Nucleolus isolation

Proteins are part of a dynamic network of biomolecules that interact to regulate their localization and function within the cell. Disruption of this physical and chemical system of interactions has become the first paramount step in performing protein analysis by shotgun proteomics. Protein isolation techniques, for instance, have allowed understanding of the complex dynamics of proteins among cellular subcompartments, such as the nucleolus or the mitochondrion (6). Membrane-embedded proteins (7) and DNA-binding transcription factors (8) are two other prominent examples where inadequate protein extraction may hamper further analysis by LC-MS. 

Fig. 2 Acidic toluidine blue staining of isolated human peripheral blood lymphocytes after 0 (a), 24 (b), and 48 hr (c) stimulation with phytohemagglutinin. The resting lymphocyte has a small ring-shaped nucleolus (arrowhead in a) that increases in size and staining with continued stimulation. Scale bar, 5 /im.

Because the activity of the nucleolus is closely linked to the overall metabolic state of the cell, a number of model systems have been developed to study activation of the nucleolus from a minimal resting state. One of the best studied systems is the isolation of human peripheral blood mononuclear cells by means of a Ficoll gradient and the subsequent culture in media to which has been added the mitogenic plant lectin phytohemagglutinin (Busch and Smetana, 1970 Wachtler et ai, 1980 Ochs and Smetana, 1989). Under these culture conditions, T lymphocytes are induced to grow and eventually divide, starting from a resting... 

However, in eukaryotic cells at least one variation of this pattern is suggested by the presence of multiple forms of RNA polymerase which are associated with different regions of the cell nucleus. Each form may even show different initiator site specificities (Roeder and Rutter, 1969 1970 Kedinger et ah, 1970). One form (I or A) is located in the nucleolus (Roeder and Rutter, 1970) and is involved in the synthesis of the GC-rich (ribosomal) RNA at low ionic strength by isolated nuelei. This form is insensitive to the toxin a-amanitin. The seeond form (II or B) is less dependent on the presence of Mg and is found predominantly in the nucleoplasm, presumably in association with the euchro-matin. In the in vitro synthesis of RNA by isolated nuclei in high ionic strength in the presence of Mn, the newly synthesized product is predominately DNA-like RNA. The synthesis of this RNA is sensitive to low concentrations of a-amanitin. A third form has been detected in rat liver and sea urchins (Roeder and Rutter, 1969, 1970), but the function of this form has not yet been delineated. 

However, Spear and Gall (1973) questioned whether somatic magnification does occur in Drosophila. rRNA—DNA saturation hybridization experiments with DNA isolated from diploid tissues revealed that XX flies contain about twice as much rDNA than XO flies. Thus, in diploid cells, the rRNA genes are present in amounts proportional to the number of nucleolus organizers. In contrast to the situation in diploid cells, polytene chromosomes of the salivary glands from XX and XO flies, respectively, contain the same amount of rDNA. Since it is known that adult Drosophila flies do contain polytene chromosomes, the rDNA increase in XO flies reported by Tartof (1971, 1973) could result from the relatively higher amount of rDNA present in polytene chromosomes... 

At first, information on the chemical composition of the nucleoli was obtained by histochemical methods. For example, the treatment of such fixed preparations with ribonuclease demonstrated that ribonucleic acid is an important component of the nucleolus. Vincent and Baltus [15] made major contributions to our knowledge of the chemical composition of nucleoli when they isolated the structure from both the mature starfish and ungerminated embryos. The nucleolus of the starfish embryonic cell is so big that the experimenter need only disrupt the cell in appropriate media, usually isotonic sucrose, and then centrifuge the preparation by a process of differential centrifugation to sediment a pellet composed mainly of nucleoli. 

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