The Viral Thymidine Kinase Gene as a Tool for the Study of Mutagenesis in Trypanosoma Brucei

• Published in: Nucleic acids research 1996-05, Vol.24 (10), p.1809-1815
• Main Authors: Valdés, Jesús, Taylor, Martin C., Cross, Michael A., Ligtenberg, Marjolijn J. L., Rudenko, Gloria, Borst, Piet
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Drug Resistance - genetics; Frameshift Mutation; Ganciclovir; Genetic Markers; Molecular Sequence Data; Mutation; Plasmids; Point Mutation; Simplexvirus - enzymology; Simplexvirus - genetics; Thymidine Kinase - genetics; Transfection; Trypanosoma brucei; Trypanosoma brucei brucei - genetics
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/24.10.1809

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 Varicellazoster 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.