Messenger ribonucleoprotein particles in developing sea urchin embryos

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)⁺ mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm³ in Cs₂SO₄. Poly(A)⁺ mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm³, while EDTA caused a shift in density to 1.55 g/cm³, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55–1.66 g/cm³, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [³H]uridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs₂SO₄ gradients at a density of 1.60–1.66 g/cm³, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10% protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.     

Equilibrium centrifugation of measles virus

Measles virus has been centrifuged on different density gradients. It sediments at densities of 1,20 g/cm³ in K-tartrate, of 1,18–1,21 g/cm³ in sucrose, 1,19–1,23 g/cm³ in CsCl and 1,19 g/cm³ in metrizamide gradients. Metrizamide reduced measles virus infectivity. In sucrose gradients sometimes more than one infectious peak was observed. Control Vero cells produced particles of the same densities as measles virus peaks. These peaks did contain actin as the major protein. The relevance of this finding in relation to the presence of actin in measles virus is discussed.     

Messenger RNA Nuclear Particles and their Attachment to Cytoplasmic Membranes in Krebs Tumour Cells

THE observation that some nuclear particles contain DNA-like RNA (D-RNA) supports the idea that messenger RNA (mRNA) species bind to specific proteins to form ribonucleoprotein complexes^1–4. The mechanism by which the nuclear particles are transported and eventually bind to ribosomes in a translation complex is still uncertain^5–9. We now present evidence that D-RNA nuclear particles of about 40–50S contain mRNA species which are transported into the cytoplasm and assemble on the network of the endoplasmic reticulum (ER). Ribosomes then bind to this membrane-bound mRNA to form a translation complex. The concept of membrane-bound mRNA has been suggested before¹⁰ but has not been conclusively demonstrated.     

Purification and partial characterization of virus-like particles from Schneider line 2 Drosophila cells

A procedure has been developed for the purification of virus-like particles (VLPs) from Schneider line 2 Drosophila cells. The VLPs were precipitated with polyethylene glycol from the cytoplasmic fraction of lysed cells and further purified by equilibrium centrifugation in CsCl density gradients, in which they band at a density of 1.366 g/ml. Electron micrographs of these preparations revealed polyhedral particles with a diameter of 310–330 Å. We have also found particles of this size in thin sections of the intact cells. Sedimentation of the VLPs through 10–70% sucrose gradients yields a sedimentation coefficient of 235 S. Preliminary studies show that the VLPs contain double-stranded RNA species of 10 S, 14.5 S, 16 S, and 18 S.     

ISOLATION AND CHARACTERIZATION OF A DNA VIRUS INFECTING FELDMANNIA SIMPLEX (PHAEOPHYCEAE)1

We report on the isolation and characterization of a virus that is formed in modified zoidangia of the marine brown alga Feldmannia simplex (Crouan) Hamel (Ectocarpales, Phaeophyceae). Isolated virus particles had a buoyant density of about 1.35 g·mL^?1 in CsCl equilibrium gradients. They contained one major polypeptide (MW = 55,000) and at least six additional polypeptides (MW = 15,000–120,000). Four of these proteins were glycosylated. The viral genome consisted of double-stranded DNA and formed two freely migrating fractions in pulsed-field-gel electrophoresis, namely linear DNA with a size of 220 kilobase pairs, and fragments of 10–60 kilobase pairs. However, electron microscopic examination revealed that a substantial fraction of the viral DNA occurred as closed circles. We suggest that the viral DNA in native particles is circular but tends to break at random sites during the preparation.     

Separation of B and C Type Virions by Centrifugation in Gentle Density Gradients

Murine type B particles were separated from type C (Rauscher leukemia virus) by means of gentle (low-increment rate) density gradients. The best separation was obtained when the density ranged from 1.13 to 1.20 g/cm³ when sucrose was used and from 1.12 to 1.28 g/cm³ with CsCl. The buoyant densities of the B and C particle bands in sucrose were 1.18 and 1.16 g/cm³, respectively. The CsCl gradient gave a better separation with the B particles banding at a density of 1.20 g/cm³ and with the C particle density little different from its value in sucrose.     

