Cytoplasmic distribution of pulse-labelled poly(A)-containing RNA,particularly 26 S RNA,during myoblast growth and differentiation

Total poly(A)-containing RNA in different polysomal and supernatant cytoplasmic fractions was analysed after pulse-labelling in dividing myoblasts and fused myotubes. In particular, the peak of 26 S RNA (putative messenger for the large subunit of myosin) is located in a light region of the gradient coinciding with the monosome-trisome fractions prior to fusion, and is found in the heavy polysomes only after fusion. These heavy polysomes are free (i.e. not membrane bound). Treatment of the light part of the polysome gradient with EDTA shows that the 26 S RNA found here does not exist as part of a polysomal complex, but is present as a ribonucleoprotein particle cosedimenting in this region. Previous experiments had indicated that in actively dividing myoblasts 26 S RNA has a relatively short half-life but that it becomes “stable” after the cessation of mitosis just prior to fusion. RNA chase experiments performed in the present study show that the “short-lived” 26 S RNA from dividing myoblasts, which is present as a ribonucleoprotein particle, does not enter the heavy polysomes. In contrast, the more stable 26 S RNA also initially present as a ribonucleoprotein, just prior to and in the early stages of fusion, can be shown by chase experiments to enter the heavy polysomes later in fusion. Hence accumulation of 26 S RNA seems to precede its activation as a messenger.     

Electron microscopic visualization of a discrete class of giant translation units in salivary gland cells of Chironomus tentans

Lysates were made of whole salivary glands from Chironomus tentans and electron microscopic spreads prepared according to Miller and Beatty (1969). The sedimented material consisted mainly of ribosomes, most of which were present in giant polysomes which showed gradients of material protruding laterally from the ribosomes, interpreted as nascent polypeptide chains. Each giant polysome contained one such gradient. On the basis of the presence of a small group of terminal ribosomes without nascent chains at the 3' end, 19 apparently complete polysomes were selected, representing a discrete class of polysomes containing between 66 and 92 ribosomes (79 +/- 7, m +/- s.d.). Arguments are presented that they constitute the translation units for giant secretory proteins coded by RNA from the large Balbiani rings, BR1 and BR2.     

Control of tyrosinase gene expression and its relationship to neural crest induction in Rana pipiens. I. Isolation and characterization of amphibian tyrosinase mRNA

Rana pipiens tyrosinase mRNA was isolated from Stage 22 (tailfin circulation) embryos by indirect immunoprecipitation of embryonic polysomes using highly specific rabbit anti-tyrosinase and goat-(anti-rabbit) immunoglobulins. Analysis on sucrose gradients indicated that anti-tyrosinase bound specifically to embryonic polysomes of the 300-350 S class coincident with the location of nascent tyrosinase enzyme activity and tyrosinase mRNA. These same anti-tyrosinase-bound polysomes were fully immunoprecipitated by the addition of goat-(anti-rabbit) IgG. Poly(A+) RNA was obtained from phenol-extracted antibody. polysome complexes by sequential passage over oligo(dT)-cellulose. The final purification of tyrosinase mRNA was achieved by preparative sucrose gradient fractionation. Tyrosinase mRNA sedimented as a single 13 S peak in 5-30% sucrose gradients and tracked on sodium dodecyl sulfate-polyacrylamide gels as a single band of 4.5 X 10(5) Da (1275 nucleotides). When assayed in a cell-free translation system, this mRNA directed the synthesis of a single 35,000-Da protein which co-migrated with native tyrosinase on sodium dodecyl sulfate-polyacrylamide gels and which was greater than 98% immunoprecipitable by anti-tyrosinase immunoglobulin. Final purification was 4103-fold over the starting polysomal RNA.     

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.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

Polysome disassembly in cell extracts of cricket male accessory gland during sucrose gradient centrifugation

The stability of polysomes in cell extracts of cricket (Acheta domesticus) male accessory gland has been examined by sedimentation through a variety of step and linear sucrose gradients. After prolonged centrifugation there is a considerable decline in polysome content with a concurrent increase in monosomes. The extent of the reduction is more severe in step gradients, although the polysomes that remain show a typical profile on linear gradients.Evidence is presented which indicates that the reduction in polysome content is not due to nuclease action. The presence of detergents can affect the extent of disassembly but is not the principal cause. Comparison of [³H]leucine pulse-labelled gluteraldehyde-fixed and unfixed polysomes subjected to extended centrifugation reveals a release of nascent label near the top of the gradients in unfixed preparations. At least part of this displaced material is present as peptidyl-tRNA, suggesting that forced dissociation of polysomes rather than premature termination of nascent chains occurs as a consequence of sedimentation pressures. Comparison of the distribution of polyadenylic acid (poly(A)) sequences in sucrose gradients following short- and long-term centrifugation shows a shift of poly(A) containing RNA out of the polysome and into the pre-monomer region. It is concluded that sucrose gradient sedimentation results in the disassembly of a portion of the polysome population in the tissue examined. The implications with regard to the study of nonpolysomal messenger ribonucleoprotein and monomeric ribosomes are discussed.     

