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Nuclear mnonolayers of WI-38 cells prepared by the method of Tsai and Green were used to determine RNA synthesis in isolated nuclei in situ. In nuclear monolayers, incorporation of [1HJUTP into RNA is dependent on the presence of the other three nueleotide triphosphates' and is abolished by actinomycin D. The extent of RNA synthesis under these conditions was measured in density-inhibited WI-38 human diploid fibroblasts at various intervals after cell proliferation was stimulated by a change of medium.RNA synthesis increases 15 mip after the nutritional change and reaches a peak at 18 hr, which is also the peak of DNA synthesis. Thereafter RNA synthesis declines. Essentially similar results are obtained whether the endogenous RNA polymerase or a bacterial polymerase is used. Replacement of the stimulating medium by conditioned medium stops the increase in RNA synthesis that occurs in-cultures subject to continuous stimulation. Finally, RNA synthesis in nuclear monolayers, using the endogenous RNA polymerase, occurs by chain elongation only, while re-initiation occurs with the bacterial RNA polymerase.In confluent monolayers of chick, rodent, or human fibroblasts in which DNA synthesis and mitosis have all but ceased (1, 2), proliferation can be induced by applying appropriate stimuli, such as neuraminidase (3), insulin (4), proteolytic enzymes (5, 6), hyaluronidase (7), or cortisol (8), or by replacing the old medium with fresh medium containing 10% serum (9-12).One of the earliest events observed in stationary WI-38 human diploid fibroblasts stimulated to divide by a nutritional change (12) is an increase in chromatin template activity which is detectable within one hour after stimulation (13) and is noticeable whether a homologous or a bacterial (Escherichia coli) DNA-dependent RNA polymerase is used (14). Although an increase in chromatin template activity has also been reported in several other situations in which quiescent cells are stimulated to proliferate (15-21), these findings are subject to criticism, ranging from the use of an exogenous polymerase (22-28) The cell cultures were free of contamination by pleuropneumonia-like organisms, as checked by autoradiography. The cells were used for experiments 7 days after plating (33). The confluent monolavers were stimulated to proliferate by replacing the old medium with medium containing-1% (w/v) fetal-calf serum. Two controls were used: cultures in which the old medium was not changed and cultures in which it was replaced by fresh medium containing 0.3% fetal-calf serum.Isolation of E. coli RNA polymerase. RNA polymerase was isolated from E. coli K 12, as described by Burgess (34), by DEAE-cellulose chromatography and two subsequent glycerol density gradient centrifugations.Preparation of Nuclear Monolayer. After the medium was removed, the cell monolayer was washed twice with 2.0 ml of phosphate buffered-saline and then 1.5 ml of 0.5% Triton X-100 in assay buffer containing 5 mM M9gC2, 6 mM KCl, 50 mM Tris-HCl (pH 7.4), 75 mM (NH4)2SO4, and 25%/...

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Nucleoli isolated from HeLa cells are functionally active but contain large amounts of RNA and proteins (RNA/DNA ratio 1:1; protein/DNA ratio 7:1). We have isolated from the nucleolus a DNA-protein complex that has the characteristics of nucleolar chromatin (RNA/DNA ratio <0.05:1; protein/DNA ratio 1.7:1). This nucleolar chromatin has most of the transcriptional activity of the intact nucleolus and, as assayed by circular dichroism and dye binding, has largely preserved its structure. The isolation of a transcriptionally active fragment of chromatin, which constitutes only a small part of the total genome and codes for only one recognizable product, offers several advantages for the study of chromatin structure and function.The isolated nucleolus offers several advantages in a study of chromatin structure and function. It comprises less than 5% of the total genome of mammalian cells (1-4), and it can be isolated easily in a highly reproducible manner while maintaining its functional activity (3,5,6). The only gene product of the nucleolus that has been identified thus far is the precursor molecule of rRNA (6). Precursor synthesis constitutes 30-40% of total nuclear RNA synthesis (7), although the genes for rRNA represent only 0.05% of the total genome (2). The rest of nucleolar DNA is apparently satellite DNA (8, 9), which is not transcribed in vivo; and in vitro, as chromatin it is not even transcribed by an exogenous bacterial RNA polymerase (10). Thus, the nucleolus contains very active genes next to totally inactive ones, a unique combination for the study of chromatin structure and function.Unfortunately, the mammalian nucleolus, as isolated by standard procedures (3, 5) has a protein/DNA ratio 7-10:1 and an RNA/DNA ratio of 1:1 (3,4,8), both clearly in excess of the values obtained for whole nuclear chromatin that range from 1.15 to 2.7 to 1 for protein/DNA ratio and less than 0.17:1 for RNA/DNA ratio (11). To study nucleolar chromatin, it would be desirable to remove the excess RNA and proteins (presumably ribonucleoprotein particles, ref. 12). In this paper, we report the isolation of a nucleolar chromatin with a protein/DNA ratio of 1.7:1, which seems to have preserved most of the functional characteristics of the intact nucleolus. Preparation of HeLa Cell Nucleoli. HeLa cells growing in logarithmic phase, in suspension, were harvested in a Sorvall 2RC centrifuge at 800 X g, washed twice with phosphatebuffered saline at pH 7.2, resuspended in a buffer consisting of 10 mM Tris-HCI at pH 8.0,3 mM CaCl2, 2 mM Mg acetate, and 0.5 mM dithiothreitol, and lysed with a Teflon homogenizer. Triton X-100 in the same buffer was added to a final concentrationof 0.25% (wt/vol) for 3 min. The nuclei were washed at least twice in a 10 mM Tris-HCl at pH 7.4, 0.25 M sucrose, and 3.3 mM MgCl2 buffer, again resuspended in 15 ml of this buffer, layered onto 35.0 ml of 0.88 M sucrose in 10 mM Tris.HCl at pH 7.4, and 0.5 mM Mg acetate, and pelleted at 1200 X g for 10 min. Nuclei thus prepared were free of cytoplasmic...