Yield Of Polysomes Research Articles

Changes in Polysomes and RNA in Developing Seeds of Chickpea (Cicer arietinum L.)

In the present investigation changes in polyribosomes and RNAs in the developing seeds of chickpea (Cicer arietinum L.) have been studied. The total polysome yield was higher in the early stages of development and declined at the later stages. The maximum level of polyribosomes was obtained at 18 days after flowering and a drastic decrease was noticed at maturity. The total RNA yield correlated with the polysomal yield. Northern hybridization with a heterologous probe (pea legumin cDNA) gave distinct hybridization with the mRNA coding for legumin proteins at different stages of seed development. Hybridization showed a direct relation between mRNA levels and seed weight accumulation.

Alzheimer's disease-associated reduction of polysomal mRNA translation

Polysomes from the frontal cortices of individuals who had histopathologically confirmed Alzheimer's disease were compared with polysomes from individuals who exhibited no neuropathological conditions. The cytosolic polysome yield from Alzheimer's disease frontal cortex was reduced 40% compared with that obtained from control frontal cortex. The translational activity per unit polysome of the Alzheimer's disease polysomes was only 50% of control in a reticulocyte lysate in vitro translation assay in which human polysomes do not undergo reinitiation. These differences exhibited brain region specificity in that polysomes isolated from Alzheimer's disease cerebellum were not different from control cerebellar polysomes. Thus, the disruptions are not due to a secondary and general response of the entire brain to the disease. These reductions were reflected by similar decreases in the translation of the mRNA for high molecular weight neurofilament polypeptide. Thus, the inhibition of polysomal mRNA translation is a mechanism by which gene expression is impaired in pathologically involved brain regions of individuals afflicted by Alzheimer's disease.

Time course of changes in protein synthesis in marcaine-induced skeletal muscle regeneration

The time course of the regeneration of rat skeletal muscle has been examined after injection of the myotoxic drug, Marcaine, to induce regeneration. Muscle wet weight decreases during the initial phase of the regeneration process while the ability of the regenerating muscle to incorporate [ 35S]methionine into protein, the yield and activity of muscle polysomes and the yield of total and poly(A) + RNA all increase initially. Following the initial changes, these parameters return to near control values by 30 days after Marcaine injection. Theoretical calculations suggest that the changes in polysome yield and activity are sufficient to account for the changes in the ability of muscle fragments to synthesize protein during the regeneration process. The specific activity of total muscle RNA in the wheat germ cell-free system decreases initially during the early stages of the regeneration process. This decrease may reflect the fact that while the yields of both total and poly(A) + RNA increase during the early stages of regeneration, the percentage of the total RNA which is poly(A) + decreases initially.

Cell‐Free Synthesis of Myelin Basic Proteins in Normal and Dysmyelinating Mutant Mice

Total polyribosomes were isolated from the brains of 16-20 day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shil/+, mld, and jp littermates) and translated in a homologous, cell-free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 +/- 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close-to-normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and less than 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an "assembly" or "synthesis" defect.

Ageing studies in rat liver II. Patterns of cellular and cell-free protein synthesis from one to ten months of age

Potential changes in liver protein biosynthesis were investigated in 1- to 10-month-old male Fischer F344 rats by isolation and translation of polysome-derived poly(A)-containing RNA and by incubation of isolated hepatocytes in Swim's S77 medium supplemented with [ 3H]leucine. Labeled protein products were analyzed by one- and two-dimensional polacrylamide gel electrophoresis followed by fluorography. The protein profiles revealed that the translation products of poly(A)-containing RNA derived from the livers of 1-month-old animals lacked a major 22 000 dalton band which was present in comparable profiles from livers of older animals. In parallel experiments, gel profiles of products from cultured hepatocytes isolated from 1-month-old animals were missing an 18 000 dalton band which was seen in preparations from older animals. With livers from rats between 2 and 10 months of age, however, no age-related changes in the pattern of protein biosynthesis were detected either in the products of poly(A)-containing RNA translated in vitro or in the cellular and secreted proteins of intact hepatocytes. Furthermore, the polysome content ( A 260 I cm/g tissue) was constant between 1 and 10 months of age, providing a constant amount of tissue was processed. A procedure for maximizing the yield of polysomes is described. These results suggest that the decline in protein synthesis observed in rat liver and in cultured hepatocytes during the first year of life is not due to changes in mRNA content or the variety of specific products synthesized, but results primarily from an overall decrease in protein biosynthesis, probably at the level of peptide chain elongation.