Membrane-bound mRNAs are recruited from preinitiated ribonucleoprotein particles in injected Xenopus oocytes

Messenger RNA injected Xenopus oocytes exhibit a differential capacity for translation. mRNAs translated in the free cytoplasm are translated efficiently whereas mRNAs translated on the rough endoplasmic reticulum (RER) membrane are translated inefficiently. If mRNA injected oocytes are injected additionally with proteins isolated from the RER, enhanced translation of RER-bound mRNAs is observed. When examined by sucrose gradient centrifugation and RNA dot blots, most of the injected RER-bound mRNA sediments less than or equal to the 80 S monosome. The RER proteins recruit these preinitiated mRNAs onto polysomes as evidenced by a shift in sedimentation to the polysome region of a sucrose gradient. When examined by immunoblotting, the RER proteins are shown to contain a protein which reacts specifically with an antibody directed against docking protein (SRP-receptor protein). However, this putative docking protein does not appear to be the protein which actually recruits the preinitiated mRNAs onto polysomes.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

An RNA-binding protein from Xenopus oocytes is associated with specific message sequences

Monoclonal antibodies directed against an RNA-binding protein from Xenopus oocytes were used to immunoselect messenger ribonucleoprotein (mRNP) particles. RNA was extracted from both the immunoselected and nonselected fractions and was used to direct the synthesis of oligo (dT)-primed 32P-cDNA. These two cDNA preparations were then used to probe Xenopus stage-1 oocyte cDNA libraries to identify sequences that had been specifically coimmunoselected by the antibodies. Three cDNA clones were shown to be derived specifically from the antibody-selected mRNPs. During very early oogenesis (stage 1-2), the RNA-binding protein and the three coselected mRNAs sediment in the nontranslating mRNP region of a sucrose gradient. By oocyte stage 6, the binding protein concentration decreases by as much as 22-fold relative to polyadenylated RNA. At this stage of development, the three mRNAs are found predominantly in the polysome region of a sucrose gradient. These data demonstrate that Xenopus oocytes contain an RNA-binding protein which binds specific message sequences and may regulate their expression.     

Identification of Saint Louis encephalitis virus mRNA.

Saint Louis encephalitis (SLE) virus-specific RNA was recovered from infected HeLa cells by sodium dodecyl sulfate (SDS)-phenol-chloroform extraction, and the molecular species were resolved by SDS-sucrose gradient centrifugation and agarose gel electrophoresis. Sucrose gradient centrifugation revealed the presence of a 45S species, minor 20 to 30S heterogeneous species, and an 8 to 10 S RNA species in the cytoplasmic extract. Analysis of the same samples by electrophoresis on agarose gels, under both nondenaturing and denaturing conditions, revealed only two virus-specific RNA molecules, the 45S genome-sized RNA and an 8 to 10S species. Varying the gel concentration to facilitate analysis of nucleic acids with molecular weights ranging from 25,000 to 25 X 10(6) failed to reveal additional RNA species, although low levels of a putative double-stranded replicative form could conceivably have escaped detection. From our observations it appears that the heterogeneous RNA species and presumably the 20S RNase-resistant species reported in other investigations of flavivirus RNA are degradation products or conformers of the 45S molecule. Polysomes from SLE virus-infected cells were prepared and separated from contaminating nucleocapsid by centrifugation on discontinuous sucrose gradients. RNA extracted from these polysome preparations was analyzed by sucrose gradient centrifugation and agarose gel electrophoresis. The 45S SLE virus genome-size molecule was found to be the only RNA species associated with the polysomes. This molecule was sensitive to RNase digestion and was released from polysomes by EDTA and puromycin treatment. These findings provide direct evidence that the 45 S SLE virus RNA serves as the messenger during virus replication, in contrast to the 26S RNA species which functions as the predominant messenger during alphavirus replication.     

