Expression of herpes virus thymidine kinase in Neurospora crassa.
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.
Sachs
M S
MS
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
Selker
E U
EU
Lin
B
B
Roberts
C J
CJ
Luo
Z
Z
Vaught-Alexander
D
D
Margolin
B S
BS
D000998:Antiviral Agents / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects; D005467:Floxuridine / Q000494:pharmacology; D015774:Ganciclovir / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics*; D006918:Hydroxyurea / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods; D009492:Neurospora crassa / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012045:Regulatory Sequences, Nucleic Acid; D013937:Thymidine Kinase / Q000096:biosynthesis* / Q000235:genetics
D000998:Antiviral Agents; D011993:Recombinant Fusion Proteins; D005467:Floxuridine; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D015774:Ganciclovir; D006918:Hydroxyurea