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.     

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.     

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.     

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.     

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.     

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.     

Characterization of the coat protein mRNA of southern bean mosaic virus and its relationship to the genomic RNA

RNA isolated from southern bean mosaic virions contains, in small amount, a subgenomic RNA (molecular weight, 0.38 × 10⁶) that serves in vitro as an mRNA for southern bean mosaic virus coat protein. The RNA has a 5′-linked protein indistinguishable from the protein linked to the 5′ end of full-length genomic RNA. Its base sequence, determined to 91 bases from the 3′ end, is identical to the 3′-terminal sequence of the genomic RNA. The results suggest that the coat protein messenger sequence exists as a “silent” cistron near the 3′ end of the genomic RNA.     

Inactivation of rubella virus by gamma radiation

The Gilchrist and M-33 strains of rubella virus exposed in the frozen state to ¹³⁷Ce or ⁶⁰Co were inactivated exponentially according to “one hit” kinetics. There was no difference in the radiosensitivity of the two strains. Experimental D₃₇ values for both strains ranged from 1.9 × 10⁵ to 2.9 × 10⁵ rads, and computed radiosensitive molecular weights ranged from 2.6 × 10⁶ to 4.0 × 10⁶ daltons.     

Herpes Simplex Virus: Genome Size and Redundancy Studied by Renaturation Kinetics

Herpes simplex virus subtype 1 deoxyribonucleic acid (DNA) was sheared in a French press to uniform fragments, denatured by heating, then allowed to reassociate. The renaturation reaction followed second-order kinetics with a single rate constant indicating that at least 95% of the genome was unique and that repetitive sequences, if present, were not detectable by this technique. The kinetic complexity of the herpes simplex genome was determined by DNA renaturation kinetics to be (95 ± 1) × 10⁶ daltons. Since this value is in excellent agreement with the molecular weight of viral DNA [(99 ± 5) × 10⁶ daltons] obtained from velocity sedimentation studies, it is concluded that virions contain only one species of double-stranded DNA molecules 95 × 10⁶ to 99 × 10⁶ daltons in molecular weight.     

Physical Map of the Channel Catfish Virus Genome: Location of Sites for Restriction Endonucleases EcoRI, HindIII, HpaI, and XbaI

The overall arrangement of nucleotide sequences in the DNA of channel catfish virus has been studied by cleavage with four restriction endonucleases. Physical maps have been developed for the location of sites for EcoRI, HindIII, HpaI, and XbaI. The sum of the molecular weights of fragments generated by each restriction enzyme indicates a molecular weight of approximately 86 × 10⁶ for the channel catfish virus genome. Fragments corresponding to the molecular ends of channel catfish virus DNA have been identified by their sensitivity to exonuclease treatment. The distribution of restriction sites in the genome shows that sequences included in a 12 × 10⁶-molecular weight region at one end are repeated with direct polarity at the other end, and that the overall genomic sequence order is nonpermuted.     

The Molecular Weight and Other Biophysical Properties of Bromegrass Mosaic Virus

Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 × 10⁶ was calculated from the sedimentation coefficient, S°_20,w = 86.2S, the diffusion coefficient, D_20,w = 1.55 × 10^-7 cm²/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 × 10⁶ atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described.     

Determination of the sedimentation coefficient of vaccinia virus DNA by analytical ultracentrifugation

By releasing intact molecules of vaccinia virus DNA, it has been possible to determine the sedimentation coefficient by analytical ultracentrifugation. The value found was 92S, which corresponds to a molecular weight of 161×10⁶.     

Maize mitochondria: purification and characterization of ribosomes and ribosomal ribonucleic Acid

Mitochondria were prepared from etiolated maize shoots (Zea mays L. var. McNair 508) by homogenization followed by differential centrifugation and equilibrium banding in discontinuous sucrose or Renografin-sucrose gradients. Mitochondria prepared by sucrose banding showed better physiological integrity than those prepared by renografin-sucrose banding, although both procedures yielded mitochondria that showed respiratory control and coupling of oxidation to phosphorylation of ADP. Mitochondria prepared by Renografin-sucrose banding were free of dectectable cytoplasmic ribosomal RNA, while sucrose banding resulted in a low level of contamination. Ribosomes isolated from mitochondria sedimented at about 78S, with subunits sedimenting at 60 and 44S. Using Escherichia coli ribosomal RNA as internal standards, the molecular weights of mitochondrial ribosomal RNAs were found to be 0.74 to 0.75 and 1.26 × 10⁶ daltons by polyacrylamide gel electrophoresis, before or after denaturation in formaldehyde. Cytoplasmic ribosomal RNA molecular weights were 0.70 and 1.26 × 16⁶ before denaturation, and 0.68 and 1.5 × 10⁶ after denaturation, suggesting an unusual reaction of the heavy ribosomal RNA to formaldehyde.     

