The viral thymidine kinase gene as a tool for the study of mutagenesis in Trypanosoma brucei

Abstract

We have tested the use of thymidine kinase as a negative selection system for Trypanosoma brucei. To this end we have targeted a construct containing a Herpes simplex virus thymidine kinase (TK) gene into the ribosomal DNA array of procyclic T.brucei. This resulted in TK activity 30-50-fold above background and in susceptibility to the nucleoside analogues ganciclovir, ethyl-deoxyuridine and 1-[2-deoxy,2-fluoro-8-D-arabinofuranosyl]-5-iodouracil, all of which have no effect on wild-type trypanosomes. TK+ trypanosomes, however, reverted to a ganciclovir resistant phenotype at a rate of 10-6 per cell-generation. A similar reversion rate was observed using the Varicella-zoster virus TK gene. Loss of TK activity was not due to detectable DNA rearrangements or a decrease in TK mRNA. Sequence analysis of the revertant genes demonstrated, however, the occurrence of point mutations and frameshifts. One revertant line had a mutation in the thymidine binding site leading to the substitution of a conserved arginine by a glycine. Other mutations included single base insertion, single base deletion and the introduction of a premature termination codon by point mutation.