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There have been many attempts to establish the temporal relationships of histone and DNA synthesis. By histochemical techniques, both are synthesized concurrently in liver and muscle fibroblasts;' Prescott,2 using elegant autoradiographic technique, has also found concurrence in the unicellular organism Euplotes eurystomus. In amethopterin-synchronized cells, however, histone synthesis may begin prior to DNA synthesis;3 and there is evidence that their synthesis is similarly dissociated in regenerating liver.4' 5 In other studies on HeLa cells synchronized with thymidine or amethopterin,6 synthesis of "basic nuclear proteins (histones)" is detected throughout interphase, but markedly increases during DNA replication. These conflicting results probably reflect the varying techniques used for synchronization, the different cell systems examined, and the frequently inadequate means used to identify histones. Although the spatial relationships of histone and DNA synthesis have received less attention, it is generally assumed that histones are synthesized in the nucleus, since they have not been found in the cytoplasm;7 and the synthesis of a lysine-rich histone fraction by isolated thymus nuclei has recently been reported.8 However, this finding does not rule out the cytoplasmic synthesis of histones, and Bloch9 has presented suggestive evidence for such cytoplasmic synthesis in developing grasshopper spermatids. The present report describes the time and locus of histone synthesis in synchronized HeLa populations obtained by selective detachment of mitotic cells. '0 Materials and Methods.-Cells: HeLa cells, subline S3, maintained in Eagle's suspension culture medium1' were used throughout. Since this cell grows equally well in monolayer and suspension culture, large populations of synchronized cells from monolayers were routinely obtained with minimal trauma, as previously described.'0 Histone isolation and characterization: Logarithmically growing HeLa cells, 2 X 107, were centrifuged from spinner culture and resuspended in 50 ml of lysine-tryptophan-deficient medium containing 0.5 jc/ml H3-lysine and 2.0 pc/ml of C'4-tryptophan plus 2% fetal calf serum. In some instances H3-labeled arginine (0.5 uc/ml) was substituted for lysine. The specific activity of the isotopes was the highest commercially available but varied from one batch to the next. After 20 min at 37°C, cells were washed in Earle's salt solution, swollen in hypotonic Tris buffer,"2 and broken with a precalibrated Dounce-type glass homogenizer. Nuclei were then collected by centrifugation in the cold at 600 X g for 5 min. Following globulin extraction with isotonic NaCl,5 the histones and certain other proteins were solubilized from washed nuclei with 0.5 ml of 0.25 N HCl at 0°C for 30 min. Sodium dodecylsulfate (SDS) was added to the extract to a final concentration of 2%, and after 12 hr dialysis against 0.01 M phosphate buffer (pH 7) containing 0.1% SDS, samples were electrophoresed on 7.5% acrylamide gels of various lengths for 3-12 hr, depending on the desired resolution."3 Gels were mechanically fractionated"3 into Packard scintillation vials containing 1 ml of H20, allowed to soak overnight, and then diluted with 10 ml of Bray's fluid. Isotope distribution was measured in the Tri-Carb liquid scintillation counter. Histones were characterized on the acrylamide gel electropherograms as those HCO soluble nucleoproteins wh-ch showed active lysine uptake and no tryptophan incorporation above background levels. DNA and histone synthesis during the cell life cycle: HeLa cells, 2 X 108, were harvested in

THE CYTOPLASMIC SYNTHESIS OF HISTONES IN HELA CELLS AND ITS TEMPORAL RELATIONSHIP TO DNA REPLICATION