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 ba...
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Published in | Nucleic acids research Vol. 24; no. 10; pp. 1809 - 1815 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
15.05.1996
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | Present address: Departamento de Fisiologia, CINVESTAV-México, Apartado Postal 14-740, México DF 07000, México To whom correspondence should be addressed ark:/67375/HXZ-HPJ8W438-W istex:83FECF52C4F50A341EAD52707ECD4C31A6802856 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0305-1048 1362-4962 1362-4962 |
DOI: | 10.1093/nar/24.10.1809 |