Isolation of ribonuclease-free polysomes from human placenta

Human placenta is known to have a high level of polysome-bound ribonuclease which has hindered the isolation of intact polyribosomes from this tissue. We describe conditions for preparing polysomes devoid of apparent ribonuclease activity from both first trimester and term placenta. This stable preparation was achieved by utilizing buffer at low pH containing 300 mM LiCl and precipitating the polysomes chemically with 200 mM MgCl 2. The yield of polysomes obtained by this procedure is 2–2.5 fold greater than that obtained by the conventional method of preparing placental polysomes. The polysomes are considered pure as judged by the ratios of absorbance 260 280 and 260 235 . Moreover, the yield of polysomes obtained is greater than 95% of the tissue content and the profile of the polysomes is probably representative of the in vivo population. This is concluded from experiments in which a known amount of labelled chick polysomes was added to fresh placental tissue and the recovery of label and its distribution was analyzed.

Isolation and characterization of polysomes from an insect cell line of Spodoptera frugiperda (Lepidoptera; Noctuidae)

In postmitochondrial supernatants prepared from Spodoptera frugiperda tissue culture cells, only monosomes can be detected. These monosomes are probably not produced by RNase-activity but by run-off, as the addition of cycloheximide (a run-off inhibitor) resulted in the formation of polysomes. The addition of RNase inhibitors remained without result. The highest yield of polysomes is obtained when 25 μg/ml cycloheximide is administered to the cell culture medium 8 hr before the isolation of the polysomes. The optimum pH and Mg 2+ concentration for the isolation of the polysomes are pH 9.0 and 1–5 mM Mg 2+ respectively. Some observations on the sedimentation behaviour of the ribosomes and their subunits under different conditions are discussed.

An improved method for the preparation of undegraded polysomes and active messenger RNA from immature chicken erythrocytes

A simple method for the large-scale isolation of undegraded polysomes and active mRNA from immature hen erythrocytes is described. The method is based on the removal of white cells from heparinized blood by adsorption on sulfoethyl- or sulfopropyl-Sephadex. Red cells thus purified can be stored frozen and lysed in the presence of nonionic detergents. The yield of polysomes is about twice that obtained by the usual mild lysis procedures designed to prevent lysis of lysosomes from contaminating white cells.

Large-sized polysomes in Chironomus tentans salivary glands and their relation to Balbiani ring 75S RNA.

Polysomes from the salivary glands of Chironomus tentans were investigated to determine whether Balbiani ring 75S RNA is incorporated into polysomal structures, and thus probably acts as messenger RNA. A new extraction technique for obtaining ribonucleoproteins was applied that gives a high yield of polysomes with only moderate degradation of the cytoplasmic, high molecular weight RNA. The polysomes sedimented in a broad region (200-2,000S) with a peak value of about 700S, which suggested that they were partly of very large sizes. This was confirmed by visualization of the polysomes in the electron microscope: 400S polysomes contained mainly 11-16 ribosomes, and 1,500S polysomes about 60 ribosomes per polysome. However, polysomes containing 100 or more ribosomes were also observed. It was further established that most of the cytoplasmic 75S RNA was located in polysomes, preferentially in the most rapidly sedimenting ones. From the available information on Balbiani ring RNA in cytoplasm and the present demonstration of 75S RNA molecules in polysomes, it was concluded that at least some Balbiani ring RNA, generated as 75S RNA within the Balbiani rings, eventually enters polysomes without being measurably changed in size. The present information on the potential amino acid coding sequences in 75S RNA is discussed in relation to the large size of the polysomes observed.

Effect of silica-liberated macrophage factors on protein synthesis in cell-free systems

The stimulatory effect of a silica-liberated macrophage factor on collagen synthesis in incubated granuloma slices was confirmed and the site of action located in that subcellular fraction which sediments through 38% sucrose. The SiO 2-liberated macrophage factor also increased the rate of collagen synthesis in a cell-free system which contained polysomes from granulation tissue, S-30 supernatant from wheat germs and the necessary cofactors. The yield of polysomes from granuloma slices which had been incubated in the presence of the SiO 2-liberated macrophage factor was larger than that from respective control slices. On the basis of incorporation experiments with labelled precursors it is suggested that the SiO 2-liberated macrophage factor retards the degradation of polysomal RNA.

