Ribonuclease H: a Ubiquitous Activity in Virions of Ribonucleic Acid Tumor Viruses

Ten ribonucleic acid (RNA) tumor viruses grown in five different host cell species and three non-oncogenic viruses from three different virus groups have been examined for ribonuclease H content. Three different substrates were used to assay ribonuclease H: calf thymus [(3)H]RNA-deoxyribonucleic acid (DNA) hybrid prepared with denatured calf thymus DNA and Escherichia coli DNA-directed RNA polymerase, (3)H-polydenylic acid [(3)H-poly(A)] complexed to polydeoxythymidylic acid [poly(dT)], and (3)H-polyuridylic acid [(3)H-poly(U)] complexed to polydeoxyadenylic acid [poly(dA)]. All ten RNA tumor viruses contained ribonuclease H activity which degraded the RNA of both the calf thymus hybrid and poly(A)-poly(dT), whereas only the ribonuclease H in the Moloney strain of murine sarcoma-leukemia virus and in RD-feline leukemia virus hydrolyzed the RNA strand of poly(U)-poly(dA). No appreciable ribonuclease H activity was detected in influenza, Sendai, or vesicular stomatitis virus. The ribonuclease H and RNA-directed DNA polymerase activities in Moloney murine sarcoma-leukemia virus were inseparable by phosphocellulose chromatography or glycerol gradient centrifugation, but appeared to be partially separated by diethylaminoethyl-cellulose chromatography.     

Properties of Maedi Nucleic Acid and the Presence of Ribonucleic Acid- and Deoxyribonucleic Acid-Dependent Deoxyribonucleic Acid Polymerase in the Virions

Maedi virus contains a ribonucleic acid (RNA) which can be resolved into three major components, namely, 62S, 33S, and 13S, by sucrose gradient centrifugation. The presence of RNA- and deoxyribonucleic acid (DNA)-dependent DNA polymerase in virions of maedi virus was demonstrated. The enzyme product could be converted into acid-soluble form by pancreatic deoxyribonuclease, but was resistant to digestion by pancreatic ribonuclease and to hydrolysis by NaOH.     

Purification of Large Amounts of Murine Ribonucleic Acid Tumor Viruses Produced in Roller Bottle Cultures

Murine ribonucleic acid tumor viruses and C-type virus particles are produced in relatively large quantities in roller bottle cultures. The viruses present in large volumes of culture fluids can be purified by a simple two-step procedure involving polyethylene glycol precipitation and equilibrium centrifugation in sucrose density gradients.     

Deoxyribonucleic Acid Polymerase Associated with Avian Tumor Viruses: Secondary Structure of the Deoxyribonucleic Acid Product

The products of the deoxyribonucleic acid (DNA) polymerase associated with Rous sarcoma virus and avian myeloblastosis virus were characterized by correlative analyses with equilibrium centrifugation and stepwise elution from hydroxyapatite. The initial enzymatic product consists of nascent DNA chains which are hydrogen-bonded to 70S viral ribonucleic acid (RNA), whereas the final enzymatic product is double-stranded DNA. Appreciable amounts of free single-stranded DNA were not detected at any point during the course of the enzymatic reaction, but the data in this regard are not decisive. The time course of synthesis of DNA:RNA hybrids and double-stranded DNA has been analyzed. It is concluded that the synthesis of double-stranded DNA is a sequel to and is probably dependent upon the synthesis of DNA:RNA hybrid.     

Transfer Ribonucleic Acid Nucleotidyltransferase and Transfer Ribonucleic Acid in Sendai Virions

Sendai virions contain both transfer ribonucleic acid (tRNA) nucleotidyltransferase and its substrate, tRNA missing its CCA-OH end.     

