Thymidine Kinase as a Selectable Marker for Studying the Biogenesis of Glycosomes inTrypanosoma brucei

Abstract

The import of proteins into the glycosome ofT. bruceihas 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 inT. bruceifor isolating glycosome-deficientT. bruceimutants 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 thetkgene (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 theT. brucei427 procyclic cells by electroporation, and the transformants were selected under hygromycin B (50 μg/ml) and cloned. The thymidine kinase activity in the crude extracts of transformants was determined. Differential digitonin treatment of the transformants indicated that thetk-SKL protein was apparently localized to the glycosomal fraction, as expected, whereas thetkprotein was found in the soluble fraction. [methyl-3H]Thymidine was incorporated into the nucleic acids of the transformant 427/ptkto a level twice as high as that incorporated into 427/ptk-SKL and the wild type. In the presence of 100μMganciclovir, the growth of 427/ptkwas totally inhibited, whereas growth of 427/ptk-SKL and the wild type was unaffected. When 150 μMtrimethoprim was added to the culture medium, growth of 427/ptk-SKL and the wild type was completely arrested while that of 427/ptkremained normal. We have thus established the methodology for both positive and negative selection of potential glycosome-deficient mutants ofT. brucei427/ptk-SKL.