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.     

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.     

Relationship between uridine kinase activity and rate of incorporation of uridine into acid-soluble pool and into RNA during growth cycle of rat hepatoma cells

Uridine kinase activity measured in cell-free extracts of Novikoff rat hepatoma cells grown in suspension culture fluctuates about 10 fold during the growth cycle of the cells. Maximum specific activity (units/10⁶ cells) is observed early in the exponential phase and then decreases progressively until the stationary phase. The rate of incorporation of uridine into the acid-soluble pool by intact cells fluctuates in a similar manner and both the rate of uridine incorporation by intact cells and the uridine kinase actvity of the cells increase several fold before cell division commences following dilution of stationary phase cultures with freshmedium. Regardless of the stage of growth, uridine is rapidly phosphorylated to the triphosphate level by the cells. The rates of incorporation of uridine into the nucleotide pool and into RNA by intact cells fluctuate in a similar manner during the growth cycle. However, evidence is presented that indicates that alterations in the rate of incorporation of uridine into RNA are not simply due to changes in the rate of phosphorylation of uridine, but are regulated independently. Inhibition of protein synthesis by treating cells with puromycin or actidione causes a marked inhibition of incorporation of uridine into RNA, but has little effect on the phosphorylation of uridine to UTP for several hours. Thus the depression of incorporation of uridine into RNA probably reflects a decrease in the rate of RNA synthesis as a result of inhibition of protein synthesis. Inhibition of RNA synthesis by treating cells with actinomycin D does not affect the rate of conversion of uridine to UTP and thus results in the accumulation of labeled UTP in treated cells.     

Ribosome degradation in growing bacteria

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.     

Effect of essential amino acids on the phosphorylation of a 40S ribosomal protein and protein synthesis in Acanthamoeba castellanii

Reversible and multiple phosphorylation of a 40S ribosomal protein is observed in a variety of eukaryotic cells. In the primitive eukaryote Acanthamoeba, one or three phosphorylated S3 derivatives are observed during growth phase in nondefined nutrient medium (ND cells) or in chemically defined nutrient medium (D cells), respectively. In both cases, stationary phase cells exhibit nonphosphorylated S3; however, transfer of these cells into the respective fresh nutrient media results in a transient accumulation of four phosphorylated S3 derivatives. Transfer of D cells into nutrient medium, deficient in all or any single essential amino acids, leads to reversible inhibition of S3 phosphorylation and growth arrest. The low level of phosphorylated S3 is not simply the consequence of growth arrest, since in cells where growth is arrested differently, the level of phosphorylated S3 can be high. In response to amino acid deficiency, a number of other changes can be observed. These include a 2-3-fold decrease of total protein synthesis, 13 changes in the cellular protein pattern, and specific alterations in the ribosome absorbance profiles and in the distribution of poly-A+ RNA within subribosomal and ribosomal fractions. While the rate of total protein synthesis seems to be associated with the level of phosphorylated S3, the level of the synthesis of at least 10 of the particular proteins can be dissociated from the level of S3 phosphorylation.     

The conformation of ribonucleic acids in Escherichia coli ribosomes: Inferences from the mode of action of ribonuclease II

1. Ribonuclease II of Escherichia coli degrades pulse-labelled RNA associated with ribosomes and polyuridylic acid on ribosomes and in solution to mononucleotides. 2. Ribosomal and pulse-labelled RNA in solution and ribosomal RNA in chloramphenicol particles (protein-deficient ribosomes) are degraded to oligonucleotides. 3. Ribosomal RNA in mature ribosomes is not attacked by the enzyme. 4. From the mode of action of ribonuclease II, which is specific for single-stranded polyribonucleotides and does not attack helical forms, it is inferred that pulse-labelled RNA associated with ribosomes of E. coli exists as a single-stranded structure and that ribosomal RNA in chloramphenicol particles has a pronounced helical character. 5. The different behaviour of ribonuclease II towards newly synthesized RNA, ribosomal RNA and chloramphenicol-particle RNA in E. coli ribosomes is discussed.     

