[Polyribosome formation in animal cell cytoplasm: the kinetics of the process and characterisitics of the cytoplasmic precursors of the polyribosomes].
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Antibiotics and polyribosomes. Chlortetracycline and polyribosomes of Bacillus megaterium.
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Bacillus megaterium↗
Actinomycin D and hepatic polyribosomal disaggregation. Evidence for different half-lives of mRNA of free and membrane-boudn polyribosomes.
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Animals↗
Polyribosomes in Azotobacter vinelandii. I. Isolation, characterization and distribution of ribosomes, polyribosomes and subunits in logarithmically growing azotobacter.
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Amino Acids↗
Polyribosomes in rat tissues. 3. The response of the polyribosome pattern of rat liver to physiologic stress.
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Animals↗
The effect of various treatments in vitro and in vivo on the binding of 125I-labeled anti-rat serum albumin Fab' to rat tissue polyribosomes.
A quantitative assay was developed to permit estimation of the relative amounts of albumin-synthesizing polyribosomes in rat liver. The polyribosomes synthesizing albumin were identified by their capacity to bind anti-RSA Fab' radiolabeled with 125I. The anti-RSA Fab'-binding sites occur on the nascent peptide chains attached to liver polyribosomes. These binding sites can be saturated by preincubation of the polyribosomes with large quantities of unlabeled anti-RSA Fab'. The iodinated antibody did not react with polyribosomes isolated from a tissue which does not synthesize rat serum albumin. Pretreatment of hepatic polyribosomes with bovine pancreatic ribonuclease resulted in a 42% enhancement of binding of anti-RSA Fab'. Pretreatment of these polyribosomes with detergents or various levels of Mg2+ did not significantly affect the specific binding of the iodinated antibody. Anti-RSA Fab' associated preferentially with membrane-bound polyribosomes when compared with free polyribosomes following their isolation from animals maintained either on a 90% or a 0% protein diet fed ad libitum. Binding of anti-RSA Fab' to each A260 unit of membrane-bound polyribosomes is from 2.4 to 27 times greater than to each A260 unit of free polyribosomes. However, each A260 unit of free polyribosomes was found to associate with 1.8 times more anti-RSA Fab' when compared with the "loosely bound" subclass of membrane-bound polyribosomes. Each A260 unit of the "tightly bound" subclass of membrane-bound polyribosomes reacted with 4.3 times as much antibody as compared with free polyribosomes. Polyribosomes isolated from the livers of rats sacrificed 6 h after treatment with actinomycin D showed a 42% reduction in their capacity to bind anti-RSA Fab'. Polyribosomes from rats sacrificed 2 h after treatment with actinomycin D showed no reduction in binding capacity. Free polyribosomes from three Morris hepatomas were capable of binding anti-RSA Fab' whereas the antibody would not associate with the membrane-bound polyribosomes of the same hepatomas. Thus the binding of 125I-labeled Fab' antibody molecules to polyribosomes is a useful technique for the subcellular localization of polyribosomes synthesizing specific proteins and for the estimation of the relative proportions of such polyribosomes.
Animals↗
[Biosynthesis of collagen and other proteins on tightly and loosely bound polyribosomes from chick embryos].
Free polyribosomes and polyribosomes bound to endoplasmic membranes were isolated from 10-day-old chick embryos by differential centrifugation. The tightly and loosely bound polyribosomal fractions were isolated from the membrane-bound polyribosomes using 0,5 M KCl. The synthesis of collagen and non-collagen proteins on the polyribosomes were studied in a homologous cell-free system. It was shown that the polyribosomes tightly bound to the membranes possess a lower protein-synthesizing activity as compared to free and loosely bound polyribosomes. The amount of bacterial collagenase-cleaved polypeptides in the protein product synthesized on the polyribosomes tightly and loosely bound to the memranes and on free polyribosomes is 31, 23 and 9%, respectively. The data obtained suggest that the loosely bound polyribosomes are actively involved in collagen synthesis and that this fraction is not a contamination of free polyribosomes in the preparations of totally bound polyribosomes. The role of tightly and loosely bound polyribosomes in the formation of the membrane polyribosomal complex is discussed.