Balbiani ring RNA in the cytoplasm of Chironomus thummi salivary gland cells

In this paper experimental results on the size, transport and stability of cytoplasmic Balbiani ring RNA and on its appearance in polysomes are presented. Cytoplasmic RNA of salivary gland cells from Chironomus thummi contains two large RNA fractions of about 20×10⁶ dalton and 10×10⁶ dalton in size. These RNA fractions correspond both to Balbiani ring BR 1 RNA and BR 2 RNA and are apparently transported from nucleus into cytoplasm without a significant size reduction. Chase experiments illustrate a great stability of giant cytoplasmic Balbiani ring RNA molecules and exclude the possibility of a precursor-product relationship between these and smaller BR RNA molecules also found in cytoplasm. A part of giant cytoplasmic Balbiani ring RNA molecules is bound to poly(U)-sepharose columns and should, therefore, contain poly(A)-sequences. — Polysomes of salivary gland cells extracted by a gentle lysis procedure and centrifuged through sucrose gradients are characterized by a rather broad sedimentation profile. Polysome sizes up to about 800 S have been detected, but in no case a distinct polysome fraction corresponding in size to Balbiani ring RNA has been observed. Hybridization of polysomal RNA with salivary gland chromosomes in situ resulted in labelling of both Balbiani rings BR 1 and BR 2.     

Polysome formation and incorporation of new ribosomes into polysomes during germination of the embryonic axis of maize

Isolated axes of Zea mays L. cvs CiV₂ and CUZCO were imbibed for different periods of time, and free polysomes were extracted and analysed by centrifugation in a sucrose gradient. The amount of rRNA per axis was determined at different moments of germination. Polysome reassembly was practically completed by 8 h and 54% of the preformed ribosomes were found in the polysome fraction. An increase in the proportion of large polysomes was also observed during this period of germination. During the following period, the polysome content and the distribution of the various classes of polysomes remained unchanged.
The time of appearance of newly synthesized ribosomes into the polysomes was investigated using axes germinated in the presence of [³H]-uridine. Centrifugal analysis of EDTA-dissociated polysomes and gel electrophoretic analysis of polysomal RNA showed that new ribosomes appeared into polysomes a few hours after completion of the initial polysome assembly. When released into the cytoplasm, the new ribosomes were preferentially incorporated into polysomes rather than stored as free ribosomes.     

Characterization of polysome-associated RNA from influenza virus-infected cells.

Virus-specific polysome-associated RNA (psRNA) and RNA after dissociation of polysomes were analyzed by direct hybridization with unlabeled viral RNA (vRNA) and complementary RNA (cRNA). psRNA after a 30-min pulse with [3H]uridine contained 28% labeled cRNA, 70% host RNA, and no vRNA. After dissociation, psRNA sedimented heterogeneously. Heavy RNA (greater than 60S), ribosomal subunit RNA (rsuRNA, 30-60S), free mRNA (fmRNA, 10-30S), and light RNA (less than 10S) contained 16%, 54%, 70% and 28% cRNA, respectively, but no vRNA. When actinomycin D (AcD) was added at 2 h postinfection, the nature of the psRNA depended on the concentration of AcD and the condition of the labeling. At AcD concentrations of 1 mug or more per ml, no detectable vRNA or cRNA was associated with polysomes. At 0.2 mug of AcD per ml (a concentration that partially inhibited cRNA synthesis) and 2 h of labeling at 2.5 h postinfection, psRNA contained 40% viral-specific RNA, which included both vRNA and cRNA in almost equal amounts. When polysomes were dissociated, however, viral-specific fm RNA from AcD-treated cells contained exclusively cRNA and no detectable vRNA. Increasing amounts of labeled vRNA were present in the heavy region of the gradient (and in the pellet), which also contained varying amounts of cRNA. The labeled vRNA appears to be associated with polysomes in a cesium chloride density gradient (rho = 1.525 g/ml). Although we have ruled out the trivial explanation of viral ribonucleoprotein contamination,the nature of the complex containing both polysomes and vRNA is unknown.     

Post-mortem decay of rat skeletal muscle polysomes]

Post-mortem changes of cytoplasmic RNP particles from rat skeletal muscles are studied using sucrose density gradient. The polysome destruction is found to beign within 1-2 hours after the animal death. However, a part of polysomes turned to be stable at least within 48-60 hours after the death. These polysomes incorporated 14C-leucine into acid insoluble fraction in cell-free system and were decomposited by RNase treatment (10 mkg/ml). The data suggest that within 24-48 hours of the after-death destruction the decay begins to free monoribosomes while there are still active polysomes in muscles.     

