Nucleic Acids Research, Vol 25, Issue 12 2389-2395, Copyright © 1997 by Oxford University Press
MS Sachs, EU Selker, B Lin, CJ Roberts, Z Luo, D Vaught-Alexander and BS Margolin
The expression of thymidine kinase in fungi, which normally lack this
enzyme, will greatly aid the study of DNA metabolism and provide useful
drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase
gene ( tk ) was expressed in Neurospora crassa. tk was expressed as a
fusion to N.crassa arg-2 regulatory sequences and as a hygromycin
phosphotransferase-thymidine kinase fusion gene under the control of
cytomegalovirus and SV40 sequences. Only strains containing tk showed
thymidine kinase enzyme activity. In strains containing the arg-2 - tk
gene, both the level of enzyme activity and the level of mRNA were reduced
by growth in arginine medium, consistent with control through arg-2
regulatory sequences. Expression of thymidine kinase in N.crassa
facilitated radioactive labeling of replicating DNA following addition of
[3H]thymidine or [14C]thymidine to the growth medium. Thymidine labeling of
DNA enabled demonstration that hydroxyurea can be used to block replication
and synchronize the N.crassa mitotic cycle. Strains expressing thymidine
kinase were also more sensitive than strains lacking thymidine kinase to
anticancer and antiviral nucleoside drugs that are activated by thymidine
kinase, including 5-fluoro-2'- deoxyuridine,
1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5- iodouridine and
trifluorothymidine. Finally, expression of thymidine kinase in N. crassa
enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to
separate newly replicated DNA from old DNA using equilibrium
centrifugation.
ARTICLES
Expression of herpes virus thymidine kinase in Neurospora crassa
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, PO Box 91000, Portland, OR 97291- 1000, USA. msachs@admin.ogi.edu
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