Polysomal and non-polysomal messenger RNA in neuroblastoma cells: Lack of correlation between polyadenylation or initiation efficiency and messenger RNA location

Neuroblastoma cytoplasm was fractionated on sucrose gradients into polysomes (>90 S) and non-polysomal particles (<90 S). Purified RNA from these fractions was translated using a wheat germ lysate and translation products were compared by two-dimensional gel electrophoresis. Non-polysomal messenger RNA directed the synthesis of a specific subset of polysomal mRNA translation products. Careful comparison of individual translation products demonstrated that specific mRNAs were not randomly distributed between polysomes and the non-polysomal fraction.Fractionation of both RNA populations into polyadenylated (poly(A)⁺) and non-adenylated (poly(A)^?) species indicated that specific, abundant non-polysomal mRNAs were not less adenylated than their polysomal counterparts. Furthermore, comparison of translation products from assays of subsaturating and supersaturating RNA concentrations demonstrated that no simple correlation could be made between the relative initiation efficiency of a specific mRNA and its distribution between polysomes and non-polysomal particles.     

Utilization of enzyme markers to determine the location of plasma membrane from Pisum epicotyls on sucrose gradients

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. Alaska) epicotyls have been fractionated and examined for marker enzyme activity. The coincidence of three reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K⁺-stimulated Mg²⁺-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with electron microscopic evidence reported by G. Shore and G. Maclachlan (J. Cell Biol. 64, 557–571; 1975), indicates that plasma membrane of pea epicotyl has a buoyant density of about 1.13 g/cm³. This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is, however, shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10–1.11 g/cm³), Golgi (1.12 g/cm³) and mitochondria (1.18 g/cm³). Furthermore, other recent literature indicates that the 1.13 g/cm³ buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. Our data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g. glucan synthetase and K⁺-stimulated Mg²⁺-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm³ density, but with particles of higher densities as well). Particulate cellulase was found to be associated only with particles with equilibrium densities of about 1.13 g/cm³. Cellulase thus proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm³ particulate components with minimal contamination by particles of higher densities.     

ISOLATION AND CHARACTERIZATION OF GOLGI FROM COCCOLITHUS PELAGICUS (PRYMNESIOPHYCEAE)1

Cells of the marine alga Coccolithus pelagicus (Wal-lich)J. Schiller grown in axenic cultures were homogenized and fractionated. The distribution of organelle markers was assessed enzymatically after centrifugation through zonal, density, and flotation gradients made with sucrose, sorbitol, or Percoll. Mitochondria (1.19 g·cm^-3) and chloroplasts (1.15 g·cm^-3) were recovered in sucrose gradients at densities similar to those observed for higher plants and most algae. The position of endoplasmic reticulum and plasma membrane in the gradients was monitored by NADPH cytochrome c reductase and vanadate-sensitive Mg²⁺-ATPase, respectively. Higher plant Golgi markers, latent undine diphosphatase (UDPase) and glucan synthase I, were colocalized at a density range including two peaks of activity at 1.13–1.15 g·cm^-3. Bound calcium was associated with high density (1.15 g·cm^-3) membranes. Ca²⁺-stimulated ATPase was found at high levels on membranes that did not coisolate with the latent UDPase-containing membranes. The Ca²⁺-stimulated ATPase, a possible participant during calcification, was associated with a chloroplast-enriched fraction in all the organelle separation systems. However, about 30% of the total activity was separated from both the chloroplasts and Golgi on 0–70% Percoll gradients containing 0.4 M sucrose. The possible relationship of the Golgi and the high-density organelle exhibiting Ca²⁺-stimulated ATPase to coccolithogenesis and the process of calcification and crystal formation is discussed.     

A precursor to hemoglobin mRNA in nuclei of immature duck red blood cells

A method is described for sedimenting RNA on a preparative scale in the presence of 85% formamide. It is likely that the RNA is fully denatured, since under the same conditions ribosomal RNA develops full hyperchromicity, and formaldehyde-treated and untreated ribosomal and nuclear RNA sediment in an identical manner.RNA from immature duck red blood cells was fractionated on sucrose gradients. The amount of hemoglobin mRNA and poly(A) in each fraction was measured by hybridization with complementary DNA and poly(U), respectively. We observed that hemoglobin mRNA forms aggregates in the presence of phenol, which are dispersed on formamide gradients. Nuclear RNA became more heavily aggregated, and again the aggregates were dispersed by formamide. A possible nuclear precursor of hemoglobin mRNA was identified. The molecular weight of the precursor is 6–7 × 10⁵, three times as long as hemoglobin mRNA, and it is attached covalently to a poly(A) sequence.     

