Lye L, Wang C C
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Exp Parasitol. 1996 Mar;82(2):211-7. doi: 10.1006/expr.1996.0026.
The import of proteins into the glycosome of T. brucei has been studied as a potential target for antitrypanosomal chemotherapy. We have previously reported on the C-terminal tripeptides, such as SKL and its analogs, which function as targeting signals in the import of proteins into glycosomes. Recently, we tested the herpes simplex virus thymidine kinase gene (tk) as both a potential positive and a negative selectable marker in T. brucei for isolating glycosome-deficient T. brucei mutants in the procyclic form and complementation studies to identify genes involved in glycosomal biogenesis. The transforming vectors that we have constructed contained the hygromycin phosphotransferase gene (hyg) coupled either to the tk gene (ptk) or to the gene encoding the thymidine kinase fused with the glycosomal targeting signal (ptk-SKL) at the C-terminus. Individual constructs were introduced into the T. brucei 427 procyclic cells by electroporation, and the transformants were selected under hygromycin B (50 micrograms/ml) and cloned. The thymidine kinase activity in the crude extracts of transformants was determined. Differential digitonin treatment of the transformants indicated that the tk-SKL protein was apparently localized to the glycosomal fraction, as expected, whereas the tk protein was found in the soluble fraction. [methyl-3H]Thymidine was incorporated into the nucleic acids of the transformant 427/ptk to a level twice as high as that incorporated into 427/ptk-SKL and the wild type. In the presence of 100 microM ganciclovir, the growth of 427/ptk was totally inhibited, whereas growth of 427/ptk-SKL and the wild type was unaffected. When 150 microM trimethoprim was added to the culture medium, growth of 427/ptk-SKL and the wild type was completely arrested while that of 427/ptk remained normal. We have thus established the methodology for both positive and negative selection of potential glycosome-deficient mutants of T. brucei 427/ptk-SKL.
布氏锥虫蛋白质向糖体的转运已作为抗锥虫化疗的潜在靶点进行了研究。我们之前报道过C端三肽，如SKL及其类似物，它们在蛋白质向糖体的转运中作为靶向信号发挥作用。最近，我们测试了单纯疱疹病毒胸苷激酶基因（tk）作为布氏锥虫中潜在的正选择和负选择标记，用于分离前循环形式的糖体缺陷型布氏锥虫突变体，并进行互补研究以鉴定参与糖体生物合成的基因。我们构建的转化载体包含潮霉素磷酸转移酶基因（hyg），其与tk基因（ptk）或与在C端与糖体靶向信号融合的胸苷激酶编码基因（ptk-SKL）相连。通过电穿孔将各个构建体导入布氏锥虫427前循环细胞，在潮霉素B（50微克/毫升）存在下选择转化体并进行克隆。测定了转化体粗提物中的胸苷激酶活性。对转化体进行不同的洋地黄皂苷处理表明，正如预期的那样，tk-SKL蛋白明显定位于糖体部分，而tk蛋白存在于可溶部分。[甲基-3H]胸苷掺入转化体427/ptk的核酸中的水平是掺入427/ptk-SKL和野生型的两倍。在100微摩尔更昔洛韦存在下，427/ptk的生长完全受到抑制，而427/ptk-SKL和野生型的生长不受影响。当向培养基中添加150微摩尔甲氧苄啶时，427/ptk-SKL和野生型的生长完全停止，而427/ptk的生长保持正常。因此，我们建立了对布氏锥虫427/ptk-SKL潜在的糖体缺陷型突变体进行正选择和负选择的方法。
