Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : III. Effect of Cycloheximide on the Recovery from B12-Induced Unbalanced Growth

When cycloheximide is added to (B12)-deficient cultures before or after replenishment of the cells with B₁₂, reversion of these cells is inhibited. This inhibition is not caused by interference of the inhibitor in the uptake of B₁₂ as measured by division kinetics. Cycloheximide does not inhibit the initial increase in the rate of DNA synthesis caused by B₁₂ replenishment, but within 30–45 min the rate decreases and DNA synthesis ceases. Cycloheximide added to replenished deficient cells after completion of DNA duplication inhibits cell division. The total cellular protein and RNA in replenished cells treated with cycloheximide does not change. B₁₂ added to deficient cells does not stimulate the incorporation of [¹⁴C]leucine into protein during resumption and completion of DNA duplication. However, there is a large increase in [¹⁴C]leucine incorporation into the protein of these cells soon after completion of DNA duplication and before resumption of cell division. The addition of cycloheximide to B₁₂-replenished or to nonreplenished deficient cells rapidly inhibits the incorporation. We suggest that the addition of B₁₂ accelerates the rate of DNA synthesis in the deficient cells and that possibly no new protein synthesis is required except for mitosis. However, protein synthesis is needed for continuous DNA synthesis.     

Protein metabolism in senescing wheat leaves : determination of synthesis and degradation rates and their effects on protein loss

Wheat leaves (Triticum aestivum L.) at the moment of their maximum expansion were detached and put in darkness. Their protein, RNA and DNA contents, as well as their rates of protein synthesis and degradation, were measured at different times from 0 to 5 days after detachment. Rates of protein synthesis were measured by incorporation into proteins of large amounts of [³H]leucine. Fractional rates of protein degradation were estimated either from the difference between the rates of synthesis and the net protein change or by the disappearance of radioactivity from proteins previously labeled with [³H]leucine or [¹⁴C]proline.

Protein loss reached a value of 20% during the first 48 hours of the process. RNA loss paralleled that of protein, whereas DNA content proved to be almost constant during the first 3 days and decreased dramatically thereafter.

Measurements of protein synthesis and degradation indicate that, in spite of a slowdown in rate of protein synthesis, an increased rate of protein breakdown is mainly responsible for the observed rapid protein loss.

     

The β-exotoxins of Bacillus thuringiensis: III. Effects on in vivo synthesis of macromolecules in an insect system

Heat stable β-exotoxin, purified from fermentations of Bacillus thuringiensis, induced malformed mouthparts in adult cabbage loopers, Trichoplusia ni, when mature larvae were injected with 9 ng β-exotoxin per 280 mg larva. Larvae injected with 26 ng β-exotoxin produced 50% adults with malformed mouthparts. Protein and nucleic acid synthesis was inhibited by presence of β-exotoxin. Specifically, β-exotoxin inhibited in vivo incorporation of ¹⁴C-valine, ¹⁴C-uracil, and ¹⁴C-thymidine into protein, RNA, and DNA, respectively. Percent inhibition, 120 min post-injection of 50 μg β-exotoxin per larva, was 53% (protein), 36% (RNA), and 41% (DNA). The asymptote for inhibition of synthesis was reached ∼30-min after injection of β-exotoxin.     

Induction of deoxyribonucleic Acid synthesis in potato tuber tissue by cutting

Incorporation of ³²P-orthophosphate was found in the DNA fraction of aerobically incubated potato discs when examined by methylated albumin kieselguhr column chromatography. The estimation of DNA content of the discs was by a method developed for starchy tissues and showed that the incorporation of ³²P was due to net synthesis of DNA. The DNA content of a disc rapidly increased after a lag period of about 12 hours. The increase continued during the entire test period although at a lower rate during the later period of aging. DNA synthesis was further examined by measuring the rate of incorporation of ³H-thymidine. The striking similarity which was found between changes in the rate of DNA accumulation and in the rate of ³H-thymidine incorporation indicates that the incorporation of ³H-thymidine actually represents the net synthesis of DNA. Although the experiments with microautoradiography revealed that DNA synthesis occurred exclusively in nuclei, no signs of cell division were detected by microscopic observation. DNA synthesis in potato discs was further examined by using inhibitors of protein and RNA synthesis and was sensitive to those inhibitors. The significance of the present results is discussed in relation to the role of wounding in the induction of DNA synthesis.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

Aspects of DNA, RNA and protein synthesis during somatic embryogenesis in a carrot cell suspension culture

