METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

Protein migration from transplanted nuclei in Amoeba proteus. I. The relation to the cell cycle and RNA migration, as studied by autoradiography

Autoradiography has been used to examine the migration of proteins from a radioactively labelled amoeba nucleus following transplantation into an unlabelled homophasic amoeba. Nuclei were transferred at three times in the cell cycle coinciding with DNA synthesis (4 h post-division); a peak of RNA synthesis (25 h); and a relative lull in synthetic activity (43 h). Six amino acids were added individually to the culture medium to label the nuclear proteins. Migration of the proteins from the donor nucleus was found to be greatest following the transfer of [3H]aspartic acid-labelled nuclei and least with protein labelled with the basic amino acids. All amino acids exhibited the greatest extent of migration following the 25-h transfers, i.e., coinciding with a peak of RNA synthesis at 26-27.5 h. Actinomycin D (actD) inhibition of RNA synthesis reduced, but did not eliminate the extent of protein migration from the transplanted nucleus, thus indicating the existence of two classes of migratory proteins. Firstly, proteins, associated with RNA transport, which migrated mainly into the host cytoplasm. The second class migrated into the host nucleus from the transplanted nucleus, irrespective of RNa synthesis. The shuttling character of the latter class of proteins is consistent with a role of regulation of nuclear activity.     

The effect of amino acid starvation on nucleoside uptake and RNA synthesis in Tetrahymena

The uptake of nucleosides and the synthesis of RNA in Tetrahymena thermophila were examined following amino acid starvation. Omission of leucine, phenylalanine, or arginine from the medium resulted in a rapid decrease in the incorporation of [3H]uridine into the acid-soluble pool and acid-insoluble material (RNA). Amino acid starvation inhibited the uptake of all ribo- and deoxyribonucleosides tested but did not affect the uptake of amino acids or glucose. In addition, under the conditions used, the omission of an amino acid did not result in a large decrease in amino acid incorporation into total protein. Treatment of cells with cycloheximide or emetine gave results similar to the effects of amino acid starvation, but in these experiments the inhibition of protein synthesis was essentially complete. Nucleotide pool sizes were also measured following amino acid starvation. ATP and UTP levels were essentially unchanged, but the dTTP pool size was decreased by 40%. The decrease in RNA synthesis in vivo in the absence of an essential amino acid was reflected in the endogenous RNA synthetic activity of isolated nuclei. However, when solubilized RNA polymerase activity was measured with calf thymus DNA as template, no significant difference was observed between control and amino acid-starved cells.     

CYTOPLASMIC SYNTHESIS OF NUCLEAR PROTEINS : Kinetics of Accumulation of Radioactive Proteins in Various Cell Fractions after Brief Pulses

The synthesis of cytoplasmic and nuclear proteins has been studied in HeLa cells by examining the amount of radioactive protein appearing in the various subcellular fractions after labeling for brief periods. Due to the rapid equilibration of the amino acid pool, the total radioactivity in cytoplasmic protein increases linearly. The radioactivity observed in the cytoplasm is the sum of two components, the nascent proteins on the ribosomes and the completed proteins. At very short labeling times the specific activity of newly formed proteins found in the soluble supernatant fraction (completed protein) increases as the square of time, whereas the specific activity of the ribosomal fraction (nascent protein) reaches a plateau after 100 sec. The kinetics of accumulation of radioactive protein in the nucleus and the nucleolus is very similar to that of completed cytoplasmic protein, which suggests that the proteins are of similar origin. The rate of release and migration of proteins from the ribosomes into the nucleus requires less time than the synthesis of a polypeptide, which is about 80 sec. The uptake of label into nucleolar proteins is as rapid as the uptake of label into proteins of the soluble fraction of the cytoplasm, while nuclear proteins, including histones, tend to be labeled more slowly. The same results are obtained if protein synthesis is slowed with low concentrations of cycloheximide. The kinetics of incorporation of amino acids into various fractions of the cell indicates that the nucleus and the nucleolus contain few if any growing polypeptide chains, and thus do not synthesize their own proteins.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

