Activation of Protein Synthesis upon Dilution of an Arachis Cell Culture from the Stationary Phase

When a stationary phase cell culture of Arachis hypogaea L. is diluted into fresh media, there occurs a 10-fold increase in the rate of protein synthesis. The kinetics of the activation of amino acid-incorporating capacity show a lag of 10 to 15 minutes with maximal activity reached at 2 hours after dilution. The activation of protein synthesis is oxygen-dependent and is accompanied by a 2- to 4-fold increase in polyribosome content, as well as by a 3- to 4-fold increase in the rate of mRNA synthesis. Ribosomal function, as ascertained by determination of ribosomal transit time, is about 2.5 times more efficient in 2-hour diluted cultures as in cells immediately after dilution. These observations indicate that a very early response in the transition of plant cell cultures from the stationary state is an increased capacity for protein synthesis. At a molecular level, this increase in protein synthetic capacity is due in part to an increased mobilization of mRNA into polyribosomes and in part to a more efficient ribosomal translational capacity.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : I. Production of Stable, Reversible G1-Arrested Populations in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO) were grown to stationary phase (approximately 8–9 x 10⁵ cells/ml) in F-10 medium. Cells remained viable (95%) for at least 80 hr in stationary phase, and essentially all of the cells were in G₁ Upon resuspension or dilution with fresh medium, the cells were induced to resume traverse of the life cycle in in synchrony, and the patterns of DNA synthesis and division were similar to those observed in cultures prepared by mitotic selection. Immediately after dilution, the rates of synthesis of RNA and protein increased threefold. This system provides a simple technique for production of large quantities of highly synchronized cells and may ultimately provide information on the biochemical mechanisms regulating cell-cycle traverse.     

Regulation of RNA synthesis in plant cell culture: delayed synthesis of ribosomal RNA during transition from the stationary phase to active growth

Ribosomal RNA synthesis was studied during the early phases of growth activation in a cell suspension culture derived from peanut (Arachis hypogaea, L.) cotyledon. Upon dilution from stationary phase, these cells show a characteristic lag of 3 days before the commencement of cell division. An analysis of the nature of RNA synthesized during this early period of growth showed that the cells obtained immediately upon dilution from stationary phase synthesize primarily messenger RNA and essentially no ribosomal RNA. The synthesis of ribosomal RNA is delayed for about 24 hr after which it rises sharply resulting in a 2- to 3-fold accumulation of ribosomal RNA per cell during the subsequent 24-hr period. Both the messenger RNA and the ribosomal RNA were characterized by their cellular localization; by sucrose and CsCl gradient analyses, and by the determination of their base ratios.It would appear that a major facet of the lag phase in the cell growth is the diversion of a significant part of the RNA biosynthetic apparatus from the synthesis of messenger RNA to that of ribosomal RNA.     

Polyribosome metabolism in Escherichia coli starved for an amino acid

Most polyribosomes are inferred to be inert in starving cells, but some are in a dynamic state, since (1) messenger RNA continues to enter polyribosomes; (2) about 20 to 40% of the polyribosomes are labile after rifampycin addition; (3) β-galactosidase can be induced with a lag period no more than three times as long as in growing cells; and (4) the apparent rate of synthesis of protein chains, judged by the distribution of pulse-labeled protein between ribosomes and soluble protein, is about half that in growing cells.     

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G₁ phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18–22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [³H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activitites was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures. J. Cell. Biochem. 71:11–20, 1998. © 1998 Wiley-Liss, Inc.     

