Somatic Embryogenesis in Carrot Cell Suspension: I. PATTERN OF PROTEIN AND NUCLEIC ACID SYNTHESIS

The role of macromolecule synthesis during the early hours ofundifferentiated growth of carrot cell suspension in a mediumcontaining the synthetic auxin, 2,4-dichlorophenoxyacetic acidand during embryogenic growth in an auxin-free medium was investigated.For each set of cultures, total protein and RNA, rate of proteinsynthesis, changes in ATP pool, and rate of RNA synthesis weredetermined. Although no changes in protein and RNA contentsof cells were noted during the first 48 h of their growth infresh media, the rate of protein synthesis in the embryogeniccells markedly increased over that of the non-embryogenic cellsas early as 2 h. ATP pool size in both types of cells stabilizedwithin 1 h and it registered only a slight decrease over a 24h period. Rate of RNA synthesis increased in the embryogeniccells from 4 to 12 h following their transfer to fresh medium,after which it decreased. Transfer of carrot cells of zero embryogenicpotential to an auxin-free medium did not however elicit themacromolecule synthetic pattern characteristic of the highlyembryogenic cells.     

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

Daughter cells of the chlorococcal algaScenedesmus quadricauda were incubated under photosynthesizing conditions in a sulphur-free medium. The course of the cell cycle under these conditions was changed in daughter cells which differed in their stage of development. In absence of sulphur, advanced daughter cells with two nuclei and 2 or 4 genomes passed a cycle identical with that of control in sulphur containing medium. Each cell yielded eight binuclear daughter cells. With less advanced daughter cells (one nucleus and 1 or 2 genomes) restriction of RNA synthesis occurred near to the end of the cell cycle and protein synthesis ceased two hours later (practically at the time of the protoplast fission). The last round of DNA replication found in the control culture was not initiated in sulphur-starved culture and uninuclear daughter cells with one genome were released. If the daughter cells coming from the starved populations were kept further in the sulphur-free medium, macromolecular syntheses were dramatically restricted. Only photosynthesis continued to produce starch at a similar rate as in normally grown cells. Thus, a very large amount of starch accumulated. Supported by these reserves, starved cells refed with sulphur passed an entire cell cycle in the dark and divided into eight daughter cells. In sulphur-supplied cells, both in the dark and in light, RNA, protein and DNA synthesis started without any delay in a similar way as in the control culture. Competition for sulphur reserves occurred between the growth and division processes; the former were preferred in the light and the latter in the dark.     

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.     

Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line

The actions of insulin, hydrocortisone, prolactin and growth hormone on the synthesis of macromolecules in MCF-7 cells was determined in a serum-free defined medium. The inclusion of the polyamine spermidine in the medium was shown to enhance the insulin stimulation of the rate of [3H]uridine incorporation into RNA in a manner similar to that demonstrated for hydrocortisone. Spermidine, in addition to insulin and hydrocortisone, was also essential for prolactin to manifest a stimulation of the rate of [3H]uridine incorporation; this effect of spermidine was optimal with spermidine concentrations between 1 and 5 mM. Prolactin also stimulated the rate of [3H]leucine incorporation into total cellular protein and into an isoelectrically precipitable (pH 4.6) phosphoprotein fraction. The actions of prolactin on total protein and phosphoprotein synthesis were only expressed if spermidine, in addition to insulin and hydrocortisone, was contained in the culture medium. All of the prolactin responses were observed employing physiological concentrations of prolactin. Specificity of the prolactin responses was established by demonstrating that porcine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells.     

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.     

Application of a nonradioactive method of measuring protein synthesis in industrially relevant chinese hamster ovary cells

Due to the high medical and commercial value of recombinant proteins for clinical and diagnostic purposes, the protein synthesis machinery of mammalian host cells is the subject of extensive research by the biopharmaceutical industry. RNA translation and protein synthesis are steps that may determine the extent of growth and productivity of host cells. To address the problems of utilization of current radioisotope methods with proprietary media, we have focused on the application of an alternative method of measuring protein synthesis in recombinant Chinese hamster ovary (CHO) cells. This method employs puromycin as a nonradioactive label which incorporates into nascent polypeptide chains and is detectable by western blotting. This method, which is referred to as SUnSET, successfully demonstrated the expected changes in protein synthesis in conditions that inhibit and restore translation activity and was reproducibly quantifiable. The study of the effects of feed and sodium butyrate addition on protein synthesis by SUnSET revealed an increase following 1 h feed supplementation while a high concentration of sodium butyrate was able to decrease translation during the same treatment period. Finally, SUnSET was used to compare protein synthesis activity during batch culture of the CHO cell line in relation to growth. The results indicate that as the cells approached the end of batch culture, the global rate of protein synthesis declined in parallel with the decreasing growth rate. In conclusion, this method can be used as a “snapshot” to directly monitor the effects of different culture conditions and treatments on translation in recombinant host cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1043–1049, 2013     