Protein Synthesis in a Cell-Free Extract from Staphylococcus aureus

Cell-free Staphylococcus aureus extracts have been prepared which actively incorporate amino acids into protein. The requirements for amino acid incorporation of this preparation were strongly suggestive of de novo protein synthesis, since it showed an absolute requirement for ribosomes, 105,000 × g supernatant fluid, energy source, and magnesium ion. The stability of these extracts was greatly improved by use of dithiothreitol instead of mercaptoethanol as a sulfhydryl protecting reagent. Data were presented to show that the binding of aminoacyl-soluble ribonucleic acid to ribosomes did not require guanosine triphosphate and supernatant enzyme. The major characteristic which distinguishes this system from other cell-free systems is the much higher magnesium concentration required to maintain ribosomes intact and to obtain the maximal incorporation of amino acids. Addition of polyuridylic acid, polyadenylic acid, or polycytidylic acid caused about 60-fold, 30-fold, or 4-fold stimulation of the incorporation of phenylalanine, lysine, or proline, respectively. Studies by density gradient sedimentation indicated that radioactive polyuridylic acid or polyadenylic acid was associated with the monosomes. This complex can actively synthesize polypeptides. On the other hand, the nascent protein synthesized under the direction of endogenous messenger ribonucleic acid was associated with both polysomes and monosomes.     

Effect of Actinomycin D on the Transfer of Ribonucleic Acid from Nucleus to Cytoplasm in Lactobacillus acidophilus

After starvation for deoxyribosides, the deoxyribonucleic acid (DNA) of Lactobacillus acidophilus is restricted to a localized region of the cell. (3)H-uracil is first incorporated into such a restricted region but subsequently is found throughout the cell. This spread occurs despite the absence of protein synthesis and a major reduction in the rate of ribonucleic acid (RNA) synthesis. However, blocking RNA synthesis with actinomycin D restricts incorporation to a localized region of the cell. It is concluded that uracil is first incorporated into RNA in the bacterial nucleus from which it subsequently spreads through the cell. Actinomycin D could prevent this spread by preventing the completion of RNA molecules, which therefore do not dissociate from the DNA template.     

Effects of Phenethyl Alcohol on Phospholipid Metabolism in Escherichia coli

The incorporation of labeled precursors into the deoxyribonucleic acid, ribonucleic acid (RNA), proteins, and phospholipids of Escherichia coli cultured in the presence of phenethyl alcohol (PEA) was determined. PEA inhibited the uptake of labeled uracil to the same extent in cells exhibiting relaxed and stringent control of RNA synthesis. This indicates that PEA does not primarily affect amino acid synthesis or activation. Uptake of labeled acetate into the phospholipid fraction was more sensitive to inhibition by low concentrations of PEA than was the uptake of labeled precursors into the macromolecules. Thymine starvation or the addition of nalidixic acid (10 mug/ml) had no effect on acetate incorporation. Chloramphenicol (25 mug/ml) was a much less effective inhibitor of acetate incorporation than was PEA. The distribution of labeled acetate incorporated into phospholipids was markedly affected by the presence of PEA. The uptake of acetate into phosphatidylethanolamine and phosphatidylglycerol was inhibited, whereas the uptake of acetate into the cardiolipin fraction was unaffected. Since acetate incorporation into phospholipid was quite sensitive to PEA, we suggest that the PEA-sensitive component required for the initiation of replication may be a phospholipid(s).     

THE EFFECT OF ACTIDIONE ON MITOSIS IN THE SLIME MOLD PHYSARUM POLYCEPHALUM

Actidione, reportedly a specific inhibitor of protein synthesis, was found to reduce the incorporation of labeled amino acids into proteins of the slime mold Physarum polycephalum without drastically inhibiting the incorporation of nucleic acid precursors into RNA. This inhibitor was found to completely block the ensuing mitosis if it was added at any time between telophase and late prophase. Plasmodia given Actidione in late prophase (about the time of nucleolar dissolution) went on through telophase to reconstruction even though nuclear amino acid incorporation was drastically reduced during that period.     

