Protein Synthesis by Isolated Pea Nucleoli

Abstract

A new method is described for the preparation of active, nucleus-free nucleoli and chromatin in relatively high purity and in sufficient quantities to permit biochemical and electron microscopic investigation. This method consists of disintegrating previously isolated nuclei by grinding with glass beads in an isotonic medium thus liberating structurally intact nucleoli and chromatin threads. Nucleoli and chromatin are then purified by differential centrifugation in Ficoll solutions. A study of the chemical composition, submicroscopic structure, and biological activity of the nucleolar preparation has been made. An equivalent study of the chromatin material has also been carried out in order to assess the significance of chromosomal contamination in nucleolar protein synthesis. The isolated nucleoli rapidly incorporate leucine-C 14 into acid and base stable compounds in vitro. Such incorporation lasts for 20 minutes at 37°C and is enhanced by the addition of an energy-regenerating system and a complete amino acid mixture. It is independent of the nuclear pH 5 enzymes. The bulk of the incorporated label is recovered in the residual, ribosome-like nucleolar protein fraction and a small percentage is found in the acid-extractable basic proteins. The rate of protein synthesis by isolated nucleoli is more rapid than that occurring in the chromatin fraction. This is taken as an additional proof that the nucleolus is the principal site of protein synthesis in the interphase pea nucleus. I N T R O D U C T I O NIn recent years intranuclear protein synthesis has attracted considerable attention. In particular, the role of the nuclcolus and the chromatin has been under scrutiny. Thus Ficq (1) and others found the nucleolus to be thc most active site for protein synthesis. This finding has been disputed, notably by Carneiro and Leblond (2), and has led to considerablc controversy. A discussion of these results, obtained by the tcchniques of autoradiography, is found in recent reviews by Sirlin (3, 4).From the work of Allfrey and his coworkers (5-10), Rendi (11), and Wang (12), who have studied isolated nuclei and extracts from such nuclei, much has been learned about the biochemical apparatus of nuclear protein synthesis and about its basic similarities to the cytoplasmic ribosomal apparatus. In many laboratories the combination of the cytological and biochemical approach has been achieved by isolating subnuclear components such as nucleoli (13-17), but these methods have not yet been extended to protein synthesis.In previous publications we have described amino acid incorporation by isolated pea nuclei (18). Fractionation of such nuclei (19) has established the nucleolus as the principal site of nuclear protein synthesis (20,21). We now wish to show that nucleus-free preparations of isolated nucleoli of relatively high purity are able to incorporate labeled amino acids into protein and that such in-41 on

Bioinformatic analysis of the nucleolus

“…However, there is no evidence that this is related to the controversial idea of mRNA translation in the nucleus [25,26]. Yet, it should be noted that, during the 1960s, data were published reporting that amino acids could be incorporated into peptides within isolated nucleoli [27][28][29]. The high protein complexity of the nucleolus implies that either the biogenesis of ribosomes is a surprisingly complex process and/or that the nucleolus carries out additional functions, consistent with the theory of a plurifunctional nucleolus [30,31].…”

Translating DRiPs: MHC class I immunosurveillance of pathogens and tumors

“…Pioneering studies localizing incorporation of very brief pulses of radiolabeled amino acids by electron and light microscopy identified the nucleoplasm and particularly, the nucleolus as fractionally minor but clear sites of protein synthesis [142][143][144][145]. A classic study from Wu and Warner [146] showed that most rapidly labeled nuclear proteins are synthesized in the cytoplasm.…”

The Organization of the Nucleolus in Meristematic Plant Cells

“…According to data recently compiled by Vincent et al, (51), for instance, nucleoli from a wide variety of sources consist mostly of proteins (85-90%), RNA representing only 5-10% of the mass of these organelles. The situation is a great deal more uncertain as regards the actual concentration of DNA within nucleoli, and relevant data, therefore, differ markedly with authors and sources of material (5,8,9,34,37,38,51). These widely divergent reports undoubtedly result in part from the difficulty of preparing nucleoli free of contaminating chromatin.…”