Basic amino acid inhibition of cell division and macromolecular synthesis in Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae on poor nitrogen sources such as allantoin or proline was totally inhibited by addition of a non-degradable basic amino acid to the medium. Cells treated with lysine contained greatly reduced quantities of histidine and arginine. Conversely, lysine and histidine were severely reduced in arginase-deficient cells treated with arginine. When all three basic amino acids were present in the culture medium, growth was normal suggesting that synthesis of all three basic amino acids was decreased by an excess of any one of them. Inhibition of growth was accompanied by a fivefold increase in the observed ratio of budded to unbudded cells. These morphological changes suggested that DNA synthesis was inhibited. Consistent with this suggestion, addition of a basic amino acid to the culture medium substantially reduced the ability of the cells to incorporate [14C]uracil into alkali-resistant, trichloroacetic acid-precipitable material. RNA and protein synthesis, although decreased, were less sensitive to the effects of such additions.     

Kinetics of growth and development of Mycobacterium rubrum under conditions of periodic culturing

The kinetics of growth, development, carotenogenesis and changes in the composition of the reaction medium were studied in the course of Mycobacterium rubrum batch cultivation in synthetic and complex media. The culture growth was found to be composed of five phases in the synthetic medium and of four phases in the complex medium. When the culture was grown in the synthetic medium, the phenomenon of distinct diauxy was recorded by changes in the specific growth rate as well as the economic and metabolic coefficients. These growth changes were shown to be associated with the fact that the culture started to assimilate sucrose instead of glucose at the subsequent growth phases, and carbotenogenesis was found to be uncoupled with the period of active biomass and protein synthesis.     

Changes in RNA metabolism and accumulation of presumptive messenger RNA during transition from the growing to the quiescent state of cultured mouse fibroblasts.

Mouse fibroblasts maintained in tissue culture regulate total protein and ribosomal RNA synthesis co-ordinately with changes in the cellular growth state. Here we show that changes in the rate of synthesis of nuclear non-polyadenylic acid-containing RNA and the rate of accumulation and breakdown of cytoplasmic ribosomal RNA also accompany the transition from the resting to the growing cellular growth state, while the rate of synthesis of nuclear poly (A)-containing RNA and the rates of accumulation and breakdown of cytoplasmic poly(A) containing RNA (presumptive messenger RNA) are, however, only marginally changed. The small net increase (20% to 30%) in the amount of presumptive mRNA is considerably less than the observed increase in protein synthesis (two to threefold) during this transition. We also isolated and characterized extra-polysomal poly(A)-containing ribonucleoprotein particles from quiescent cultures that were similar to those particles obtained by treatment of polyribosomes with EDTA. These experiments suggest that the early increase in protein synthetic activity when quiescent, cultured cells are induced to grow is partially caused by an increased attachment of pre-existing mRNA molecules to free ribosomes.     

Model for the feedback control system of bacterial growth. II. Growth in continuous culture

A mathematical model is developed that describes substrate limited bacterial growth in a continuous culture and that is based upon the conceptual framework elaborated in a previous paper for describing the feedback control system of cell growth [S. Bleecken, (1988). J. theor. Biol. 133, 37.] Central to the theory are the ideas that the limiting substrate is converted into low molecular weight building blocks of macromolecular synthesis which again are converted into biomass (RNA and protein) and that the rates of RNA and protein synthesis are controlled by the intracellular concentration of building blocks. It is shown that a continuous culture can be simulated by two interconnected feedback control systems the actuating signals of which are limiting substrate concentration and the intracellular concentration of building blocks, respectively. Three types of steady-states are found to appear in a continuous culture, besides the well-known stable steady-state of the whole culture there exist two batchlike steady-states of the biotic part of the culture which are metastable. The model is used to analyse the steady-states and their stability properties as well as the dynamic responses of biomass, RNA, protein, building block and substrate concentrations to changes in environmental conditions. Especially the inoculation of a continuous culture and the effects of step changes in dilution rate, inlet substrate concentration and growth temperature are studied in detail. Relations between the growth behaviour of a single cell and that of a continuous culture are derived. The RNA to protein ratio is introduced as a rough measure of the physiological state of cells and it is shown that a cell reacts to environmental changes with a simple pattern of basic responses in growth rate and physiological state. There are reasons to assume that the model presented is the minimal version of a structured model of bacterial growth and represents an optimum compromise between biological relevance and mathematical practicability.     

