The isolation and characterization of nuclear ghosts from cultured HeLa cells.
Macromolecular complexes, which appear as ghosts when viewed by phase contrast microscopy, have been isolated from the nuclei of HeLa cells grown in culture. The preparation of these ghosts involves a detergent wash which removes the unit membranes of the nuclear envelop structure but leaves intact both the nuclear pores and the dense structure conferring nuclear margins (possibly the dense lamella). Detergent-washed nuclei are subsequently treated with 0.5 M MgCl2 and fractionated on continuous sucrose gradients containing 0.5 M MgCl2. The ghosts are recovered as a sharp band at an apparent sucrose density of 47-52% and consist of 72% protein, 10% phospholipid, 14% DNA, And 4% RNA. The release of the majority of intranuclear components is indicated by the large loss of nuclear DNA (95%), RNA (71%), and protein (87%) contrasted to the small loss of phospholipid (27%) druing the conversion of detergent washed nuclei to isolated ghosts. Sodium dodecyl sulfate-polyacrylamide gel patterns of the ghost proteins consist of two major bands with approximate molecular weights of 20,000 and 35,000. The isolation of ghosts with a similar density and protein composition from nondetergent-washed nuclei indicates that the ghost is not an artifact induced by the detergent treatment. The absence of cytoplasmic contamination in the preparations of detergent washed nuclei and nuclear ghosts was demonstrated by chemical, enzymatic, and electron microscope studies. We suggest that the isolated ghosts represent a structural macromolecular complex which underlies and is probably attached to the inner nuclear membrane of intact nuclei. The possible additional presence of intranuclear network proteins has not been excluded.
D000596:Amino Acids / Q000378:metabolism; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000032:analysis; D003840:Deoxycholic Acid / Q000494:pharmacology; D005643:Fucose / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000032:analysis; D006367:HeLa Cells / Q000648:ultrastructure; D006801:Humans; D008566:Membranes / Q000187:drug effects / Q000648:ultrastructure; D008722:Methods; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D008970:Molecular Weight; D010168:Palmitates / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D011136:Polysorbates / Q000494:pharmacology; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis; D013936:Thymidine / Q000378:metabolism
D000596:Amino Acids; D010168:Palmitates; D010743:Phospholipids; D011136:Polysorbates; D011506:Proteins; D012335:RNA, Ribosomal; D003840:Deoxycholic Acid; D005643:Fucose; D012313:RNA; D004247:DNA; D005969:Glutamate Dehydrogenase; D013936:Thymidine