Insertional Tagging, Cloning, and Expression of the Toxoplasma gondii Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase Gene

doi:10.1074/jbc.271.24.14010

Insertional Tagging, Cloning, and Expression of the Toxoplasma gondii Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase Gene

USE AS A SELECTABLE MARKER FOR STABLE TRANSFORMATION*

From the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the
^‡ Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201

^∥ National Science Foundation Presidential Young Investigator with support from the MacArthur Foundation and Merck Research Laboratories. To whom correspondence should be addressed:
Dept. of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.
Tel.: 215-898-2118; Fax: 215-898-8780; E-mail: droos{at}sas.upenn.edu

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.

Footnotes

↵^§ Recipient of an N. L. Tartar Trust Fellowship from the Medical Research Foundation of Oregon.
↵^¶ Recipients of Burroughs Wellcome Awards in Molecular Parasitology.
↵* This work was supported by Grants AI-28724, AI-31808, and AI-23682 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

DHFR-TS

dihydrofolate reductase-thymidylate synthase

6-TX

6-thioxanthine

FUDR

5-fluorodeoxyuridine

HXGPRT

hypoxanthine-xanthine-guanine phosphoribosyl transferase

MPA

mycophenolic acid

UPRT

uracil phosphoribosyl transferase

bp

base pair(s)

PCR

polymerase chain reaction

kb

kilobase pair(s)

PrPP

phosphoribosylpyrophosphate

nt

nucleotide(s)

PRT

phosphoribosyl transferase.
↵² A. L. C. Moulton and D. S. Roos, unpublished observations.
↵³ R. G. K. Donald, D. Carter, B. Ullman, and D. S. Roos, unpublished observations.
- Received January 26, 1996.