Sensitivity of Ribonucleic Acid and Deoxyribonucleic Acid Viruses to Different Species of Interferon in Cell Cultures

Although two deoxyribonucleic acid (DNA) viruses, pseudorabies (PsRV) and vaccinia, are as susceptible as a ribonucleic acid (RNA) virus, vesicular stomatitis (VSV), to interferon when tested in chicken or mouse cells, they are refractory to inhibition in interferon-treated primary rabbit kidney cells and in a continuous line (RK-13) of rabbit kidney cells. Superinfection with VSV of RK-13 cells first infected with PsRV completely blocks the replication of PsRV with no effect on VSV yield. When the same experiment is carried out in RK-13 cells pretreated with 1,000 units of interferon, VSV replication is inhibited, which permits PsRV to replicate normally. These findings demonstrate that in the same cell one virus (PsRV) can be refractory to interferon and a second virus (VSV) can be susceptible. These experiments show that rabbit kidney cell cultures are deficient in the synthesis of resistance factors active against the DNA viruses tested and raise the possibility that separate resistance factors may exist for RNA and DNA viruses. In the case of sequential infection of interferon-treated RK-13 cells with vaccinia and VSV, it was found that not only was vaccinia replication refractory to inhibition by interferon, but also that prior infection with vaccinia was able to partially reverse the effect of the inhibitor on the replication of the VSV used for superinfection. On the basis of these and other data it is postulated that a vaccinia virion component or a replication product of vaccinia virus, or both, enables VSV to escape the inhibiting action of interferoninduced resistance factors.     

Large-Scale Purification of Ribonucleic Acid Tumor Viruses by Use of Continuous-Flow Density Gradient Centrifugation

This report describes a 200- to 300-fold increase in the quantity of ribonucleic acud tumor virus particles previously isolated at one time by zonal centrifugation.     

Developmental Changes in Multiple Forms of Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase in Soybean Hypocotyl

The relative levels of multiple RNA polymerases were determined in soybean (Glycine max L. var. Wayne) hypocotyl during various stages of development. The meristematic region of the hypocotyl contains more total polymerase activity per gram fresh weight and a greater proportion of polymerase I relative to II than the differentiated regions. The fully elongated tissue comprising the lower half of the hypocotyl contains mainly RNA polymerase II. The hook region contains a polymerase activity peak which is completely sensitive to alpha-amanitin and partially sensitive to rifamycin SV. This peak is not detectable in other regions of the hypocotyl. Polymerase I is reproducibly separated into a major and a minor component, both being resistant to alpha-amanitin. The two components elute at salt concentrations of 0.2 m and 0.23 m KCl, respectively, while the alpha-amanitin-sensitive polymerase (II) elutes at 0.3 m KCl. The polymerase activity peak which is detectable only in the hook region elutes at approximately 0.5 m KCl. Polymerase levels were also determined in water-stressed tissue and in tissue which was harvested after three days of growth instead of the usual four days.     

Inhibition of Bacterial Conjugation by Ribonucleic Acid and Deoxyribonucleic Acid Male-Specific Bacteriophages

Both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) male-specific phages, with an F-specific host range, inhibited the bacterial mating process of Escherichia coli. DNA phages prevented the formation of mating pairs but had no effect on mating pairs once they were formed. A step in RNA phage infection, prior to RNA penetration, prevented the formation of mating pairs and, in addition, prevented a fraction of existing mating pairs from completing the mating process. These findings are compatible with the hypothesis that donor cells have a single surface structure involved in both conjugation and male-phage adsorption and that this element is the F pilus.     

Ribonucleic Acid Transcriptases in Sendai Virions and Infected Cells

Sendai virions contain an enzyme which catalyzes the incorporation of ribonucleotides into ribonucleic acid (RNA). Enzyme activity was optimal at pH 8.0 and 28 C; otherwise conditions were similar to those reported for Newcastle disease virion (NDV) RNA polymerase. The initial rate of RNA synthesis by the Sendai virion enzyme was about 10 pmoles per mg of protein per hr, but after 3 hr of incubation the rate increased about fivefold. The virion enzyme was compared with an RNA polymerase in the microsomal fraction of infected cells. Both enzymes made predominantly single-stranded RNA which was complementary in base sequences to 50S virion RNA. Most of the RNA synthesized by the virion polymerase sedimented at 16S, but the product of the microsomal enzyme sedimented at about 8S.     

Ultraviolet-Induced Cross-Links in the Deoxyribonucleic Acid of Single-Stranded Deoxyribonucleic Acid Viruses as a Probe of Deoxyribonucleic Acid Packaging

Deoxyribonucleic acid (DNA) from ultraviolet (UV)-irradiated phiX174 sediments in alkali at rates up to 1.7 times that of unirradiated phiX174 DNA and is observed as a condensed, cross-linked structure when examined in the electron microscope by the formamide spreading technique. This structure appears to result from multiple cross-links induced in the tightly coiled DNA contained within the spherical phiX174 capsid. In contrast, the DNA extracted after UV irradiation of the filamentous bacteriophage M13 is not strikingly altered in its sedimentation properties and appears by electron microscopy to be rod-shaped as a result of side-to-side association of the circular DNA. The differences in these UV-induced structures reflect the differences in the packaging of the single-stranded DNA in the two virions.     

