An improved method for the isolation of polysomes from synchronous macroplasmodia of Physarum polycephalum

An improved method for the isolation of polysomes from synchronous macroplasmodia of Physarum polycephalum is described. The procedure involves preincubation and homogenization of the macroplasmodia in high ionic strength media supplemented with high concentrations of Mg²⁺ and ethylene glycol bis (β-aminoethyl ether) N,N′-tetraacetic acid. The polysomes recovered from plasmodial postmitochondrial lysates or heavy particle fractions prepared in this way are sensitive to RNase and EDTA and more than 90% of the total single ribosome population is present as polysomes. The polysomes obtained could also be utilized as a beginning point for the isolation of poly(A)-containing RNA which showed a mass average sedimentation value of 18 S. The development of a satisfactory procedure for the isolation of intact polysomes and poly(A)-containing RNA from macroplasmodia should facilitate studies on mRNA metabolism and translational activity during a naturally synchronous mitotic division cycle.     

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)⁺RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)⁺RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules.     

Isolation and in vitro translation of polysomes from mature rye leaves

Cytoplasmic polysomes have been prepared from mature leaves of winter rye (Secale cereale L. cv Puma). This is the first time a method has been developed for isolation of highly polymerized polysomes from mature leaves. The degree of intactness of isolated plant polysomes has been determined by two independent but complementary methods: size class distribution by sucrose gradient centrifugation and in vitro translation. The polymerization of isolated polysomes was estimated by the ratio of the proportion of large polysomes to the proportion of small polysomes obtained from the profiles. Our results show that the composition of the optimal polysome isolation buffer for mature rye leaves is different from that reported for young tobacco and pea leaves. Polysomes were translated in vitro with the S-105 wheat germ fraction. The degree of polysome polymerization has a significant effect on their in vitro translation since both the incorporation of amino acid and the presence of high molecular weight polypeptides are proportional to the large polysomes/small polysomes ratio. This study emphasizes the need to evaluate isolation conditions carefully before proceeding with polysome studies in any particular tissue or in tissues under different physiological status.     

Translation of mRNA associated with monosomes and residual polysomes following disaggregation of brain polysomes by LSD and hyperthermia

Intravenous administration of LSD to young adult rabbits induces a transient disaggregation of brain polysomes and a relocalization of mRNA from polysomes to monosomes. To analyze the spectrum of mRNA molecules which were associated with either the residual polysomes or the translationally inactive monosome complex, these two fractions were isolated on sucrose gradients and translated in a reticulocyte cell-free system. Analysis of [³⁵S]methionine labeled translation products by one and two dimensional gel electrophoresis revealed that a full spectrum of mRNA molecules was relocalized from polysomes to monosomes following drug induced polysome disaggregation. The only exception was the mRNA coding for the LSD-induced 74K protein which was associated with the residual polysome fraction and not with the monosome complex. This brain protein is similar in molecular weight to one of the major heat shock proteins which are induced in tissue culture cells following elevation of ambient temperature and disaggregation of existing polysomes. The mRNA coding for the 74K brain protein was not observed in polysomes isolated following blockage of LSD-induced hyperthermia but it was noted when hyperthermia was induced by elevation of ambient temperature. The mRNA species coding for the 74K protein was polyadenylated.     

Translational alterations in maize leaves responding to pathogen infection, paraquat treatment, or heat shock : polysome dissociation and accumulation of a 57 kilodalton protein

