Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity in Cells Infected with Influenza Virus

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase activity was assayed on nuclear preparations of chick embryo fibroblast cells at various times after infection with an influenza A virus (fowl plague virus) and was compared with the activity of uninfected cells. Polymerase activity was increased by about 60% by 2 hr after infection, and this increase coincided with an increase in RNA synthesis in infected cells, as determined by pulse-labeling with uridine. No difference could be detected between the polymerases of infected and uninfected cells as to their requirements for DNA primer, divalent cations, and nucleoside triphosphates, and they were equally sensitive to addition of actinomycin D to the reaction mixture. It is possible that host cell DNA-dependent RNA polymerase is involved in the replication of influenza virus RNA.     

Synthesis and Intracellular Localization of Vaccinia Virus Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.     

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.     

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.     

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.     

Role of Subunits of 60 to 70S Avian Tumor Virus Ribonucleic Acid in Its Template Activity for the Viral Deoxyribonucleic Acid Polymerase

Heating the 60 to 70S ribonucleic acid (RNA) of Rous sarcoma virus (RSV) destroys both its subunit structure and its high template activity for RSV deoxyribonucleic acid (DNA) polymerase. In comparative analyses, it was found that the template activity of the RNA has a thermal transition of 70 C, whereas the 60 to 70S structure dissociates into 30 to 40S and several distinct small subunits with a T(m) of 55 C. Analysis by velocity sedimentation and isopycnic centrifugation of the primary DNA product obtained by incubation of 60 to 70S RSV RNA with RSV DNA polymerase indicated that most, but perhaps not all, DNA was linked to small (<10S) RSV RNA primer. Sixty percent of the high template activity of 60 to 70S RSV RNA lost after heat dissociation could be recovered by incubation of the total RNA under annealing conditions. The template activity of purified 30 to 40S subunits isolated from 60 to 70S RSV RNA was not enhanced significantly by annealing. However, in the presence of small (<10S) subunits also isolated from 60 to 70S RNA, the template activity of 30 to 40S RNA subunits was increased to the same level as that of reannealed total 60 to 70S RNA. It was concluded that neither the 30 to 40S subunits nor most of the 4S subunits of 60 to 70S RSV RNA contribute much as primers to the template activity of 60 to 70S RSV RNA. The predominant primer molecule appears to be a minor component of the <10S subunit fraction of 60 to 70S RSV RNA. Its electrophoretic mobility is similar to, and its dissociation temperature from 60 to 70S RSV RNA is higher than that of the bulk of 60 to 70S RSV RNA-associated 4S RNA. The role of primers in DNA synthesis by RSV DNA polymerase is discussed.     

Deoxyribonucleic Acid Polymerase Activity in a Deoxyribonucleic Acid Polymerase I-Deficient Mutant of Bacillus subtilis Infected with Temperate Bacteriophage SPO2

Increased deoxyribonucleic acid (DNA) polymerase activity is found in soluble extracts from a polymerase I-negative mutant of Bacillus subtilis after infection with temperate phage SPO2, or after induction of SPO2 prophage in lysogenic derivatives of this mutant. No increased enzyme activity is found after SPO2 infection in the presence of chloramphenicol. Infection of the polymerase-negative mutant with the DNA-negative sus mutant SPO2 L244 gives no increased enzyme activity, whereas infection with DNA-negative sus mutant SPO2 J385 gives enzyme activities comparable to those found in wild-type infected cells. These findings suggest that SPO2 determines a DNA polymerase activity essential for synthesis of phage DNA.     

Chloroplast Development in Etiolated Peas: Reformation of Prolamellar Bodies in Red Light without Accumulation of Protochlorophyllide

Chloroplast development and accumulation of chlorophylls werestudied in etiolated peas (Pisum sativum L., cv. Green feast)exposed for 24 h to one of three intensities of red light orto a corresponding intensity of white light producing a similarhigh medium or low terminal chlorophyll content. Chloroplastdevelopment was assessed by counts of grana and partitions pergranum in electron micrographs. Chlorophylls were partitionedand protochlorophyllide measured in the unphytylated fraction.More sensitive estimates of protochlorophyllide were made byfluorescence. In the high- and medium-intensity red-light treatmentsconsiderable reformation of prolamellar bodies took place duringdevelopment. This occurred at a stage when no protochlorophyllidewould be detected. In the low-intensity treatments reformationwas accompanied by an accumulation of protochlorophyllide ata higher level than that obtaining in the dark.     

