Comparative characteristics of protein-synthesizing apparatus of diploid and aneuploid human embryo fibroblasts

The time of average polypeptide chain synthesis (tc), distribution of synthesized polypeptides according to their molecular masses, the ratio of translating and nontranslating ribosomes and polyribosomes of different size have been analyzed for diploid and aneuploid strains of fibroblasts. The magnitude of tc as well as the size of polypeptide chains synthesized were found to be similar for both kinds of fibroblasts. The relative cellular content of the translating ribosomes has been shown to decrease during the transition of cells from both strains to the stationary growth phase. The relative content of heavy polyribosomes is lower in aneuploid cells as compared with that in diploid cells. The process of translation in aneuploid fibroblasts is concluded to have no essential deviations from normal.     

Reciprocal changes in the levels of functionally related folate enzymes during the culture cycle in human fibroblasts

The activities of 6 folate enzymes were measured in extracts of human diploid skin fibroblasts during the lag, log and stationary phases of the culture cycle. The levels of 4 folate enzymes involved in nucleic acid biosynthesis, $\text{viz.}$, folate reductase, serine hydroxymethyltransferase, thymidylate synthetase and 10-formyl-THF synthetase, increased from 2–20 fold during the log phase of growth. In contrast, the levels of 2 enzymes, $\text{viz.}$, methylene-THF reductase and 5-methyl-THF: homocysteine methyltransferase, involved in regulating the levels of 5-methyl-THF, the major tissue and serum folate compound, decreased 3–4 fold during log growth, returning to high levels again only after the cells had been in the stationary phase for 5 and 20 days respectively. This reciprocal pattern of change is consistent with the known or postulated functions of these folate enzymes.     

Complementation of translational defect for growth of human adenovirus type 2 in Simian cells by a Simian virus 40-induced factor

The defective step which leads human adenovirus type 2 infection of African green monkey kidney cells (clone C14) to be abortive and its complementation in simian virus 40-transformed cells (clone T22) were studied by comparing the synthesis and function of macromolecules in these cell lines. Neither a quantitative nor a qualitative difference was detected in virus DNA replication and in virus mRNA synthesis in these cells, while a definite difference was observed in protein synthesis. The capsid proteins, such as hexon or penton, were synthesized in T22 cells but not in C14 cells. Inability of polyribosomes to synthesize the capsid proteins in C14 cells infected with adenovirus type 2 may not be due to a defect in elongation of nascent polypeptides or their release, since nascent polypeptides pulse-labelled with [³H]leucine were completely released from polyribosomes after the chase. The electrophoretic analysis of proteins synthesized in vitro with polyribosomes from either infected T22 or C14 cells using the pH 5 enzyme and S100 fraction from T22 cells revealed that hexon was synthesized with polyribosomes from T22 cells but not from C14 cells, thereby suggesting that the defect is not ascribed to a component in the pH 5 enzyme and S100 fraction, but resides in polyribosomes. The analysis of late adenovirus mRNA associated with polyribosomes in the infected T22 and C14 cells by hybridization competition or by sedimentation revealed that all the species of virus mRNA were present in the cytoplasm of these cells; however, certain species of virus mRNA larger than 20 S were absent in polyribosomes of the infected C14 cells. Sedimentation analysis of late adenovirus mRNA following separation on poly(U)-Sepharose or by membrane filtration gave the same results. These results suggest that the defect of C14 cells to support growth of adenoviruses is due to the inability of ribosomes to associate with certain species of late virus mRNA to form polyribosomes and suggest that a factor complementing this defect is induced by simian virus 40.     

Biochemical Alterations of Dermatophytes during Growth

Alterations in the biochemical constituents of mycelia were studied during the growth, development, and starvation of Microsporum quinckeanum. On the basis of dry weight, growth of this dermatophyte could be divided into four phases: lag, log, stationary, and death. The percentage of total nitrogen, inorganic phosphorus, ribonucleic acid (RNA), and protein increased rapidly during the lag phase. The percentage of protein remained constant after the initial increase; however, inorganic phosphate and RNA decreased in older mycelia. Acid-soluble materials in the cells increased in concentration as the organism aged. Chitin was present in the spores at a much higher concentration than in the mycelia. The percentage of this compound decreased rapidly until the end of the lag phase. An increase and subsequent decrease in per cent chitin occurred during the log phase. Inorganic phosphorus in the mycelia increased from the value in the spore stage to a maximum in the early log phase, and then decreased rapidly during the remainder of the growth cycle. Compounds involved in protein synthesis increased rapidly during the lag phase of growth. Changes in chemical composition of the mold during starvation indicate that carbohydrate does not form the principal endogenous reserve of M. quinckeanum, whereas lipids may represent the primary reserve material.     

