Toxoplasma gondii: characterization of a mutant resistant to 6-thioxanthine.
6-Thioxanthine caused 50% inhibition of the growth of Toxoplasma gondii in human fibroblasts at a concentration of 5 micrograms/ml. A mutant induced by treatment with ethylnitrosourea (ThxR-1) was 20-fold more resistant than the wildtype. Wild-type parasites grown in Lesch-Nyhan fibroblasts efficiently incorporated hypoxanthine, guanine, and xanthine, but ThxR-1 incorporated each of these precursors less than 2% as well as the wildtype did. Soluble extracts of wild-type parasites had potent phosphoribosyltransferase activities for hypoxanthine, guanine, and xanthine, while extracts of ThxR-1 had barely detectable activity with any of these substrates. The basis for the resistance of ThxR-1 to 6-thioxanthine is, therefore, the lack of the enzyme hypoxanthine-guanine phosphoribosyltransferase. Thus, salvage pathways that employ this enzyme are not essential for the acquisition of purines, which the parasite must obtain from the host cell. Incubation in a medium containing mycophenolic acid and xanthine allowed the efficient recovery of wild-type T. gondii in the presence of many ThxR-1 parasites. Together with the use of 6-thioxanthine to detect resistant mutants in the presence of many wild-type parasites, this procedure provides a simple selection and back-selection for mutations that affect the hypoxanthine-guanine phosphoribosyltransferase gene of T. gondii.
Pfefferkorn
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Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755-3842.
Borotz
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D000818:Animals; D002478:Cells, Cultured; D005038:Ethylnitrosourea; D005347:Fibroblasts / Q000469:parasitology; D006147:Guanine / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D007042:Hypoxanthines / Q000378:metabolism; D016296:Mutagenesis; D014122:Toxoplasma / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D019820:Xanthine; D014970:Xanthines / Q000378:metabolism / Q000494:pharmacology*
D007042:Hypoxanthines; D014970:Xanthines; D019820:Xanthine; C054700:6-thioxanthine; D019271:Hypoxanthine; D006147:Guanine; D007041:Hypoxanthine Phosphoribosyltransferase; D005038:Ethylnitrosourea