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.     

Fluctuations of DNA-dependent RNA polymerase and synthesis of macromolecules during the growth cycle of Novikoff rat hepatoma cells in suspension culture

The transition of suspension cultures of Novikoff rat hepatoma cells from the exponential to the stationary phase is accompanied by decreases of over 90% in the rates of synthesis of RNA, DNA and protein, a 90% loss of the apparent DNA-dependent RNA polymerase activity of the cells, and a disaggregation of the polyribosomes with a concomitant accumulation of 80 S and 110 S ribosomal structures. The cells also attain a minimum content of DNA, RNA and protein and a minimum size. Upon dilution of stationary phase cultures with fresh medium, the rate of protein synthesis begins to increase immediately and this correlates with a rapid reformation of the polyribosomes. The initial re-formation of polyribosomes is little affected by the presence of actinomycin D. RNA polymerase activity also begins to increase immediately after dilution and an increase in rate of RNA synthesis becomes apparent shortly thereafter. The increase in polymerase activity is inhibited by treating the cells with puromycin or actidione. Cell division commences only 9–13 hours after dilution and the rate of DNA synthesis begins to increase about midway through the lag period. During the lag period the average cellular content of protein increases about 80% and that of RNA and DNA about 30%. These increases are accompanied by a marked increase in the average size of the cells. Upon continued incubation of stationary phase cultures, the cells become irreversibly damaged physiologically before gross morphological damage becomes apparent. The irreversible physiological damage is recognized by the fact that the cells fail to recover when suspended in fresh medium.     

Reversible inhibition of ribosomal RNA synthesis in HeLa by lucanthone (Miracil D) with continued synthesis of DNA-like RNA

Ribosomal RNA synthesis was selectively inhibited in HeLa cells by lucanthone, a clinically useful schistosomicide which shares many of the properties of Actinomycin D. Synthesis of DNA-like RNA continued during complete inhibition of ribosomal RNA synthesis. Under these conditions newly synthesized DNA-like RNA accumulated normally in polyribosomes of the cell cytoplasm; most of it appeared to be messenger RNA. DNA synthesis was partially inhibited by lucanthone but protein synthesis was undisturbed. Synthesis of ribosomal RNA promptly resumed after removal of lucanthone and cell survival was not affected if exposures to the drug were limited to two hours.     

Human cytomegalovirus stimulates host cell RNA synthesis.

Human cytomegalovirus infection of human fibroblast cells (WI-38) induced cellular RNA synthesis. The RNA synthesis in infected cultures preceded the synthesis of viral DNA and progeny virus by approximately 24 h. RNA species synthesized in infected cells included ribosomal 28S and 18S; and 4S transfer RNA; all were markedly increased in comparison to uninfected cells. This induction of host cell RNA synthesis was dependent upon a protein(s) that was synthesized during the early stages of infection.     

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.     

Changes in RNA Synthesis during the Culture Growth Cycle of Tetrahymena

During logarithmic growth of the holotrichous ciliate Tetrahymena pyriformis , as well as in most cell systems, characteristic growth parameters are constant and highly reproducible. If the cells are allowed, however, to perform the complete culture growth cycle, their entry into the stationary phase is accompanied by events too complex to be attributed to "cessation of proliferation". At that stage we observed a sharp increase in uptake and incorporation of tritiated uridine. This seems to be caused by the increase in specific radioactivity of the external precursor supply following consumption of the non-radioactive counterpart. Density gradient centrifugation of pulse-labelled RNA revealed the cessation of ribosomal RNA synthesis at stationary phase to be due to a transition of pulse-labelled RNA from ribosomal to heterogenous low molecular weight RNA profiles. The decrease in ribosomal and the appearance of heterogenous RNA are discussed in terms of degradation and density-dependent selective inhibition of ribosomal RNA.
Evidence of a density-dependent inhibition of culture growth would indicate principles of growth control functioning in a similar way in unicellular organisms as observed in epithelial cells.     

RNA synthesis in the ovary and oocytes of the starfish

Qualitative studies on the in vitro uptake and incorporation of tritiated uridine into RNA of the somatic and germinal elements of the starfish ovary were carried out prior to and during hormone-induced oocyte maturation and spawning.Autoradiography of nonhormone-treated ovaries indicated that the outer ovarian wall contained the highest concentration of label, with lesser amounts in the follicle cells and least in the oocytes. Oocytes and follicle cells localized at the periphery of the ovary were labeled first, and both cells became progressively labeled throughout the ovary with time; the label first appeared localized in the nucleolus of the oocyte.Sucrose gradient analysis of the separated cellular components of prelabeled hormone-treated ovaries indicated that RNA synthesis occurred in all segments of the ovary and that the spawned oocyte fraction was the least active. Synthesis of ribosomal RNA was detectable after a lag period of approximately 4 hr. Oocytes incubated in ³H-uridine during and subsequent to 1-methyladenine-induced spawning and maturation synthesized 15–19 S and low molecular weight RNA but not ribosomal RNA. Synthesis of the 15–19 S RNA was inhibited with ethidium bromide and to a limited extent by actinomycin D. Isolated mitochondrial fractions contained most of the labeled 15–19 S RNA. These data suggest the mitochondrial origin of most, if not all, of this intermediate-weight RNA. On the basis of these studies, it appears that starfish oocytes and follicle cells are metabolically active at the transitional period from growth to maturational stages in oocytes. Synthesis of RNA furthermore apparently continues in the cytoplasm subsequent to germinal vesicle breakdown and spawning.     