The use of metrizamide as a density-gradient medium in studies of rat-liver polysomes.

The behaviour of rat liver cytoplasmic ribonucleoproteins in metrizamide has been studied to determine whether the iodinated compound would offer any advantage over other centrifugation media for studies of polysome structure and function. 1. Polysomes had a density of 1.295--1.300 g/cm3 in metrizamide gradients which was also the density of glycogen. However, the polysaccharide reached its equilibrium more rapidly than the polysomes. Thus a short centrifugation of a polysome suspension from non-starved rats over a 40% metrizamide cushion was sufficient to eliminate more than 85% of the glycogen with a polysome yield of about 75%. 2. Ribosomal subunits had neighbouring densities (1.23 and 1.20 g/cm3 for large and small EDTA-derived subunits; 1.23 and 1.21 g/cm3 for large and small KCl/puromycin-derived subunits, respectively). Polysomal messenger ribonucleoproteins were heterogeneously distributed (phi = 1.12 to 1.23 g/cm3) and overlapped with subunits in a similar manner as in sucrose gradients. 3. Analysis of a post-mitochondrial supernatant in metrizamide showed a clear separation of free polysomes, rough membranes and the soluble phase.

Creatine kinase and myofibrillar proteins in hereditary muscular dystrophy and vitamin E deficiency

Although the myopathy of vitamin E deficiency imitates many features of hereditary muscular dystrophy, the molecular basis of these similarities remains unexplained. Unlike the water-soluble vitamins that act as coenzyme precursors in mammalian tissues, the fat-soluble vitamins appear to regulate the synthesis of specific proteins required by highly differentiated organisms. To test the hypothesis that vitamin E does regulate the synthesis of specific proteins required for normal muscle function, a series of studies has been carried out in my laboratory to examine general protein synthesis and the synthesis of creatine kinase and selected myofibrillar proteins in vitamin E deficiency and hereditary muscular dystrophy in animals. Creatine kinase was selected for a number of studies because it has served as a marker for the onset of dystrophy in a variety of animals and man. It was shown by isotope-labeling studies of the purified enzyme from muscle in vitamin E-deficient rabbits, that the turnover rate was twice as great as that for the normal enzyme. Electron-microscopic studies of muscle from vitamin E-deficient rabbits, furthermore, showed that myofibrillar degradation and synthesis was occurring in the presence of an enhanced number of ribosomes. It was found that the yield of polysomes per gram of vitamin E-deficient muscle was 4 times higher than that from corresponding controls. Total muscle RNA levels were 2 to 3 times higher with increased muscle RNase. There appeared to be no differences in the intrinsic protein synthetic activity between normal and vitamin E-deficiency ribosomes. Because of reports of abnormalities in isoenzyme distribution of creatine kinase in human dystrophy and in hereditary dystrophy in the mouse, it was decided to investigate the nature of the creatine kinase occurring in vitamin E deficiency and in hereditary muscular dystrophy in the New Hampshire Red chick. The enzyme was purified to homogeneity from each source. It was found that the essential SH groups of the enzyme from dystrophic animals was highly labile to oxidation during the isolation process of the enzyme in contrast to vitamin E-deficient and normal chicks. This lability could be prevented by addition of dithiothreitol. Enzymes from normal, vitamin E-deficient, and dystrophic animals were similar in size, shape, charge, number of essential SH groups and kinetic properties. There were, however, some differences in the nature of the tryptic peptides obtained from the dystrophic enzyme.

Influence of zinc deficiency on synthesis and cross-linking of rat skin collagen

1. (1) The yield of salt-soluble collagen was significantly reduced in zinc-deficient rats, while the acid-soluble fraction was increased by approximately 20%. 2. (2) The incorporation of [2- 14C]glycine into the α 1 and α 2 chains and of L-[U- 14C]proline into the salt-soluble collagen fractions was reduced 30–50% in the zinc-deficient animals. 3. (3) The incorporation of [ Me- 3H]thymidine into skin DNA was 60–80% lower than that from zinc-supplemented animals. 4. (4) Muscle collagen synthesis per mg polyribosomal RNA was approximately the same in both groups. The polysome yield and consequently total muscle collagen synthetic activity per 100 g muscle was, however, 30% lower in the animals on a zinc-deficient diet. 5. (5) The content of β-dimers in the salt-soluble collagen from zinc-deficient skins was 1 3 higher than that from the control. No difference in β-subunits from the acid-soluble fraction was apparent. 6. (6) The aldehyde content of the salt-soluble collagen was markedly higher and that from insoluble collagen lower in the zinc-deficient group.

