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 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.
Valdés
J
J
Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan, Amsterdam.
Taylor
M C
MC
Cross
M A
MA
Ligtenberg
M J
MJ
Rudenko
G
G
Borst
P
P
D000818:Animals; D001483:Base Sequence; D004351:Drug Resistance / Q000235:genetics; D016368:Frameshift Mutation; D015774:Ganciclovir; D005819:Genetic Markers; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D017354:Point Mutation; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D005819:Genetic Markers; D013937:Thymidine Kinase; D015774:Ganciclovir