Nucleic Acids Research, 1985, Vol. 13, No. 14 5111-5126
© 1985
Articles |
Inhibition of the herpes simplex virus thymidine kinase gene transfection in Ltk- cells by potential Z-DNA forming polymers
Institute of Cancer Research, Columbia University College of Physicians and Surgeons, Departments of Biochemistry and Molecular Biophysics, and Division of Environmental Sciences 701 W 162 St, New York, NY 10032, USA
Received April 30, 1985. Accepted June 24, 1985.
It has been demonstrated that certain alternating purine and pyrimidine sequences may assume a left-handed Z-DNA conformation. In order to evaluate the possibility that Z-DNA is involved in the modulation of gene expression, we examined the ability of various synthetic DNA polymers to affect the transfectlon of herpes simplex virus thymidine kinase (HSVtk) gene in Ltk- cells using the DNA-calcium phosphate cotransfection technique. We found that potential Z-DNA forming polymers such as, poly(dGm5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dc), cotransfected with the tk gene decreased the level of Tk+ transformed colonies. In contrast, cotransfection of the tk gene with polymers which do not assume Z-conformation such as, poly(dG).poly(dC) or poly(dA-dT).poly(dA-dT) showed no effect on the number of colonies formed. About 50% inhibition of the Tk+ colony formation was obtained by 0.4 µg of poly(dG-m5dC).poly(dG-m5dC), or by 2 µg of poly(dG-dC).poly(dG-dC). DNA uptake into Ltk- cells was not significantly affected by any of these polymers. Approximately 20–42 base pairs (bp) long alternating dG-dC sequence linked at either the 5'-end or 3'-end of tk gene were cloned into plasmids. These recombinant plasmids, however, showed no remarkable effect upon the transfection of Ltk- cells. The DNAs of Tk+ colonies obtained by transfecting these recombinant plasmids were digested with BssH II and analyzed by Southern blotting. We demonstrated that the dG-dC sequences proximal to the tk gene were integrated into cellular DNA. All the presented results indicate that only larger polymers with the potential to assume a Z-DNA conformation may affect tk gene transfection either by inhibiting transcription or more probably by affecting the stable integration of the tk gene into the host chromosome.