Isolation of chromatin DNA tightly bound to the nuclear envelope of HeLa cells.
Recent discovery of the role of nuclear pores in transcription, predicted by our early DNA-membrane complex (DMC) model, makes membrane-bound DNA (MBD) isolation from the cell nucleus and analysis of the MBD actual. The method of MBD isolation proposed by us retains DMC integrity during isolation. We used HeLa cells for DMC extraction. Changing the ionic composition of the isolation medium and replacing DNase I, used commonly for chromatin destruction, with a set of restriction enzymes allowed us to isolate the MBD. Treatment of a nuclear membrane with proteinase K and ultrasound has been used to increase the yield of MBD. Electron microscopic analysis of the purified fraction of isolated DMC supports our previous model of nuclear envelope lipid-chromatin interaction in the nuclear pore assembly.
Department of Reception Mechanisms, Institute of Cell Biopysics, Russian Academy of Sciences, Institutskaya, Pushchino, Moscow region, Russia, VVKuvichkin@gmail.com
Vasily Vladimirovich
VV
Kuvichkin
D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D022022:Nuclear Pore / Q000648:ultrastructure
D002843:Chromatin; D046911:Macromolecular Substances; D004247:DNA