Synthesis of macromolecules by Escherichia coli near the minimal temperature for growth

When a culture of Escherichia coli ML30 growing exponentially at 37 C in a glucose minimal medium was shifted abruptly to 10 C, growth decreased for about 4.5 hr. There was no net synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. The cells, however, respired at a rate characteristic of cells growing in the steady state at 10 C and were able to accumulate alpha-methyl-d-glucoside. When growth recommenced at 10 C, protein synthesis started at 4 hr, RNA synthesis, with a burst at 6 hr, and DNA synthesis, with a burst at 7 hr. One synchronous division occurred at about 11 hr after shifting to 10 C. There was no alteration in the steady-state RNA to protein ratio. The results are discussed in relation to other reported effects of shifts in environmental conditions. The lag at 10 C was dependent on prior conditions of growth at 37 C. Growth at 37 C under conditions giving catabolite repression were necessary for the lag to be established on shifting to 10 C.     

Mechanism of α Factor Biosynthesis in Saccharomyces cerevisiae

The biosynthesis of alpha factor, a mating-type-specific regulatory oligopeptide which is secreted by Saccharomyces cerevisiae cells of alpha mating type, was studied. In batch cultures only small amounts of the peptide were synthesized during the exponential growth phase. During the stationary phase, alpha factor was produced at a constant rate and accumulated in the culture medium. Inhibition of translation in wild-type cells by cycloheximide, or in mutant strains under conditions which blocked protein or ribonucleic acid (RNA) synthesis completely inhibited the production of alpha factor. These results indicate that the factor is produced by ribosomal translation of a specific messenger RNA and not by an extraribosomal mechanism of peptide synthesis.     

Regulation of aromatase in estrogen-producing cells

Human adipose stromal cells in monolayer culture aromatize androstenedione to estrone. The rate of aromatization is stimulated 20- to 30-fold by glucocorticoids when fetal calf serum is present in the culture medium and by dibutyryl cyclic AMP in the absence of serum. The action of dibutyryl cyclic AMP to stimulate aromatase activity is potentiated markedly by phorbol esters and inhibited by growth factors, such as EGF. In order to investigate the mechanisms underlying this multifactorial regulation, we have prepared polyclonal and monoclonal antibodies specific for aromatase cytochrome P-450. By use of these antibodies it was demonstrated that the action of these various factors to regulate aromatase activity was caused by alterations in the rate of synthesis of aromatase cytochrome P-450, whereas the synthesis of the reductase component of the aromatase enzyme complex was relatively unaffected. The changes in the rate of synthesis of aromatase cytochrome P-450 were, in turn, reflective of changes in the levels of translatable mRNA specific for this protein. In order to analyze the levels of aromatase cytochrome P-450 mRNA directly, we have isolated a cloned cDNA insert complementary to the mRNA encoding aromatase cytochrome P-450, by screening a lambda gt 11 human placental cDNA library utilizing the polyclonal anti-aromatase P-450 IgG. Use of this cDNA probe in Northern analysis of RNA extracted from human adipose stromal cells revealed that the changes in translatable mRNA resulting from incubation of the cells with the various regulatory factors were due to changes in the absolute levels of mRNA encoding this protein.     

Ribonucleic Acid and Protein Synthesis in a Mutant of Bacillus subtilis Defective in Potassium Retention

A mutant of Bacillus subtilis 168 (strain 168 KL), which had lost its normal capacity to accumulate K(+), was used to explore the interrelationship between protein and ribonucleic acid (RNA) synthesis. In contrast to the wild type, the growth rate of strain 168 KL was markedly dependent on the K(+) concentration in the medium. K(+) uptake in the mutant strain was identical to that in the parent, but the mutant was unable to retain and accumulate K(+). Protein synthesis was markedly dependent on the K(+) concentration in the medium, whereas RNA synthesis was relatively unaffected by changes in the level of K(+). Most of the RNA synthesized during K(+) depletion was ribosomal RNA; it appeared in crude extracts in the form of ribonucleoproteins particles with sedimentation values between 4S and 30S. These particles were converted into mature ribosomes when growth was allowed to resume by the addition of K(+). Simultaneous synthesis of RNA and protein was necessary for the quantitative conversion of the ribonucleoprotein particles into ribosomes. During recovery from K(+) depletion, ribosomal protein was synthesized in preference to the other proteins of the cell.     