Growth and cell division of Mycobacterium avium

The rates of cell division and of protein, DNA and RNA synthesis upon transition of Mycobacterium avium to and from rich medium were examined. The changes in cell morphology (elongation) were also examined by optical and electron microscopy. Upon transfer from poor to rich medium, the rate of synthesis of RNA increased rapidly, followed by an increase in protein synthesis within 3 h and by an increase in DNA synthesis within 7 h; cell division began after a lag of about 10 h. Upon transfer from rich to poor medium, the preshift rates for protein and DNA synthesis changed to postshift rates after 3 h and 7 h, respectively; RNA synthesis stopped immediately, there was a transient fall in total RNA, and synthesis was resumed at a new rate only after 24 h. After the period of adjustment to new medium, the bacteria entered the postshift growth in which cell size, the increase in cell mass (absorbance at 650 nm) and viable counts, and the rates of synthesis of protein, DNA and RNA were constant. Ultrastructural examination of elongated cells during the adjustment period showed that they had septa at different stages of formation, but no evidence of fragmentation was found. It was concluded that cell division in M. avium was by binary fission, and that the notion of a life-cycle was not supported by present findings.     

Macromolecular syntheses and the course of cell cycle events in the chlorococcal algaScenedesmus quadricauda under nutrient starvation: Effect of nitrogen starvation

Daughter cells of the chlorococcal algaScenedesmus quadricauda incubated under photosynthesizing conditions in a nitrogen-free medium did not make any progress in the cell cycle. Photosynthetic starch formation continued for a period corresponding to a half of the cell cycle and then levelled off. Protein synthesis was very slow and it did not surpass double the initial amount. RNA content decayed from the start of treatment and approached about 2 pg/cell. When a synchronous population was deprived of nitrogen or of light in the middle of the cell cycle RNA synthesis stopped immediately or very soon afterwards and, in spite ofabundant intracellular nitrogen reserves, RNA content slowly declined. This degradation was much extensive in nitrogen starved cells where, eventually, the RNA content attained about half the starting value. In both experimental variants, DNA replications started at the same time as in control culture, but the final amount of DNA attained only half the control value. Protein synthesis stopped immediately in the dark. In the nitrogen-starved cells, it continued for several hours and protein content increased about 70 % of the amount present at the start of starvation. The number of daughter cells formed was proportional to the final protein content in the nitrogen-and light-deprived cells (corresponding division numbers were 6 and 4, respectively). Upon refeeding of daughter cells formed under nitrogen starvation, RNA synthesis started immediately, while protein synthesis displayed a lag of about 5 h. DNA replications were triggered at the time when the ratio of RNA to DNA content attained the same value as in the control culture.     

Protein synthesis in BHK-21 cells infected with semliki forest virus.

[3H]leucine-labeled proteins synthesized in BHK-21 cells infected with Semliki Forest virus were fractionated by polyacrylamide gel electrophoresis (PAGE). Cellular and virus-specific proteins were identified by difference analysis of the PAGE profiles. The specific activity of intracellular [3H-A1leucine was determined. Two alterations of protein synthesis, which develop with different time courses, were discerned. (i) In infected cultures an inhibition of overall protein synthesis to about 25% of the protein synthesis in mock-infected cultures develops between about 1 and 4 h postinfection (p.i.). (ii) The relative amount of virus-specific polypeptides versus cellular polypeptides increases after infection. About 80% of the proteins synthesized at 4 h p.i. are cellular proteins. Since significant amounts of nontranslocating robosomes in polyribosomes were not detected up to 7 h p.i., the inhibition of protein synthesis is not caused by inactivation of about 75% of all polyribosomes but by a decreased protein synthetic activity of the majority of polyribosomes. Indirect evidence indicates that an inhibition of elongation and/or release of protein synthesis develops in infected cells, which is sufficient to account for the observed inhibition of protein synthesis. Inhibition of over-all protein synthesis developed when virus-specific RNA began to accumulate at the maximal rate. This relationship was observed during virus multiplication at 37, 30, and 25 C. A possible mechanism by which synthesis of virus-specific RNA in the cytoplasm could inhibit cellular protein synthesis is discussed. Indirect evidence and analysis of polyribosomal RNA show that the increased synthesis of virus-specific protein is brought about by a substitution of cellular by viral mRNA in the polyribosomes.     

