Isolation of ribosome particles from meningopneumonitis organisms

In ribonucleic acid (RNA) extracted by phenol and sodium dodecyl sulfate from purified reticulate bodies of meningopneumonitis (MP) organisms, 21S, 16S, and 4S RNA were found by sucrose density gradient sedimentation analysis. When purified reticulate bodies were homogenized by sonic treatment or by treatment with sodium deoxycholate and were fractionated by differential centrifugation, more than 50% of the RNA was recovered in the fraction which was sedimented by centrifugation at 105,000 x g for 2 hr, but not at 13,000 x g for 20 min. From homogenates prepared in this manner, 50S and 30S particles containing RNA were isolated by sucrose density gradient centrifugation. These 50S and 30S particles were also found in lysates of cytoplasmic fractions of infected cells which were labeled by (32)P during 17 to 17.5 hr or 15 to 18 hr after infection. The synthesis of 50S and 30S particles was not inhibited by actinomycin D. When infected cells were homogenized in the presence of 0.01 or 0.02 m MgCl(2), 70S particles were isolated instead of 50S and 30S particles. When dialyzed against low concentrations of MgCl(2), the 70S particles dissociated to 50S and 30S particles. The base ratio of the 70S particles is very similar to that of 16S plus 21S RNA. The characteristics of the 70S, 50S, and 30S particles suggest that these are ribosome particles, similar to bacterial ribosomes.     

Virus-like particles from killer, neutral, and sensitive strains of Saccharomyces cerevisiae.

Procedures were developed for purification of virus-like particles (VLPs) from killer, neutral, and sensitive strains of Saccharomyces cerevisiae. Morphologically similar spherical VLPs measuring 40 nm in diameter were extracted from all three phenotypes. The particles were partially purified by high-speed centrifugation through a layer of CsCl (1.26 g/cm3) and sucrose density gradient centrifugation. Gradient-purified preparations contained three centrifugal species that sedimented at approximately 43, 102, and 162S. The 43S component is considered to be an artifact. Preparations from killer strains contained three double-stranded RNA (ds-RNA) components with molecular weights of 1.19 x 10(6), 1.29 x 10(6) and 2.54 x 10(6). VLPs from neutral and sensitive strains contained only the largest ds-RNA species. VLP preparations were subsequently separated into two major density components by CsCl equilibrium gradient centrifugation. The light component banding at 1.28 to 1.30 g/cm3 was void of nucleic acid, and the heavy component banding at 1.40 g/cm3 contained only the largest ds-RNA species.     

香蕉束顶病毒的纯化及理化特性

从具有典型香蕉束顶病(BBTD)症状的香蕉病组织中提纯了香蕉束顶病毒(Banana bunchy top virus,BBTV)。电镜下可观察到直径为18nm的球形病毒颗粒。最高紫外吸收在255nm,最低紫外吸收在240nm,A_(260)/A_(280)为1.30。用标准BBTV抗体通过ECL-Western转印法测定其外壳蛋白分子量为21kDa。其核酸经DNaseI、RNaseA和Mung Bean Nuclease分析,表明是约1kb的ssDNA。结果与国外文献报道一致。     

Electron microscopic demonstration and isolation of ribosomes in mesosomes from Staphylococcus aureus

Mesosomes of Staphylococcus aureus 209P were observed to be extruded as tubules upon protoplast formation by electron microscopy and isolated under hypertonic conditions to maintain their structural integrity by differential centrifugation followed by sucrose density gradient centrifugation. Isolated mesosomes were composed of long, branched tubules of irregular sizes and they were shortened during purification. Thin sections of isolated mesosomes showed that the mesosomal tubule was surrounded by a triple-layered membrane and contained ribosome-like particles in diameter of about 15 to 20 nm. These particles were isolated from purified mesosomal preparation by disrupting the mesosomal tubule with deoxycholate and Triton X-100 under hypotonic conditions followed by a linear sucrose density gradient centrifugation. Negatively stained preparations of the isolated particles revealed the same appearance as those of the ribosomes isolated from the cytoplasm. The mesosomal particles sedimented at 70S in sucrose gradients in the presence of 10 mM Mg2+, but they were dissociated into two subparticles, 50S and 30S subunits, upon lowering the Mg2+ concentration to 1 mM. These findings indicate that the mesosomal tubule is packed with ribosomes.     

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.     