Synthetic Capabilities of Plasmolyzed Cells and Spheroplasts of Escherichia coli

Effects of plasmolysis and spheroplast formation on deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, and phospholipid synthesis by Escherichia coli strain THU were studied. RNA and protein synthesis were severely diminished. DNA and phospholipid synthesis were inhibited, but less so; they could be partly restored. DNA synthesis could be restored by replacing thymine in the medium with thymidine, and phospholipid synthesis, by adding back small quantities of soluble cell extract. Plasmolysis effected marked reductions in rates of growth and macro-molecule synthesis, and temporarily reduced culture viability. Plasmolysis also caused an anomalous stimulation of phospholipid synthesis. Spheroplasts and plasmolyzed cells synthesized small amounts of ribosomal RNA that sedimented normally. However, this ribosomal RNA was very inefficiently packaged to ribosome subunits. Spheroplasts were unable to carry out induced synthesis of beta-galactosidase, and plasmolyzed cells were delayed in this function. Radioautographs examined in an electron microscope showed that DNA synthesis in plasmolyzed cells and spheroplasts was performed by a substantial fraction of the culture populations. That DNA and membrane were associated in the spheroplasts used in this study was suggested by formation of M-bands containing membrane and most of the cell's DNA. The results are discussed in terms of alterations of membrane structure and conformation attending plasmolysis and spheroplasting.     

Coordination of macromolecular synthesis in the slime mould Physarum polycephalum.

Microplasmodia of P. polycephalum were grown either in batch culture, in both complex and defined media to give a 3-4 fold variation in growth rate, or in a chemostate. The protein/DNA ratio of batch cultures was almost invariant, whilst the RNA/DNA ratio increased as a non-linear function of growth rate. The amount of ribosomal RNA, expressed as a fraction of total RNA, showed little variation and this was also true for the proportion of ribosomes found in polyribosomes. Calculation of the rate of protein synthesis per ribosome shows that this parameter increases by approximately 50% over the range of growth rates studied, although it should be emphasized that the effect of protein turnover has not yet been taken into account. Enrichment of batch cultures growing in a defined medium produced an increase in the rate of RNA synthesis. Data obtained with chemostat cultures differed in several respects from those described above for batch cultures, especially at low growth rates, and are discussed in relation to the early stages of differentiation of microplasmodia to spherules.     

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.     

Sensitivity of 3T3 cells to low serum concentration and the associated problems of serum withdrawal.

The sensitivity of cells to serum deprivation depends upon whether they are transformed. Most supplies of 3T3 cells are of this type and are considerably less sensitive than untransformed cells. In addition, the apparently simple manoeuvre of reducing serum levels has considerable effects on cell fragility, viability, growth rate and metabolism, which were found to be due to small changes in pH, substrate availability, cell density and other parameters, many of which cannot be attributed to the absence of growth factors from the medium. Supplementation of medium with bovine serum albumin (BSA) to compensate for low normal serum protein did not aid growth nor offset the disturbances caused by low serum levels themselves. Problems associated with the altered precursor availability for DNA, RNA and protein synthesis are also discussed.     

Dynamics in oxygen-induced changes in S-layer protein synthesis from Bacillus stearothermophilus PV72 and the S-layer-deficient variant T5 in continuous culture and studies of the cell wall composition.

Stable synthesis of the hexagonally ordered (p6) S-layer protein from the wild-type strain of Bacillus stearothermophilus PV72 could be achieved in continuous culture on complex medium only under oxygen-limited conditions when glucose was used as the sole carbon source. Depending on the adaptation of the wild-type strain to low oxygen supply, the dynamics in oxygen-induced changes in S-layer protein synthesis was different when the rate of aeration was increased to a level that allowed dissimilation of amino acids. If oxygen supply was increased at the beginning of continuous culture, synthesis of the p6 S-layer protein from the wild-type strain (encoded by the sbsA gene) was immediately stopped and replaced by that of a new type of S-layer protein (encoded by the sbsB gene) which assembled into an oblique (p2) lattice. In cells adapted to a prolonged low oxygen supply, first, low-level p2 S-layer protein synthesis and second, synchronous synthesis of comparable amounts of both types of S-layer proteins could be induced by stepwise increasing the rate of aeration. The time course of changes in S-layer protein synthesis was followed up by immunogold labelling of whole cells. Synthesis of the p2 S-layer protein could also be induced in the p6-deficient variant T5. Hybridization data obtained by applying the radiolabelled N-terminal and C-terminal sbsA fragments and the N-terminal sbsB fragment to the genomic DNA of all the three organisms indicated that changes in S-layer protein synthesis were accompanied by chromosomal rearrangement. Chemical analysis of peptidoglycan-containing sacculi and extraction and recrystallization experiments revealed that at least for the wild-type strain, a cell wall polymer consisting of N-acetylglucosamine and glucose is responsible for binding of the p6 S-layer protein to the rigid cell wall layer.     

Stimulation of guanylate cyclase and RNA polymerase II activities in HeLa cells and fibroblasts by biotin

HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10^–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.