Ribonucleic Acid Polymerase Activity in Sendai Virions and Nucleocapsid

After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the (3)H-RNA reaction product revealed two components, a 45S component with properties of double-stranded RNA and 4 to 6S component which appeared to be mostly single-stranded RNA.     

Role of Ribonucleic Acid Synthesis in Replication of Deoxyribonucleic Acid

An experiment previously interpreted to show a ribonucleic acid requirement for propagation of deoxyribonucleic replication is reexamined and the earlier interpretation is shown to be incorrect.     

Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase of Caulobacter crescentus

Deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) was purified from the dimorphic bacterium Caulobacter crescentus at three stages in development. Enzyme from pure populations of stalked cells, as well as populations enriched in swarmer and predivisional cells, appeared identical in subunit structure and template requirements. The molecular weights of the enzyme subunits were 165,000, 155,000, 101,000, and 44,000, respectively. By analogy with RNA polymerase from other bacterial sources, they are considered to be components of the C. crescentus holoenzyme, beta', beta, sigma, and alpha, respectively. The C. crescentus enzyme appeared similar to the Pseudomonas aeruginosa enzyme and unlike the Escherichia coli enzyme with respect to subunit molecular weights and failure to separate into core and sigma components upon phosphocellulose chromatography. In addition, the effects of ionic strength on the time course of polymerization varied both with the sources of bacterial polymerase and bacteriophage DNA.     

Ribonucleic Acid-Dependent Deoxyribonucleic Acid Polymerase in Visna Virus

A ribonucleic acid-dependent deoxyribonucleic acid polymerase was found in virions of visna virus. The enzyme product was resistant to ribonuclease and alkaline hydrolysis but susceptible to the digestion of deoxyribonuclease.     

Specific Inhibition of Mammalian Ribonucleic Acid C-Type Virus Deoxyribonucleic Acid Polymerases by Rat Antisera

Inhibition of the ribonucleic acid (RNA)- and deoxyribonucleic acid (DNA)-dependent DNA polymerase activities of mammalian C-type viruses was obtained with sera from rats bearing murine leukemia virus-induced transplant tumors. Polymerase activities of nonmammalian (viper) C-type virus and murine mammary tumor virus were not inhibited by such sera nor by serum from a rat immunized with the DNA polymerase of feline leukemia virus purified by isoelectric focusing. The latter serum appeared to inhibit preferentially the DNA-dependent DNA polymerase activity of mammalian C-type viruses showing no inhibition of RNA-dependent DNA synthesis.     

Nuclear Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerases from Saccharomyces cerevisiae

Two deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerases (I, II) have been solubilized from isolated Saccharomyces cerevisiae nuclei. The enzymes can be separated by chromatography on O-diethylaminoethyl Sephadex. Both enzymes are active with high-molecular-weight nuclear yeast DNA, although RNA polymerase I has a higher affinity for polydeoxy-adenylic-thymidylic acid and RNA polymerase II for denatured DNA. RNA polymerase I is active only with manganese. alpha-Amanitin inhibits only the activity of RNA polymerase II.     

Double-Stranded Ribonucleic Acid in Viruses of Penicillium citrinum

[This corrects the article on p. 811 in vol. 34.].     

Analysis of Arbovirus Ribonucleic Acid Forms by Polyacrylamide Gel Electrophoresis

Viral ribonucleic acid (RNA) from Semliki Forest virus- and Sindbis virus-infected cells was analyzed by electrophoresis on polyacrylamide gels. In contrast to earlier results obtained by sucrose density gradient centrifugation, all of the known viral RNA forms (i.e., the 42S, 26S, replicative form, and replicative intermediate) were very clearly separated. The high resolution of the electrophoretic method permitted the identification of two new single-stranded RNA species. In addition, the replicative form was shown to be heterogeneous and to consist of at least two forms. The results suggested that the replicative forms occur in vivo although in relatively small amounts.