Nucleic Acid of Rubella Virus and Its Replication in Hamster Kidney Cells

Ribonucleic acid (RNA) has been isolated from partially purified rubella virus preparations and fractionated by rate zonal centrifugation in sucrose density gradients. The bulk of the RNA sedimented as a sharp band with a sedimentation coefficient of 38S. Rubella virus RNA appears to be single-stranded on the basis of its sensitivity to the degrading action of ribonuclease. Fractionation by precipitation with 1 m NaCl, followed by chromatography on cellulose columns, and by rate zonal centrifugation in sucrose density gradients of labeled RNA isolated from actinomycin D-treated and infected baby hamster kidney cells revealed the presence of the following virus-specific types of RNA: (i) single-stranded RNA with a heterogeneous sedimentation pattern, the 38S viral RNA becoming the predominant species only after long periods of labeling late after infection; (ii) double-stranded RNA with a sedimentation coefficient of 20S; (iii) RNA apparently composed of 20S double-stranded RNA and single-stranded branches. On the basis of their properties, the last two species were tentatively identified as the replicative form and the replicative intermediate of rubella virus RNA. Rubella virus RNA was infectious.     

Preparation and Chemical Composition of the Cell Membranes of Developmental Reticulate Forms of Meningopneumonitis Organisms

The outer limiting membranes of developmental reticulate forms of the meningopneumonitis organism were purified by a combination of differential centrifugation, trypsin digestion, and sodium dodecyl sulfate treatment, and their physical and chemical properties were compared with those of outer envelopes of mature dense forms of this organism. Reticulate bodies were easily disrupted by short periods of sonic treatment and were lysed by trysin digestion, in contrast to the dense bodies which were resistant to these treatments. In electron micrographs, reticulate body membranes were seen as very thin, flattened structures, whereas dense-body envelopes showed folding rigid membranes. The results of chemical fractionation of (32)P-labeled purified preparations indicated that reticulate body membranes have smaller amounts of phospholipid, and are more dense than cell walls of the mature forms. The analysis of amino acid composition of reticulate body cell membranes showed that they do not contain cystine or methionine, both of which were found in cell walls of dense bodies. These results clearly show that there are significant differences in the chemical and physical properties of the outer envelopes of the developmental and mature forms of this organism.     

Effect of Penicillin on the Multiplication of Meningopneumonitis Organisms (Chlamydia psittaci)

Although formation of infectious particles of meningopneumonitis organism in L cells was completely inhibited by 1 or more units of penicillin per ml, multiplication of reticulate bodies was observed, by light microscopy, in the presence of 200 units of penicillin per ml in stained smears of infected cells. When reticulate bodies were purified from cultures containing penicillin after 18, 30, and 45 hr of incubation, continuously increasing yields were obtained. When penicillin was added to infected cultures 0 to 15 hr after infection, no increase in infectivity was observed at 40 hr, but when antibiotic was added between 20 and 35 hr, partial synthesis of infectious particles was observed at 40 hr. On the other hand, removal of penicillin from an infected culture before 15 hr after infection did not affect the final yields of infectivity when assayed at 40 hr, but elimination of penicillin after 20 hr resulted in a decrease in infectivity. In suspensions of (32)P-labeled purified reticulate bodies grown in cultures containing penicillin and harvested 18 and 40 hr after infection, the (32)P distributions obtained by acid fractionation were similar to those of reticulate bodies from penicillin-free cultures. Cell membranes of reticulate bodies were also prepared from 40-hr cultures with penicillin. The size and shape of purified membranes, as seen by electron microscopy, and their amino acid compositions were similar to membranes prepared from reticulate bodies grown without penicillin, except that very small structures were observed in membranes from cultures containing penicillin. These results indicated that penicillin does not inhibit reproduction of reticulate bodies and formation of their cell membranes, but does inhibit the formation of elementary body cell envelopes.     

Toward an in vitro system for picornavirus assembly: purification of mengovirus 14S capsid precursor particles.

Mengovirus 14S subviral protein particles generated in infected L cells and in a cell-free translation system primed with mengovirus RNA were purified by sucrose gradient centrifugation and immunoaffinity chromatography. The preparations from both sources contained essentially pure proteins epsilon, alpha, and gamma, as was demonstrated in terms of virus-specific proteins (by autoradiography) and total protein content (by silver staining of sodium dodecyl sulfate-polyacrylamide electrophoresis gels). These purified proteins sedimented as discrete particles at the 14S position when recentrifuged in sucrose gradients. Although their assembly properties have not yet been studied in detail, preliminary results indicate that during incubation with virion RNA the 14S particles purified from infected cells can form a structure cosedimenting with mature mengovirus.     