Phage formation in Staphylococcus muscae cultures. XI. The synthesis of ribonucleic acid,desoxyribonucleic acid,and protein in uninfected bacteria

1. The synthesis of ribonucleic acid, desoxyribomicleic acid, and protein in S. muscae has been studied: (a) during the lag phase, (b) during the early log phase, and (c) while the cells are forming an adaptive enzyme for lactose utilization. 2. During the lag phase there may be a 60 per cent increase in ribonucleic acid and protein and a 50 per cent increase in dry weight without a change in cell count, as determined microscopically, or an increase in turbidity. 3. During this period, the increase in protein closely parallels the increase in ribonucleic acid, in contrast to desoxyribonucleic acid, which begins to be synthesized about 45 minutes after the protein and ribonucleic acid have begun to increase. 4. The RNA N/protein N ratio is proportional to the growth rate of all S. muscae strains studied. 5. While the RNA content per cell during the early log phase depends upon the growth rate, the DNA content per cell is fairly constant irrespective of the growth rate of the cell. 6. Resting cells of S. muscae have approximately the same RNA content per cell irrespective of their prospective growth rate. 7. While the cells are adapting to lactose, during which time there is little or no cellular division, there is never an increase of protein without a simultaneous increase in ribonucleic acid, the RNA N/protein N ratio during these intervals being approximately 0.15. 8. Lactose-adapting cells show a loss of ribonucleic acid. The purines-pyrimidines of the ribonucleic acid can be recovered in the cold 5 per cent trichloroacetic acid fraction, but the ribose component is completely lost from the system. 9. The significance of these results is discussed in relation to the importance of ribonucleic acid for protein synthesis.     

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.     

RNA synthesis in starved deciliated Tetrahymena pyriformis.

Tetrahymena pyriformis which has been starved for 20 h by incubation in buffer, and then deciliated, can regenerate its cilia in about 90 min while still in suspension in non-nutrient medium. The process of reciliation is accompanied by protein synthesis which begins a few minutes after deciliation and by synthesis of ribosomal and messenger RNAs during a period extending from about 1 h to about 3 h after deciliation. Although net synthesis of RNA remains at a very low level until 1 h after deciliation, a qualitative change in the translatable poly(A)-containing messenger RNA content of deciliated cells, and in particular, formation of beta-tubulin mRNA can be detected almost immediately after deciliation.     

Characterization of RNA synthesized during germination of Blastocladia ramosa zoospores

The synthesis of RNA was studied during the synchronous germination of Blastocladia ramosa zoospores. Comparison of RNA synthesis during germination of B. ramosa and Blastocladiella emersonii zoospores revealed that B. ramosa has a longer lag time before RNA synthesis is initiated and, in addition, the rate of RNA synthesis is ten-fold lower in B. ramosa. Zoospores of B. ramosa were shown to contain pre-formed messenger RNA but this messenger RNA directs only a portion of the protein synthesis which occurs during early germination. The conclusion that the remainder of the protein synthetic activity of the germinating spores is due to new message synthesis was supported by demonstrating that the timing of the initation of protein synthesis on new messages correlates with the time RNA synthesis is initiated. New message synthesis was also demonstrated by the incorporation of label into RNA which contains a poly (A) fragment. Synthesis of all classes of RNA including ribosomal, messenger, and transfer RNA was shown to be initiated at the same time. The implications of this observation are discussed.     

Thiopyronine-sensitized photodynamic effect on cell growth, RNA and DNA synthesis of Chinese hamster ovary cells

After treatment of Chinese hamster ovary (CHO) cells with very low concentrations of thiopyronine (TP; 1 microgram/ml) and visible light, a delay in growth of cell cultures (prolongation of the lag phase] was observed. The lengthened lag phase, however, was followed by normal growth of the cells. The length of the lag period is dependent on the irradiation dose applied. A similar effect on DNA and RNA synthesis could be seen after photodynamic treatment with TP in CHO cells: the maxima of RNA and DNA synthesis occur later but are not significantly reduced after treatment with low concentrations of TP and irradiation with visible light. This result is further evidence that the photodynamic effect with TP does not involve attack on nuclear DNA in eukaryotic cells.     

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.     

Synthesis of two DNA-dependent RNA polymerases in yeast

Specific activities of Saccharomyces cerevisiae RNA polymerases I and II were measured in cells growing under different nutrient conditions and throughout the mitotic cell cycle. The specific activity of RNA polymerase I (possibly the ribosomal polymerase) does not vary during the yeast cell cycle. In contrast the specific activity of RNA polymerase II (messenger polymerase) increases during the first third of the cycle and thereafter declines. The independent regulation of synthesis of these two enzymes is further emphasised by observations on the response to different nutrient conditions. Shifting cells from minimal to rich medium led to enhanced RNA polymerase I activity but very little change in activity of RNA polymerase II. Furthermore the activity of RNA polymerase I varies directly with change in growth rate whereas the activity of RNA polymerase II is approximately constant over a range of growth rates. From this data it is suggested: (i) The synthesis of these two enzymes is independently regulated; (ii) RNA polymerase I is synthesised continuously throughout the cycle whereas RNA polymerase II is synthesised periodically early in the cell cycle.