Translational alterations occur in maize (Zea mays L.) leaves stressed by pathogen infection or herbicide paraquat treatment. These translational changes include: (a) dissociation of large polysomes to small polysomes, monosomes, and subunits; (b) a decreased rate of total protein synthesis; and (c) a reduced synthesis of several proteins by polysomes in vitro. The polysome dissociation was neither due to an extraction artifact nor to degradation of RNA by RNase. The protein patterns of polysomes isolated from leaves inoculated with Bipolaris maydis at 6 to 48 hours showed an increase in the intensity of a 57 kilodalton protein. When inoculated with less virulent pathogens, such as B. zeicola, Exserohilum turcicum, or Colletotrichum graminicola, the protein was accumulated in polysomes of leaves at 24 to 48 hours after inoculation. The 57 kilodalton protein was also accumulated in polysomes of maize leaves responding to heat shock or herbicide paraquat treatments. The purified 57 kilodalton protein reassociated with polysomes isolated from healthy leaves and inhibited polysomal translation in vitro. Since the 57 kilodalton protein is rapidly accumulated in maize polysomes in response to various biological and environmental stresses and may affect protein synthesis, it may be involved in translational regulation of maize leaves during stress response.     

PROTEIN SYNTHESIS IN PANCREAS OF FASTED PIGEONS

The regulation of protein synthesis in the pigeon has been studied by comparing the capability of cell-free amino acid incorporating systems of membrane-bound and membrane-free polysomes prepared from fasted and fed birds. New methods were developed for isolating polysomes since techniques used for other tissues did not provide quantitative recovery of polysomal RNA. The sucrose gradient profile of polysomes from pigeon pancreas showed a predominance of trisome species. Although initiation factors are present on polysomes, it was found that polysomes in cell-free systems would not initiate protein synthesis without exogenous initiation factors. This suggested the presence of an inhibitor or regulator of protein synthesis. These studies show that fasting resulted in: (a) decreased amounts of polysomes; (b) disaggregation of polysomes to monosomes; (c) decreased capability of polysomes to synthesize nascent peptides and to initiate additional synthesis, apparently not related to concentration of initiation factors.     

The termination of the synthesis of proteins in vitro with extracts from the undeveloped cyst of Artemia salina

Soluble fractions (S-100) from both undeveloped cysts and developing embryos of Artemia salina promoted elongation of polypeptides initiated in vivo on polysomes of developing embryos or nauplius larvae. The ability of the extract from the undeveloped cyst to terminate correctly the synthesis of polypeptides has been determined indirectly from the distribution of polysomes before and after in vitro translation and, more directly, from the nature of the protein product released from rabbit reticulocyte polysomes. The extract from the undeveloped cyst and also, as expected, that from the developing embryo catalyzed a reduction in the amount of the polysomes of larger size and an increase in the amount of 80 S ribosomes. The soluble extract from the undeveloped cyst can terminate the synthesis of rabbit globin on reticulocyte polysomes. The major polypeptide product released from the polysomes had an electrophoretic mobility identical with that of the subunit of isolated rabbit globin. This indicated that the cyst contained the components necessary to complete and terminate the synthesis of polypeptides correctly and that the released protein product was not predominantly as a result of premature chain termination. The size distribution of Artemia salina proteins released from polysomes from developing embryos was similar when the synthesis was directed by the S-100 at each stage of development.     

Variations in the amount of polysomes in mature oocytes of Drosophila melanogaster.

Quantitative measurements of polysomes and ribosomes of Drosophila melanogaster egg chambers, mature oocytes, and embryos were done using sucrose gradient analysis. The amount of polysomes per egg chamber increases about 20 times from stage 5 to 13, and then remains constant up to the end of embryogenesis. The percentage of ribosomes in polysomes is fairly constant during oogenesis and embryogenesis (56 ± 7%). Depending on the fly population, the percentage of ribosomes in polysomes of mature oocytes varies from 10 to 70%. It is shown that the percentage of polysomes in mature oocytes decreases with the time of retention of the mature oocytes in the ovary. Twenty-four- to thirty-six-hour-old flies kept in optimal conditions retain their mature oocytes for 2–3 hr. These mature oocytes still contain 40–60% ribosomes in polysomes. Conditions are given which allow the obtainment of reproducibly high amounts of polysomes from mature oocytes of Drosophila.     

