Greater Length of Ribonucleic Acid Synthesized by Chromatin-bound Polymerase from Auxin-treated Soybean Hypocotyls

An investigation has been made of the RNA synthesized by chromatin-bound RNA polymerase from soybean hypocotyls (Glycine max var. Wayne). Polymerase activity is 4- to 5-fold higher with chromatin from tissue treated with 2,4-dichlorophenoxyacetic acid, a synthetic auxin, compared to untreated tissue. Thin layer chromatography of the RNA hydrolysis products and acrylamide gel electrophoresis of the RNA synthesized by the chromatin show that increased activity induced by 2,4-dichlorophenoxyacetic acid is due primarily to the production of longer RNA chains, with only 20 to 50% increase in the number of RNA chains. The observation that 2,4-dichlorophenoxyacetic acid treatment leads to greater rates of RNA synthesis, producing longer chains in unit time, suggests that one manifestation of auxin activity is in activation of RNA polymerase I (ribosomal RNA polymerase).     

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.     

The Influence of Auxin and Ethylene on Chromatin-directed Ribonucleic Acid Synthesis in Soybean Hypocotyl

Soybean seedlings treated with ethylene exhibited small increases in ribonucleic acid content in the elongating section of the hypocotyl. Chromatin isolated from the elongating section of ethylene-treated seedlings showed a 35 to 60% increase in the capacity for RNA synthesis. The ethylene-induced response was saturated at 1 microliter/liter of ethylene and was fully expressed after 3 hours. Auxin caused marked accumulation of RNA and DNA in the elongating and basal tissue of the hypocotyl. Chromatin isolated from these auxin-treated tissues showed an 8- to 10- fold increase in RNA synthetic capacity as measured in vitro. Ethylene added with auxin reduced the auxin enhancement of nucleic acid synthesis in the elongating and basal tissues. Both ethylene and auxin treatment of the seedlings inhibited nucleic acid accumulation and chromatin activity in the apical tissue. Ethylene did not appear to mediate the auxin effects on nucleic acid synthesis in soybean hypocotyl with the possible exception of inhibition in the apical tissue.     

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.     

Ribonucleic Acid Polymerase Catalyzing Synthesis of Double-stranded Arbovirus Ribonucleic Acid

The large-particle fraction from the cytoplasm of chick embryo fibroblasts infected with Semliki Forest virus was found to catalyze the incorporation of the 5'-triphosphates of guanosine, adenine, cytidine, and uridine into an acid-insoluble alkali-labile product. The conditions affecting the preparation and assay of this enzyme were investigated. The ribonucleic acid (RNA) polymerase was not present in uninfected cells, and it appeared in infected cells at the time of rapid viral RNA synthesis. The polymerase was found to catalyze the synthesis of a species of RNA which was resistant to ribonuclease and which exhibited the sedimentation properties, buoyant density, and thermal transition temperature of the double-stranded RNA found in vivo in chick cells infected with Semliki forest virus. Attempts to demonstrate that the reaction product of this enzyme also included single-stranded viral RNA were not successful. Although other interpretations are possible, these results give some support to the suggestion that more than one enzyme may be involved in the replication of viral RNA.     

Transcription of the Influenza Ribonucleic Acid Genome by a Virion Polymerase I. Optimal Conditions for In Vitro Activity of the Ribonucleic Acid-Dependent Ribonucleic Acid Polymerase

In vitro reaction conditions have been determined for the maximal synthesis of product ribonucleic acid by the influenza (WSN) virion ribonucleic acid polymerase. The reaction requires the presence of all four triphosphates, Mg(2+) and Mn(2+) ions, monovalent cations, nonionic detergent, and ribonucleoside triphosphates at concentrations above certain threshold values. The optimum pH for the reaction is around 8.0 to 8.2 and the kinetics of product synthesis are linear through at least 6 hr when incubated at 31 to 33 C.     

Separation of Replicative Intermediate from Single-stranded Ribonucleic Acid by Sedimentation at Low Ionic Strength

By sedimentation in 2.5 x 10(-4)m sodium ethylenediaminetetraacetate at 25 C, replicative intermediate could be separated from single-stranded ribonucleic acid, both viral and ribosomal. All of the label in the replicative intermediate after a brief pulse of titrated uridine was resistant to ribonuclease.     

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.     