Changes in DNA, RNA and protein content, incorporation of ³H-thymidine, ¹⁴C-uridine and ³H-leucine and template activity of chromatin were investigated in the early process of somatic embryogenesis in a carrot (Daucus carota L. cv. Kurodagosun) cell suspension culture using a synchronous system. An embryogenetic culture in a medium containing 10^-7M zeatin was compared with a non-embryogenetic culture in a medium containing 10^-7M zeatin and 5 x 10^-7M 2,4-D. DNA was synthesized very actively prior to and during the formation of globular embryos in the embryogenetic culture. The RNA and protein content per tube increased at an almost constant rate in both cultures, while the rate of incorporation of labelled precursors of RNA and protein rose much more prior to active DNA synthesis in the embryogenetic culture than in the non-embryogenetic culture. Template activity of chromatin was high in the early stage of embryogenesis in the embryogenetic culture. The results obtained here showed that synthesis and turnover of RNA and protein became active prior to active DNA synthesis in the early stage of embryogenesis, and that these changes at macromolecular levels may play important roles in embryogenesis.     

Induction of deoxyribonucleic Acid synthesis in potato tuber slices: role of protein synthesis

Timing of protein synthesis which is a prerequisite to DNA synthesis induced in potato tuber tissue (Solanum tuberosum L.) by cut injury has been studied using cycloheximide. The induction of DNA synthesis which was measured by incorporation of ³H-thymidine was completely inhibited when the inhibitor was applied to the tuber discs immediately after slicing. When the application of cycloheximide was delayed for 6 hours or more after slicing, DNA synthesis was observed but its rate was reduced to 20% of control. The inhibitory effect of cycloheximide, however, rapidly decreased when the inhibitor was applied at 6 or less hours immediately prior to determination of DNA synthesis. The effect of cycloheximide on the incorporation of ¹⁴C-leucine suggests that the change in the effect of cycloheximide on the induction of DNA synthesis is not due to incomplete inhibition of protein synthesis. Cycloheximide did not have significant effects on either uptake or phosphorylation of ³H-thymidine in the discs. Inhibition of both protein and DNA synthesis by cycloheximide was reversed by washing and further incubation of the discs. Almost no qualitative difference was detected by buoyant density analysis between DNA formed under inhibition of protein synthesis of the later stage and DNA synthesized under normal conditions. These results suggest that DNA synthesis induced in potato tuber tissue by cut injury requires continuous synthesis of new protein molecules in a characteristically programmed sequence.     

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.     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

Schistosoma mansoni: utilization of exogenous metabolites by eggs in vitro

Schistosoma mansoni eggs were isolated from the livers of mice infected for 8 wk and were purified by a series of settling and sieving procedures. Aliquots of eggs were suspended in saline with added Eagle's minimal essential medium supplemented with NaHCO₃, glutamine, penicillin, and streptomycin to which a variety of radioisotope labelled metabolites were added. The uptake and utilization of tritiated thymidine demonstrated a high rate of DNA synthesis, particularly in the more immature eggs studied. RNA synthesis as shown with tritiated uridine was also significant. Large amounts of ¹⁴C-labelled isoleucine and arginine were incorporated into protein. Little glycolytic activity was demonstrated on prolonged incubation with ¹⁴C-labelled glucose. A high rate of catabolism of amino acids to CO₂ was observed, as was a very high rate of acetate metabolism. Degradation of radiolabelled glutamate after incubation of eggs with ¹⁴C acetate revealed labelling consistent with metabolism via the Krebs cycle. Thus, Schistosoma mansoni eggs utilize a wide variety of exogenous metabolites. They show active DNA and RNA metabolism, incorporation of amino acids into protein, and intermediary metabolism characterized by a low rate of glycolysis and an active Krebs cycle.     

Nutrient media for plant-parasitic nematodes. VI. Nucleic acids content and nucleotide synthesis in Aphelenchoides rutgersi

The nucleic acids content of Aphelenchoides rutgersi, Hooper and Myers, was 0.9% DNA and 2.6% RNA dry weight. The DNA contained 29.5% adenine, 29.3% thymine, 22.5% guanine, and 18.8% cytosine, while the RNA was composed of 22.8% adenine, 23.0% uracil, 31.4% guanine, and 22.9% cytosine on a molar basis.The nematodes needed folic acid for reproduction regardless of the presence or absence of nucleic acid supplements in the culture medium. This was shown by including aminopterin, a folic acid antagonist in the culture medium. A 2-hr incubation of nematodes with glycine-¹⁴C (U) and orotic-5-³H acid resulted in the incorporation of ³H-label into both DNA and RNA. Only the RNA fraction contained a significant amount of ¹⁴C-label. When this RNA was fractionated, the adenine and guanine accounted for the ¹⁴C-label, while cytidylic and uridylic acids contained the ³H-label, thereby demonstrating purine and pyrimidine synthesis by A. rutgersi. The incorporation of orotic acid into the pyrimidines was 8 times higher than that of glycine into purines.