The herpes simplex virus thymidine kinase gene as a conditional negative-selection marker gene in Arabidopsis thaliana.
The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as a conditional lethal marker in mammalian cells. The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors. The utility of HSVtk as a conditional negative-selection marker was explored in Arabidopsis thaliana (L.) Heynh. HSVtk was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants were morphologically indistinguishable from wild type and exhibited normal fertility. Ganciclovir at 10(-5) to 10(-4) M drastically reduced shoot regeneration on transgenic, HSVtk+ root explants or callus formation on HSVtk+ leaf explants but did not affect the wild-type cultures. There was a 35-fold reduction in shoot regeneration 8 d after transfer to shoot-induction medium. Negative selection against HSVtk activity along with kanamycin selection was also efficient in Agrobacterium-mediated gene transfer experiments. Shoot regeneration was 25 times lower on double-selective (ganciclovir plus kanamycin) plates than in the kanamycin control. This regeneration rate in double-selective plates is in the range of the frequency of shoots normally escaping kanamycin selection in Arabidopsis cultures.
Czakó
M
M
Department of Biological Sciences, University of South Carolina, Columbia 29208.
Márton
L
L
D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics*; D015774:Ganciclovir / Q000494:pharmacology; D005804:Genes, Lethal; D005814:Genes, Viral; D005819:Genetic Markers; D006801:Humans; D007612:Kanamycin / Q000494:pharmacology; D030821:Plants, Genetically Modified; D012231:Rhizobium / Q000235:genetics; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014170:Transformation, Genetic
D005819:Genetic Markers; D007612:Kanamycin; D013937:Thymidine Kinase; D015774:Ganciclovir