Characterization of cells expressing RNA polymerase II tagged with green fluorescent protein: Effect of ionizing irradiation on RNA synthesis

Abstract

Purpose: To isolate and to characterize cells expressing RNA polymerase II tagged with green fluorescent protein for analyses of the effects of ionizing radiation on transcription in living cells.

Materials and methods: We introduced an α-amanitin-resistant mutation into a vector encoding the largest subunit of RNA polymerase II tagged with green fluorescent protein (GFP-pol). Cell lines stably expressing functional GFP-pol were isolated under selection with α-amanitin from a Chinese hamster cell line, CHO-K1, and a radiation-sensitive mutant CHO cell line, XR-1.

Results: We tested the functionality of the fusion protein in vivo by determining RNA synthesis activity by incorporation of nucleoside analogues. Both CHO-K1 and XR-1 cells expressing GFP-pol had properties similar to those of their respective parental cell lines, indicating that GFP-pol is functional.

Conclusions: These stable lines might prove useful for analyses of the roles of transcription after ionizing radiation.

Keywords:

• Green fluorescent protein
• α-amanitin
• ionizing radiation
• RNA polymerase
• transcription
• transcription-coupled repair