Hydrangea mosaic virus, a new ilarvirus from Hydrangea macrophylla (Saxifragaceae)

A previously unrecognised virus isolated from Hydrangea macrophylla with chlorotic mosaic leaf symptoms in West Sussex was named hydrangea mosaic virus (HydMV). HydMV was mechanically transmitted without difficulty to four of 16 species from three of five families, and was seed transmitted in Chenopodium quinoa, but was not aphid transmitted. Although relatively unstable in vitro, HydMV was purified by clarifying leaf extracts by emulsification with chloroform and acidification with citric acid, followed by differential centrifugation and sucrose density gradient centrifugation. Purified virus incompletely separated on sucrose density gradients into three components (T. M and B) with sedimentation coefficients (s^o_20w) of 86, 97, and 105 S respectively, but all particles had buoyant densities in caesium chloride of 1.37 g/cm³. Virus contained a single polypeptide species (mol. wt 26.4 times 10³), appeared quasiisometric to ovoid or elliptical, and measured c. 28 times 30 (T), 30 times 30 (M) or 30 times 32–38 nm (B). Single-stranded RNA species or mol. wt 1–25, 1–08, 0–83, 0–36 and 0–27 (RNA-1 to 5 respectively) were obtained from virus preparations but mixtures containing RNA-1 to 3 plus either RNA-4 + 5 or the coat protein, were infective. These properties suggest that HydMV has affinities with ilarviruses, but it showed no serological relationship to any of six ilarviruses or 42 other viruses.     

Ribonucleoproteins containing mRNA in the cytoplasm of mouse plasmacytoma cells]

The rapidly labelled postribosomal ribonucleoprotein (RNP) found in the cytoplasm of mouse plasmacytoma cells were investigated. It has been shown that 45S and 80S particles contain relatively high molecular weight (approximately 12-17S) pulse-labelled RNA similar to the polyribosomal mRNA. No other postribosomal RNP was found which would contain an RNA with similar sedimentation characteristics. In CsC1 density gradients, the postribosomal RNP gives two peaks. One of them, the rapidly labelled component (rho 1.52 g/cm3) is found only in 45S RNP. The other rapidly labelled component (rho 1.36-1.41 g/cm3) is revealed in all investigated regions of sucrose gradients. The latter contains relatively low molecular weight RNA (approximately7-9S). These RNP are supposed to be informosome-like particles. The components with a buoyant density of 1.52 g/cm3 may represent an mRNP-45S subparticles complex. The rapidly labelled mRNA of 80S particles is released after EDTA treatment in the form of mRNP with a buoyant density of 1.45-1.47 g/cm3.     

Host range, purification and some properties of rubus Chinese seed-borne virus

A previously undescribed virus, for which the name rubus Chinese seed-borne virus (RCSV) is proposed, was isolated from a single, symptomless plant of an unidentified Rubus species grown from seed collected in the wild in the People's Republic of China, Experimentally RCSV infected 23 out of 39 spp. in six out of eight families. The virus was seed-transmitted in Chenopodium quinoa (100%) and Nicotiana bigelowii (27%). RCSV was not transmitted by the nematodes Xiphinema diversicaudatum or X. index. The particles of RCSV were isometric, c. 30 nm in diameter with some penetrated by negative stains. In thin sections particles were found in double walled tubular structures with an outer membrane enclosing one or more tubules. In crude extracts some particles were found within single-walled tubules. Two virus-associated bands were seen in sucrose density gradients of purified preparations. The upper band was not infective and consisted of penetrated particles apparently devoid of nucleic acid. The lower, infective band was resolved into two components, of density 1.452 and 1.461 g/ml, in caesium chloride isopycnic gradients. There were two polypeptides (mol. wts c. 47 000 and 25 200 daltons) and two nucleic acid species (one of mol. wt c. 1.4 × 10⁶ daltons; the second was poorly defined by the methods used but was of higher molecular weight). RCSV was distantly related serologically (6–7 SDI) to the type isolate of strawberry latent ringspot virus (SLRV) and also reacted with antisera to serologicaly distinct grape and olive isolates of SLRV. It did not react with antisera to 10 other isometric viruses.     

Characterization of poly(A+)RNA in free messenger ribonucleoprotein and polysomes of mouse Taper ascites cells

Cytoplasmic extracts of mouse Taper ascites cells were centrifuged on sucrose gradients to give 0–80 S, monosome, and polysome fractions. CsCl equilibrium density centrifugation of formaldehyde-fixed material from the 0–80 S fraction demonstrated that the messenger RNA in the 0–80 S fraction was in the form of free ribonucleoprotein. The size of the poly(A⁺)RNA and the size of the poly(A) segments of these molecules were shown to be very similar in both the free mRNP² and polysome fractions. The labeling kinetics of the free mRNP poly(A⁺)RNA was similar to that of the polysomal poly(A⁺)RNA.The free mRNP poly(A⁺)RNA efficiently stimulated protein synthesis in the wheat germ cell-free system, supporting the view that it was mRNA. Two-dimensional gel electrophoresis was used to analyze the proteins whose synthesis was directed by free mRNP and polysomal poly(A⁺)RNA. The free mRNP poly(A⁺)RNA directed the synthesis of a simpler set of abundant protein products than did the polysomal poly(A⁺)RNA. Most of the free mRNP abundant protein products were also present in the polysomal products, though obvious quantitative differences were evident, indicating that each individual mRNA had its own characteristic distribution between polysomes and the translationally inactive RNP form.     

