Nuclear Structure and Function by Miguel Berrios (Eds.)

By Miguel Berrios (Eds.)

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Has become increasingly detailed [see Shaw and Jordan (1995), for cites of recent reviews and historical background]. , 1996). Over the past 10 years, a great deal of progress in understanding nucleolar function has been made in the yeast Saccharomyces cerevisiae. Our understanding of pre-rRNA processing has advanced the farthest and has been reviewed (Venema and Tollervey, 1995). An aspect of nucleolar function that has lagged behind, relatively speaking, is our understanding of the macromolecular assembly events that accompany the prerRNA processing steps.

Remove the needle from the syringe and expel nuclei into a 50 ml-tube on ice. Mix nuclei to create a uniform suspension. Note: If nuclei will not be used to prepare nucleoli, one may elect to perform only one Ficoll step gradient (omit the next step), and harvest less nuclei (-6 ml) at this step to achieve a reasonable level of enrichment of nuclei. However, do not attempt to prepare nucleoli starting with nuclei isolated from only one Ficoll step gradient. 15. Repeat the Ficoll step gradient using two Ficoll gradients.

By about 1 hr of digestion spheroplasts will clump, but will separate by about 2 hr. The digestion is complete when spheroplasts appear round and individual, with no clumps. Clumps of cells will not be lysed effectively, which will result in a lower yield of nuclei. 6 X 10'" cells per gram wet weight of cell pellet. 7) X 10"' cell per gram wet weight. 25 ml Glusulase, 50 p1 10 mg/ml Zymolyase 100T. , 1995), the digestion step should be followed by a recovery step in which spheroplasts are incubated in osmotically stabilized medium.

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