Animals↗
Polyribosomes under developing spine synapses: growth specializations of dendrites at sites of synaptogenesis.
We have previously reported that there is a dramatic increase in polyribosomes associated with dendritic spines during periods of synapse growth induced by denervating lesions. We suggested that polyribosomes at the postsynaptic site may somehow be involved in the growth of synapses. To evaluate this hypothesis further, the present study determines whether synapses which are growing in the developmental period also have accumulations of polyribosomes. We examined the dentate gyrus of the developing rat electron microscopically at 7, 10, 15, 20, and 28 days of age, which spans the major period of synaptogenesis in this structure. Qualitative observations revealed dramatic accumulations of polyribosomes under spine synapses in the youngest animals (7 and 10 days of age). With synapse development, the accumulations of polyribosomes became less dramatic, so that by 28 days of age, the neuropil of the dentate gyrus appeared qualitatively mature. To determine the relationship between polyribosomes under spine synapses and synapse development, quantitative electron microscopic methods were use to evaluate synapse density (number of synapses/100 micron 2), and the incidence of polyribosome-containing spines (proportion of spine synapses with underlying polyribosomes) in the neuropil of the dorsal blade of the dentate gyrus at each age. An inverse relationship was found between synapse density and the proportion of spines with polyribosomes. Synapse density increased in an almost linear fashion between 7 and 28 days of age to levels which were actually somewhat higher than in mature rats, whereas the incidence of polyribosome-containing spines was highest at the youngest ages and decreased with development. Thus, polyribosomes were most prominent under spine synapses during the period of maximal synapse growth. These results, together with our previous observations of increased numbers of polyribosomes under spines during lesion-induced growth, suggest that the polyribosomes represent a structural specialization of dendrites at sites of synapse construction. We propose that they produce protein(s) that are involved in synapse growth.
Animals↗
The distribution of nascent polypeptide chains among intact polyribosomes from Chlamydomonas reinhardi.
A model of polyribosome function based on tape theory has been applied to the analysis of intact polyribosomes from Chlamydomonas reinhardi. The distribution of nascent polypeptide chains found on polyribosomes does not conform to the expected pattern in which small polypeptides are synthesized on small polyribosomes and large polypeptides on correspondingly large polyribosomes. This discrepancy was revealed in the analysis of specific activity of polyribosomes (radioactivity in nascent chains per ribosome) versus polyribosome size at labeling saturation. It was found that the specific activity of small polyribosomes was higher than predicted and that of large polyribosomes was lower. This finding was validated by measuring the sizes of nascent chains from various polyribosome size classes by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of large polypeptides on small polyribosomes could be partially accounted for by the synthesis of polypeptides on chloroplast (chloramphenicol-sensitive) polyribosomes. A maximum peptide interval time of 10 s was estimated from the labeling kinetics of the nascent chains of mid-sized (cytoplasmic) polyribosomes. This rate of translation is comparable to that reported in other eucaryotic cells.
Arginine↗
Protein-synthetic machinery at postsynaptic sites during synaptogenesis: a quantitative study of the association between polyribosomes and developing synapses.
