Polyribosomes and protein synthesis in after-ripening pear seeds

Polyribosomes have been isolated from pear seeds which show a capacity for in vitro protein synthesis. Translational capacity increased during stratification with a peak after 13 days of stratification. This increase was greatly diminished in seeds which were incubated at 25°C (“warm stratification”). The peak in protein synthetic activity coincides with the period of stratification required for normal development of excised embryos.     

Helical inclusions with nucleopore-like characteristics in the cytoplasm of swiss 3T3 Cells: A preliminary communication.

Helical inclusions have been observed at sites remote from the nucleus in the cytoplasm of Swiss 3T3 fibroblasts. Because of the interest shown in their unusual nucleopore complex-like nature, this preliminary communication briefly reports their existence and describes their essential ultrastructural features. Although they could be found singly, most formed collections of up to about 30 inclusions in which different planes of section were represented. TS profiles with hexagonal packing showed apparently identical eight-fold symmetry and dimensions to pores in the nuclear envelope of 3T3 cells. No membranes were associated with the inclusions, nor was there any suggestion of the strict vertical plate-like stacking found in annulate lamellae (AL). Furthermore, the inclusions were undoubtedly helical, and in all probability composed of several strands.     

Transition of Xwnt-11 mRNA from inactive form to polyribosomes in frogs during early embryogenesis

The distribution of Xwnt-11 mRNA between polysomes and informosomes was studied in Xenopus laevis and Rana temporaria during early embryogenesis. The ratio between polysomes and informosomes suggests their involvement in translation of these mRNAs. In eggs and immediately after fertilization the Xwnt-11 mRNAs are mostly positioned in informosomes. During the cleavage stage, these mRNAs have also been recognized in polysomes. Just before the onset of zygote genome functioning (at the stage of mid blastula), Xwnt-11 mRNA rapidly appears in polysomes of Rana embryos. However, in Xenopus, Xwnt-11 mRNA appears in polysomes only at the end of gastrula. Before this stage, the Xwnt-11 mRNA in Xenopus can be found mostly in informosomes.     

Translationally distinct populations of NMDA receptor subunit NR1 mRNA in the developing rat brain

The translational activity of the NMDA subunit 1 (NR1) mRNA was examined in the developing rat brain by sucrose gradient fractionation. One translationally-active pool of NR1 mRNA was associated with large polyribosomes (polysomes) over the entire developmental period examined. A second NR1 mRNA pool, approximately half of the NR1 mRNA at post-natal day 4, sedimented only within the two to three ribosome range, indicating that it was translationally blocked during early brain development despite active translation of mRNAs coding for the NR2 subunits of the receptor. At post-natal day 4, both NR1 mRNA pools were distributed throughout the brain and contained similar profiles of NR1 mRNA splice variants, except that NR1-3 appeared to be present only in the translationally-blocked NR1 pool. After post-natal day 8, the translationally-blocked NR1 mRNA pool became progressively active within a background of globally-decreasing brain translational activity.     

Translation and the cytoskeleton: a mechanism for targeted protein synthesis

This review describes the critical evidence that in eukaryotic cells polyribosomes, mRNAs and components of the protein synthetic machinery are associated with the cytoskeleton. The role of microtubules, intermediate filaments and microfilaments are discussed; at present most evidence suggests that polyribosomes interact with the actin filaments. The use of non-ionic detergent/deoxycholate treatment in the isolation of cytoskeletal-bound polysomes is described and the conclusion reached that at low salt concentrations this leads to mixed preparations of polysomes derived from both the cytoskeleton and the endoplasmic reticulum. At present the best approach for isolation of cytoskeletal-bound polysomes appears to involve extraction with salt concentrations greater than 130 mM after an initial non-ionic detergent treatment. Such polysomes appear to be enriched in certain mRNAs and thus it is suggested that they are involved in translation of a unique set of proteins. The evidence for mRNA localisation is presented and the role of the cytoskeleton in transport and localisation of RNA discussed. Recent data on the role of the 3 untranslated region in the targeting of mRNAs both to particular regions of the cell and for translation on cytoskeletal-bound polysomes is described. The hypothesis is developed that the association of polysomes with the cytoskeleton is the basis of a mechanism for the targeting of mRNAs and the compartmentalization of protein synthesis.Abbreviations CBP cytoskeletal-bound polysomes - FP free polysomes - MBP membrane-bound polysomes - ER endoplasmic reticulum     