Proteins associated with purified human cytomegalovirus particles.

Virion-associated proteins isolated from purified human cytomegalovirus particles (strain AD169) were used as substrates for chemical sequence analysis. Extracellular virions, noninfectious enveloped particles, and dense bodies were purified by negative viscosity-positive density gradient centrifugation, and their component proteins were separated by denaturing polyacrylamide gel electrophoresis. The deduced amino acid sequence of individual protein bands was used to identify six corresponding viral genes whose products have not previously been identified as virion constituents: UL47, UL25, UL88, UL85, UL26, and UL48.5. In addition, a 45-kDa cellular protein was identified, and the protein fragments sequenced have a high degree of amino acid identity with actin. However, antiactin monoclonal and polyclonal antibodies did not react with a specific protein in the virus preparations, suggesting that this 45-kDa protein is an immunologically distinct isoform of actin. The newly identified viral and cellular proteins were resistant to protease treatment of purified virions, suggesting that they are unlikely to be contaminants of the viral preparations.     

Particle Size Distribution of Serratia marcescens Aerosols Created During Common Laboratory Procedures and Simulated Laboratory Accidents

Andersen air samplers were used to determine the particle size distribution of Serratia marcescens aerosols created during several common laboratory procedures and simulated laboratory accidents. Over 1,600 viable particles per cubic foot of air sampled were aerosolized during blending operations. More than 98% of these particles were less than 5 mu in size. In contrast, 80% of the viable particles aerosolized by handling lyophilized cultures were larger than 5 mu. Harvesting infected eggs, sonic treatment, centrifugation, mixing cultures, and dropping infectious material produced aerosols composed primarily of particles in the 1.0- to 7.5-mu size range.     

Cytoplasmic Ribonucleoprotein Components of the Novikoff Hepatoma

Prominent nucleoprotein sedimentation boundaries were demonstrable in cytoplasmic extracts of Novikoff hepatoma. Fractionation of the homogenates by differential centrifugation or a density gradient method revealed that 65 to 75 per cent of the cytoplasmic ribonucleic acid was present in the form of free ribonucleoprotein particles. After purification by differential centrifugation in dilute buffer, the particles contained 37 per cent RNA, very little lipid, and no demonstrable membrane material. Ultracentrifugal boundaries corresponding to those seen in the original extracts were present, the main component having an s20, _w of 81 S. Upon exposure to chelating agents, the particles dissociated through an intermediate component with sedimentation rate of 56 S to a final stage in which 46 and 28 S subunits were present in a weight ratio of 2:1. ATP and pyrophosphate were equally effective in causing dissociation. ADP was considerably less effective. Treatment of the purified particles with deoxycholate removed one-third of the protein and significantly altered the ultracentrifugal pattern. The particles now dissociated directly to the 46 and 28 S subunit when exposed to chelating agents. Upon electron microscopy, the 81 S particle appeared as an oblate spheroid 24 mµ in diameter. The 46 and 28 S subunits also appeared spheroidal.     

Characterization of mesosomes of Micrococcus luteus: isolation and properties of mesosomal ribosomes, and localization of penicillin-binding proteins in mesosomal membranes

Mesosomes were isolated and purified from Micrococcus luteus under hypertonic conditions throughout preparation processes. The purified mesosomal preparation was composed of closed tubules and vesicles. Electron-dense ribosome-like particles were observed within the isolated mesosomal vesicles by electron microscopy. The ribosome-like particles were isolated from the purified mesosomes by a procedure involving solubilization of the membranes with detergents followed by centrifugation on a linear density gradient of sucrose. The isolated particles have a sedimentation coefficient of 70S in the presence of 10 mM Mg2+, when Mg2+ concentration was lowered to 0.1 mM, the particles were dissociated into two sub-particles of 30S and 50S. The 70S particles had the same appearance as cytoplasmic 70S ribosome particles upon observations of negatively stained preparations. These findings indicate that mesosomal tubules contain ribosomes. The isolated mesosomal ribosomes had the ability for protein synthesis when polyuridylic acid-directed polyphenylalanine synthesis was assayed. The sensitivity of mesosomal ribosomes to inhibitors, chloramphenicol and streptomycin, for protein synthesis was significantly lower than that of both cytoplasmic and cytoplasmic membrane-bound ribosomes. In addition, three penicillin-binding proteins were detected in the mesosomal membranes. One of these was localized predominantly in the mesosomal membranes and the other two were distributed almost equally in both mesosomal and cytoplasmic membranes.     