Previous studies have revealed dramatic accumulations of polyribosomes under growing spine synapses, suggesting a critical role for protein synthesis at the postsynaptic site during synaptogenesis. The present study quantitatively analyzes the distribution of polyribosomes under synapses during developmental synaptogenesis in the rat's dentate gyrus. The middle molecular layer of the suprapyramidal blade of the dentate gyrus was examined electron-microscopically at 1, 4, 7, 10, 15, 20, and 28 d of age. At each age, we evaluated (1) synapse density (the number of synapses/100 micron2 of neuropil), (2) the width of the molecular layer, (3) the proportion of spine synapses with underlying polyribosomes, and (4) the number of polyribosome-containing synapses/1000 micron2 of neuropil. From the first two measures, an estimate was obtained of the total extent of synaptogenesis, taking into account both the increase in synapse density and the increase in total area of neuropil. At 1 d of age, very few synapses were found in the molecular layer of the dentate gyrus, and those that were present were quite immature in appearance. Synapse density increased about 140-fold between 1 and 28 d of age, from an average of 0.36 synapses/100 micron2 at 1 d of age to 49 synapses/100 micron2 at 28 d of age. An inverse relationship was found between synapse density and the proportion of synapses with polyribosomes. Between 1 and 7 d of age, about 60% of the spine synapses had one or more polyribosomes under the spine base. Thereafter, the proportion of spines with polyribosomes decreased as synapse density increased. Similarly, the proportion of shaft synapses with underlying polyribosomes was greatest between 1 and 7 d postnatal, and decreased thereafter. While the proportion of synapses with polyribosomes was greatest between 1 and 7 d, the actual number of polyribosome-containing synapses/1000 micron2 of neuropil was negligible at 1 d, increased to a peak at 7 d of age, and then decreased as synapse density increased. Qualitatively, the most dramatic accumulations of polyribosomes were also found at 7 d of age. We conclude that spine-associated polyribosomes represent a structural specialization of dendrites at sites of synapse construction and as such may represent a marker for growing synapses. We propose that these elements produce protein(s) that are critically involved in the formation of the synaptic contact.
Animals↗
Synthesis of human placental lactogen and human chorionic gonadotropin by polyribosomes and messenger RNA's from early and full term placentas.
Synthesis of human placental lactogen (hPL) and of human chorionic gonadotropin (hCG) by membrane-bound and free polyribosomes from early and from full term human placentas was investigated by in vitro release of the nascent hormone peptides, followed by immunoprecipitation and electrophoresis in sodium dodecyl sulfate gels, and by specific binding of 125I-labeled hPL antibody to nascent peptide chains. In addition, messenger RNA'S Were extracted from total, free, and membrane-bound placental polyribosomes and their capacities for hPL and hCG synthesis were measured in a heterologous cell-free system prepared from wheat germ. Membrane-bound polyribosomes from full term placentas were several times more active in the synthesis of both peptide hormones than were free polyribosomes. By binding 125I-labeled hPL antibody to nascent chains on the polyribosomes, it was determined that hPL is made by clusters of seven to nine ribosomes. About 8% of the nascent peptide chains released by incubation of polyribosomes from full term placentas was accounted for by hPL, and 2% by hCG. In contrast, no chains of hPL were released by polyribosomes from 10-week placentas, whereas 11% of the total released chains were accounted for by hCG. When messenger RNAs prepared from the polyribosomes of 20- and 40-week placentas were used to stimulate protein synthesis in a wheat germ system, hPL accounted for 0.4 and 2%, respectively, of total protein synthesis, while hCG was 8 and 2%, respectively. This confirmed the relative proportions observed for nascent chains on the polyribosomes of early and late placentas. Unexpectedly, translation of mRNA from free polyribosomes yielded as much hPL and two-thirds as much hCG as did translation of mRNA from bound polyribosomes. We conclude, that the decreased blood levels of hCG and increased blood levels of hPL with advancing gestational age reflect the relative in vitro rates of synthesis of these hormones by placental polyribosomes, the abundance of which is determined by availability of their respective messenger RNAs at different times in gestation.
Animals↗
Poly (A)-containing polyribosomal RNA in sea urchin embryos: changes in proportion during development.