Morphological changes in Krebs II ascites tumour cells induced by insulin are associated with differences in protein composition and altered amounts of free,cytoskeletal-bound and membrane-bound polysomes

A three-step sequential detergent/salt extraction procedure was used in order to isolate three distinct subcellular fractions containing free (FP), cytoskeletal-bound (CBP) and membrane-bound polysomes (MBP), respectively, from Krebs II ascites cells (Vedeler et al., Mol Cell Biochem 100: 183–193,1991). The purpose was to study changes in the distribution of polysomes in these three fractions during long-term incubation with insulin under either stationary conditions or in roller suspension culture- Insulin caused a redistribution of polysomes between FP, CBP and MBP fractions. The hormone appeared to promote an entry of ribosomes into polysomes both in CBP and MBP populations. When cells were grown in stationary culture in the presence of insulin and thus promoted to attach to the substratum and undergo morphological changes, a diversion of ribosomes from CBP into MBP was observed. The level of protein synthesis was apparently very high in this latter fraction since more then 70% of ribosomes were in polysomes. Morphological changes observed following insulin treatment were accompanied by a shift of certain proteins among subcellular fractions (for example actin and p35). The fibronectin content was about 20% higher in attached compared to non-attached cells. The results suggest that morphological changes induced by stimulation with insulin are associated with an increased activity of MBP, presumably reflecting a requirement for an increased synthesis of membrane proteins. (Mol Cell Biochem 118: 131–140, 1992)     

Red-Far Red Reversible Effect on Polysome Formation in the Embryos of Pinus thunbergii Seeds

Polysome formation in the embryos of Pinus thunbergii seeds was studied. Free ribosomes were dissociated to smaller subunits in a high salt buffer, but the complex ribosomes were not. The free ribosomes could be distinguished from monomer ribosomes derived from polysomes after RNase treatment. The monomer ribosomes present in the embryos of the dark-imbibed seeds were predominantly free ribosomes; very small quantities of polysomes could be detected in the embryos from dark-imbibed seeds. Such polysomes remained at a very low level during dark imbibition at least for a month. The level of polysomes increased 4 hours after a brief exposure to red light. The effect of red light on polysome formation was partially reversed when followed by far red light irradiation.     

Movement of ribosomes over messenger RNA in polysomes of rel + and rel - Escherichia coli strains

The in vitro movement of ribosomes over messenger RNA was studied in both the presence and the absence of protein synthesis. For this purpose, labeled polysomes were extracted from rel⁺ and rel^? strains of Escherichia coli grown in the presence of radioactive uracil and incubated in a cell-free system containing tRNA, amino acids, soluble enzymes and a source of energy. The gradual conversion of the labeled polysomes into monosomes and ribosomal subunits was followed by subjecting the reaction mixture to sucrose gradient sedimentation after various incubation times and measuring the radioactivity present in the three relevant ribosomal fractions.It was found that when the conditions of incubation allow protein synthesis to occur, polysomes extracted from rel⁺ and rel^? cells are converted mainly into free monosomes, which can be made to dissociate into subunits by high-sodium or low-magnesium ion concentrations. Under conditions in which protein synthesis cannot occur because a mutant aminoacyl-tRNA synthetase has been rendered inactive, polysome conversion still occurs, though to a reduced extent. When the products of such residual run-off are examined, however, a difference is manifest between polysomes extracted from rel⁺ and from rel^? strains: whereas the polysomes from the rel^? strain are still converted into free monosomes even in the absence of protein synthesis, the polysomes from the rel⁺ strain are now converted mainly into subunits. It can be inferred, therefore, that ribosomes from rel^? bacteria, but not those from rel⁺ bacteria, continue movement over messenger RNA in the absence of protein synthesis.Studies of mixed extracts from rel^? and rel⁺ bacteria have shown that the character of the run-off process does not depend on the source of tRNA and soluble enzymes; the proportions of monosomes and subunits among the run-off products formed in the absence of protein synthesis depend only on the source of the polysomes. It is suggested that the mutation of the rel gene alters the functional architecture of ribosomes.     