Associations of maize protein bodies with cytoskeleton, membranes, and ribosomes in the endosperm of wild type and opaque-2 mutant

Maize endosperm was homogenized in a cytoskeleton-stabilizing buffer, filtered and layered on gradients of 20–80% sucrose and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient, at about 60–65% sucrose (density of approximately 1.3 g·ml⁻¹). Biochemical, fluorescence microscopic, and immunoblot analyses of this peak showed that it consisted of protein bodies associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2 mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized microfilaments and microtubules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton.     

In Vitro Synthesis of the Four Mouse Myelin Basic Proteins: Evidence for the Lack of a Metabolic Relationship

Abstract: Studies on the synthesis of the four immunologically related mouse myelin basic proteins (MBPs) were carried out to determine if these proteins were metabolically related. Two in vitro systems were used: (a) a homologous brain system consisting of free polysomes, pH 5 enzymes, and initiation factors; and (b) a reticulocyte lysate system directed with mRNA and supplemented with brain factors. Incorporation of [³⁵S]methionine into the four MBPs (14K, 17K, 18.5K, and 21.5K) was detected by immunoprecipitation of the in vitro products of synthesis followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The four MBPs were identified by cross-reactivity with purified anti-MBP antibodies and their apparent molecular weights in SDS gels. Synthesis of all four proteins was detected in both systems soon after the incubations were begun. The kinetics of the labeling of the proteins showed no evidence of a precursor-product relationship (i.e., 21.5K→ 18.5K; 17K → 14K) in either system. Inhibition studies with puromycin and "chase" experiments with unlabeled methionine demonstrated that neither system contained posttranslational "processing" activity. Thus, the 21.5K and 17K proteins were not being processed into the 18.5K and 14K MBPs by either . in vitro system. Detection of the synthesis of all four proteins in the reticulocyte system programmed with brain mRNA indicates that the four proteins are probably coded for by separate mRNAs. This conclusion was supported by studies using polyribosomes separated into different size classes, which suggest that the mRNAs for the four proteins may be translated on proteins of differing size class. It is proposed, therefore, that the four MBPs are the primary translation products of independent brain mRNAs and are not metabolically related.     

Localization and Organization of the Translational Apparatus in Mitochondrial Membranes of Astasia longa

In isolated mitochondrial membranes of Astasia longa, large and small circular substructures were observed with diameters of 1.30 and 0.35 m and thickness of 0.08 and 0.03 m, respectively. Such substructures were isolated by membrane treatment with proteolytic enzymes (proteinase K, trypsin) or by lipid solubilization with Triton X-100. After the removal of surface protein layer, we uncovered circular polyribosomes with similar diameters as those of original substructures. Polyribosomes were identified on the basis of their morphology, positive staining with uranyl acetate, a capacity for chloramphenicol-sensitive incorporation of ¹⁴C-amino acids into polypeptides, and from their buoyant density as estimated by equilibrium centrifugation in the CsCl density gradient. The conclusion is that, in mitochondrial membranes of A. longa, the translational apparatus is organized similarly to that in the membranes of chloroplasts and cyanobacteria, e.g., large and small circular polyribosomes situated within the membrane ring-shaped substructures are the basics for the formation of the latter.     

Isolation of undegraded polysomes from radish cotyledons: Use of proteinase K and cycloheximide

A procedure for isolating undegraded polysomes from radish cotyledons is described. The current method for plant polysome preparation using buffers of high pH and high ionic strength was unable to prevent the breakdown of large polysomes occurring during the extraction. Proteinase K was very efficient in protecting polysomes from degradation. However, this yielded a high monosome content in the preparations. When proteinase K was combined with cycloheximide, a more satisfactory recovery of polysomes was achieved. This procedure could facilitate investigations of the in vitro protein synthesis activity and mRNA isolation in tissues possessing a high ribonuclease content.