Rat livcr nuclei, after prcliminary isolation in 2.2 molar sucrose solution, were separated into density classcs by ccntrifugation at 95,000 g for 45 to 85 minutes in a sucrose dcnsity gradient (density range, 1.28 to 1.33). Nuclei from normal liver separated into thrce bands with averagc DNA phosphorus content per nucleus of 0.67, 0.84, and 0.93 picogram for top, middle, and bottom bands, rcspectivcly. Nuclei from rcgcncrating liver (26 hours after oncthird hepatectomy) yiclded thrce bands and a pellet fraction with average DNA phosphorus content per nucleus of 0.76, 1.02, 1.38, and 1.51 picograms (top to bottom of tube). This method appears capable of yielding nuclei which have increased their DNA content prior to mitosis, and this procedure should be valuable in studies of biochemical changes which occur in nuclei prcparing for mitosis.In a preliminary communication (7) we reported the isolation of rat liver nuclei with high deoxyribonucleic acid (DNA) content by density gradient centrifugation of nuclei from regenerating liver. In the interim, Falzone and coworkers (6) have reported a successful fractionation of normal rat liver nuclei into diploid and tetraploid classes by a density gradient technique which differs considerably from that used in this laboratory. The present paper is a more detailed report of our work on this subject.Normally, individual ceils within a tissue are dividing in a rather random manner. However, considerable synchronization of cell division can be obtained in liver during short intervals following partial hepatectomy. At a given time, one should find more cells preparing to divide and thus a greater proportion of nuclei with increased DNA content in regenerating tissue than in normal tissue. Furthermore, since DNA has a higher density than other nuclear constituents, such as protein or lipid (with the exception of ribonucleic acid (RNA), which has higher density but lower concentration than DNA), one might expect that those cells or nuclei with increased DNA content would have higher densities than those with lesser DNA content, when other factors remain constant.A physical technique useful for the separation of particles of various densities is available in density gradient centrifugation. With this technique nuclei of normal and regenerating liver of the rat can be separated into various density classes which differ in DNA content.
MATERIALS AND METHODSMale Wistar strain rats, approximately 5 to 7 months old and weighing 275 to 400 grams, were used in these experiments. For each experiment, three rats were selected and subjected to one-third hepatectomy by removal of the left lateral lobe. After 26 hours, the animals were sacrificed, and the liver was homogenized in 0.25 molar sucrose containing 0.0018 molar calcium chloride (9) and centrifuged at 600g for 15 minutes in a refrigerated centrifuge. The crude nuclear fraction was suspended in 2.2 molar sucrose 231 on