Purification of maize streak virus and its relationship to viruses associated with streak diseases of sugar cane and Panicum maximum

Maize streak virus (MSV) was purified by homogenising infected leaf tissue in 0·01 m pH 3·9 phosphate buffer and clarifying the extract with n-butanol (7 ml/100 ml extract). Purified preparations contained particles 20 nm in diameter, some occurring singly, but most occurring in pairs, forming structures of 30 × 20 nm. The sedimentation coefficients of single and paired particles were 54 and 76 S respectively. When centrifuged in sucrose density gradients preparations made by extracting leaves at pH 3·9 gave a single intense light-scattering zone containing paired particles. Preparations made at pH 5·9 or 7·9 gave one or two additional upper zones containing single particles and fragmented material. Preparations treated with 0·05 or 0·1 m ethylene diamine tetra-acetic acid, disodium salt, (EDTA) contained no paired particles, few single particles and much fragmented material. In immunoelectrophoresis, the major component in preparations without EDTA migrated to the cathode whereas that in EDTA-treated preparations migrated to the anode. Virus isolates from streak-diseased sugarcane and guinea grass (Panicum maximum) were serologically related to MSV and had similar particles with identical sedimentation coefficients. No such particles were seen in purified preparations of healthy maize, sugarcane, or guinea grass. The viruses from sugarcane and guinea grass are probably host-adapted and are referred to correctly as the sugarcane and guinea grass strains of MSV. MSV probably contains single-stranded RNA, and the cryptogram is (R)/1:*/*:S/S:S/Au.     

Analysis of cytoplasmic 19 S ring-type particles in Drosophila which contain hsp 23 at normal growth temperature

Cytoplasmic 19 S particles were isolated from postpolysomal supernatants of 25 degrees C Drosophila embryos and culture cells. The particles were purified by salt extraction and sucrose gradient centrifugation. Electron microscopic investigation showed that the 19 S particles possess a ring-shaped morphology with an outer diameter of 12 nm and a hollow core of 3 nm. Biochemically the particles are characterized by a group of 16 polypeptides within the molecular weight range of 35 to 23 kDa, and small RNA molecules in the size range of 200 to 60 nucleotides. The RNP character of the particles is also shown by their buoyant density in Cs2SO4 of rho = 1.29 g/cm3 and their susceptibility to uv crosslinking and density in CsCl of rho = 1.38 g/cm3. Antibodies were raised against the proteins of the 19 S particles isolated from 25 degrees C cells and tested by immunoblotting after one- and two-dimensional gel-electrophoresis. Two of the antibodies raised cross react with the small heat-shock proteins hsp 28/27 and hsp 23. Comparative protease V8 cleavage of hsp 23 and the 23-kDa particle protein demonstrates that these two proteins are identical and that the small hsp of Drosophila must be a genuine part of the 19 S cytoplasmic ring-shaped complexes at normal growth temperature. The data support the idea of a general developmental role of some of the so-called heat-shock proteins.     

Structural characterization of polysomal poly(A)-protein particles in rat liver

Poly(A)-protein particles were prepared from rat liver polyribosomes, washed with 0.5 M KCl or unwashed, after digestion with pancreatic ribonuclease and ribonuclease T1 by two successive rounds of sucrose gradient centrifugation. The particles were sedimented in a range of 5--13 S with a peak at about 9 S. The KCl wash of polysomes had no effect on the sedimentation properties of the particles. The particles isolated in this manner were 99% resistant to further pancreatic ribonuclease treatment and contained about 96% adenylic acid. The length of the poly(A) molecules prepared from the poly(A)-protein particles showed a broad distribution of about 70--290 nucleotides with a peak around 130 nucleotides, as measured by polyacrylamide gel electrophoresis. In CsCl density gradient the poly(A)-protein particles banded in a density range of 1.30--1.42 g/cm3 with a peak at 1.36 g/cm3, which amounts to about 80% of the protein content. Sodium dodecyl sulfate/polyacrylamide and urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated six polypeptides with molecular weights of 50 000, 54 000, 58 000, 63 000, 76 000 and 90 000 in the poly(A)-protein particles, but the main components were dependent on the method. The treatment of polysomes with KCl resulted in a loss of the 90 000-molecular-weight component. Amino acid analysis of the polypeptides bound to poly(A) revealed that they contained a relatively large amount of aspartic plus glutamic acid (21.6%) as well as hydrophobic amino acids (41.4%). Digestion of glutaraldehyde-fixed particles with ribonuclease T2 showed that about 50% of poly(A) was accessible to the enzyme, thus this part of poly(A) was located on the surface of the particles. In the electron micrographs the shadowed poly(A)-protein particles appeared in a globular, somewhat elongated form and were mostly 14-18 nm in diameter. On the basis of the results a model for the 'average' 9-S particles was constructed.     

