Alteration of Coding Properties of Polysome-Associated Messenger RNA in Potato Tuber Slices during Aging

Kinetics of polysome formation, translational capacity, and coding properties of polysome-associated messenger RNA were investigated in potato tuber tissue discs during aging. Polysome content rapidly increased immediately after slicing from 14% of total ribosomes in freshly sliced discs to 55% within 12 hours of aging. The amount of polysomal RNA also increased 5-fold during this period. Translational capacity of polysome-associated messenger RNA increased in parallel with the increase in content of polysomal RNA of the tissue discs when measured in a wheat germ cell-free system. Analysis of the in vitro translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the majority of polypeptides coded by the messenger RNA did not vary greatly during the period of rapid polysome formation. Three types of messenger RNA were found to change in amount during that period: those which appeared only after aging, those which disappeared during aging, and those which disappeared early but reappeared later in the aging period.     

RNA synthesis in the early stage of aerobic incubation of potato tuber discs

RNA synthesis of potato tuber discs during the early periodof their aerobic incubation was investigated by feeding thediscs with ³H-uridine. The rate of total RNA synthesis increasedin two steps during the incubation. The increase during thefirst 2 to 3 hr was small, but that after 3 hr was large. Thelabeled RNAs were separated into poly(A) containing RNA [poly(A)(+) RNA] and poly(A) lacking RNA [poly(A) (–) RNA] bythe use of a poly(U)-Sepharose column. Poly(A) (+) RNA was synthesizedeven in the freshly prepared discs which incorporated little¹⁴C-leucine into a protein fraction, and the synthetic rateof poly(A) (+) RNA increased by about 50% during the first 3hr incubation period, then gradually decreased thereafter. Synthesisof poly(A) (–) RNA continued to increase up to 7 hr afterslicing. When the discs were pulse labeled, the proportion ofradioactivity in poly(A) (+) RNA to that in the total RNA wasmaintained at about 50% until about 3 hr after slicing, butit abruptly decreased between 3 and 5 hr to about 35% whichwas maintained up to 9 hr after slicing. (Received October 12, 1977; )     

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.     

Massive synthesis of ribonucleic Acid and cellulase in the pea epicotyl in response to indoleacetic Acid, with and without concurrent cell division

Measurements were made over a 4-day period of the effect of added indoleacetic acid (IAA), puromycin, actinomycin D and 5-fluorodeoxyuridine (FUdR) on growth and the levels of total DNA, RNA, protein and cellulase in segments of tissue at the apex of decapitated etiolated epicotyls of Pisum sativum, L. var. Alaska.

The hormone induced swelling of parenchyma cells and cell division. By 3 days after IAA application, the amounts of DNA and protein were approximately double, RNA triple and cellulase 12 to 16 times the levels in controls. All of these changes were prevented by both puromycin and actinomycin D. FUdR prevented DNA synthesis and cell division but not swelling or synthesis of RNA, protein and cellulase.

It is concluded that IAA-induced RNA synthesis is required for cellulase synthesis and lateral cell expansion, whether or not cell division takes place.

     

Fat Metabolism in Higher Plants. XXXIV. Development of Fatty Acid Synthetase as a Function of Protein Synthesis in Aging Potato Tuber Slices

Experiments with inhibitors of protein synthesis (actinomycin D, puromycin, actidione) showed that the increase and the change in fatty acid synthetase activity, observed during the aging of potato disks, were accompanied by and related to a temporary rise in the rate of protein and RNA synthesis. These results concur with the earlier suggestion by Click and Hackett that the aging process involves a type of derepression. A possible course of events during aging, and possible derepression mechanisms are suggested and discussed.     

Enhancement of polyribosome formation and RNA synthesis of gibberellic Acid in wounded potato tuber tissue

As part of a more detailed study on plant tumorigenesis, the action of gibberellic acid (GA₃) in wounded potato tuber tissues as a model system has been evaluated. GA₃ stimulates total RNA synthesis in wounded tissues, the optimal concentration being 0.1 micromolar. The responsiveness of the tissue toward the hormone develops with time after wounding. Whereas freshly wounded tissue does not respond at all to the hormone, it becomes competent after about 6 hours, the competence being maximal after 1 day of wound healing.     

