Isolation of Polyribosome from Tobacco Plants Infected with Tobacco Mosaic Virus

Polyribosomes have been isolated from tobacco leaves. Upon infection with TMV, a new polyribosome has appeared which is specific for infection, and TMV-antigenic protein is formed on this polyribosome. This new polyribosome has an S-value of 360–380. From these results, it is suggested that this TMV specific polyribosome is an aggregate of 60–80 ribosomes which is bound by TMV-RNA as messenger, and that TMV-RNA is translated polycistronically.     

Polyribosome metabolism, growth and water status in the growing tissues of osmotically stressed plant seedlings

Relationships between growth of osmotically stressed intact seedlings and polyribosome levels and water status of growing tissues were examined. Sudden exposure of barley (Hordeum vulgare L. cv. Arivat) roots to a solution of ?0.8 MPa polyethylene glycol caused leaf growth to stop almost immedately, but growth resumed at a much lower rate after 0.5–1 h. In the growing region of leaves, the polyribosome: total ribosome ratio of free (non-membrane-bound) ribosomes was significantly reduced after 15 min stress, but a decrease in the large polyribosome:total polyribosome ratio occurred only after 1–2 h. Membrane-bound and free polyribosome levels both decreased to 70% of unstressed control values after 4 h stress. Recovery of total polyribosomes occurred within 1 h after relief of 4 h stress, but required 3 h after relief of 24 h stress. Stress detectably reduced the water potential and osmotic potential of growing tissue within 0.5–1.0 h, and osmotic adjustment continued for up to 10 h. Recovery of water status was incomplete after 1 h relief of a 4 h stress. In contrast, expanded blade tissues of stressed plants underwent minor changes in water status and slow decreases in polyribosomes levels. These results confirm that growing tissues of barley leaves are selectively responsive to stress, and suggest that changes in growth, water status and polyribosome levels may be initiated by the same signal. Measurements of seedling growth, polyribosome levels and water status of growing tissues of barley and wheat (Triticum aestivum L. cv. Zaragoza) leaves, etiolated pea (Pisum sativum L. cv. Alaska) epicotyl and etiolated squash (Cucurbita pepo L. cv. Elite) hypocotyl stressed with polyethylene glycol solutions of ?0.3 to ?0.8 MPa for 12 h or more showed that polyribosome levels were highly correlated with seedling growth rate as well as with tissue water and osmotic potentials, while turgor remained unchanged. These results suggest that long-term growth of osmotically stressed plants may be limited by a reduced capacity for protein synthesis in growing tissues and is not dictated by turgor loss.     

在条锈菌侵染早期小麦初生叶内多聚核糖体水平与蛋白质合成能力的变化

小麦初生叶接种条锈菌毒性生理小种（ＣＹ２９）及其弱毒突变菌系（ＣＹ２９－ｍｕｔ３）后,呈不亲和反应的寄主叶片可溶性蛋白质合成能力在接种后２４ｈ显著高于未接种对照,但其后逐渐降低,直至接近对照;而呈亲和性反应的寄主叶片可溶性蛋白质合成在侵染早期与对照相近,但与膜结合蛋白质在９６ｈ时大大高于对照。对接种叶中核糖体的密度梯度分析证实：呈不亲和反应寄主叶片游离多聚核糖体及亲和反应的寄主内与膜结合多聚核糖体均有特异性增加。上述结果表明寄主的抗病和感病反应均与蛋白质合成能力的变化有关。     

Phytochrome-mediated assembly of polyribosomes in etiolated bean leaves. Evidence for post-transciptional regulation of development.

Light operating through phytochrome controls the proportion of total ribosomes present as polyribosomes in etiolated leaves of Phaseolus vulgaris. Similar responses but with slightly different time courses are elicited by brief red light treatment and by continuous far-red or white light. The increase in polyribosome proportions after red light treatment reaches a maximum within 2 h, after which the proportion steadily declines. Light treatment appears to lead to increased proportions of polyribosomes in higher size classes. This is most evident with continuous white light, but probably also occurs with red and far-red light. The increase in polyribosomes is due principally to cytoplasmic ribosomes although proportionately greater effects are observed in chloroplast ribosomes. Although cordycepin inhibits RNA synthesis by 85-90% within 3 h there is no depression of the light-mediated increase in polyribosome proportions when leaves are preincubated in the inhibitor for 3 h. The data therefore indicate that phytochrome is capable of controlling protein synthesis, and thus development, at a post-transciptional level.     

Within-day Changes in the Polyribosome Content and in Synthesis of Proteins in Leaves of Capsicum annuum L

Capsicum annuum cv. California Wonder was grown in controlled environment with a 12-hour photoperiod. The polyribosome content of leaves varied from 60 to 72% of total ribosomes with the highest level occurring in the middle of the photoperiod and the lowest in the middle of the dark period. The variation was accounted for by changes in the content of large polyribosomes (hexamers and larger). There was no indication of an immediate effect on polyribosome content of light-on or light-off.

The synthesis of proteins at two times in the 24-hour cycle was compared using a dual isotope technique. Statistically significant results were obtained that suggested that protein(s) with molecular weights of 26,000 daltons were preferentially synthesized in the photoperiod compared to the dark period. No evidence was found for the differential synthesis of proteins within the photoperiod.