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 Polymerase Induced in Cells Infected with Sendai Virus

A ribonucleic acid (RNA)-dependent RNA polymerase was induced in chick embryo fibroblast cells after infection with Sendai virus (parainfluenza 1 virus). The enzyme was associated with the microsomal fraction of infected cells and reached maximum detectable activity at 18 hr after virus infection. The activity of the enzyme in vitro was dependent on the presence of added magnesium ions and all four nucleoside triphosphates and was not inhibited by actinomycin D. The RNA synthesized by the enzyme in vitro was sensitive to ribonuclease and consisted of a complex mixture of RNA species including 34S, 24S, and 18S components. Similar RNA components were detected in the microsomal fraction of Sendai virus-infected cells by labeling with (3)H-uridine from 17 to 18 hr postinfection in the presence of actinomycin D. Of the RNA synthesized by Sendai virus-induced RNA polymerase in vitro, 98% became insensitive to ribonuclease after annealing with RNA extracted from purified Sendai virus particles.     

Inhibition of Auxin-induced Deoxyribonucleic Acid Synthesis and Chromatin Activity by 5-Fluorodeoxyuridine in Soybean Hypocotyl

Rootless soybean (Glycine max) seedlings were used as a test system to examine the action of auxin on chromatin-directed RNA synthesis. Chromatin from the basal tissue of rootless seedlings (both control and auxin-treated) had RNA synthetic capacity similar to that of chromatin from comparably treated intact seedlings. When DNA synthesis normally induced in the basal tissue by auxin was blocked in the rootless seedlings by 5-fluorodeoxyuridine, the auxin enhancement of chromatin activity was inhibited 70%. This level was still three times the control level, indicating that auxin influenced the synthetic activity of existing DNA template. Experiments with Escherichia coli RNA polymerase revealed that chromatin from both auxin- and auxin plus 5-fluorodeoxyuridine-treated tissue saturated at higher levels than chromatin from control tissue.     

The Isolation and Characterization of Adenosine Monophosphate-rich Polynucleotides Synthesized by Soybean Hypocotyl Cells: Their Relation to Messenger Ribonucleic Acid

Plant ribonucleic acids which have high adenosine monophosphate concentrations were studied. Purified deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid fractions both contained poly-adenosine monophosphate sequences (those from the latter being longer than those from the former); without these poly-adenosine monophosphate sequences their base compositions were the same. The average poly-adenosine monophosphate sequence from purified tenaciously bound ribonucleic acid was 160 residues long, as measured by gel electrophoresis. However, base hydrolysis and chromatography indicated one 3′-nucleoside (adenosine) per 71 nucleotides, giving a chain length of 72 residues. The dominant species in the cytoplasm, as measured by radioactive precursor incorporation, was tenaciously bound ribonucleic acid, whereas deoxyribonucleic acid-like ribonucleic acid was present in greater amounts in the nucleus. This work provides evidence that deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid represent forms of messenger ribonucleic acid in soybean, with deoxyribonucleic acid-like ribonucleic acid residing in the nucleus, perhaps as the messenger ribonucleic acid precursor, and tenaciously bound ribonucleic acid residing, as the active messenger ribonucleic acid, in the cytoplasm.     

Characterization of a Ribonucleic Acid Polymerase Activity Associated with Purified Cytoplasmic Polyhedrosis Virus of the Silkworm Bombyx mori

Purified cytoplasmic-polyhedrosis virus has been found to have associated with it a polymerase activity capable of catalyzing the synthesis of virus-specific, single-stranded ribonucleic acid (RNA) from the double-stranded RNA genome.     

The Structural Organization of Ribonucleic Acid Associated with Soybean Microsomes

The microsomal fraction of soybean (Glycine max) hypocotylswas characterized using analytical and electron microscope techniques.Two microsomal sub-fractions (smooth and rough vesicles) wereseparated by untracentrifugation. Analysis showed that the ribonucleicacid (RNA) of the microsomal fraction cannot be completely accountedfor by the ribosomes, and that some of the RNA is associatedwith the membranes in a non-ribosomal form. A similar conclusionwas reached by using ethylenediaminetetra-acetic acid (EDTA)to disperse the ribosomal material. Although no visible robosomesremained after the treatment with EDTA, the microsomal fractionretained a quarter of its RNA. Ribonuclease removed all themocrosomal RNA without visibly altering the membrane structureexamined in cross section. When hypocotyl tissue sections wereincubated with ¹⁴C amino-acids, the microsomes incorporatedthe radioactive label more rapidly than any other subcellularfraction. This was especially true in the rapidly growing zoneof the hypocotyl. Smooth microsomes were 75 per cent as activeas rough microsomes. These observations are discussed with referenceto the structural organization of the microsomal RNA and itsrole in protein synthesis.     

Encephalomyocarditis Virus Ribonucleic Acid Polymerase Associated with 150S Cytoplasmic Particles

Cytoplasmic particles which sedimented at 150S were the smallest structures containing detectable viral ribonucleic acid polymerase in mouse cells infected with encephalomyocarditis virus.     

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.     

Ribonucleic Acid Polymerase Mutant of Escherichia coli Defective in Flagella Formation

Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.