Reactivation of stationary Tetrahymena : A contribution to the question of G0 state

Stationary cells of Tetrahymena were reactivated to exponential growth phase by transfer to fresh medium. The sequence of resuming cell cycle events was analysed by scoring the division index, the labelling index for macro- and micronuclei and the increase in cell number. By long-term labelling it was found that all cells replicate in stationary phase cultures. They also divide eventually. Upon transfer to fresh medium a small fraction of cells (about 3%) divide immediately, whereas the rest divide 3 h later after having replicated their macronuclear DNA. The kinetics of entry into the S phase indicates that these cells have a lag period of about 2 h before they resume progress through the cell cycle. It takes more than 1 h until all cells have begun replication. These data show that in stationary cultures all cells proceed through the events of the cell cycle. The cell cycle phases are extended differentially, G1 taking the largest part. During G2 cells pass very slowly through a certain stage close to division. Under the present conditions there is no indication for cells being in a resting state that is not part of the cell cycle, from which they can be restimulated and which has been called the G0 state. The criteria to demonstrate a resting state of this nature are discussed.     

Transient Stimulation of Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase and Histone Acetylation in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Apparent Induction of Host Ribonucleic Acid Synthesis

The synthesis of cell-specific ribonucleic acid (RNA) appeared to be stimulated in human embryonic kidney (HEK) cultures infected with adenovirus 2 or 12. Deoxyribonucleic acid (DNA)-RNA hybridization experiments revealed that by 44 to 70 hr after infection with either virus, the relative amount of pulse-labeled RNA capable of hybridizing with HEK cell DNA increased considerably; such RNA was detected in both nuclear and cytoplasmic fractions. The main increase in apparent host RNA synthesis was preceded by (i) a relatively early transient stimulation of the DNA-dependent RNA polymerase activity in isolated nuclei, and (ii) a small but consistently observed increase in the rate of acetylation of lysine-rich and arginine-rich histone fractions. The Mn²⁺-(NH₄)₂SO₄ and Mg²⁺-activated RNA polymerase reactions measured in nuclei isolated from cells infected with adenovirus 2 or 12 were stimulated at about the same time; a rapid loss of polymerase activity followed. The augmentation of the two RNA polymerase reactions found in adenovirus 12-infected cells was independent of protein synthesis. After the initial increase, the acetylation rate of histones of cells infected with adenovirus 2 or 12 declined, until late in infection it was approximately 40 to 70% of the control cell rate.     

NUCLEOLAR AND BIOCHEMICAL CHANGES DURING UNBALANCED GROWTH OF TETRAHYMENA PYRIFORMIS

Numerous nucleoli can be observed in the macronucleus of the logarithmically growing ciliated protozoan Tetrahymena pyriformis; at late log phase the nucleoli aggregate and fuse. In stationary phase this fusion process continues, leaving a very few large vacuolated nuclear fusion bodies in the nucleus. When these stationary phase cells are placed into fresh enriched proteose peptone medium, the large fusion bodies begin to disaggregate during the 2.5-hour lag phase before cell division is initiated. By 3 to 6 hours after inoculation the appearance of the nucleoli in many cells returns to what it was in logarithmic cells. In view of the possible role of nucleoli in ribosome synthesis, attempts were made to correlate the morphological changes to changes in RNA and protein metabolism. The beginning of an increased RNA synthesis was concomitant with the beginning of disaggregation of the large fusion bodies into nucleoli, which was noticed in some cells by 1 hour after the return to fresh enriched proteose peptone medium. Increased protein synthesis then followed the increased RNA synthesis by 1 hour. The supply of RNA precursors (essential pyrimidines) were removed from cultures which were grown on a chemically defined synthetic medium, in order to study the relation between nucleolar fusion and synthesis of RNA and protein. Pyrimidine deprivation drastically curtailed RNA and protein synthesis, but did not cause fusion of nucleoli. When pyrimidines were added back to this culture medium, RNA synthesis was immediately stimulated and again preceded an increased protein synthesis by 1 hour. These studies suggest the involvement of unfused nucleoli in RNA and protein synthesis and demonstrate the extreme plasticity of nucleoli with respect to changes in their environment.     