In Vivo Distribution of Ribonucleic Acid Polymerase Between Cytoplasm and Nucleoid in Escherichia coli

Three forms of ribonucleic acid polymerase can be distinguished in exponentially growing Escherichia coli cells: (i) active, (ii) inactive, inside the nucleoid, and (iii) inactive, free in the cytoplasm.     

Effect of Rifamycins and Related Antibiotics on the Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase of Vaccinia Virus Particles

A number of compounds related to rifampin which act as expected in the Escherichia coli system have been tested for their ability to inhibit the vaccinia particle deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase in vitro. Some compounds are inactive even at concentrations of 500 mug/ml, others are able to produce partial inhibition, and others strongly inhibit the enzyme activity at 150 mug/ml or less. The inhibition, where present, operates immediately but appears to be at least partially reversible. At least one compound which is without effect against bacterial RNA polymerase is a potent inhibitor of the viral RNA polymerase. As the enzyme activity of rifampin-resistant mutants of vaccinia virus is inhibited to the same extent as that of the wild type, the observed in vitro effect on vaccinia virus RNA polymerase is not identical with the in vivo effect specifically directed against a vaccinia-specified protein.     

Effect of Far Red and Red Light on a Pelletable Peroxidase Activity in Extracts from Spinach Leaves

The effect of in vitro irradiations on ihe pelletability of peroxitlase activity was tested in extracts prepared from leaves of light-grown spinach (Spinacia oleracea). It appeared that far red light increased the pelletable activity and that red light reversed this effect. This phytochrome-mediated effect occurred in a particulate fraction which banded at 39% (w/w) sucrose on an isopycnic density gradient.     

Increased Activity of Chromatin-bound Ribonucleic Acid Polymerase from Soybean Hypocotyl with Spermidine and High Ionic Strength

Optimal activity of chromatin-bound RNA polymerase from soybeans is obtained with 1 mm Mn²⁻, but only when high ionic strength or polyamines are included in the medium. Such inclusion does not increase the Mg²⁺ activation of the polymerase, but it does lower the concentration needed for optimum activity from 10 mm to 1 mm. Mg²⁻ activation is inhibited by added Mn²⁺, and the inhibition is relieved by high ionic strength or spermidine. The RNA polymerase with either cation is almost entirely polymerase I at low and high ionic strength as evidenced by insensitivity to α-amanitin. Treatment of soybean seedlings with 2,4-dichlorophenoxyacetic acid does not change these characteristics; although the activity rises 3- to 4-fold.     

Control of Deoxyribonucleic Acid and Ribonucleic Acid Synthesis in Pyrimidine-Limited Escherichia coli

The effects of pyrimidine limitation on chromosome replication and the control of ribosomal and transfer ribonucleic acid syntheses were investigated. Chromosome replication was studied by autoradiography of (3)H-thymine pulse-labeled cells. Pyrimidine limitation did not affect the fraction of cells incorporating radioactive thymine during a short pulse, indicating that when growth is limited by the supply of pyrimidine, the time required for chromosome duplication increases in proportion to the time required for cell duplication. Control of ribosomal RNA and transfer RNA syntheses was examined by chromatographing cell extracts on methylated albumin kieselguhr columns. When growth was controlled by carbon-nitrogen limitation, the ratio of tRNA to total RNA remained roughly constant at growth rates above 0.5 doublings per hour. During pyrimidine limitation, however, the control of rRNA synthesis was apparently dissociated from the control of tRNA synthesis: the ratio of tRNA to total RNA increased as the growth rate decreased.     

