Pfefferkorn E R, Borotz S E
Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755-3842.
Exp Parasitol. 1994 Nov;79(3):374-82. doi: 10.1006/expr.1994.1099.
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.
6-硫代黄嘌呤在浓度为5微克/毫升时可抑制人成纤维细胞中弓形虫生长的50%。用乙基亚硝基脲处理诱导产生的突变体（ThxR-1）比野生型的抗性高20倍。在莱施-奈恩成纤维细胞中生长的野生型寄生虫能有效地摄取次黄嘌呤、鸟嘌呤和黄嘌呤，但ThxR-1摄取这些前体的量均不到野生型的2%。野生型寄生虫的可溶性提取物对次黄嘌呤、鸟嘌呤和黄嘌呤具有很强的磷酸核糖基转移酶活性，而ThxR-1的提取物对任何这些底物的活性几乎检测不到。因此，ThxR-1对6-硫代黄嘌呤产生抗性的基础是缺乏次黄嘌呤-鸟嘌呤磷酸核糖基转移酶。这样，利用该酶的补救途径对于寄生虫必须从宿主细胞获取的嘌呤的获取并非必不可少。在含有霉酚酸和黄嘌呤的培养基中培养，可在存在许多ThxR-1寄生虫的情况下有效地回收野生型弓形虫。连同使用6-硫代黄嘌呤在存在许多野生型寄生虫的情况下检测抗性突变体，该程序为影响弓形虫次黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因的突变提供了一种简单的选择和反选择方法。
