Abstract
Here we describe a new, rapid method for isolating nucleoli from Ehrlich tumor cells that preserves their morphological integrity and high transcriptional activity. Until now, methods for isolation of nucleoli were generally assumed to empty one of their three main compartments, the fibrillar center, of its contents. This new method consists of sonicating cells in an isotonic medium containing MgSO4, spermidine, and spermine, followed by separation of nucleoli through a Percoll density gradient. Using the nonisotopic approach of labelling with BrUTP, we have further investigated the dynamics of nascent ribosomal RNAs (rRNAs) within morphologically intact isolated nucleoli at the electron microscope level. We show that ribosomal transcripts are elongated in the cortex of the fibrillar center and then enter the surrounding dense fibrillar component.
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Acknowledgments
The authors acknowledge the skilful technical assistance provided by F. Skivée. This work received financial support from the Fonds National de la Recherche Scientifique Belge (grant n° 3.4540.06) to M.T., and from the Ligue Régionale de l’Aube and the Association pour la Recherche sur le Cancer (grant N° 4497) to D.P.
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Thiry, M., Ploton, D. (2008). Isolation of Nucleoli from Ehrlich Ascites Tumor Cells and Dynamics of Nascent RNA within Isolated Nucleoli. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 463. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-406-3_8
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DOI: https://doi.org/10.1007/978-1-59745-406-3_8
Publisher Name: Humana Press, Totowa, NJ
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