Biosynthesis of Novel Exopolymers by Aureobasidium pullulans

Aureobasidium pullulans ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 × 10⁶ to 2.12 × 10⁶ to 0.85 × 10⁶ to 0.77 × 10⁶ with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 ± 3 to 29 ± 2 mol%, and the molecular weight increased from 2.73 × 10⁶ to 4.86 × 10⁶. There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 ± 3:13 ± 3 to 28 ± 2:72 ± 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.     

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.     

Physicochemical properties of tipula iridescent virus

The molecular weight of Tipula iridescent virus, based on sedimentation and diffusion coefficients, was 5.51 × 10⁸, with hydration of 0.57 g of water per g of virus. Deoxyribonucleic acid content, based on total inorganic phosphorus liberated, was 19 ± 0.2%. At 260 mμ, the virus gave an uncorrected absorbance of 18.2 cm²/mg of virus and a light-scattering corrected absorbance of 9.8 cm²/mg of virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus. The possibility is discussed that the four iridescent insect viruses reported to date bear a strain relationship.     

The Enzymatic Synthesis of Rubber Polymer in Parthenium argentatum Gray

Washed rubber particles isolated from stem homogenates of Parthenium argentatum Gray by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain rubber transferase which catalyzes the polymerization of isopentenyl pyrophosphate into rubber polymer. The polymerization reaction requires Mg²⁺ isopentenyl pyrophosphate, and an allylic pyrophosphate. The K_m values for Mg²⁺, isopentenyl pyrophosphate, and dimethylallyl pyrophosphate were 5.2 × 10⁻⁴ molar, 8.3 × 10⁻⁵ molar, and 9.6 × 10⁻⁵ molar, respectively. The molecular characteristics of the rubber polymer synthesized from [¹⁴C]isopentenyl pyrophosphate were examined by gel permeation chromatography on three linear columns of 1 × 10⁶ to 500 Ångstroms Ultrastyragel in a Waters 150C Gel Permeation Chromatograph. The peak molecular weight of the radioactive polymer increased from 70,000 in 15 minutes to 750,000 in 3 hours. The weight average molecular weight of the polymer synthesized over a 3 hour period was 1.17 × 10⁶ compared to 1.49 × 10⁶ for the natural rubber polymer extracted from the rubber particles. Over 90% of the in vitro formation of the rubber polymer was de novo from dimethylallyl pyrophosphate and isopentenyl pyrophosphate. Treatment of the washed rubber particles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilized the rubber transferase. The solubilized enzyme(s) catalyzed the polymerization of isopentenyl pyrophosphate into rubber polymer with a peak molecular weight of 1 × 10⁵ after 3 hours of incubation with Mg²⁺ and dimethylallyl pyrophosphate. The data support the conclusion that the soluble preparation of rubber transferase is capable of catalyzing the formation of a high molecular weight rubber polymer from an allylic pyrophosphate initiator and isopentenyl pyrophosphate monomer.     

Characterization of a Small RNA-Containing Virus in Field-Collected Larvae of the Tussock Moth, Lymantria ninayi, from Papua New Guinea

Field-collected larvae of the tussock moth, Lymantria ninayi, a major pest of exotic pines in Papua New Guinea, were found to contain a small RNA-containing virus with a diameter of 29 nm and a buoyant density of 1.32 g/ml. The RNA was single stranded, had a molecular weight of 2.8 × 10⁶, and was polyadenylated. Virion RNA stimulated an in vitro translation system, and high-molecular-weight proteins were produced. Purified virions contained four structural proteins with molecular weights of 43,000, 38,000, 33,000, and 32,000. The virus reacted positively with antisera raised against a strain of Drosophila C virus. The properties of this virus indicate that it should be placed in the family Picornaviridae.     

Concentrations,Source and Risk Assessment of Polycyclic Aromatic Hydrocarbons in Soils from Midway Atoll,North Pacific Ocean

This study was designed to determine concentrations of polycyclic aromatic hydrocarbons (PAHs) in soil samples collected from Midway Atoll and evaluate their potential risks to human health. The total concentrations of 16 PAHs ranged from 3.55 to 3200 µg kg⁻¹ with a mean concentration of 198 µg kg⁻¹. Higher molecular weight PAHs (4–6 ring PAHs) dominated the PAH profiles, accounting for 83.3% of total PAH mass. PAH diagnostic ratio analysis indicated that primary sources of PAHs in Midway Atoll could be combustion. The benzo[a]pyrene equivalent concentration (BaP_eq) in most of the study area (86.5%) was less than 40 µg kg⁻¹ BaP_eq and total incremental lifetime cancer risks of PAHs ranged from 1.00×10⁻¹⁰ to 9.20×10⁻⁶ with a median value of 1.24×10⁻⁷, indicating a minor carcinogenic risk of PAHs in Midway Atoll.     

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.