An efficient procedure for the isolation of polyribosomes from tissue culture.

In this paper an efficient procedure is described for the isolation of polysomes from tissue culture cells. Optimal yields, especially of the heavier classes of polysomes can only be obtained at a rather high KC1 concentration in the presence of nonionic detergent. Evidence is provided that the lower polysomal yield after extraction at low salt concentration is not due to breakdown of the polysomes by RNAase.Furthermore, the yield of polysomes is pH dependent only at low salt concentration, while at high salt concentration no influence of the pH is observed. Refreshing of growth medium several hours before harvesting the cells, increases the ratio of polysomes to 80 S monomers.

Factors affecting protein synthesis in rat kidney ribosome preparations.

Kidney ribosomes that were prepared from the postmitochondrial supernatant or the microsomal pellet of homogenates by deoxycholate treatment consisted mainly of monomers and dimers rather than polysomes. The addition of liver postmicrosomal supernatant to kidney homogenates or to kidney microsomes did not increase the polysome yield or the incorporation of 14C-leucine into hot acid-insoluble protein. In contrast to kidney, liver ribosomes that were prepared in the presence of liver supernatant (containing ribonuclease inhibitor) had an increased proportion of polysomes and an increased incorporation of 14C-leucine into protein compared to liver ribosomes prepared in the absence of supernatant.The incorporation of 14C-leucine into protein in kidney ribosome preparations was considerably lower than it was in liver ribosome preparations. The incorporation depended on the concentration of Mg2+, GTP, and pH 5 enzymes. Following preincubation of kidney ribosomes to eliminate endogenous mRNA, the incorporation of 14C-phenylalanine into peptide using either phenylalanine or phenylalanyl-tRNA was found to depend on the addition of poly U.Ribonuclease activity in kidney supernatant and pH 5 enzyme preparations was much greater than it was in liver preparations, and may partly account for the lower polysome yield and protein synthesis in kidney.

Effect of magnesium chloride and potassium chloride on the sedimentation characteristics of ribonucleoprotein isolated from dog pancreas

Ribonucleoprotein particles were isolated from dog pancreas under conditions of invariant ionic composition and analyzed by zone sedimentation in sucrose gradients. On the basis of calculated sedimentation coefficients, the particle population was considered as three fractions: monomeric ribosomes and subunits, S20,w 0 to 100; polysomes, S20,w 100 to 500; and large ribosomal aggregates, S20,w greater than 500. The distribution of the particle population was studied under conditions of variable magnesium ion concentration in one series and of variable potassium chloride concentration in another. Monomeric ribosomes dissociated into 57 s and 38 s subunits at concentrations of magnesium ion below 3 m M , The 57 s particle became the predominant species at 0·5m M -Mg2+ . The polysomal pattern, however, remained relatively stable in this range. The release of subunits from polysomes occurred at magnesium ion concentrations below 1 m M and was responsible for a decreased yield of this fraction. The yield of large aggregates increased markedly above 3 m M -Mg2+. The data indicate that ribosomes possess a heterogeneous range of magnesium ion dissociation constants. Magnesium ion concentration was demonstrated to play a dual part in this population of particles; (1) dissociation of monomers at low concentrations; (2) association of the particles into very large aggregates at high concentrations. The yield of the large aggregates was also increased at low concentrations of potassium chloride. Sedimentation analysis of the post-mitochondrial supernatant fraction in the absence of deoxycholate showed 60% as many monomeric ribosomes as in its presence, but the yield of polysomes was greatly reduced. The results suggest that most of the polysomes are bound to the endoplasmic reticulum in the cells of this tissue. Variations in magnesium ion concentration altered the sedimentation pattern of the post-mitochondrial supernatant fraction similarly to that of the isolated particles. The data support the conclusion that polysomes are not random aggregates, since the amount of the latter can be varied independently of that of polysomes by alterations in the ionic composition of the medium.