Changes in the synthesis of ribosomal ribonucleic acid and of poly(A)-containing ribonucleic acid during the differentiation of intestinal epithelial cells in the rat and in the chick.

Epithelial cells were isolated from rat and chick small intestine by techniques which separated subpopulations of differentiating villus and upper crypt cells from each other and from populations of mitotically dividing lower crypt cells. Incorporation of precursors into epithelial-cell DNA, cytoplasmic rRNA and cytoplasmic poly(A)-containing RNA occurred in the lower crypt cells in vivo when precursor was supplied from the vascular system of the intestine. Incorporation of precursor into 28S and 18S rRNA continued in the upper crypt cells, but occurred to only a very slight extent (if at all) in villus cells, whereas incorporation into poly(A)-containing RNA continued (at a diminishing rate) as the differentiating cells migrated along the villi. When precursor was supplied from the intestinal lumen, its incorporation into DNA and into rRNA of crypt cells was not very different from that observed with the other mode of precursor administration, but incorporation into villus-cell poly(A)-containing RNA then occurred at essentially the same rate in all intestinal epithelial cells in vivo. Cytoplasmic poly(A)-containing RNA appeared to turn over in rat crypt cells with a half-life not exceeding 24 h; crypt-cell rRNA showed no turnover and no evidence could be found for the presence of 'metabolic DNA'.     

RELATIONSHIP BETWEEN RNA SYNTHESIS, CELL DIVISION, AND MORPHOLOGY OF MAMMALIAN CELLS : I. Puromycin Aminonucleoside As An Inhibitor of RNA Synthesis and Division in HeLa Cells

Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P³² and H³-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division within the period studied (3 generation times). The cell content of RNA returned to normal when the inhibitor was removed.     

Effect of puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

Aurintricarboxylic acid (ATA) and DNA synthesis. II. Effect of ATA on structure and function of the crypts of the small intestine.

ATA affects only slightly DNA synthesis of continuously replicating cells. A single injection of the drug reduces the incorporation of (3H)-thymidine into DNA of crypt cells to only 62% of the control. The effect on DNA synthesis is preceded by a slight inhibition of protein synthesis, and by a partial decrease in the number of dividing cells. On the contrary, the incorporation of (3H)-uridine into RNA was enhanced. Electron microscopic studies revealed no cytologic abnormalities in ATA-treated animals. In view of the fact that ATA at the same concentration inhibits DNA synthesis of growth stimulated cells to 100% (Novi, 1976), it was suggested that the drug may become an useful tool in inducing a preferential inhibition of growth stimulation.     

Incorporation of [2-14C orotic] acid into chromatin RNA of tissues of animals of various ages

The structure and biological activity (the level of the labelled precursor incorporation into RNA) of active and repressed chromatin of the liver and small intestine mucosa were studied in adult (6-8 months) and old (24-26 months) rats. The content of repressed chromatin fraction in both tissues is found to increase with age. In the liver of old rats the level of [14C[ orotic acid incorporation into RNA of chromatin fractions decreases, radioactivity of the acid-soluble fraction being unchanged. In the small intestine mucosa a high leve of [14C] orotic acid incorporation into chromatin RNA with ageing is due to an increase in permeability of the mucosa cells.     

Evidence that alterations in small molecule permeability are involved in the Clostridium perfringens type A enterotoxin-induced inhibition of macromolecular synthesis in Vero cells