Inhibition of mammalian DNA replication by dichlorobenzimidazole riboside

We have investigated the effect of the nucleoside analogue 5,6-dichloro-1-β- -ribofuranosyl-benzimidazole (DRB) on DNA synthesis in the L-929 line of mouse fibroblasts. Earlier studies have shown that this compound selectively inhibits the synthesis of a major fraction of nuclear heterogeneous RNA (hnRNA). At 60–75 μM, DRB decreases the rate of nuclear hnRNA synthesis by two-thirds and prevents the appearance in the cytoplasm of almost all poly(A)-containing messenger RNA (mRNA). At similar dose levels, DRB inhibits the overall rate of DNA synthesis as measured by thymidine incorporation by only 20% (after correction for decreased thymidine uptake into total cellular material). Analyses of density gradients of BUdR and [³H]thymidine-substituted DNA and of autoradiograms of extended fibers of [³H]thymidine-labeled DNA confirm this inhibition and suggest that DRB acts on DNA solely by reducing the rate of replication fork movement. The apparent size of replication units is unchanged by drug treatment. DRB also inhibits the transport of thymidine into cells. As determined by kinetic studies of thymidine uptake, transport is inhibited competitively by drug treatment. Several other aspects of thymidine metabolism are not affected by DRB. It does not alter the size of the total dTTP pool, and there is no breakdown of template DNA in DRB-treated cells.     

T-Even Bacteriophage-Tolerant Mutants of Escherichia coli B II. Nucleic Acid Metabolism

T-even bacteriophage-tolerant mutants are strains of Escherichia coli which can adsorb T-even phages but cannot support the growth of infective virus. Under some conditions, the infected cells are not killed. Mutant cells infected by phage T6 are able to carry out several metabolic functions associated with normal virus development, including arrest of bacterial nucleic acid and protein synthesis, incorporation of isotopic precursors into viral nucleic acids and proteins, synthesis of early enzymes of deoxyribonucleic acid (DNA) metabolism, formation of rapidly sedimenting DNA intermediates, and formation of normal levels of early and late messenger ribonucleic acid species. Phage are unable to mutate to forms capable of growth on these mutants. The nature of the biochemical alteration leading to tolerance is still unknown.     

Effect of Endotoxin and Cortisone on Synthesis of Ribonucleic Acid and Protein in Livers of Mice

The effect of cortisone and endotoxin, singly and in combination, on ribonucleic acid (RNA) synthesis in livers of adrenalectomized mice was determined. This was accomplished by measuring the incorporation either of inorganic (32)P or of (14)C-orotic acid into the RNA. Under similar conditions, the effect of these agents on the rate of protein synthesis was examined with the use of (14)C-leucine. Bacterial endotoxin was found to augment the uptake of isotope in the RNA and in the protein of the liver. These reactions did not appear to be mediated via the pancreatic hormone insulin, which was found to depress the incorporation of the radioactive compounds into RNA. Cortisone increased the uptake of isotope in liver RNA but depressed the incorporation of leucine into hepatic protein. These results indicate that the previously observed ability of endotoxin to prevent the hormone induction of hepatic enzymes, such as tryptophan oxygenase, is not associated with impaired synthesis of liver RNA or protein.     

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.     

Effect of “osmotic stress” on protein and nucleic acid synthesis in isolated tobacco protoplasts

The incorporation of labeled precursors into RNAs and proteins of isolated tobacco (Nicotiana tabacum L.) leaf protoplasts decreases with increasing osmotic pressure in the incubation medium. The incorporation of precursors into RNA and proteins is linear for 15–18 h after the isolation of the protoplasts, irrespective of the osmolarity of the culture media. The uptake of precursors is also affected by the osmolarity of the medium. However, the osmotic stress-induced inhibition of incorporation of precursors into RNA and proteins is also apparent if the differences in uptake are taken into consideration in the calculation. Incorporation of ³²P into TMV-RNA is also inhibited by osmotic stress. As assayed by the double labeling ratio technique, osmotic stress has less unequivocal effect on TMV protein synthesis.Abbreviations PP protoplast - RNase ribonuclease - rRNA ribosomal ribonucleic acid - SDS sodium dodecyl sulfate - SSC 0.1 M Na-acetate in 0.15 M NaCl - TCA trichloroacetic acid - TMV tobacco mosaic virus     

Increase of protein synthesis after isolation in dormant and afterripened Agrostemma githago L. embryos