Polyhydric alcohol production and intracellular amino acid pool in relation to halotolerance of the yeast Debaryomyces hansenii

Changes in polyol production and the intracellular amino acid pool were followed during the growth cycle of Debaryomyces hansenii in 4 mM and 2.7 M NaCl media. The intracellular levels of polyols were markedly enhanced by high salinity, the dominant solutes being glycerol in log phase cells and arabinitol in stationary phase cells. At low salinity arabinitol was the most prominent intracellular solute throughout the growth cycle. There were no major changes in the composition of the total amino acid pool with changes in cultural salinity. The amount of total free amino acids related to cell dry weight was 15–50% lower in cells cultured in 2.7 M NaCl as compared to 4 mM NaCl media.After subtraction of contributions from intracellular polyols the calculated cellular C/N ratio was found to be unaffected by cultural age and salinity during the late log and early stationary phase. On prolonged incubation of stationary phase cells, this ratio decreased, particularly at high salinity. The sensitivity of cells towards exposure to high salinity was measured in terms of the length of the lag phase after transference to 2.7 M NaCl media. This lag phase decreased with increasing intracellular polyol concentrations. At a given polyol content, stationary phase cells were considerably less sensitive than were log phase cells.When cultured at high salinity the mutant strain, 26-2b, grew more slowly and retained less of the total polyol produced during the early growth stages than did the wildtype. Exogenously supplied mannitol, arabinitol, and glycerol stimulated the growth of the mutant in saline media. Erythritol was without effect.Abbreviations GLC gas-liquid chromatography - TCA trichloroacetic acid     

The kinetics of the incorporation of newly synthesized ribonucleic acid and protein into the ribosomes of the uterus of the oestrogen-stimulated immature rat.

The kinetics of the synthesis of the components of polyribosomes was investigated in the uterus of the immature rat responding to the administration of oestradiol-17 beta. The hormone brings about a rapid stimulation of the association of newly synthesized mRNA with uterine ribosomes, which is maximal 2-4 h after oestradiol administration and causes the aggregation of pre-existing ribosomes into polyribosomes. Despite the striking stimulation of rRNA synthesis 2-4 h after hormone treatment [Knowler & Smellie (1971) Biochem. J. 125, 605-614], the accumulation of new rRNA into ribosomes does not reach a peak until 12 h after administration. At this time, the incorporation of new ribosomal protein is also maximal. A second peak of incorporation of newly synthesized mRNA into polyribosomes follows the peak of ribosome synthesis and coincides with the oestrogen-activated synthesis of DNA.     

Occurrence, isolation, and characterization of polyribosomes in yeast

This report details the procedural requirements for preparing cell-free extracts of yeast rich in polyribosomes. This enabled us to demonstrate the occurrence of polyribosomes in yeast, to show their role in protein synthesis, and to devise methods for their resolution and isolation. When certain precautions are met (the use of log phase cells, rapidly halting cell growth, gentle methods of disruption, sedimentation through exponential density gradients, etc.), individual polyribosome size classes ranging up to the heptosome can be fractionated and separated from their nearest neighbors. Larger size classes are resolved partially among themselves, free of smaller polyribosomes. This was confirmed by extensive electron micrographic studies of material from the various fractions obtained upon density gradient centrifugation of yeast extracts. Modifications of the gradients and procedure should allow fractionation and isolation of the larger polyribosomes, including those containing polycistronic messages. Yeast polyribosomes are disaggregated to single ribosomes by longer term grinding, cell disruption by the French pressure cell, the Hughes press, or by incubation with dilute RNAse. Yeast polyribosomes are active in the incorporation of amino acids into polypeptide; the single ribosomes exhibit only slight activity. The latter activity is probably due to the presence of a small fraction of monosomes still containing mRNA. Poly-U stimulates amino acid incorporation only in the single ribosomes.     

Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.     

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.     

Rates of synthesis and degradation of ribosomal ribonucleic acid during differentiation of Dictyostelium discoideum.

Synthesis of ribosomes and ribosomal ribonucleic acid (RNA) continued during differentiation of Dictyostelium discoideum concurrently with extensive turnover of ribosomes synthesized during both growth and developmental stages. We show here that the rate of synthesis of 26S and 17S ribosomal RNA during differentiation was less than 15% of that in growing cells, and by the time of sorocarp formation only about 25% of the cellular ribosomes had been synthesized during differentiation. Ribosomes synthesized during growth and differentiation were utilized in messenger RNA translation to the same extent; about 50% of each class were on polyribosomes. Ribosome degradation is apparently an all-or-nothing process, since virtually all 80S monosomes present in developing cells could be incorporated into polysomes when growth conditions were restored. By several criteria, ribosomes synthesized during growth and differentiation were functionally indistinguishable. Our data, together with previously published information on changes in the messenger RNA population during differentiation, indicate that synthesis of new ribosomes is not necessary for translation of developmentally regulated messenger RNA. We also establish that the overall rate of messenger RNA synthesis during differentiation is less than 15% of that in growing cells.     

