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


METHODS
We introduced an alpha-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 alpha-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.
Hongo
Etsuko
E
Radiation Effect Mechanisms Research Group, National Institute of Radiological Sciences, Inage-ku, Chiba, Japan.
Ishihara
Yoshie
Y
Sugaya
Keiko
K
Sugaya
Kimihiko
K
D053959:Alpha-Amanitin / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000494:pharmacology; D036681:Fluorescence Recovery After Photobleaching; D015971:Gene Expression Regulation, Enzymologic / Q000528:radiation effects*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000096:biosynthesis*; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D014158:Transcription, Genetic / Q000528:radiation effects*; D014965:X-Rays
D053959:Alpha-Amanitin; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012313:RNA; D012319:RNA Polymerase II