Isolated embryos are more suitable for in vivo study of protein synthesis than non-isolated embryos because, after isolation, the uptake of labeled amino acids is about 1000 times higher. However, isolation also stimulates protein synthesis: Up to 4 h after isolation, the capacity to incorporate labeled amino acids increased 7 times. Therefore, data on incorporation obtained with isolated embryos cannot be extended to non-isolated embryos. The increase of protein synthesis was not due to synthesis of specific proteins, but was a general increase. Furthermore, ripening, dormant, and afterripened embryos showed the same degree of increase. Isolation therefore stimulates protein synthesis nonspecifically. When embryos were kept under anaerobic conditions after isolation, protein synthesis did not increase. Therefore, higher oxygen consumption after removal of the seedcoat is probably the cause of the higher incorporation capacity. Furthermore, the activation of protein synthesis lagged several hours behind the increase of oxygen consumption.Abbreviations A afterripened - D dormant - pre-rRNA precursor of ribosomal RNA     

Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver

1. The livers of rats were perfused in situ with medium containing mixtures of amino acids in multiples of their concentration in normal rat plasma. The incorporation of labelled amino acid into protein of the liver and of the perfusing medium increased with increasing amino acid concentration. During 60min. perfusions, labelling of liver protein reached a plateau, and labelling of medium protein was inhibited when the initial concentration of the amino acid mixture was more than ten times the normal plasma value. 2. Examination of polysome profiles derived from livers perfused without amino acids in the medium showed that the number of large aggregates was decreased and the number of small aggregates, particularly monomers and dimers, was increased with time of perfusion. The addition of amino acids to the perfusion medium reversed this polysome shift to an extent that was dependent on the initial concentration of amino acids. Polysome profiles derived from livers perfused for 60min. with ten times the normal plasma concentration of amino acids were essentially the same as the polysome profiles of normal non-perfused livers. 3. The ability of ribosome preparations from perfused livers to incorporate amino acids into protein in vitro decreased with increasing time of perfusion when no amino acids were added to the medium, but increased as the concentration of amino acids in the perfusion medium was increased. 4. The ability of cell sap from perfused livers to support protein synthesis in vitro was not influenced by the amino acid concentration of the perfusion medium. 5. Livers were perfused for 60min. with medium containing amino acid mixtures at ten times the normal plasma concentration but deficient in one amino acid. Maximal incorporation of labelled amino acid into liver protein, the stability of the polysome profile and the ability of ribosome preparations to incorporate amino acids into protein were found to depend on the presence of 11 amino acids: arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine. A mixture of these 11 amino acids, at ten times their normal plasma concentration, stimulated the incorporation of labelled amino acid into liver protein, stabilized the polysome profile and increased the ability of ribosome preparations to incorporate amino acids into protein to the same extent as the complete mixture. 6. It is concluded that the availability of certain amino acids plays an important role in the control of protein synthesis, possibly by stimulating the ability of ribosomes to become, and to remain, attached to messenger RNA.     

Modulation of lymphocyte nuclear matrix organization in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study

Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti-NM antibodies to concanavalin A-stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double-labelling showed, furthermore, that snRNPs and the internal staining component of PI1 were largely co-localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H-uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H-uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A- and DRB-induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery.     

Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.     

The effect of chick-liver ribonucleic acid in amino acid-incorporation systems from rat liver

1. Rat-liver microsomes, ribonucleoprotein particles and a fraction mainly consisting of microsomal membranes were tested for their ability to incorporate amino acids into protein in the presence of ATP, GTP, phosphoenolpyruvate and pyruvate kinase. Addition of polyuridylic acid or of ribonucleic acid from rat-liver nuclei stimulated the incorporating activities. 2. These protein-synthesizing systems were found to be susceptible to ribonucleic acid from chick-liver nuclei as well. The biological activity of the ribonucleic acid from chick liver was measured by its capacity to stimulate amino acid incorporation. 3. In the presence of chick-liver ribonucleic acid, the ribonucleoprotein particles from rat liver showed an increased radioactivity in ribosomal units with a sedimentation constant higher than 70s. 4. This was investigated by sucrose-gradient centrifugation or by column chromatography on agarose suspensions.