Proportion Of Polyribosomes Research Articles

The use of transcription inhibitors in the study of the mechanism of abscisic acid action in germinating triticale caryopses

The study was conducted on germinating triticale (var. Grado) caryopses. The purpose of the experiments was to compare the effect of selected inhibitors of transcription with the action of abscisic acid during germination of caryopses. The following inhibitors were used: α-amanitin, cordycepin, cycloheximide and 5-fluorouracil. Studied were the synthesis of total and polyribosomal RNA, the process of polyribosome formation and the synthesis of ribosomal proteins. The effect of exogenous ABA, especially in the early stages of germination, was not similar to any of the four above inhibitors of transcription. After 12 h of imbibition at a lowered temperature and 3 h of germination, ABA caused a relatively low level of inhibition of RNA synthesis, whereas all of the inhibitors used halted RNA synthesis in embryos by about 50-60%. After 6 h of germination, the same proportion of polyribosomes in the total ribosome fraction (46%) was found in both the embryos from the control sample and treated with ABA. The use of inhibitors brought this figure down to below 40%. The conclusion is drawn that in the early stages of germination, regulation of protein synthesis by ABA in triticale caryopses must occur on a level other than transcription.

Precocious germination and its regulation in embryos of triticale caryopses

Triticale var. Lasko embryos, isolated from grain gathered at milk ripeness, the beginning of wax ripeness and at full ripeness, were allowed to germinate for 48 h on agar with glucose. The highest incorporation of tritiated adenosine into polyribosomal RNA during germination was found in the ribosome fractions from embryos of grain gathered at full ripeness, lower incorporation was in preparations from embryos of milk ripe grain and the lowest in preparations from embryos of wax ripe grain. Different tendencies were observed in respect to the synthesis of ribosomal proteins. The highest incorporation of <sup>14</sup>C-amino acids into ribosomal proteins was found in preparations of ribosome fractions from embryos of milk ripe grain, lower in preparations of embryos from fully ripe grain, the lowest in preparations of embryos from wax ripe grain. ABA (10<sup>-4</sup> M) completely inhibited the external symptoms of germination of immature embryos, while its inhibition of the synthesis of polyribosomal RNA and ribosomal proteins was greater the more mature the embryos that were germinated. The greatest stimulation of precocious germination by exogenous BA and GA<sub>3</sub> was demonstrated in the least mature embryos isolated from milk ripe grain. Under the influence of both stimulators, an increase of the proportion of polyribosomes in the total ribosome fraction occurred in this sample, as did a rise in the intensity of ribosomal protein synthesis. The incorporation of <sup>3</sup>H-adenosine into polyribosomal RNA, however, was lower than in the control sample. The results obtained suggest that the regulation of precocious germination of triticale embryos by phyto-hormones is not directly related to transcription.

Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentate gyrus

Electron microscopic studies of the dentate gyrus of the rat have revealed an apparent association between polyribosomes and dendritic spines. The present study was designed to elucidate the nature of this association. Our qualitative observations revealed that polyribosomes appeared primarily in two locations within the dendrite: (1) beneath the base of identified spines just subjacent to the intersection of the spine neck with the main dendritic shaft and (2) beneath mounds in the dendritic membrane which had the appearance of the base of a spine which extended out of the plane of section. To quantitatively define the nature of the apparent association, we attempted to determine (1) the proportion of spines with associated polyribosomes and (2) the proportion of the polyribosomes within dendrites which are associated with spine bases. Evaluation of profiles which were identifiable as spine neck-dendritic shaft intersections in a single section revealed that an average of 12.2% had associated polyribosomes. A serial section analysis revealed a somewhat higher incidence, however. Of a collection of 34 through-sectioned spines, 29% had polyribosomes which were revealed in one or more of the sections comprising the series. To evaluate what proportion of polyribosomes within the dendrite was associated with spines, we evaluated a series of photographs covering approximately 1250 micrometer2 of the dentate molecular layer from five animals, identifying all polyribosomes within dendrites and scoring their location as being (1) under spines, (2) under mounds, or (3) other. An average of 9.6% of the polyribosomes were found under processes identifiable as spine neck-dendritic shaft intersections, while 71.4% of the polyribosomes were found under mounds. Only 19% were not obviously associated with spines or mounds. Spine bases and mounds comprise only 3 of 35%, respectively, of the outline of dendritic profiles, however, indicating that the high incidence of polyribosomes under these elements cannot be accounted for by chance. To attempt to determine whether the mounds represent the base of dendritic spines, 68 mounds in 21 dentritic profiles were selected from the middle of the series of 20 serial sections. Ninetine of these mounds (28%) were continuous with an identified spine, and an additional 31% were continuous with thin processes of the size and appearance of spine necks. Thus, most of the mounds probably do represent the base of spines which extend out of the plane of a single section.