Altered Deoxyribonucleic Acid Polymerase Activity in a Methyl Methanesulfonate-Sensitive Mutant of Bacillus subtilis

Of six deoxyribonucleic acid repair mutants of Bacillus subtilis assayed for deoxyribonucleic acid polymerase, only the methyl methanesulfonate-sensitive and ultraviolet light-sensitive mutant JB1-49(59) has impaired polymerase activity. Extracts prepared by sonic treatment or gentle lysis had about 10% of the wild-type activity with poly d(A-T), an alternating copolymer of deoxyadenylate and deoxythymidylate, used as template. The sensitivity to methyl methanesulfonate and ultraviolet light and the low level of polymerase activity transformed and reverted together, indicating that the two characteristics are a pleiotropic manifestation of a single mutation. Mixed extract and kinetic experiments mitigated against an altered nuclease activity as the enzymatic consequence of the mutation. Also, the mutant and wild type activities were stimulated equally by Escherichia coli exonuclease III. The residual activity in the mutant showed several differences from the wild-type activity: it purified differently, was more sensitive to sulfhydryl reagents, and displayed a different template specificity. We tentatively conclude that either the mutation in JB1-49(59) has introduced a qualitative as well as a quantitative change in the polymerase or the wild type contains two distinct polymerases, one of which is missing in the mutant.     

Non-Continuous Translation of Coat Protein and Ribonucleic Acid Polymerase Cistrons in MS2 Bacteriophage Ribonucleic Acid

In an MS2 phage ribonucleic acid (RNA)-directed in vitro protein-synthesizing system, the coat protein cistron and the adjacent RNA polymerase cistron are translated non-continuously. The ribosomes which have completed the synthesis of coat protein dissociate from the MS2 RNA and do not read through the intercistronic gap. Translation of the adjacent RNA polymerase cistron requires ribosomes other than those translating the coat protein cistron.     

Hybridization of Rous Sarcoma Virus Deoxyribonucleic Acid Polymerase Product and Ribonucleic Acids from Chicken and Rat Cells Infected with Rous Sarcoma Virus

Rous sarcoma virus (RSV)-specific ribonucleic acid (RNA) in virus-producing chicken cells and non-virus-producing rat cells infected with RSV was studied by hybridization with the endogenous deoxyribonucleic acid (DNA) product of the RSV virion DNA polymerase system. By hybridizing the total DNA product with excess virion RNA, the product DNA was separated into hybridized (“minus”) and nonhybridized (“plus”) DNA. The “minus” DNA was complementary to at least 20% of the RNA from RSV which remained of high molecular weight after denaturation. A maximum of approximately 65% hybridization was observed between “minus” DNA and RSV RNA or RSV-infected chicken cell RNA. A maximum of about 60% hybridization was observed between “minus” DNA and RSV-infected rat cell RNA. RSV-infected chicken cells contained RSV-specific RNA equivalent to about 6,000 virions per cell. RSV-infected rat cells contained RSV-specific RNA equivalent to approximately 400 virions per cell. Neither cell type contained detectable RNA complementary to virion RNA. The RSV-specific RNA in RSV-infected rat cells did not appear to be qualitatively different from that in RSV-infected chicken cells.     

Attempts to Detect Deoxyribonucleic Acid from Agrobacterium tumefaciens and Bacteriophage PS8 in Crown Gall Tumors by Complementary Ribonucleic Acid/Deoxyribonucleic Acid-Filter Hybridization

Labeled ribonucleic acid (RNA) complementary to Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA (cRNA) were used in a systematic study of the sensitivity of cRNA/deoxyribonucleic acid (DNA)-filter hybridization for detection of small amounts of phage or bacterial DNA immobilized on filters. A. tumefaciens cRNA of specific activity 10(6) to 2 x 10(6) counts per min per mug reacted to a significant extent when the DNA-filter contained 1% A. tumefaciens DNA in a salmon DNA background, but 0.1% A. tumefaciens DNA was not detectable. PS8 phage cRNA of the same specific activity reacted to a significant extent when the DNA-filter contained as little as 0.01% PS8 DNA in a salmon DNA background. Both kinds of cRNA were found to bind to tobacco crown gall tumor DNA-filters. Similar reaction was found with control normal callus DNA-filters but not with tobacco seedling DNA-filters. The "hybrids" formed by cRNA with normal callus and tumor DNA-filters had low thermal stability. Attempts to purify the tumor and normal callus DNA prior to immobilization on the filter resulted in elimination of this spurious binding. No evidence was found for bacterial or phage DNA in crown gall tumor DNA.     

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.