Metabolic requirements for the in vitro augmentation of mouse natural killer activity by interferon

The metabolic processes involved in the in vitro augmentation of mouse natural killer (NK) activity by interferon (IF) were studied. Augmentation occurred after a very brief (5–10 min), temperature-independent exposure of spleen cells to IF. This binding of, or triggering by, IF was independent of protein synthesis, since treatment with puromycin before or during contact with IF failed to block augmentation. Spleen cells, however, required new RNA and protein synthesis in the first few hours after contact with IF to develop the boosted reactivity, as shown by their susceptibility to inhibition by actinomycin D, emetine, pactamycin, and puromycin. Mitomycin C did not interfere with the boosting, suggesting that a pool of NK cells exists which rapidly responds to IF without cell proliferation. The need for new RNA and protein synthesis may be for the differentiation of pre-NK cells to functionally active cells or for the increase in lytic efficiency of already active NK cells.     

Analysis of secretory processes in Dipteran salivary glands

Summary This study indictes the complexity of cell function in one tissue. Cells of the larval salivary gland produce their secretion both by synthesis of some proteins and extraction of different proteins from the hemolymph by selective uptake and concentration. Uptake and transport are not dependent on de novo protein synthesis, at least for several hours and, as would be expected, are also not immediately dependent on new RNA synthesis. De novo synthesis of secretory proteins by the gland is almost completely inhibited by puromycin but occurs on RNA templates which are stable for at least 12 hr. Both the large cells forming most of the gland and the smaller cells forming the duct and the base of the duct are capable of taking up hemal, proteins, but synthesis of secretory proteins probably occurs only in the large cells.     

The Fate of Patatin and Its mRNA in Slices of Potato Tuber

The fate of patatin mRNA was investigated in slices of potatotuber since this mRNA appeared to be a potential example ofa preexisting mRNA that is involved in the rapid formation ofpolysomes which occurs in such slices. Levels of patatin, whichis the major storage protein in mature potato tubers, decreasedslightly during the first 4 h after slicing but remained constantfor the next 44 h. Analysis of products of in vitro translationshowed that patatin mRNA was present and stable in the tubercells even after several months of storage. The translationalactivity of patatin mRNA relative to total translational activityin total cellular RNA fraction increased transiently duringthe first hour and then decreased rapidly to undetectable levelswithin 6 h. By contrast, the activity in polysomal RNA fractiondecreased immediately after slicing. The difference betweenthe relative activities of patatin mRNA in total and polysomalRNA fractions during the first hour suggests that the extentof incorporation of patatin mRNA into polysomes was not in directproportion to its abundance in the cells of the slices. Additionof actinomycin D to the slices did not prevent the transientincrease in the translational activity of patatin mRNA in totalRNA fraction at 1 h, indicating that the transient increasewas not due to synthesis of patatin mRNA de novo after slicing.However, the inhibitor prevented the degradation of patatinmRNA in the slices. This result indicates that the synthesisof new mRNAs is necessary for the degradation of patatin mRNA. ¹Present address: Aburahi Laboratories, Shionogi and Co., Ltd.,Koka-cho, Shiga, 520-34 Japan (Received June 30, 1989; Accepted May 9, 1990)     

Mitochondrial RNA and protein synthesis in enucleated African green monkey cells

The behavior of extramitochondrial protein synthesis and of mitochondrial RNA and protein synthesis was examined in the cytoplasts of African green monkey kidney cells (TC-7 subline) at different times following enucleation by cytochalasin B. The rate of incorporation of [³H]isoleucine into protein of the soluble cytoplasmic fraction decreased in an approximately exponential fashion, with a half-life of about five hours, during the first 26 hours after enucleation. Discrete mitochondrial 16 S, 12 S and 4 S RNA components were identified among the products of cytoplast RNA synthesis. The rates of [³H]uridine incorporation into the 16 and 12 S RNA components as well as into total RNA declined progressively after enucleation to a barely detectable level by the 20th hour. By contrast, the rate of chloramphenicol-sensitive [³H]isoleucine incorporation into protein (due to mitochondrial protein synthesis) did not undergo a substantial decline for at least 20 hours in TC-7 cytoplasts; instead, a reproducible transient stimulation occurred in the first hours following enucleation. The products of mitochondrial protein synthesis pulse-labeled in nucleated cells and in cytoplasts 24 hours after enucleation exhibited similar electrophoretic profiles.     

THE EFFECT OF PUROMYCIN ON INTRANUCLEAR STEPS IN RIBOSOME BIOSYNTHESIS

Inhibition of protein synthesis by puromycin (100 γ/ml) is known to inhibit the synthesis of ribosomes. However, ribosomal precursor RNA (45S) continues to be synthesized, methylated, and processed. Cell fractionation studies revealed that, although the initial processing (45S → 32S + 16S) occurs in the presence of puromycin, the 16S moiety is immediately degraded. No species of ribosomal RNA can be found to have emerged from the nucleolus. The RNA formed in the presence of puromycin is normal as judged by its ability to enter new ribosomal particles after puromycin is removed. This sequence of events is not a result of inhibition of protein synthesis, for cycloheximide, another inhibitor of protein synthesis, either alone or in combination with puromycin allows the completion of new ribosomes.     