The mechanism by which Clostridium perfringens enterotoxin (CPE) simultaneously inhibits RNA, DNA, and protein synthesis is unknown. In the current study the possible involvement of small molecule permeability alterations in CPE-induced inhibition of macromolecular synthesis was examined. Vero cells CPE-treated in minimal essential medium (MEM) completely ceased net precursor incorporation into RNA and protein within 15 minutes of CPE treatment. However, RNA and protein synthesis continued for at least 30 minutes in Vero cells CPE-treated in buffer (ICIB) approximating intracellular concentrations of most ions. Addition of intracellular concentrations of amino acids to ICIB (ICIB-AA) caused a further small but detectable increase in protein synthesis in CPE-treated cells. ICIB did not affect CPE-specific binding levels or rates. Similar small molecule permeability changes (i.e., 86Rb-release) were observed in cells CPE-treated in either ICIB or in Hanks' balanced salt solution. Collectively these findings suggest that CPE-treatment of cells in ICIB-AA ameliorates CPE-induced changes in intracellular concentrations of ions and amino acids and permits the continuation of RNA and protein synthesis. These results are consistent with and support the hypothesis that permeability alterations for small molecules are involved in the CPE-induced inhibition of precursor incorporation into macromolecules in Vero cells.     

Effect of 72 Hz pulsed magnetic field exposure on macromolecular synthesis in CCRF-CEM cells.

Cells from the T-lymphoblastoid cell line, CCRF-CEM, have been exposed in vitro to a quasirectangular, asymmetric electromagnetic field pulsed at 72 Hz at 37 degrees for periods of 30 min to 24 h. RNA synthesis, assessed by incorporation of 3H-uridine, increased (relative to control cells) 2-fold after 30 min in exposed cells and achieved its greatest increase of 3.2-fold relative to controls after 2 h exposure. Increased precursor incorporation was observed at all subsequent exposure times up to 24 h. Synthesis of mRNA was similar, but not identical to that observed with total cellular RNA. Additionally, protein synthesis, determined by incorporation of radioactive precursor into acid-precipitable material, was increased 2.8-fold, compared to controls, after 2 h exposure. Longer exposure times resulted in an exponential decrease in precursor incorporation to 1.1-times control levels after 24 h. Using a dye reduction assay, mitochondrial activity was also found to be increased over a 24 h exposure period. No effect of electromagnetic field exposure was found on cellular synthesis of DNA. These data are generally consistent with other reports documenting effects of electromagnetic field exposure on macromolecular synthesis in vitro.     

Effects of cytosine arabinoside, 6-aminonicotinamide, and 6-mercaptopurine riboside on ectoderm and mesoderm of mouse limb buds.

The effects of cytosine arabinoside, 6-aminonicotinamide, and 6-mercaptopurine riboside on the incorporation of [14C] glucose moieties and [32P] phosphate into acid-soluble material and lipids, RNA, DNA, and protein were measured in the dissected mesoderm and ectoderm of mouse limb buds at the 42-45 (day 11) somite stage. Due to the different proliferative capacities of the two tissues the incorporation of the precursors into mesodermal cells was considerably higher the than into ectodermal ones. Cytosine arabinoside inhibited the incorporation of the precursor moieties only into DNA, but very early after its application. This effect was more obvious in mesoderm than ectoderm. 6-Aminonicotinamide interfered only with glucose metabolism, whereas the incorporation of phosphate was not affected. 14C radioactivity in the various cell components was similarly reduced in mesoderm and ectoderm. 6-mercaptopurine riboside caused an increased incorporation of precursor material in all fractions studied in the mesoderm as well as in the ectoderm during the first 12 hours. This was succeeded by a dramatic decrease of incorporated 14C and 32P radioactivity. Differences of response in the tissues could not be detected with this drug. It is suggested that the malformations of the extrmities caused by these antimetabolites may be predominantly attributed to changes in the cell function rather than to gross effects on cell metabolism.     

THE TIMING OF DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE CELL CYCLE OF SACCHAROMYCES CEREVISIAE

Randomly dividing cultures of Saccharomyces cerevisiae were briefly exposed to radioactive adenine and then treated successively with dilute acid, ribonuclease, buffered formaldehyde, and NaOH. This treatment was shown to remove virtually all the radioactivity of the labelled cells other than that in DNA. Thus, in subsequent autoradiographs, only cells which had been synthesizing DNA during exposure to the precursor were labelled. The ages of these individuals within the cell cycle were estimated by measuring their sizes. This revealed that incorporation into DNA occurred almost exclusively during the first quarter of the cell cycle, starting with the initial appearance of the bud. This behaviour agreed closely with that of cells growing in artificially synchronized cultures.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