Poly(A) RNA metabolism during change of physiological state of Tetrahymena pyriformis cells

In the present work the metabolism of poly(A)+ RNA was investigated in cells of Tetrahymena pyriformis derived either from stationary cultures or from starved suspensions that were initiating growth. Under these circumstances the organisms derived from stationary cultures synthesize ribosomal and poly(A)+ RNA and form polysomes. In the presence of actinomycin D (actD) the observed expansion of the polysomal population is arrested. Pre-starved cells, on the other hand, start making polysomes in the virtual absence of ribosomal and poly(A)+ RNA synthesis soon after being transferred to peptone medium. In this case polysome formation is only partially sensitive to actD. These results have been interpreted as indicating that, in the beginning of growth, cells derived from stationary cultures are dependent on RNA synthesis for polysome formation, whereas pre-starved cells use pre-synthesized RNA for the same purpose.     

Physiological Changes of Carrot Cells in Suspension Culture during Growth and Senescence

Changes in the synthetic activities of nucleic acids and protein and in the amount of adenine nucleotides in Daucus carota cells have been examined during a period of rapid cell division and during the stationary phase. Cell growth and the syntheses of those macromolecules in the logarithmic phase were significantly enhanced by increasing the concentration of phosphate in the medium. In a phosphate-rich medium, RNA and protein rapidly degraded and the number of cells with denatured cytoplasm increased when the culture entered into a stationary phase because of exhaustion of glucose. On the other hand, when the growth ceased by exhaustions of phosphate and nitrogen, the cells slowly underwent physiological changes leading to cellular senescence. The value of adenylate energy charge during the growth period was about 0.8–0.9 irrespective of the growth rate. After cessation of growth the value declined to about 0.5.     

CELL CYCLE COMPARTMENT ANALYSIS OF CHINESE HAMSTER CELLS IN STATIONARY PHASE CULTURES

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G₁ and G₂ portions of the cycle; the relative proportion of these cells in G₁ increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G₂ than in G₁. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.     

Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1

The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose. The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e. from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium. The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle. The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle. The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle. Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.     

Inhibition of protein synthesis stimulates intracellular protein degradation in growing yeast cells

In exponentially growing cells of Saccharomyces cerevisiae, cycloheximide stimulated intracellular protein degradation to the same extent as did starvation for required amino acids. By using inhibitors of macromolecular synthesis and temperature-sensitive mutants defective in different steps of RNA and protein synthesis it could be demonstrated, that this stimulation of protein degradation was directly related to the inhibition of protein synthesis per se, but not connected to the cessation of ribosomal RNA synthesis or to the inhibition of cell growth.     

Ribosome Production and Protein Synthesis in the Preimplantation Rabbit Embryo

Biochemical and radioautographic data show that protein synthesis is increased markedly at the morula stage of rabbit development (60 h embryo). In the late morula an increase in cytoplasmic ribosomes is observed, suggesting that ribosome availability may be rate-limiting for protein synthesis during cleavage. Incorporated ³H-amino acids become highly localized within the nucleoli of late morulae which have been pulse-labelled for 10 min. This localization suggests that ribosomal protein synthesis is increased at the same time as ribosomal RNA synthesis has been shown to increase. Changes in both the incorporation of ³H-amino acids and cytoplasmic ribosome density were found to occur 'synchronously' in all embryonic cells during the cleavage and early blastocyst period (84 h of development). Between 84 h and 108 h, considerable differences in the number of ribosomes per unit area of cytoplasm become apparent among the cells of the blastocyst.     

Density-dependent regulation of cell growth: An example of a cell-cell recognition phenomenon

Summary Cell-to-cell contact can result in a variety of changes in the cell's physiology. For different cell types, this may include both the initiation as well as the cessation of cell growth and changes in the state of differentiation. This review examines in detail one such phenomenon, density-dependent inhibition of growth, which is observed with many fibroblasts in culture. Data are summarized which demonstrate that the cessation of growth at high cell density is in part a consequence of cell-to-cell contact. An approach to the study of the molecular basis of this phenomenon is presented based on the demonstration that plasma membranes, when bound to sparse growing cells, mimic contact inhibition of growth. The present status of attempts to purify plasma membrane proteins responsible for this effect are summarized, and the properties of these membrane proteins are compared to those of previously described soluble proteins that inhibit cellular growth.     