Cerebral ribosomal protein phosphorylation in experimental hyperphenylalaninaemia.

Investigations were carried out on the effects of phenylalanine loading on ribosomal protein phosphorylation in cerebral cortices of infant rats. Administration of L-phenylalanine intraperitoneally, in doses of 1 or 2 mg/g body wt., resulted within 30 min in a significant decrease in incorporation of radioactivity from intracisternally administered [32P]Pi into constitutive ribosomal proteins of the cerebral 40S subunit. This phenomenon was not accompanied by significant variations in 32P uptake into the cerebral cytosol. Incorporation of radioactivity into ribosomal proteins of the cerebral 60S subunit exhibited only minor variations under these circumstances. Alterations in the phosphorylation state of cerebral 40S ribosomal proteins induced by phenylalanine loading involved principally the S6 protein, which exists in multiple states of phosphorylation. The proportions of the more highly phosphorylated congeners of this protein were markedly decreased, as detected by two-dimensional electrophoretograms and autoradiographs of the cerebral 40S ribosomal proteins. Phenylalanine loading also altered the relative extent of phosphorylation of the S6 protein in cerebral polyribosomes and monoribosomes. In control animals, the specific radioactivity of 40S proteins in cerebral polyribosomes was five to ten times that of 40S proteins in the monoribosome population. At 1 h after phenylalanine administration, the specific radioactivities of 40S proteins in the two ribosome populations tended to approach equality. These alterations in ribosomal protein phosphorylation were accompanied by a decrease in the proportion of polyribosomes in purified ribosome preparations isolated from cerebral cortices of phenylalanine-treated infant rats. In animals given the higher dose of phenylalanine (2 mg/g body wt.), subsequent administration of a mixture of seven neutral amino acids, which resulted in partial recovery of polyribosomes, also tended to reverse the changes in ribosomal protein phosphorylation. Variations in the activities of ribonuclease enzymes in the cerebral cytosol were also observed under these conditions. Administration of phenylalanine increased the activities of cerebral ribonucleases, whereas subsequent treatment with the amino acid mixture partly reversed this effect. The results suggest that alterations in cerebral ribosomal protein phosphorylation, ribosome aggregation and ribosome function are interrelated in experimental hyperphenylalaninaemia.

Insulin Dependent and Independent Actions of Dietary Protein on in Vitro Protein Synthesis in Skeletal Muscle of Rats

Insulin dependency of the effect of dietary protein on in vitro protein synthesis in skeletal muscle was studied in rats. Re-feeding fasted rats with an adequate protein diet caused an increase in protein synthetic activity of skeletal muscle within only 5 hours with a concomitant increase in concentration of polyribosomes. During re-feeding of a protein-free diet muscle protein synthesis increased significantly but it was somewhat lower than in rats fed an adequate protein diet. The concentration of insulin in plasma increased promptly and markedly when the rats were re-fed either of these diets. However, the degree of increase was slightly less in rats fed a protein-containing meal. Re-feeding normal rats with egg albumin alone, after fasting 1 day, produced a marked increase in protein synthetic activity of skeletal muscle with a parallel increase in proportion of polyribosomes in the tissue. The stimulatory action of dietary protein on muscle protein synthesis was not abolished even in streptozotocin-diabetic rats. However, there was no appreciable change in ribosomal profile in the diabetic rats. These results indicate that the function of dietary protein in regulating muscle protein synthesis is not mediated entirely by insulin.

The Influence of Axis Removal on Protein Metabolism in Cotyledons of Pisum sativum L.

The protein metabolism of cotyledons attached to the embryonic axis has been compared with that in cotyledons removed from the axis at the initiation of a 6-day imbibition. Total protein declined in the attached but not in the detached cotyledons. Concurrent with the decline in protein level in the intact cotyledons there was an increased capacity to incorporate exogenously supplied leucine into protein. In contrast, detached cotyledons showed a restricted capacity for protein synthesis. It was demonstrated that ribosomal preparations from cotyledons of intact seedlings contained an increasing proportion of polyribosomes as germination progressed and such ribosomes were active in in vitro amino acid incorporation. Ribosomal preparations from detached cotyledons contained few polyribosomes and had a restricted capacity to incorporate amino acids in vitro. The in vitro incorporation of phenylalanine was stimulated by polyuridylic acid with the stimulation being greatest in ribosomal preparations from detached cotyledons. The results suggest that an axis component may regulate the availability of messenger RNA in the cotyledons during germination.