Protein Synthesis in BHK-21 Cells Infected with Semliki Forest Virus

[³H]leucine-labeled proteins synthesized in BHK-21 cells infected with Semliki Forest virus were fractionated by polyacrylamide gel electrophoresis (PAGE). Cellular and virus-specific proteins were identified by difference analysis of the PAGE profiles. The specific activity of intracellular [³H]leucine was determined. Two alterations of protein synthesis, which develop with different time courses, were discerned. (i) In infected cultures an inhibition of overall protein synthesis to about 25% of the protein synthesis in mock-infected cultures develops between about 1 and 4 h postinfection (p.i.). (ii) The relative amount of virus-specific polypeptides versus cellular polypeptides increases after infection. About 80% of the proteins synthesized at 4 h p.i. are cellular proteins. Since significant amounts of nontranslocating ribosomes in polyribosomes were not detected up to 7 h p.i., the inhibition of protein synthesis is not caused by inactivation of about 75% of all polyribosomes but by a decreased protein synthetic activity of the majority of polyribosomes. Indirect evidence indicates that an inhibition of elongation and/or release of protein synthesis develops in infected cells, which is sufficient to account for the observed inhibition of protein synthesis. Inhibition of over-all protein synthesis developed when virus-specific RNA began to accumulate at the maximal rate. This relationship was observed during virus multiplication at 37, 30, and 25 C. A possible mechanism by which synthesis of virus-specific RNA in the cytoplasm could inhibit cellular protein synthesis is discussed. Indirect evidence and analysis of polyribosomal RNA show that the increased synthesis of virus-specific protein is brought about by a substitution of cellular by viral mRNA in the polyribosomes.     

Effects of Elevated Temperatures on Mengovirus Ribonucleic Acid Synthesis and Virus Production in Novikoff Rat Hepatoma Cells

The production of mengovirus in Novikoff rat hepatoma cells is progressively reduced with an increase in incubation temperature of the cells from 34 to 40 C, in spite of the fact that about the same amounts of single-stranded and double-stranded viral ribonucleic acid (RNA) are synthesized at 34, 37, and 40 C; the rate of overall protein synthesis is as high at 40 C as at 37 C. At 40 C, progeny viral RNA accumulates in an undegraded form without being incorporated into virus particles. The results suggest that virus maturation is preferentially inhibited at supraoptimal temperatures. At 42 C, on the other hand, no viral RNA is produced and no viral RNA polymerase activity is detectable in cell lysates. Failure of infected cells to form viral RNA polymerase at 42 C is probably due to an impairment of protein synthesis since most of the polyribosomes are rapidly lost during incubation at 42 C and the rate of amino acid incorporation into protein is 70% lower at 42 C than at 37 C. When infected cells are shifted from 37 to 42 C during the period of active viral RNA synthesis, viral RNA polymerase activity is rapidly lost from the cells, and viral RNA synthesis ceases within 45 min. In contrast, the RNA polymerase is as active in vitro at 42 C as at 37 C, and the activity is relatively stable at 42 C.     

Modulation of protein synthesis during the growth cycle of a culture of scarlet rose.