PROTEIN TURNOVER DURING MATURATION OF MOUSE BRAIN TISSUE

The measurement of protein turnover involves the product of the rates of protein synthesis and degradation. It is the dynamic balance between these two components that determines the measured net rate of protein synthesis. The data reported here show that brain cells from newborn animals incorporate arginine-¹⁴C into acid-insoluble protein at a rate 10-fold greater than the rate for brain cells obtained from 15-day old animals. This difference in incorporation occurred even though the rate of arginine accumulation and the resulting pool size of radioactive precursor were similar for both ages. The measurement of protein turnover in brain cell suspensions prepared from 1-day old animals was shown to be complex and to exhibit a cyclic phenomenon in regard to arginine-¹⁴C incorporation into and release from protein. The variation in half-life calculations (0.5–3.5 hr) due to this cyclic phenomenon is discussed. Although puromycin was added in an attempt to amplify the rate of degradation by preventing the synthesis of new protein, it was found that degradation was inhibited as well, suggesting a relationship between protein synthesis and degradation.     

DNA replicatiion and cell-cycle progresssion of cultured mouse FM3A cells after treatment with 8-methoxypsoralen plus near-UV radiation

To investigate the response of cells to one type of DNA damage — namely DNA crosslinks — cell-cycle progression and macromolecular synthesis were studied with cultured mouse FM3A cells. Treatment of the cells with low doses of 8-methoxypsoralen (8-MOP) plus near-UV radiation (0.1 μg/ml plus 5 kJ/m² or 1.0 μg/ml plus 1–2.5 kJ/m²)_halted the progression of cells through the cell cycle temporarily for the first several hours. Then the cells resumed progression through the cell cycle, and most of the cells reached, and were finally arrested at, the G2 phase of the cycle. There was a rapid decrease of incorporation of [³H]thymidine into cellular DNA immediately after the treatment. Then, after 8 h of incubation, the incorporation of [³H]thymidine recovered to some extent depending on the dose of 8-MOP plus near-UV radiation. Thus the decrease and recovery of the incorporation of [³]Hthymidine were correlated with the halt and resumption in the cell-cycle process.Synthesis of RNA and protein was measured by determination of the amounts in the cells or by the incorporation of radioactive precursors after treatment. RNA and protein synthesis were stimulated by low doses of 8-MOP plus near-UV radiation, but inhibited severely by high doses.     

Protein-carbohydrate interaction. A turbidimetric study of the interaction of concanavalin A with amylopectin and glycogen and some of their enzymic and chemical degradation products

1. RNA and protein synthesis was studied during the incubation of excised radish cotyledons in nitrate, conditions that induced nitrate reductase activity in the tissue. 2. Synthesis of total RNA and protein, as measured by the incorporation of radioactive precursor, was significantly stimulated in the presence of nitrate (compared with chloride control), but was decreased in the presence of ammonium nitrate, which induced higher enzyme activity. 3. Synthesis of RNA and protein was required for induction of enzyme activity, as determined by using the inhibitors actinomycin D, puromycin and cycloheximide. 4. On the basis of 5-fluorouracil inhibition, the synthesis of only DNA-like RNA was required for induction, but no differences, either quantitative or qualitative, were observed in DNA-like RNA synthesis in the presence or absence of induction. 5. A 100-fold purification of the nitrate reductase activity showed no increase in nitrate reductase protein, nor any increased incorporation of radioactive precursor into nitrate reductase protein in the induced versus the control system. Such results suggested that the protein synthesis required for induction may be for a protein other than nitrate reductase.     

DNA synthesis in the elongating nondividing cells of the lentil epicotyl and its promotion by gibberellin

Two-day-old lentil seedlings, (Lens culinaris Med.) were incubated for a 48-hour period with and without gibberellin (GA) in the presence and absence of 5-fluorodeoxyuridine (FUDR). The number of cells per epicotyl did not increase during this period. Growth of the epicotyl was thus due to cell elongation alone.