In vitro translation of nitrate reductase messenger RNA from maize and tobacco and detection with an antibody directed against the enzyme of maize

NADH-nitrate reductase (NR) from maize leaves was purified by affinity chromatography on blue sepharose and used for the raising of a rabbit polyclonal antibody. Polyadenylated (Poly A⁺) RNA was isolated from maize leaves, fractionated by centrifugation on sucrose density gradients and translated in vitro. Among translation products, a 105 kDa polypeptide could be immunoselected specifically by the anti-nitrate reductase antibody. This polypeptide was shown to comigrate, in denaturing conditions, with the subunit of the purified enzyme. Similar results were obtained with mRNA preparations from tobacco.     

Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation.

The stored mRNP particles of Xenopus oocytes contain protein kinase activity and two major phosphoproteins of 60 kDa (pp60) and 56 kDa (pp56). These proteins can be phospholabelled in the particles either in vivo or in vitro and then isolated by SDS-PAGE. On renaturing pp60 in the presence of globin mRNA, a stable RNA-protein complex is formed. The complex has a uniform density in Cs salt gradients, corresponding to the binding of about 10 protein molecules to each mRNA, probably at the poly(A) sequence. Compared with uncomplexed mRNA, the RNP complex is translated poorly both in vitro and in vivo. Translation of the complex can be regained after treatment with protein phosphatase. It is shown that dephosphorylation destabilizes the binding of protein to RNA, making the mRNA accessible for translation. Studies with native mRNP particles show that their translation also can be enhanced by dephosphorylation.     

Partial characterisation of a new virus in brusca from the Venezuelan lowlands

Flexuous thread‐like virus particles c. 650–700 nm in length were isolated from brusca (Senna pallida) plants showing stunting, mosaic, vein yellowing and leaf malformation. The virus was mechanically transmitted to healthy Senna pallida, Cassia obovata and Cassia emarginata L. plant species. Virus particles sedimented in sucrose density gradients as one component, with a bouyant density of 1.2 g cm^?3 in caesium chloride equilibrium gradients. Virions contained a molecule of ssRNA with an apparent size of 6.4 kb. The dsRNA pattern showed one main band of about 12 kb, and two subgenomic dsRNA of c. 10 and c. 5.4 kb. Analyses of purified virus preparations by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) resolved two coat protein subunits, with mol. wt of c. 28 000 and 26 000 daltons. In Western blotting the virus coat proteins reacted with an homologous polyclonal antiserum and with an antiserum to Lettuce infectious yellow virus. Electron microscopic observations of cells from infected plants showed the accumulation of cytoplasmic vesiculate inclusion bodies and crystalline aggregates of virus particles within phloem tissue. Some of the physicochemical and ultrastructural properties of this virus resemble those of a Closterovirus; however, differences show it to be sufficiently distinct from any previously reported viruses. We proposed the name of Senna chlorotic stunt (SeCSV) for this virus.     

Casein and alpha-lactalbumin messenger RNAs during the development of mouse mammary gland. Isolation, partial purification, and translation in a cell-free system

Messenger RNAs for the milk proteins, casein and α-lactalbumin, were isolated and partially purified from lactating mouse mammary glands by oligo(dT)cellulose chromatography followed by sucrose density gradient centrifugation. The translation of poly(A)⁺ mRNA in a wheat germ cell-free system yielded three casein polypeptides and a putative precursor form of α-lactalbumin which were precipitated by specific antibodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The casein polypeptides synthesized in vitro had a molecular weight that was no greater than that of the caseins in mouse milk. The presence of individual casein mRNAs coding for these polypeptides was demonstrated by the translation of various fractions of mRNA obtained by sucrose density gradient centrifugation of poly(A)⁺ mRNA. Casein mRNA activity increased about 250-fold between midpregnancy and the 10th–12th days of lactation, amounting to 50–60% of the total mRNA activity in that tissue. A similar study of α-lactalbumin mRNA showed an increase during lactation amounting to 0.2–0.4% of the total mRNA activity, which corresponds to the percentage of α-lactalbumin in total mouse milk protein.