Some Properties of a Phloem-Limited Non Mechanically-Transmissible Grapevine Virus

Abstract Grapevine phloem-limited isometric virus (GPLIV) is the name proposed for a non mechanically-transmissible virus found in Italian and Tunisian grapevines. In density gradient centrifugation purified virus preparations sedimented as two components: T, made up of empty protein shells, and B, composed of intact nucleoprotein particles. B particles had a buoyant density of 1.45 g/cm³ at equilibrium in CsCl and contained 35% RNA consisting of a single molecule with an apparent size of 7.4 kb. The coat protein consisted of a single species with a mol.wt of 28,000 daltons. Purified virus preparations did not infect herbaceous hosts by manual inoculation. A specific antiserum with a titre of 1: 64 raised in rabbits, was used for identification of, GPLIV in field-grown Tunisian grapevines and in leafroll-affected Italian vines before and after heat treatment. Although heat treatment eliminated the virus from the majority of the plants, leafroll symptoms persisted in several GPLIV-free vines, indicating that there is no clear-cut relationship between GPLIV and this disease.     

Studies on the Proteinaceous Subcellular Particles in Rice Endosperm: Electron-Microscopy and Isolation

Electron-microscopic examination of rice endosperm revealed the existence of protein-aceous subcellular particles, 1 to 4 µ in diameter and spherical or oval in shape. Isolation of the particles was effected by differential centrifugation in density gradient medium after mechanical or enzymic disintegration of endosperm cells. The isolated particles were predominantly composed of protein, and residual constituents were mainly lipid and carbohydrate. Their shape and behaviors were similar to those found in the endosperm. These facts show that the subcellular particles concerned are “protein bodies” There seemed to be several kinds of protein bodies different with respect to their protein and lipid contents.     

Cytomegalovirus proteins. I. Polypeptides of virions and dense bodies.

Cytomegalovirus virions and dense bodies were purified by sucrose velocity and equilibrium centrifugation from the medium of fibroblasts infected with the strain AD169. The final virus preparations were purified more than 228-fold with respect to cellular proteins as determined by double-isotopic labeling and at least 1,600-fold on the basis of changes in the ratio of total protein to virus particles. The protein content of purified particles approximated that found for purified preparations of other herpesviruses. Twenty polypeptides ranging from 22,000 to greater than 230,000 molecular weight were detected in purified virus preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polypeptides of virions and dense bodies were allocated on the basis of analyses of preparations containing differing percentages of virions and dense bodies. Six polypeptides were represented predominantly or exclusively in virions, and four polypeptides were represented predominantly or exclusively in dense bodies, whereas the remainder appeared to be shared by both types of particles. Four polypeptides were glycosylated, and at least three of these appeared to be shared by both particles. Four polypeptides were glycosylated, and at least three of these appeared to be shared by both particle types. The protein composition of cytomegalovirus differs profoundly from that of herpes simplex virus.     

兔的一种新病毒：Ⅱ.一株兔出血症...

In this paper a strain of Rabbit Hemorrhagic Disease Virus (RHDV) was isolated and purified from the diseased rabbit livers with a method of using chloroform, two-phase of polyethylene-glycol-dextran sulfate sodium and sucrose density gradient centrifugation. Purified virus was nonenveloped, icosahedeal symmetry with a triangulation number of 3, and 33-37 nm in diameter. The capsid was composed of 32 capsomeres with central holes in an outer diameter of about 9nm. Two types of viral particles having different sedimentation coefficient, 130s and 166s could be identified after sucrose density gradient centrifugation. Probably no less than four virion proteins with molecular weight of 66.4, 65.0, 63.5, 41.0 x 10(3) dalton were detected by SDS-polyacrylamide gel electrophoresis. Viral nucleic acid was extracted from purified virus by using SDS-proteinase K-phenol. Tests with diphenylamine, formaldehyde, and staining with acridine orange as well as the curves of thermal denaturation showed that this kind of virus had a single-stranded DNA. The molecular weight of the ssDNA was approx 2.1 x 10(6) dalton as determined by electron microscopy. Data indicate that the RHDV may like the parvovirus of the family Parvoviridae.