     

Polyribosome Isolation in the Presence of Diethyl Pyrocarbonate

Isolation of polyribosomes from wheat embryos and corn root tips in the presence of diethyl pyrocarbonate showed this reagent to have a protective effect on polyribosome structure. In addition, the use of diethyl pyrocarbonate allowed initial homogenization to be performed under less stringent conditions than those normally employed for polyribosome isolation. The use of the reagent is however limited, in that it is deleterious to in vitro ribosomal amino acid incorporation.     

Further studies on the oscillatory rate of hemoglobin synthesis in rabbit reticulocytes

The rate of hemoglobin synthesis in rabbit reticulocytes was found to be oscillatory after synchronization of the reticulocytes by precooling at 0 °C. Both α and β chains are synthesized in an oscillatory manner and in phase after synchronization. Experiments designed to study the effect of cooling on the polyribosomes showed a slowly proceeding shift in the polyribosome profile in favor of monosome formation. This shift proved to be reversible. After rapidly warming cooled reticulocytes, the original polyribosome profile was restored within 40–120 sec.     

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro.

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.     

Kinetics of the in vivo synthesis of the polypeptide chain; the effect of translation inhibitors]

Effects of aurinetricarbonic acid and cycloheximide on kinetics of polypeptide chain synthesis and polyribosome protile in mouse liver cell in vivo are studied. Both compounds are found to decrease the absolute rate of protein synthesis and to increase the time of polypeptide synthesis. Cycloheximide changed the ratio of translating and non-translating ribosomes and different in size polyribosome types. Aurinetricarbonic acid exerts no effect on polyribosome profile. Effects of cycloheximide and aurinetricarbonic acid on kinetic parameters of translation and a mechanism of action of these compounds are studied.     

Association of D-RNA with Polyribosomes in the Soybean Root

Ribosomes from soybean root tips were shown to consist of about 75% polyribosomes based on size distribution on sucrose gradients. Electronmicrographs showed the presence of ribosome clusters in normal preparations and only monomers and dimers in ribonuclease-treated preparations. The polyribosome preparations, but not single ribosomes, showed good in vitro amino acid incorporating activity.     

Preparation of polyribosome aminoacyl-transfer ribonucleic acid from the muscle of chick embryos.

A procedure for preparing polyribosome aminoacyl-tRNA free from contamination by supernatant aminoacyl-tRNA and free amino acids is described. Important features of the procedure are the use of acidic buffers to help protect the amino acid-tRNA linkage and the inclusion of sodium dodecyl sulphate, to inhibit ribonuclease activity. The specific radioactivity of polyribosome aminoacyl-tRNA is high within 30s and reaches a maximum in 2 1/2 min, well ahead of polyribosome peptides which, as described by Herrmann et al. (1971), attain maximum specific radioactivity in about 10 min.     

Effect of N-methyl-N'-nitro-N-nitrosoguanidine on amino acid incorporation into mucosal protein of rat stomach.

Administration of the carcinogenic N-nitroso compound, N-methyl-N'-nitro-N-nitrosoguanidine in drinking water (0.5 mg/mL) to male Wistar rats for 1 week caused impairment of in vivo and in vitro incorporation of [14C]leucine into stomach mucosal protein. This impairment gradually returned to normal after 4 weeks. Uptake of [14C]leucine into mucosal protein was significantly inhibited after in vitro treatment of stomach mucosa with the carcinogen. Addition of the N-nitroso compound in a cell-free system using postmitochondrial supernatant prepared from stomach mucosa also showed inhibition of amino acid incorporation. Using a more defined system consisting of purified polyribosome from stomach mucosa and pH 5 enzyme fraction derived from liver it was further demonstrated that the carcinogen purturbed protein synthesizing ability of polyribosome, under both in vivo and in vitro treatment conditions. In these respects this carcinogen has similar action on the target tissue of stomach as in the liver, although the in vivo effect may be related more to toxicity than carcinogenicity.     

Nucleic acid contents and polyribosome formation in wheat embryos during germination and vernalization

The contents of RNA and DNA in wheat embryo, determined by theSTS method, were higher during cold treatment than the periodof germination. The ratios of sRNA and IRNA to DNA and thatof sRNA to total RNA for both periods were similar accordingto MAK column chromatography. The polyribosome contents werehigher during cold treatment than germination. However, monosomecontents were identical for both periods. During germination,the polyribosome content was the highest in embryos germinatedfor two days and continuously decreased thereafter. During coldtreatment, polyribosome content was maximum in embryos cold-treatedfor 10 days then and decreased slowly for 60 days. (Received August 12, 1977; )     

Effects of Hypoglycemia on Rat Brain Polyribosome Sedimentation Pattern

Insulin-induced hypoglycemia provokes polyribosome disaggregation and accumulation of monomeric ribosomes in the brain of rats with hypoglycemic paresis and coma. The extent of brain polyribosome disaggregation depends on the decrease of blood glucose concentration, and in comatose animals on the duration of hypoglycemia. Cycloheximide prevents the disaggregation of brain polyribosomes induced by hypoglycemia, indicating that hypoglycemia affects brain protein synthesis, decreasing the rate of initiation relative to the rate of elongation of polypeptide chain synthesis.     