Evidence is described which suggests that a large fraction of sea urchin embryo polyribosomal RNA is not polyadenylated, and that the proportion of poly(A)-containing RNA increases during embryological development. Cleavage, early blastula, and mesenchyme blastula stage embryos were treated with cordycepin, and the effects of the drug on polyribosome appearance were determined. Polyribosomes in cleaving embryos and early blastulae were unaffected by the drug. However, there was a significant reduction in the amount of polyribosomes present and in the amount of newly synthesized RNA entering polyribosomes in mesenchyme blastulae after cordycepin treatment. Polyribosomal RNA was isolated from cleavage, early blastula, and mesenchyme blastula stage embryos, and the RNA was characterized by sucrose gradient centrifugation. Cleaving embryo polyribosomal RNA has a prominent 9 S peak and a smaller 20 S peak, and the larger RNA is heterodisperse. There is no poly(A) associated with the 9 S and 20 S material, but there is poly(A) associated with the RNA sedimenting faster than 20 S. Early blastula and mesenchyme blastula polyribosomal RNA have 22 S and 26 S peaks, respectively. This later stage RNA is quite heterodisperse and poly(A) is associated with RNA in all size classes larger than 9 S. Finally, the proportion of polyadenylated light and heavy polyribosome RNA was determined for cleavage, early blastula, and mesenchyme blastula stage embryos. Approximately 20-25% of newly synthesized light polyribosome RNA is polyadenylated and this amount is relatively constant during early development. However, the proportion of newly synthesized RNA in heavy polyribosomes that is polyadenylated increases during development: 25% at cleavage, 33% at early blastula, and 48% at mesenchyme blastula stages.
Adenine Nucleotides↗
The isolation and characterization of intact polyribosomes from a cell wall mutant of Chlamydomonas reinhardi.
A technique is described for obtaining intact polyribosomes from a cell-wall mutant of Chlamydomonas reinhardi. When cells were lysed by nonionic detergent in buffers containing high salt and Mg2+-EDTA, polyribosomes of up to 25 ribosomes per polyribosome were obtained on sucrose density gradients. Under these conditions, nascent polypeptide radioactivity was associated with the polyribosomes and not with monoribosomes, and inactive monoribosomes were dissociated to ribosomal subunits. Whole cell lysates contain a mixture of cytoplasmic and chloroplastic polyribosomes. The relative contribution of the two types of polyribosomes was evaluated by polyacrylamide gel electrophoretic analysis of ribosomal RNA extracted from polyribosomes. This analysis showed that less than 15% of the polyribosomes from detergent-lysed cells were from chloroplasts. The contribution of chloroplast polyribosomes to total polyribosomes was increased to about 30% by incubation of the cells in chloramphenicol. When cells were disrupted mechanically (in a French pressure cell) only about 10% of the resulting free polyribosomes were chloroplastic.
Arginine↗
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.
Animals↗
Antibiotics as probes of ribosome structure: binding of chloramphenicol and erythromycin to polyribosomes; effect of other antibiotics.
Antibiotics were used as probes of ribosome topology and function. Studies of [(14)C]chloramphenicol and [(14)C]erythromycin binding to ribosomes and polyribosomes revealed the following features. The requirement of high K(+) concentration (150 mM) for [(14)C]chloramphenicol binding to NH(4)Cl-washed ribosomes resulted from the washing procedure. Neither native 70S ribosomes nor polyribosomes require K(+) greater than 30 mM for [(14)C]chloramphenicol binding. Whereas [(14)C]chloramphenicol binds to both ribosomes and polyribosomes, [(14)C]erythromycin binds essentially only to ribosomes. After removal of peptidyl-transfer ribonucleic acid (tRNA) from polyribosomes, [(14)C]erythromycin could then be bound. The effects of a number of antibiotics on [(14)C]chloramphenicol binding to ribosomes and polyribosomes was assessed. It was found that most of the macrolides (erythromycin, carbomycin, spiramycin III, niddamycin, oleandomycin, and tylosin) and streptogramins A and B (vernamycin A, PA114A, vernamycin Balpha, and PA114B) inhibited chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes, but not to polyribosomes. After removal of peptidyl-tRNA from polyribosomes, [(14)C]chloramphenicol binding was then inhibited. In contrast, sparsomycin and althiomycin inhibited chloram-phenicol binding to polyribosomes, but not to ribosomes. After removal of peptidyl-tRNA from polyribosomes, sparsomycin and althiomycin were then ineffective. The presence of peptidyl-tRNA on polyribosomes apparently is required for binding of sparsomycin and althiomycin, but prevents binding of most macrolides and streptogramins. The lincosaminides (lincomycin and celesticetin) and methymycin (a small macrolide) inhibited [(14)C]chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes best, but also inhibited the binding to polyribosomes. The amino nucleosides and other antibiotics tested do not seem to interact strongly with the major chloramphenicol-binding site. These results provide knowledge of the interrelationships between antibiotic and substrate ribosome binding sites which should eventually contribute to a map of ribosomal topology.