Quantitative changes in protein synthesis during oogenesis in Xenopus laevis

Protein synthesis rates in Xenopus laevis oocytes from stage 1 through stage 6 were measured. In addition, the translational efficiencies, total RNA contents, and percentages of ribosomes in polysomes in growing oocytes at several stages were determined. Stage 1 oocytes synthesize protein at a mean rate of 0.18 ng hr⁻¹ while stage 6 oocytes make protein at a rate of 22.8 ng hr⁻¹. Polysomes from growing and full-grown oocytes sedimented in a sucrose gradient with a peak value of 300 S, corresponding to a weight-average packing density of 10 ribosomes per mRNA. Ribosome transit times of endogenous mRNAs were essentially unchanged at all stages examined. While the oocyte's total ribosomal RNA content was observed to increase about 115-fold during oogenesis, the percentage of ribosomes in polysomes remained constant at approximately 2%. Taken together, the data suggest that the 127-fold increase in protein synthesis which occurs during Xenopus oogenesis involves the progressive recruitment onto polysomes of mRNA from the maternal stockpile.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

Specificity of factors isolated from free polysomes and microsomes on in vitro protein synthesis in plasmacytoma cells

A simple procedure for measuring chain initiation in complete in vitro systems is described. The capacity of free and membrane-bound polysomes prepared by a detergent technique or by nitrogen cavitation to incorporate radioactive amino acids into proteins was compared with the capacity of these polysomes to initiate polypeptide chains.The extent of amino acid incorporation by polysomes prepared by either of these methods did not differ significantly. However, chain initiation determined by measuring the incorporation of radioactive subunits into polysomes showed that polysomes prepared by the detergent technique were less effective than polysomes made by nitrogen cavitation in chain initiation.Crude initiation factors, prepared by washing either free polysomes or microsomes with 0.5 M KCl, stimulated chain initiation and amino acid incorporation by both types of polysomes. Free polysomes were stimulated almost to the same extent by factors isolated from free polysomes or microsomes. Membrane-bound polysomes on the other hand showed a specific requirement for microsomal factors.The extent of stimulation by crude polysomal factors was dependent on the concentration of high speed supernatant in the assay system.     

A test for masked message: the template activity of messenger ribonucleoprotein particles isolated from sea urchine eggs

The hypothesis that the “masked message” of unfertilized eggs consists of nontranslatable mRNP particles was directly tested by in vitro translation of mRNPs in a system derived from wheat germ. Three classes of mRNPs were tested: particles prepared from sea urchin eggs in buffers containing 0.35 M K⁺, particles prepared from sea urchin eggs in 0.35 M Na⁺, and particles released with EDTA in 0.35 M K⁺ from polysomes of sea urchin embryos cultured in the presence of actinomycin D. The mRNA content of particles was monitored by determination of poly(A) content. The wheat germ system used is quantitatively stimulated by addition of mRNA derived from eggs or from any of the classes of mRNPs used. Particles prepared from eggs with Na⁺ or released from polysomes contain less protein than particles isolated from eggs in K⁺, and as expected these particles are fully translatable in vitro. Particles prepared from eggs in buffers containing 0.35 M K⁺ produce little or no stimulation in the in vitro system. That this lack of translation represents in vivo masking is indicated by several considerations: (1) The nontranslatable particles were prepared in 0.35 M K⁺ and 5 mM Mg²⁺, ion concentrations similar to those found in echinoderm eggs; (2) density and sedimentation rate characteristics of the particles are little changed by isolation; (3) RNA extracted from isolated particles is fully translatable; and (4) particles prepared from polysomes or under conditions which destabilize RNPs are translatable. These data support the masking hypothesis for the protein synthesis repression system of eggs.     

