Isolation of active transcription complexes from animal cell nuclei by nitrocellulose binding.
A method for the rapid isolation of active transcription complexes from animal cell nuclei is described. The method is based on the observation that, after lysis of nuclei with the detergents Sarkosyl and Triton X-100, transcription complexes are selectively bound to nitrocellulose. The nitrocellulose filters retain 80-90% o the RNA labelled briefly in vitro and about 10% of the nuclear DNA. The bulk of the retained DNA is in the size range of 20 kb. Transcription complexes involving both RNA polymerase I and II are retained by nitrocellulose. The nitrocellulose-bound transcription complexes preserve almost all of their RNA polymerase activity. The size distribution of the RNA product shows that bound transcription complexes retain also most of their growing RNA chains. The possibility to use selective retention by nitrocellulose in the analysis of transcriptionally active genes is discussed.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000302:isolation & purification; D003101:Collodion / Q000378:metabolism*; D004247:DNA / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003902:Detergents; D005374:Filtration; D008099:Liver / Q000378:metabolism; D009698:Nucleoproteins / Q000302:isolation & purification; D012313:RNA / Q000096:biosynthesis; D051381:Rats; D014158:Transcription, Genetic
D002843:Chromatin; D003902:Detergents; D009698:Nucleoproteins; D012313:RNA; D003101:Collodion; D004247:DNA; D012321:DNA-Directed RNA Polymerases