A method for measuring the content of a specific messenger ribonucleic acid in Escherichia coli

A method is described for measuring the porportion of a specific messenger RNA in the total RNA extracted from pulse-labelled cells. A model system consisting of total ribosomal RNA and Escherichia coli DNA is used to validate the method and to define the conditions under which it can be used.     

CHANGES IN GLUCOSE OXIDATION DURING GROWTH OF EMBRYONIC HEART CELLS IN CULTURE

A rapid and convenient method has been utilized to investigate glucose oxidation during growth of chick embryo heart cells in tissue culture. Primary isolates of chick embryo heart cells showed exponential growth when plated at low densities and exhibited density-inhibited growth as cultures became confluent. The density-dependent growth inhibition of chick embryo heart cells is associated with a marked decrease in the specific activity of glucose oxidation to CO₂. This decrease in glucose oxidation was observed as density increased as either a function of time in culture or as related to initial plating density. The decrease in ¹⁴CO₂ production associated with density-dependent inhibition of growth is due to a marked decrease in activity of the pentose phosphate pathway.     

Transient self-inhibition of the growth of Lactobacillus delbrueckii subsp. bulgaricus in a pH-regulated fermentor

An industrial strain of Lactobacillus delbrueckii subsp. bulgaricus was grown in a synthetic medium on lactose as carbon substrate, in a pH-regulated fermentor. Growth proceeded in two distinct phases separated by a transient stationary phase. Various experimental approaches were used to identify the cause of this growth arrest. Growth experiments in L. bulgaricus culture supernatant fluids collected at different cultivation times in fermentor, and supplemented or not with various nutritional solutions, enabled us to discard the possibility of a nutritional limitation. Tube cultures of L. bulgaricus in medium supplemented with various lactic acid concentrations showed a potential inhibition by this metabolic end product but confirmed that this inhibition was not responsible for the cessation of growth. It was concluded that at least one inhibitory compound was produced during the growth phase of the strain, and this compound disappeared from the medium in the transient stationary phase, enabling the growth to start again later in the culture. Indeed, the stoichiometric analysis of the culture showed, firstly, that unidentified carbon compounds were produced from lactose during growth, which were probably converted in lactic acid during the transient stationary phase and, secondly, that part of the amino acids consumed gave catabolic end products. Finally, bacteriocin-like compounds were not considered to be responsible for this growth arrest.     

The hybridization capacity of ribonucleic acid produced during hormone action

1. Measurements of hybridization with homologous DNA were used to assess the nature of the RNA synthesized during hormone action in several systems. 2. When increasing amounts of pulse-labelled rat liver nuclear RNA were annealed with constant amounts of DNA, saturation was not achieved even with RNA/DNA ratios of up to 180:1, which is taken to indicate great diversity in the species of labelled RNA molecules. In the converse experiment, when the DNA/RNA ratio was varied up to 20:1, a plateau of hybridization was observed, and the non-hybridizing RNA is believed to represent chiefly ribosomal and ribosomal precursor species. 3. In the livers of hypophysectomized and thyroidectomized rats treated with growth hormone and tri-iodothyronine, and in whole Xenopus larvae during induced metamorphosis, the synthesis of non-hybridizing RNA was consistently stimulated more than that of hybridizing RNA. This is interpreted as reflecting preferential synthesis of ribosomal RNA in response to these hormones.     

Control of cell multiplication in vitro. V. The dependence of cell multiplication on the ganglioside content

The concentration and composition of gangliosides in the cells of L-line and its clones--L-2 and Lebr 1/1, with values of saturation densities being 0.9, 1.6 and 3.2 X 10(5) cells/mc2, respectively, were compared. The transition of populations in the stationary growth phase was shown to be accompanied by an increment of ganglioside concentration and accumulation of di- and three-sialogangliosides. The data obtained are discussed in connection with the previously published results about changing intracellular cAMP content and conA-mediated agglutination at density-dependent growth inhibition L-cell variants.