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.     

Hormone-mediated translational control of protein synthesis in cultured cells of Glycine max

Proliferative growth in cultured soybean cells (Glycine max cv. Sodifuri, of cotyledenary origin) is regulated to a substantial degree by the plant hormone, cytokinin. Previously we reported that stationary phase soybean cells exhibit a rapid increase in polyribosome formation upon transfer to fresh cytokinin-containing medium. Cytokinin plays a significant role in this expansion of the cell's protein synthetic capacity through a heretofore unidentified effect on protein synthesis at the translational level. The present study was undertaken to identify the deficiency in the translational process of stationary phase cells which is overcome by cytokinin treatment. We have demonstrated that cytokinin-induced polyribosome formation was brought about by the recruitment of existing monoribosomes into polyribosomal aggregates. Three hours of cytokinin stimulation nearly doubled the total amount of [³H]leucine incorporated into nascent polypeptides. Average polyribosome size was nearly identical in stationary phase cells and in cells which were induced to grow by cytokinin. Cytokinin-enhanced protein synthetic capacity was not accompanied by changes in polypeptide chain elongation or termination rates. Partial cycloheximide inhibition of elongation was used to demonstrate that initiation of polypeptide synthesis was limiting to a slight degree both before and after cytokinin stimulation. These results suggest that cytokinin stimulates protein synthesis by making more message available for translation. We obtained direct evidence to support this hypothesis by showing that labeled poly(A)-containing, nonpolyribosomal RNA, which was synthesized during stationary phase but not translated, moves into polyribosomes upon cytokinin treatment.     

Transformed cells have lost control of ribosome number through their growth cycle.

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.     

Absence of translational control of histone synthesis during the HeLa cell life cycle

The cell-free synthesis of histone-like polypeptides has been achieved using a selected class of small polyribosomes as the only particulate fraction. This synthesis is prevented if the deoxyribonucleic acid (DNA) inhibitor, cytosine arabinoside, is added to the cells prior to disruption, and it is not detected when the cytoplasm used is derived from postmitotic (G₁) cells. When the 100,000 g supernate from pure metaphase populations was compared with that from S phase cells, the cell-free synthesis of histone-like polypeptides in the presence of S phase polyribosomes remained unchanged. These data suggest that, except for the histone messenger RNA-ribosome complex, the cytoplasmic factors requisite for histone synthesis are present throughout the cycle, and that the shut-off of this synthesis is not under translational control.     

INFLUENCE OF GROWTH STATUS AND NUTRIENTS ON EXTRACELLULAR POLYSACCHARIDE SYNTHESIS BY THE SOIL ALGA CHLAMYDOMONAS MEXICANA (CHLOROPHYCEAE)1

Increased levels of nitrogen in liquid growth medium bring about increased growth and a delay in extracellular polysaccharide production by Chlamydomonas mexicana Lewin on a per-cell basis. Addition of nitrogen to stationary phase cultures causes renewed growth and a temporary lag in polysaccharide synthesis until growth again ceases. Removal of nitrogen terminates growth, causing an immediate increase in polysaccharide synthesis. Phosphate-starved cells show a response similar to nitrogen-starved cells, indicating that the beginning of stationary phase and not nitrogen depletion causes the stimulation in extracellular polysaccharide synthesis. As similar results are assumed to occur on soil, the significance of this response is discussed.     

Variation in the latency of acid phosphatase and ribonuclease throughout the growth cycle of Tetrahymena pyriformis

The latency of both acid phosphatase and ribonuclease was studied throughout the growth cycle of the ciliate protozoan Tetrahymena pyriformis and was found to be remarkably low in cells in the logarithmic phase of growth. The latency increased progressively throughout the log phase until it reached a maximum just after the cells had entered the stationary phase. The specific activity of acid phosphatase remained constant throughout the whole of the growth cycle while that of ribonuclease decreased as the cells began to leave log phase. The possibility is discussed that all rapidly dividing cells have a high proportion of their acid hydrolases outside the lysosomes and that these free hydrolases may have a function in cell division.     

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.     

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.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells during their erythroid differentiation

The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post-exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis by demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin.