Electron Microscopy of Viral RNA: Molecular Weight Determination of Bacterial and Animal Virus RNAs

A method for preparation of single-stranded RNA for electron microscopy determination of molecular weight is reported. The method uses treatment with formaldehyde at elevated temperatures to remove secondary structure and spreading in a protein monolayer from 50% formamide onto a 50% formamide hypophase. Molecular weights were determined for some bacterial and animal viruses, for which conflicting values had been reported earlier. Molecular weights determined by the method, using Escherichia coli large subunit rRNA for a standard (1.1 × 10⁶), are as follows: E. coli small subunit rRNA, 0.53 × 10⁶; coliphage f2-RNA, 1.3 × 10⁶; Qβ-RNA, 1.55 × 10⁶; and Newcastle disease virus RNA, 5.78 × 10⁶.     

The effect of reaction with formaldehyde on the sedimentation rates of ribonucleic acids

It has been reported that the RNA of several bacteriophages and that of the larger ribosomal sub-units of mammalian cells sediment faster in the presence of 0·1m-sodium chloride than is expected from their estimated molecular weights. The effect of blocking the hydrogen-bonding amino groups of these and other types of RNA was studied. The RNA of phage R17 no longer sedimented anomalously fast after treatment with formaldehyde. In contrast, the larger ribosomal RNA of HeLa cells appeared more aberrant than before, sedimenting faster than tobacco-mosaic-virus RNA (mol.wt. 2×10⁶) in the presence of formaldehyde. The rapidly labelled nuclear 45s RNA of HeLa cells still sedimented faster than the larger ribosomal RNA after reaction with formaldehyde, showing no evidence of disaggregation. It is suggested that both the large ribosomal RNA and the 45s RNA of HeLa cells may have a non-linear structure.     

Molecular Weight Determination of Sendai RNA by Dimethyl Sulfoxide Gradient Sedimentation

The molecular weight of the large RNA of Sendai virus has been determined by sedimentation analysis in sucrose gradients containing 99% dimethyl sulfoxide (DMSO) to be 2.3 × 10⁶. Sendai RNA recovered from 99% DMSO was found to cosediment with nondenatured Sendai RNA at 46 to 48s in ordinary sucrose gradients. The molecular weight value of 2.3 × 10⁶ is considerably smaller than the estimates of 6 × 10⁶ to 7 × 10⁶ determined under nondenaturing conditions, suggesting a unique structure for Sendai RNA.     

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D₃₇ values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 × 10⁵ and 1.4 × 10⁵, were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 × 10⁵) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.     

Characteristics and intracellular distribution of messengerlike RNA in encysted embryos of Artemia salina

Homogenates of dormant cysts of Artemia salina were fractionated by differential centrifugation. RNA was prepared from the various fractions and tested for stimulatory activity in a [¹⁴C]leucine incorporating Escherichia coli system. The highest specific activity was found in the RNA extracted from a cytoplasmic fraction sedimenting at 15,000 g. Some activity was associated with the soluble and crude ribosomal fractions, while the RNA extracted from the crude nuclear fraction was less active.The 15,000 g sediment was purified by centrifugation in a sucrose density gradient. The active material formed a characteristic, colored band at a buoyant density of about 1.17 g/ml. The banding fraction was mainly composed of endoplasmic vesicles and mitochondria. The specific activity of the extracted RNA was further increased when the 15,000 g sediment was treated with buffered 20–100 mM EDTA (with or without 0.1% Triton X-100) before banding.Sedimentation analysis of the active RNA from the purified 15,000 g fractions revealed three distinct absorption peaks at 28 S, 18 S, and 16 S, apparently representing cytoplasmic and mitochondrial rRNA. The 28 S and 18 S peaks were reduced by EDTA treatment, but only to a certain limit. By gel electrophoresis a number of additional components were resolved, including 4 S and 5 S RNA. The template activity showed a heterodisperse distribution with a maximum at 17–20 S, not correlated with the 16 S peak. Isolated 18 S and 28 S rRNA had very low activity.The experiments suggest that in Artemia cysts an appreciable amount of messengerlike RNA is associated with mitochondria and/or endoplasmic vesicles carrying ribosomal monomers.     

Plasmid Distribution and Evidence for a Proteinase Plasmid in Streptococcus lactis C2

Five plasmids, distinguishable by their molecular weights (10⁶, 2 × 10⁶, 5 × 10⁶, 10⁷, 3 × 10⁷, respectively) were isolated from Streptococcus lactis C2. A spontaneous proteinase-negative derivative of this strain lacked the 10⁷ plasmid.     

Gene Expression During the Development of Bacillus subtilis Bacteriophage φ29 II. Resolution of Viral-Specific Ribonucleic Acid Molecules

The ribonucleic acid (RNA) specified by bacteriophage 29 was isolated under conditions which minimized physical and enzymatic degradation, reduced aggregation, and enriched for completed molecules. This RNA was fractionated both by sedimentation through sucrose density gradients and electrophoresis through polyacrylamide gels to measure the size and relative amount of each component. Early RNA consisted of six components of molecular weight 0.75 × 10⁶, 0.44 × 10⁶, 0.37 × 10⁶, 0.25 × 10⁶, 0.09 × 10⁶, and 0.04 × 10⁶, accounting for 35% of the coding capacity of 29 deoxyribonucleic acid (DNA). All of these components except the one at 0.44 × 10⁶ were detected when infection occurred in the presence of chloramphenicol. Synthesis of the major early component (0.75 × 10⁶) ceased shortly after the onset of viral DNA synthesis. The other species of early RNA were synthesized throughout the latent period. Three additional components, 1.75 × 10⁶, 0.93 × 10⁶, and 0.07 × 10⁶, appear at late times. The two large RNAs may be polycistronic messenger RNAs corresponding to the seven viral capsid proteins.     

Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg²⁺ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The ³H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs₂SO₄ equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.     

Biophysical and Biochemical Studies on Rhinovirus and Poliovirus II. Chemical and Hydrodynamic Analysis of the Rhinovirion

Chemical analysis of rhinovirus 14 revealed a ribonucleic acid (RNA) content of 29.8% and a high adenylic acid content (35%). A partial specific volume of 0.682 cm³/g was obtained for the rhinovirion. Rhinovirus and poliovirus had identical sedimentation coefficients of 158S. A diffusion coefficient of 1.71 × 10⁻⁷ cm²/sec was consistent with a hydrated diameter of 25 nm for the rhinovirion. The calculated molecular weights of the rhinovirion and its genome were 7.1 × 10⁶ and 2.1 × 10⁶ daltons, respectively. Sedimentation analysis of infectious RNA confirmed the similarity of the molecular size of the poliovirus and rhinovirus genomes.     

Double-stranded Ribonucleic Acid from Cytoplasmic Polyhedrosis Virus of the Silkworm

Ribonucleic acid (RNA) was extracted by phenol treatment from cytoplasmic polyhedrosis virus isolated from the midgut of infected silkworms. This RNA appears as threads when precipitated in alcohol. Two components having different sedimentation constants were observed. The molecular weight of the RNA preparation obtained by sedimentation coefficient (weight-averaged) and intrinsic viscosity was about 2 × 10⁶ to 3 × 10⁶. It was one-half to one-third the size of the calculated molecular weight for an entire RNA molecule in a virion. Electron micrographs of this RNA preparation showed two peaks in the distribution of contour length, at 0.4 and 1.3 μm, which would correspond to molecular weights of 10⁶ and 3 × 10⁶, respectively. The extracted RNA seemed to split into segments at a preferential breaking point. This RNA was soluble in concentrated salt solution, differing from single stranded high-molecular-weight RNA. The base composition of this RNA was complementary in the ratios of adenosine to uridine and guanosine to cytosine. It contained 43% guanosine plus cytosine. Based on its filamentous appearance by electron microscopy, typical pattern of optical rotatory dispersion and circular dichroism, sharp transition of the optical properties on heating, great hyperchromicity on degradation, nonreactivity with formaldehyde, and resistance to ribonucleases, it is concluded that this RNA is double-stranded and has regular base pairings of guanosine-cytosine and adenosine-uridine.     

A study of the effects of reaction with formaldehyde on some optical and physical properties of reticulocyte ribosomes

1. The optical rotatory dispersion and ultraviolet-absorption spectrum of ribosomal RNA in situ appear to be unchanged when the ribosome is dissociated into its RNA and protein moieties. 2. Reaction with 0·05% formaldehyde at 20° for 2hr. `fixes' ribosomes so that they remain intact in 1% sodium dodecyl sulphate. 3. The RNA moiety of the ribosome undergoes a conformational change when ribosomes in 8% formaldehyde are heated at 70° for 10min. and cooled to 20°. After this treatment no double-helical character can be detected, but neither the sedimentation coefficient nor the morphology of the ribosome determined by electron microscopy is altered. 4. It is concluded that the RNA moiety of reticulocyte ribosomes is freely accessible to formaldehyde.     

Thermal denaturation in acidic solutions of double-helical ribonucleic acid from virus-like particles found in Penicillium chrysogenum. A spectrophotometric study

1. Two species of double-helical RNA isolated from mycelium of Penicillium chrysogenum were titrated with acid at 25°C and 95°C (solvent 0.1m-sodium phosphate buffer). At 25°C denaturation occurred at about pH3. At 95°C in the denatured form cytosine residues titrated as a simple monobasic acid of pK3.9 compared with pK2.5 for the native form at 25°C. 2. On thermal denaturation in neutral and acidic solutions one species of RNA (38% rG·rC) `melted' in three distinct stages, equivalent to a mixture of three species, namely one of about 25% rG·rC, another of about 33% rG·rC and a third of about 46% rG·rC: the relative proportions were 0.25:0.35:0.40. 3. On thermal denaturation in acidic solutions the increase in the fraction of ionized cytosine residues concomitant with the `melting' of rG·rC base pair also affects the spectrum especially at 280nm and serves to enhance the contribution of rG·rC base pairs at this wavelength. The increment in ε_(P) at 280nm on `melting' an rG·rC base pair approaches 53501·mol<sup>−1</sup>·cm<sup>−1</sup> depending on pH, compared with 33501·mol<sup>−1</sup>·cm<sup>−1</sup> at pH7. In contrast ε<sub>(P)</sub> at 280nm is scarcely affected by `melting' rA·rU base pairs or by the protonization of adenine residues. 4. Changes in the spectrum of Escherichia coli rRNA on denaturation in acidic solutions were studied to yield the mole fractions of rA·rU and rG·rC base pairs `melting' at particular pH values.     

Effects of Actinomycin D and Ultraviolet and Ionizing Radiation on Pichinde Virus

Actinomycin D (0.05 μg/ml) suppresses the synthesis of ribosomal RNA of baby hamster kidney (BHK21) cells. The production of infectious Pichinde virus was enhanced in the presence of actinomycin D, although the production of virus particles was not substantially different from cultures inoculated in the absence of the drug. By prelabeling BHK21 cells with ³H-uridine and then allowing the virus to replicate in the presence of actinomycin D, it was possible to show that ribosomal RNA synthesized prior to infection was incorporated into the virion. A single-hit kinetics of inactivation of Pichinde virus was observed with ultraviolet light, suggesting that the virus contains only a single copy of genome per virion. Comparison of the inactivation kinetics by gamma irradiation of Pichinde virus with Sindbis and rubella virus indicated that the radiosensitive genome of Pichinde virus was about 6 × 10⁶ to 8 × 10⁶ daltons. This value is greater than the 3.2 × 10⁶ daltons which was estimated by biochemical analysis. One possible explanation considered is that the ribosomal RNA of host cell origin is functional and accounts for the differences in genome size estimated by the two methods.     

Diffusion Rates in Disrupted Bacterial Cells

The viscosity of the material resulting from squeezing Escherichia coli cells through an orifice in a French pressure cell has been shown to be very high and variable with temperature. Diffusion constants in this medium have been determined for sucrose, dextran, and beta galactosidase. The values found are: 1.07 × 10^-6cm²/second for sucrose, 0.36 × 10^-6cm²/second for dextran, and 0.025 × 10^-6cm²/second for beta galactosidase. The results agree with the idea that there is much interstitial space available for diffusion of small molecules in the cell medium in spite of the high viscosity, but that large molecules will be transported less readily.     

