Isolation of transcriptionally active chromatin from mammalian nucleoli.

Abstract

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...

Chapter 3 Isolation of Nuclei and Nucleoli from the Yeast Saccharomyces cerevisiae

“…Methods for isolating subnuclear fractions enriched in nucleoli ("nucleoli") from various cell types have been described and used to explore requirements for Pol I-dependent transcription in vitro, and to identify nucleolar proteins (Albanese and Studzinski, 1979;Beebee and Butterworth, 1977;Bombik et al, 1977;Jackowski et al, 1976;Spohn et al, 1985). Isolated nucleoli have been useful in the identification of ribosome subunit precursors in cultured cells (Kumar and Warner, 1972;Vaughan et al, 1967;Warner and Soeiro, 1967).…”