The elongating cells of this tissue synthesized DNA. GA promoted and FUDR inhibited cell elongation, DNA synthesis, and RNA synthesis in the tissue.

FUDR promoted uptake of thymidine and thymidine incorporation into cellular DNA, presumably by inhibiting synthesis of endogenous thymidine. Presence of GA promoted thymidine incorporation into cellular DNA and uridine incorporation into cellular RNA. In either case, there was no effect on the uptake of the precursor into the tissue.

Fractionation of thymidine-labeled nucleic acids on a MAK column showed that thymidine was exclusively incorporated into the DNA fraction. Presence of GA promoted thymidine incorporation into this fraction and also increased the amount of ribosomal RNA.

The data provide direct evidence for the conclusion that DNA synthesis is necessary for elongation of certain plant 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.     

Stimulation of incorporation of nucleic acid precursors into HeLa cells caused by provaline

1. The effect of proflavine and other acridines on the incorporation of precursors into the nucleic acids of HeLa cells was examined. 2. Relatively low concentrations (50mum) of proflavine completely inhibited incorporation of precursors into DNA, but allowed a small extent of incorporation into RNA. 3. Acridine-resistant incorporation into RNA was unaffected by actinomycin D at 2mug./ml. and persisted even at high concentrations (500mum) of many acridines. 4. A few combinations of acridine and precursor, notably 250mum-proflavine and [(14)C]adenine, caused a stimulation of incorporation. 5. The proflavine-stimulated incorporation was into alkali-stable di- and tri-nucleotides. 6. It was concluded that the effect was due to the preferential inhibition of degradation of a fraction of RNA that normally turned over, thus allowing small radioactive oligonucleotides to accumulate in the cells.     

Automatic monitoring of biochemical parameters in tissue culture. Studies on synchronously growing mouse myeloma cells

An instrument is described that will maintain a population of mammalian cells at constant cell density while automatically monitoring the growth rate of the culture and the extent of precursor incorporation into a variety of cell products. The apparatus was used in an investigation of cyclic changes in the incorporation of labelled precursors into the DNA, RNA, total protein and myeloma protein synthesized in synchronous cultures of a mouse myeloma line. The incorporation of [(3)H]uridine into trichloroacetic acid-insoluble material reveals a slight periodicity, with maxima and minima corresponding to late S phase and the mitotic phases respectively. The incorporation of [(3)H]lysine into total intracellular protein also shows a slight oscillation, with maxima and minima occurring during the respective G2 and mitotic phases. Cyclical changes in the synthesis of serologically precipitable myeloma protein were found to vary somewhat according to the conditions used to synchronize the cells. In experiments conducted with 4.0mm-thymidine, maximal incorporation of label took place during S phase or early G2 phase. Experiments with 1.0mm-thymidine revealed a significantly less marked periodicity of myeloma protein synthesis.     

NUCLEOLAR NECKLACES IN CHICK EMBRYO MYOBLASTS FORMED BY LACK OF ARGININE

The round nucleoli of chick embryo myoblasts, when grown in a culture medium devoid of arginine, unravel in several days into 5–20 µ long, beaded strands termed nucleolar necklaces (NN). Addition of arginine reverses this change. The NN contain protein, RNA, and traces of DNA as determined cytochemically by enzyme digestion and by acridine-orange fluorescent staining. When a cell containing the beaded strand is treated with agents, such as actinomycin D, that prevent rRNA polymerase action, the strand collapses and condenses into a small dense nucleolus with segregated regions of ribonucleoprotein (RNP) and deoxyribonucleoprotein (DNP). The properties of the NN appear to resemble those of the nucleolar necklaces of amphibian oocytes. Cycloheximide or puromycin inhibition of general protein synthesis does not lead to NN formation. We suggest that NN formation during arginine starvation may be a result of a singular depletion of some rapidly turning over, arginine-rich proteins that normally attach to ribosomal RNA precursor molecules during their synthesis in the processing towards maturation of the ribosomes.