Nucleic Acids Research, Vol 24, Issue 10 1809-1815, Copyright © 1996 by Oxford University Press
J Valdes, MC Taylor, MA Cross, MJ Ligtenberg, G Rudenko and P Borst
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.
ARTICLES
The viral thymidine kinase gene as a tool for the study of mutagenesis in Trypanosoma brucei
Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan, Amsterdam.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Leal, A. Acosta-Serrano, J. Morris, and G. A. M. Cross Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A J. Biol. Chem., July 9, 2004; 279(28): 28979 - 28988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Bussler, M. Linder, D. Linder, and E. Reinwald Determination of the Disulfide Bonds within a B Domain Variant Surface Glycoprotein from Trypanosoma congolense J. Biol. Chem., December 4, 1998; 273(49): 32582 - 32586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. van Leeuwen, R. Kieft, M. Cross, and P. Borst Biosynthesis and Function of the Modified DNA Base beta -D-Glucosyl-Hydroxymethyluracil in Trypanosoma brucei Mol. Cell. Biol., October 1, 1998; 18(10): 5643 - 5651. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. Blattner, S. Helfert, P. Michels, and C. Clayton Compartmentation of phosphoglycerate kinase in Trypanosoma brucei plays a critical role in parasite energy metabolism PNAS, September 29, 1998; 95(20): 11596 - 11600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cross, M. C. Taylor, and P. Borst Frequent Loss of the Active Site during Variant Surface Glycoprotein Expression Site Switching In Vitro in Trypanosoma brucei Mol. Cell. Biol., January 1, 1998; 18(1): 198 - 205. [Abstract] [Full Text]
|
|