms. Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii.

Original languageEnglish (US)
Pages (from-to)14010-14019
Number of pages10
JournalJournal of Biological Chemistry
Volume271
Issue number24
DOIs
StatePublished - Jan 1 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene: Use as a selectable marker for stable transformation'. Together they form a unique fingerprint.

  • Cite this