Rapid Changes in Levels of Polyribosomes in Zea mays in Response to Water Stress

Sucrose gradient profiles of polyribosomes from the coleoptilar node region of seedlings of Zea mays L. were obtained without pelleting and redispersion of the particles. Water stress caused a shift of ribosomes from the polymeric to the monomeric form, starting about 30 minutes after stress initiation and when the water potential of the tissue began to decrease measurably. After about 4 hours of stress (a decrease in tissue water potential of about 5 bars), most of the higher polymers of ribosomes had shifted to monoribosomes. Release of stress caused the ribosomes to revert from monomeric to polymeric form after a lag period apparently determined by the extent of prior stress. Use of bentonite and isolation of polyribosomes from combined stressed and control tissue gave results indicating that the reduced polyribosomal level was not an artifact caused by ribonuclease during isolation.Incubating roots in cycloheximide (2 micrograms per milliliter) had no effect on the proportion of polyribosomes in control roots, but it prevented the loss of polyribosomes caused by stress. Since cycloheximide inhibits the release of nascent polypeptide from polyribosomes, it appears possible that stress-effected loss in polyribosomes occurs only if polypeptides can be terminated and released.

The sedimentation of rat skeletal-muscle ribosomes. Effect of hydrocortisone, insulin and diet.

1. The influence of hydrocortisone, insulin and diet on the size distribution of ribosomes in a post-mitochondrial supernatant prepared from rat skeletal muscle was studied by sedimentation analysis with a linear 15-40% (w/v) sucrose gradient. 2. Within 4hr. after the injection of 5mg. of hydrocortisone to well-nourished rats, a decrease in the yield per g. of muscle and proportion of total RNA due to polyribosomes was observed. Similar results were obtained in rats given a protein-free diet for 3 days before administration of the hormone. 3. Insulin injection increased the yield and proportion of polyribosomes within 2hr. and decreased the proportion of the lighter ribosomal aggregates. Similar results were noted in rats given a protein-free diet for 3 days before injection. A protein-free diet given for 3 days decreased the yield and proportion of polyribosomes. Insulin did not increase the yield of polyribosomes if rats were starved for 52hr. before injection, but decreased the yield and proportion of the lighter ribosome species. 4. A 52hr. period of starvation or 2,4-dinitrophenol (15mg./kg. body wt.) given 1hr. before the rats were killed resulted in a decreased yield and proportion of polyribosomes, and, within 6hr. of re-feeding the rats with protein-free diets, an increased concentration of polyribosomes was noted. 5. The effects of a protein-free diet, hydrocortisone and insulin on the sedimentation of muscle ribosomes were found to be in accord with their net effects on muscle protein synthesis.

Initiation of haemoglobin polypeptide chains

1. 1. Ribosome fractions, prepared by differential centrifugation of a lysate of rabbit reticulocytes, contained different proportions of polyribosomes, ribosomal monomers (monosomes) and native subunits. In the cell-free system polyribosomes of all fractions synthesised on average one half polypeptide chain per constituent monomer. 2. 2. Chain synthesis de novo also occurred in the cell-free system, to a greater extent in the lighter of the fractions prepared by differential centrifugation. Since large quantities of monosomes were present in all fractions it is unlikely that monosomes are responsible for the initiation of polypeptide chains. An alternative hypothesis, that chain initiation requires the participation of native ribosomal subunits, is supported by several lines of evidence. 3. 3. The heaviest ribosome fraction, containing about 50 % polyribosomes but only 5 % subunits, had little capacity to initiate polypeptide chains. Chain initiation was increased by adding small amounts of a fraction rich in subunits. Zone sedimentation showed the stimulatory activity to sediment at about 50 S. 4. 4. Amongst a number of preparations of the ribosome fractions, a linear relationship could be demonstrated between chain initiation and subunit content, over a limited range. 5. 5. During incubation in the cell-free system the amount of ribosomal subunits declined. A parallel fall occurred in the capacity of the ribosome population to initiate polypeptide chains.