THE VIRAL THYMIDINE KINASE GENE AS A TOOL FOR THE STUDY OF MUTAGENESIS IN TRYPANOSOMA-BRUCEI

We have tested the use of thymidine kinase as a negative selection sys tem for Trypanosoma brucei. To this end we have targeted a construct c ontaining a Herpes simplex virus thymidine kinase (TK) gene into the r ibosomal DNA array of procyclic T.brucei, This resulted in TK activity 30-50-fold above background and in susceptibility to the nucleoside a nalogues ganciclovir, ethyl-deoxyuridine and deoxy,2-fluoro-8-D-arabin ofuranosyl]-5-iodouracil, all of which have no effect on wild-type try panosomes, TK+ trypanosomes, however, reverted to a ganciclovir resist ant phenotype at a rate of 10(-6) per cell-generation. A similar rever sion rate was observed using the Varicella-zoster virus TK gene, Loss of TK activity was not due to detectable DNA rearrangements or a decre ase in TK mRNA, Sequence analysis of the revertant genes demonstrated, however, the occurrence of point mutations and frameshifts, One rever tant line had a mutation in the thymidine binding site leading to the substitution of a conserved arginine by a glycine, Other mutations inc luded single base insertion, single base deletion and the introduction of a premature termination codon by point mutation.