Ribosomal RNA synthesis in mammalian cells permealized by diethylaminoethyl (DEAE)-dextran treatment.
Ehrlich ascites tumor cells were made permeable to nucleoside triphosphates by treating them with diethylaminoethyl (DEAE)-dextran. Permeable cells incorporated labeled UTP into RNA at a constant rate for at least 30 min at 37 degrees C and 100 min at 25 degrees C, whereas no incorporation was detected without DEAE-dextran treatment. This RNA synthesis is dependent on added nucleoside triphosphates and is affected differentially by different concentrations of alpha-amanitin. The results of both hybridization and S1-nuclease protection mapping experiments with pulse-labeled RNA synthesized in this system indicate that reinitiation of rRNA transcription takes place from the physiological initiation site in cells made permeable by treatment with DEAE-dextran. By using this system, we examined the kinetics of rRNA synthesis in mouse FM3A cells during inhibition of protein synthesis. The results reflected those previously obtained by analyses of nucleolar RNA synthesis in vivo during protein synthesis inhibition. This permeable cell system should provide a rapid and reproducible assay method for rRNA synthesis in mammalian cells.
Y
Y
Mishima
R
R
Kominami
M
M
Muramatsu
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003637:DEAE-Dextran / Q000494:pharmacology*; D003911:Dextrans / Q000494:pharmacology*; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014316:Tritium
D003911:Dextrans; D010761:Phosphorus Radioisotopes; D012335:RNA, Ribosomal; D014316:Tritium; D003637:DEAE-Dextran