Dilution of a stationary phase culture of Scarlet Rose results in an increased rate of protein synthesis. This study compares the time course of this increase with the changes in polyribosome content and the levels of adenine and guanine nucleotides. During the first two hours after dilution, protein synthesis increases 2- to 3-fold; much of the large monoribosome pool that characterizes the stationary state disappears and a steady state situation is reached in which 70% of the ribosomes are in polyribosomes. Between two and eight hours, there is no further change in polyribosome content although the rate of protein synthesis increases an additional 2- to 3-fold. During this initial 8-hour period there is little change in the levels of ATP and GTP. An explanation consistent with these observations is that the initial activation (within the first 2 hours), characterized by the monoribosome to polysome transition, is at the level of a component(s) of the initiation system, and that between two and eight hours, since neither mRNA availability nor energy level are primary determinants, protein synthesis is augmented by the activation of a translational component, perhaps an elongation factor. After 24 hours, there is a proliferative phase characterized by the onset of ribosome accumulation. By day 5, maximum ribosome levels, 5-fold that of 24-hour cells, are reached, but the rate of protein synthesis increases only 2.5-fold during this period. The lack of quantitative coincidence between the changes in polyribosome content and the rates of protein synthesis again suggests that factors other than mRNA availability are involved in determining the overall rate of protein synthesis. Finally at days 6–8, while the growth of the culture is still in the exponential phase, the rate of protein synthesis per unit fresh weight drops markedly concomitant with a decline in ribosome content. At days 11–12, the monoribosome to polysome ratio begins to change with the monoribosome pool increasing. Presence of either actinomycin D or cordycepin inhibits increased protein synthesis in direct relation to the ability of these compounds to inhibit RNA synthesis. This suggests that the protein synthetic processes occurring after dilution require either the synthesis of the mRNA that is being translated or of an RNA functioning in a closely linked reaction.     

Protein SH-group concentration and RNA synthesis during the process of induction of proliferation in the stationary phase of cell culture growth]

The dynamics of intracellular protein SH-group (PSH) content was studied cytochemically in the course of stimulation of cell proliferation in stationary cultures of an established Chinese hamster cell line and of human diploid embryo fibroblasts. The results were compared with the pattern of RNA synthesis during the prereplicative period. In Chinese hamster cells immediately after medium changing in stationary cultures there is an augmentation of PSH content in parallel withe the increase in RNA synthesis rate. Later on, the rate of RNA synthesis and PSH content are seen decreasing followed by a new increase in the rate of RNA synthesis correlated with the second rise in PSH content. In stationary cultures of human diploid fibroblasts, there is also an increase in the rate of RNA synthesis and in the content of SH after medium changing, but the second wave of RNA synthesis and the second rise in PSH content are not pronounced. The variation in PSH content reflects the shift in the cell metabolism during the prereplicative period and is not attributed to changes in cell protein content.     

Effect of growth conditions on the activity of ornithine decarboxylase in cultured hepatoma cells. I. Effect of amino acid supply

Ornithine decarboxylase activity in high density, stationary phase rat hepatoma (HTC) cells in suspension culture has an extremely short half-life of between 5 and 15 minutes, as measured after inhibiting protein synthesis. Following dilution of these cells into fresh medium there is a large increase in ornithine decarboxylase activity, reaching a peak often several hundred times the initial level at about four hours. At least part of this stimulation is due to an increase in the apparent half-life of the enzyme, to between 30 and 90 minutes. Evidence is presented that the supply of amino acids can control the turnover of ODC under some conditions. For example supplementing high density cells with glutamine, asparagine, serine, glycine and proline, either singly or together, increases ODC activity and decreases its apparent turnover. The stimulation by amino acids is enhanced by serum.     

Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5–8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.     

Ornithine decarboxylase induction and nucleolar RNA synthesis in Friend leukemia cells

The induction of ornithine decarboxylase and the stimulation of nucleolar RNA synthesis following dilution of stationary phase Friend Leukemia Cells into fresh medium were studied. Ornithine decarboxylase activity and the rate of nucleolar RNA synthesis reached maximum values within 4 hours after dilution, with ornithine decarboxylase levels increasing 10–20 fold and nucleolar RNA synthesis increasing by about 60% during this period. 0.5 mM putrescine effectively inhibited the rise in ornithine decarboxylase following cell transfer, but did not prevent increases in the rate of nucleolar RNA synthesis.