Inhibition of the herpes simplex virus thymidine kinase gene transfection in Ltk- cells by potential Z-DNA forming polymers.

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RESUMO

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 transfection 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(dG-m5dC) X poly(dG-m5dC) and poly(dG-dC) X 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) X poly(dC) or poly(dA-dT) X 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 micrograms of poly(dG-m5dC) X poly(dG-m5dC), or by 2 micrograms of poly(dG-dC) X 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.

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