Ricin does not act as an endonuclease on L cell polysomar RNA.

The ability of ricin to act as an endonuclease was examined using labelled polysomes isolated from mouse L cells grown in [³²P] orthophosphate. No change was detected in the electrophoretic mobility of any ribosomal RNA species whether L cells were first treated with ricin or the isolated polysomes were treated with ricin. No trichloroacetic acid soluble counts over control levels were released by ricin treatment of isolated polysomes and no changes were observed in sucrose density gradient polysome profile with ricin treatment. The action of ricin on polysomes which results in inhibition of translation does not appear to involve endonuclease activity.     

Content and Aggregation of Ribosomes during Formation, Dormancy and Sprouting of Tubers of Helianthus tuberosus

The ribosomes and their qualitative (monosomes-polysomes) and quantitative variations over a whole vegetative period of the tuber of Helianthus tuberosus L. (cv. OB 1) were examined. Tubers in different phases of growth, dormancy and sprouting or slices of dormant tubers activated with 2 x 10^-6M indol-3-acetic acid were used. The ribosomes were analyzed by a linear sucrose gradient. During flowering, polysomes of tuber disappeared almost completely and rRNA decreased in comparison with the level present at the beginning of tuber formation. After flowering, there was a new synthesis of monosomes and polysomes until the onset of dormancy; this last period was characterized by a marked increase in polysomes and a proportional increase in monosomes. The level remained almost constant till the break of dormancy. When the tubers sprouted, ribosomes, present almost exclusively as monosomes, decreased considerably; on the contrary the non-photosynthetic sprouts contained many monosomes and polysomes. The first phases of activation (3 h) of tuber slices were characterized by a RNA synthesis, which occurred during one hour, in the subunit region of the gradient. Successively (10 h of activation) the ³²P incorporation was seen also in the polysome region and increased with time. Some possible interpretations of these last results are discussed.     

Action of red and far red light on ribosome formation in cabbage seedlings

The action of light on ribosome formation was examined in the cabbage seedlings, a system extensively used in the studies of anthocyanin synthesis. Ribosomes were extracted 18 h after the beginning of the irradiation and separated by sucrose gradient centrifugation. In the cotyledons of dark-grown cabbage seedlings, a brief red light induces an increase both in total ribosomes and in the fraction present as polysomes; the effect of red light is reversed by far red light, indicating the involvement of phytochrome in polysome formation in cabbage seedlings. Continuous red and continuous far red light are about equally effective in bringing about an increase of total ribosomes and of the polysome fraction. Streptomycin, which inhibits chlorophyll synthesis and chloroplast development, and enhances anthocyanin synthesis in cabbage seedlings, causes a decrease of total ribosomes and of the fraction present as polysomes. In hypocotyls, the red-far red reversibility is evident only for the polysome content and streptomycin does not decrease the polysome/monosomo ratio as it does in cotyledons.     

Characterization of polysomes from Xenopus liver synthesizing vitellogenin and translation of vitellogenin and albumin messenger RNA's in vitro.

1. Conditions are described for the isolation of polysomes from the liver of Xenopus laevis. The method involves homogenization of liver in 0.2 M Tris-HCl pH 8.5, treatment with 2% Triton X-100 and subsequent sucrose density gradient fractionation of polysomes from a 10000 X g supernatant. 2. Vitellogenin synthesis was induced in male Xenopus liver by oestradiol treatment. Polysomes were isolated and vitellogenin-synthesizing polysomes characterized by their association with monospecific 125 I-labelled rabbit anti-vitellogenin antibody and by reaction with rabbit anti-vitellogenin immunoglobulins followed by indirect immunoprecipitation with goat anti-rabbit antibody. 3. Changes in liver polysome content following oestrogen treatment of male Xenopus are correlated with the appearance of vitellogenin synthesis using an organ culture assay. 4. RNA extracted from livers of oestradiol-treated male Xenopus and from purified polysomes is shown to code for the synthesis of vitellogenin-specific immunoprecipitable polypeptides in a rabbit reticulocyte cell-free protein-synthesizing system, a major component having a molecular weight of 210000. Xanopus liver RNA is also shown to code for the synthesis of an albumin-specific immunoprecipitable polypeptide of 74000 molecular-weight which coelectrophoresed with Xenopus albumin.