Cytokinin stimulates polyribosome loading of nuclear-encoded mRNAs for the plastid ATP synthase in etioplasts of Lupinus luteus: the complex accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space

Three of the nine subunits of the plastid ATP synthase, including the subunit of the CF(1) moiety (gene AtpC), are encoded in the nucleus. Application of cytokinin to etiolated lupine seedlings induces polyribosome association of their mRNAs. This appears to be specific as no such regulation was observed for messages for three ribosomal proteins. Cytokinin-mediated polyribosome loading was also observed for the spinach AtpC message in etiolated transgenic tobacco seedlings. Analysis of various spinach AtpC mRNA derivatives uncovered that the 5' untranslated region (5' UTR) of this message is sufficient to direct polyribosome loading, and that sequences at the 3' end of the AtpC 5' UTR, including an UC-rich motif, are crucial for this regulation. The increase in polyribosome loading of the AtpC message correlated with an increased synthesis of the polypeptide. The subunit, together with the ATP synthase complex, accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space of the etioplast. These results suggest that cytokinin promotes accumulation of the ATP synthase in the inner-envelope membrane of lupine etioplasts by stimulating the translation efficiency of their nuclear-encoded messages.     

The effect of low temperature upon the polyribosome, nucleic acid and protein content of potato leaves

Short periods of low temperature exposure, or longer periodsof cool temperature growth did not cause a decline or "run-off"of potato leaf polyribosomes. In fact, polyribosome levels werehigher in the leaves of plants grown in the cool temperatureregime. The ribosomal RNA levels were higher in cool grown leavesafter day 12 of treatment, while the protein and amino acidlevels did not exhibit a dramatic change. The results are discussedin respect to efficient plant protein synthesis in cooler climates. ¹This research was supported in part by a grant from the RockefellerFoundation. ²Scientific Journal Series Article 8745 of the Minnesota AgriculturalExperiment Station. (Received June 20, 1974; )     

Influence of leaf age, light, dark, and iron deficiency on polyribosome levels in maize leaves

The relations between leaf age and polyribosome levels werestudied with dark-and light-grown maize (Zea mays L.) seedlings.In general, polyribosome levels decline with the period of growthin darkness. Light induces an increase in the polyribosome levelin dark-grown seedlings. The response can be detected after30 min exposure to light. Seven or eight-day-old dark-growncorn seedlings, used in the present study, have high levelsof polyribosomes when greened in the light. This is indicativeof healthy seedlings, competent in protein synthesis. The polyribosomelevels in iron deficient maize plants were significantly differentfrom plants grown under complete nutrient solution, while thereis no significant difference among plants suffering differentdegrees of iron deficiency. (Received November 18, 1977; )     

Transcortin in rat kidney: subcellular distribution of transcortin-synthesizing polyribosomes

The [3H]corticosterone-transcortin complexes from kidney cytosol show elution positions on DEAE-cellulose identical to serum transcortin. The incorporation of 14C-labeled amino acids into anti-transcortin-precipitable material of kidney slices has been measured and compared with that of serum transcortin. It was established that kidney synthesized transcortin with an apparent molecular weight of 66 kDa on SDS-electrophoresis which resembles serum corticosteroid-binding globulin. Studies on the binding of [125I]anti-transcortin-IgG to membrane-bound rat kidney polyribosomes revealed an association of [125I]anti-transcortin-IgG with a discrete polyribosome fraction in the heavy polyribosome region; free polyribosomes were devoid of antigenic material able to bind antibodies to transcortin.     

Three-Dimensional Organization of Polyribosomes–A Modern Approach

Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed tworow forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes. At the same time, modern cell-free translation systems have opened the possibility of investigating polyribosomes on mRNA of a given structure to elucidate the mechanism of polyribosome structure formation, especially of circular polyribosomes. There is an increasing amount of data supporting the idea of interdependence between polyribosome structure and their translational activity. Moreover, participation of polyribosomes in mRNA transport and localization of protein synthesis in the cell has been shown. Improvement of the resolution and the development of the cryo electron tomography technique for the analysis of polyribosomes in situ will enable further progress in understanding the process of protein synthesis in cells.     

Benzyladenine-controlled protein synthesis and growth in apple cell suspensions

Prolific growth in cytokinin-requiring apple cell suspensions was achieved during 10 days of culture with appropriate concentrations of benzyladenine (BA). Unlike the controls, BA-treated cells showed a well developed sub-cellular organization and protein synthesis. Upon transfer to fresh cytokinin-free medium, however, these cells exhibited a rapid decrease in polyribosome formation, but with unchanged nuclear and nucleolar activities. Cells lacking added cytokinin showed a reduced metabolic activity and failed to divide. Deficiency in the translation process of cytokinin-deprived cells was overcome by exogenous BA-treatment. In cells maintained on a cytokinin-free medium for 5 days, BA-treatment enhanced protein synthesis from pre-existing mRNA in a specific way.