A Rapid Method for the Separation of Free and Membrane-Bound Polysomes of Rat Liver by Gel Filtration on Columns of Sepharose 4B

Abstract

A rapid method is reported for the separation of free and bound polysomes from total microsomes of rat liver on columns of Sepharose 4B, in which the total procedure from sacrifice of the animal to final preparation takes less than five hours. Both forms of polysomes are active in the incorporation of amino acids into proteins, the free polysomes having approximately twice the activity of bound polysomes. Both preparations contain polysomes at least six ribosomes in length.     

Effects of juvenile hormone on polysome integrity

Juvenile hormone inhibits protein and RNA synthesis in cell cultures from Trichoplusia ni and in the testicular germinal cysts of Hyalophora cecropia pupae in vitro. Sucrose gradient analyses revealed that the polysomes of both the T. ni cells and the germinal cysts were disaggregated almost immediately after the addition of juvenile hormone in vitro with a corresponding dose-dependent increase in monosomes. It is suggested that previous reports revealing juvenile hormone inhibition of ecdysone stimulated RNA and protein synthesis may be due to polysome disaggregation. Further studies demonstrated that the effect is not restricted to insect cells and can be elicited by several other lipids devoid of juvenile hormone morphogenetic activity. Experiments with broken cell preparations and isolated polysomes suggest the necessity of cell membrane integrity for the effect on the polysomes. Several probing studies utilizing cycloheximide, ribonuclease, and high K⁺ concentrations were conducted on the means by which juvenile hormone and other lipids may elicit polysome disaggregation.     

QUANTITATIVE ISOLATION AND PROPERTIES OF NEARLY HOMOGENEOUS POPULATIONS OF UNDEGRADED FREE AND BOUND POLYSOMES FROM RAT BRAIN1

Abstract— A procedure is described for the preparation of free and bound polysomes from whole homogenate of rat brain tissue. Brain is homogenized in a sucrose-polysome buffer medium high in KCl (250 mm). After a 12-min centrifugation at 135,000 g, the free polysomes in the supernatant are decanted and saved, while the membrane bound polysomes in the pellet are resuspended in homogenizing medium, homogenized in the presence of detergent (Triton X-100), centrifuged for 5min at 1470 g to remove nuclei, decanted, treated with deoxycholate and centrifuged for 10 min at 24,000 g to remove deoxycholate-insoluble material. Polysomes in the two supernatants are harvested by centrifugation through sucrose gradients prepared in high KCl polysome buffer, and with or without cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and largely free of the usual contaminants. Cross-contamination is minimal (>10%). The recovery of polysomes is at least 95%. The distribution of ribosomes and polysomes in rat brain is 58% free and 42% membrane-bound. The distribution of rat brain RNA is 65% ribosomal and 35% non-ribosomal. Conditions are described for the visualization and analysis of the entire complement of free and bound ribosomes. The size fractionation procedure is rapid and reproducible, requires much less ultracentrifugation than the density-gradient technique, and provides a nearly quantitative means of isolating undegraded free and bound polysomes of rat brain tissue.     

Surface biochemical changes accompanying primary infection with Rous sarcoma virus. I. Electrokinetic properties of cells and cell surface glycoprotein:glycosyl transferase activities

A special class of polysomes synthesizing tubulin was determined using embryos of the sea urchin, Hemicentrotus pulcherrimus. Three criteria were established for identification of polysomes carrying nascent tubulin, i.e., nascent tubulin on polysomes should have (i) colchicine binding activity, (ii) precipitability with vinblastine and (iii) coincidence in mobility by electrophoresis with tubulin. Two classes of polysomes had polypeptides which accorded with the three criteria. One was tetramers and the other was composed of 15–20 ribosomes. From data reported previously on the molecular weight and amino acid composition of completed microtubule proteins, it was suggested that the class of polysomes composed of 15–20 ribosomes constituted the polysome-synthesizing tubulin of sea urchin embryos. The nature of the nascent polypeptides carried by the tetramer polysomes having colchicine binding activity and precipitability with vinblastine could not be clarified.