Ribonucleic acids from barley leaves

1. The total RNA and the RNA present in 27000g pellet (probably composed of chloroplasts, nuclei and mitochondria) and in 27000g supernatant (probably composed of microsomes and soluble proteins) fractions (separated by centrifugation at 27000g of a leaf homogenate prepared in 0·5m-sucrose–0·02m-tris–HCl, pH7·6) of barley leaves were extracted by phenol–sodium lauryl sulphate and their elution profiles on Sephadex G-200 and on ECTEOLA-cellulose anion-exchanger were examined and their nucleotide compositions and the melting curves were determined. 2. The pellet and the supernatant fractions contained respectively about 55% and 20% of the total RNA, whereas 25% of the total RNA was lost during homogenization of the leaf tissue with sucrose–buffer. 3. The total RNA or the RNA from pellet or supernatant fractions, which by its behaviour on Sephadex G-200 columns was found to be predominantly of high molecular weight (i.e. of ribosomal origin), produced about 13 peaks on ECTEOLA-cellulose columns. The RNA species in the pellet and supernatant fractions probably resembled each other in molecular size or secondary structure or both. However, they were present in relatively different amounts in these fractions. 4. The T_m (i.e. the temperature at which 50% of the maximal increase in extinction had occurred) of total RNA and of RNA from pellet fraction was 64·5° whereas T_m of RNA from the supernatant fraction was 73°. The total RNA and the RNA from pellet fraction also resembled each other in nucleotide composition, and the RNA from the supernatant fraction in accordance with its high T_m had a high GMP+CMP content.     

Phycobilisome-thylakoid Topography on Photosynthetically Active Vesicles of Porphyridium cruentum

Conditions are described for isolating functional phycobilisome-thylakoid vesicles from the red alga Porphyridium cruentum. Phycobilisome-thylakoid vesicles were prepared by brief sonication and centrifugation in a medium containing 0.5 molar sucrose, 0.5 molar potassium phosphate, and 0.3 molar sodium citrate (pH 7.0). They required ferricyanide as an oxidant and had O₂ evolution rates (about 450 micromoles O₂ per hour per milligram chlorophyll) higher than whole cells (about 250 micromoles O₂ per hour per milligram chlorophyll). Energy transfer to photosystem II chlorophyll was evident from a high F695 nanometer (−196 C) emission peak. Preparations could be stored for over 24 hours and were considerably more stable than those from the cyanobacterium Anabaena variabilis (Katoh T, E Gantt 1979 Biochim Biophys Acta 546: 383-393). In electron micrographs of negatively stained material, the active thylakoid vesicles were found covered by closely spaced phycobilisomes on their external surface. The phycobilisome number in negatively stained vesicles was 450 per square micrometer, which was in the same range as the 400 per square micrometer observed in surface sections. A cell containing 1.5 × 10⁻⁶ micrograms phycoerythrin and 1.3 × 10⁻⁶ micrograms chlorophyll was found to contain 5 to 7 × 10⁵ phycobilisomes on a thylakoid area of 1.1 to 1.6 × 10³ square micrometers.     

Electron Microscopical and Biochemical Characterization of Infectious Pancreatic Necrosis Virus

An electron microscopical and biochemical examination of the properties of infectious pancreatic necrosis virus (IPN) and of its ribonucleic acid (RNA) was made. The buoyant density of IPN in CsCl was found to be 1.33 g/cm³. Electron microscopical examination of the banded virus revealed structures similar in size (74 nm) and shape to reoviruses but lacking a characteristic inner capsid structure. Polyacrylamide gel electrophoretic analysis of IPN-RNA revealed a single non-segmented component of molecular weight 3.2 × 10⁶. Its susceptibility to ribonuclease, base composition, and resistance to thermal denaturation indicated a single-stranded RNA structure. However, its sedimentation behavior (16S) independent of ionic strength in sucrose gradients, partial solubility in 2 m LiCl, and ribonuclease resistance in the presence of Mg²⁺ suggest an unusual secondary structure of unknown nature. The accumulated data indicate that IPN virus does not belong to either the picornavirus or reovirus groups and may represent a new group of viruses.