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.     

Modification of the cytokinin promotion of deoxyisoflavone synthesis in soybean tissue

Soybean tissue incubated in liquid media formed daidzin in response to cytokinin when the media contained 0.1 m sucrose but formed another, unidentified compound when the media contained 0.6 m sucrose or mannitol. The cytokinin effect in either setup was detectable only after a lag period of several hours. Addition of possible precursors of the compounds being synthesized or of related compounds did not alter the lag period to any great extent. Trans-cinnamic acid did perhaps shorten it a little as did preincubation of the tissue in the basal medium before the addition of kinetin. Several inhibitors of RNA synthesis substantially reduced the production of daidzin and the other compound. Protein synthesis inhibitors were also effective except that cycloheximide and puromycin actually promoted synthesis of the unknown compound while inhibiting that of daidzin. The results do not give a clear impression as to how the cytokinins are involved in the synthesis of the compounds, but are at least suggestive that cytokinin is involved in RNA or protein synthesis.     

SENESCENCE: HORMONAL CONTROL OF RNA AND PROTEIN SYNTHESIS IN EXCISED BEAN POD TISSUE

The kinetics of degradation of RNA, total protein, ribosomal and soluble (R-S) protein and DNA were followed over 24 hours in excised segments of Kentucky Wonder pole beans (Phaseolus vulgaris). In absence of exogenous auxin, RNA degradation proceeded from about zero time. By 15 hours, after about 20% of the RNA had degraded, the degradation of DNA, total protein and R-S protein was initiated. Exogenous auxin (25 ppm α-napthaleneacetic acid) prevented these degradations. The addition of kinetin had little or no effect. Auxin enhanced incorporation of orotic acid into RNA two to several times more than it did amino acids into protein. Actinomycin D repressed RNA synthesis about 75% including a repression of auxin-induced synthesis. It had no effect on the basal level of protein synthesis but did repress auxin-induced synthesis. The results indicate that the primary action of auxin in preventing senescence of bean endocarp tissue is at the site of RNA synthesis, and the effect of auxin on DNA and protein is a consequence of the effect of auxin on RNA metabolism.     

RNA biosynthesis in adipose tissue: effect of fasting

RNA metabolism has been examined in intact adipose tissue and isolated fat cells from rats. The lipocyte contains three species of RNA with sedimentation rates corresponding to those of ribosomal and transfer RNA. The de novo biosynthesis of RNA by adipose tissue cells in vitro was demonstrated. The base ratios of the RNA formed indicate that it was synthesized from a DNA template. Actinomycin D administered in vivo and in vitro decreased total RNA synthesis with the most marked effect on the synthesis of the heavy RNA components. Actinomycin D or puromycin added in vitro was not toxic: they did not inhibit total fatty acid biosynthesis or glucose utilization by the fat pad nor did they inhibit the immediate stimulation of fatty acid biosynthesis and glucose uptake by the addition of insulin in vitro. Starvation for 48-72 hr significantly depressed the synthesis of the heavy RNA components as measured by in vitro uridine incorporation into the individual RNA classes. Refeeding the fasted rat with glucose repaired the defect in RNA biosynthesis before the biosynthesis of monoenoic fatty acid was completely restored. Actinomycin D administered at the time of refeeding prevented the repair of monoenoic fatty acid synthesis. It is concluded that RNA metabolism is intimately involved in the control of biosynthetic reactions in adipose tissue.     

Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.     

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.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

Inhibition of estrogen-induced increases in uterine guanosine 3',5'-cyclic monophosphate levels by inhibitors of protein and RNA synthesis.

Uterine guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels are elevated significantly from 2 to 12 h after a single injection of estradiol-17 beta or diethylstilbestrol to mature, ovariectomized, or immature rats. The accumulation of cyclic GMP is greater in endometrial- than myometrial-enriched uterine tissue. The estrogen-induced increase in cyclic GMP can be prevented by administration of the protein synthesis inhibitors, cycloheximide and puromycin, or by relatively large doses of the RNA synthesis inhibitor, actinomycin D, but not by the muscarinic antagonist, atropine. The requirement for a protein with a relatively rapid rate of turnover is suggested by the demonstration that cycloheximide, when administered after estrogen, can within a 3-h period restore the estrogen-elevated levels of cyclic GMP to those of the non-estrogen-treated tissue.