Anti-Bacterial Agents↗
Transient dissociation of polyribosomes and concurrent recruitment of calreticulin and calmodulin transcripts in gravistimulated maize pulvini.
The dynamics of polyribosome abundance were studied in gravistimulated maize (Zea mays) stem pulvini. During the initial 15 min of gravistimulation, the amount of large polyribosomes transiently decreased. The transient decrease in polyribosome levels was accompanied by a transient decrease in polyribosome-associated mRNA. After 30 min of gravistimulation, the levels of polyribosomes and the amount of polyribosome-associated mRNA gradually increased over 24 h up to 3- to 4-fold of the initial value. Within 15 min of gravistimulation, total levels of transcripts coding for calreticulin and calmodulin were elevated 5-fold in maize pulvinus total RNA. Transcripts coding for calreticulin and calmodulin were recruited into polyribosomes within 15 min of gravistimulation. Over 4 h of gravistimulation, a gradual increase in the association of calreticulin and calmodulin transcripts with polyribosomes was seen predominantly in the lower one-half of the maize pulvinus; the association of transcripts for vacuolar invertase with polyribosomes did not change over this period. Our results suggest that within 15 min of gravistimulation, the translation of the majority of transcripts associated with polyribosomes decreased, resembling a general stress response. Recruitment of calreticulin and calmodulin transcripts into polyribosomes occurred predominantly in the lower pulvinus one-half during the first 4 h when the presentation time for gravistimulation in the maize pulvinus is not yet complete.
Biological Transport↗
Protein-synthetic machinery beneath postsynaptic sites on CNS neurons: association between polyribosomes and other organelles at the synaptic site.
Previous studies have demonstrated that polyribosomes are selectively positioned beneath postsynaptic sites on CNS neurons. In spine-bearing neurons, these polyribosomes are selectively localized at the base of the spines, and occasionally within spine heads. The present study evaluates whether there are relationships between the polyribosomes and other organelles of the postsynaptic cytoplasm, including membranous cisterns and spine apparatuses. Dendritic spines from the dentate gyrus and hippocampus of the rat were analyzed at the electron-microscopic level in 2 ways. First, relatively thick sections were prepared for electron microscopy, and spines were photographed in stereo using a goniometer stage. Second, conventional serial thin sections were taken, and spines were reconstructed. From the stereo photographs and serial reconstructions, we determined the proportion of polyribosomes that was associated with membranous cisterns. We also counted the number of ribosomes per cluster to determine whether there were differences between polyribosomes in different intradendritic locations, or between free polyribosomes and polyribosomes on cisternal membranes. From the serially reconstructed spines we determined the incidence of polyribosomes, membranous cisterns, and spine apparatuses, and evaluated the relationships between these organelles. We found that in both the dentate gyrus and hippocampus, about 50% of the polyribosomes that were present beneath the base of spines were associated with membranous cisterns. Polyribosomes that were present in the head of the spine were rarely associated with a cistern, however. The overall incidence of polyribosomes was similar in spines with spine apparatuses and spines without a spine apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
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.
Animals↗