Thymidine kinase as a selectable marker for studying the biogenesis of glycosomes in Trypanosoma brucei.
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.
Lye
L
L
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Wang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D015774:Ganciclovir / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D008830:Microbodies / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D013937:Thymidine Kinase / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D014162:Transfection; D014170:Transformation, Genetic; D014295:Trimethoprim / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure
D000963:Antimetabolites; D000998:Antiviral Agents; D017931:DNA Primers; D014295:Trimethoprim; D013937:Thymidine Kinase; D015774:Ganciclovir