Poly(A) synthesis by RNA polymerase II from rat liver

Multiple forms of DNA-dependent RNA polymerase were resolved by DEAE-Sephadex chromatography. In addition to RNA polymerases, an active poly(A) polymerase was also fractionated. RNA polymerases were examined for their capacity to synthesize poly(A). None of the freshly prepared enzymes could efficiently make poly(A) in presence or absence of exogenous primers. However, “aging” of polymerase II by simple incubation at 37°C resulted in the loss of RNA polymerizing activity with a corresponding increase in poly(A) synthesizing activity. Transformation of RNA polymerase to poly(A) polymerase resulted in reduced capacity to transcribe native DNA and altered chromatographic behavior. The results suggest that subunits of polymerase II obligatory to DNA-dependent RNA synthesis were degraded by “aging” and that a stable subunit of the RNA polymerase could preferentially make poly(A).     

Template requirement of maize RNA polymerase

Maize RNA polymerase utilizes heated deoxyribonucleic acid more effectively than native deoxyribonucleic acid as a template for ribonucleic acid synthesis. A ribonucleic acid-deoxyribonucleic acid hybrid accumulates in the presence of heated deoxyribonucleic acid. The amount of product formed with either native or heat-denatured deoxyribonucleic acid does not exceed the amount of deoxyribonucleic acid added as template.     

Characterization of arithmetic deoxyribonucleic acid synthesis at restrictive temperature in a dnaE mutant of Escherichia coli K-12.

The dna-293 mutation is shown to be a dnaE allele. The linear deoxyribonucleic acid synthesis previously observed in this mutant has been further characterized. The production of small deoxyribonucleic acid intermediates and their subsequent joining were identical in the mutant and its dnaE+ parent at 42.5 degrees C. Though the mutant cells continued to divide at the nonpermissive temperature, the rate of division was reduced. The data are consistent with a lack of production of replicationally active deoxyribonucleic acid polymerase III at the restrictive temperature.     

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.     

Ribonucleic Acid Polymerases of the Yeast Phase of Histoplasma capsulatum

Ribonucleic acid (RNA) polymerases of Histoplasma capsulatum (yeast phase) were fractionated by phosphocellulose chromatography and partially characterized. Three distinct, active fractions were seen. The major RNA polymerase species was inhibited strongly by α-amanitin, whereas the other two were resistant. When either slightly purified (HSE) extract or the major active component was assayed at 37 C, the incorporation of tritiated uridine monophosphate into RNA stopped after 10 to 15 min. In contrast, the synthesis continued for at least 1 h at 23 C. The other two RNA polymerase species exhibited higher rates of incorporation when tested at 37 C, and continued to synthesize RNA even after 60 min. However, by that time the levels of incorporation at 23 C were higher than at 37 C for all three enzymes. The temperature sensitivity was not affected by changing substrate concentration or employing either native or denatured calf thymus deoxyribonucleic acid as a template. These results are compared with the data obtained with RNA polymerases from different fungi and other organisms. A possible involvement of RNA polymerase(s) in morphological differentiation of H. capsulatum is discussed.     

Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435-1446. 1966.-During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation.     

Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division.

The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.     

Enhancement of deoxyribonucleic acid polymerase I-directed repair synthesis in toluene-treated Escherichia coli after growth in the presence of low levels of N-methyl-N''-nitro-N-nitrosoguanidine.

Deoxyribonucleic acid polymerase I-directed repair synthesis can be selectively measured in toluene-treated Escherichia coli cells exposed to alkylating chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine. Prior growth of the cells in the presence of a low dose of N-methyl-N'-nitro-N-nitrosoguanidine results in an enhanced deoxyribonucleic acid polymerase I-directed repair synthesis and an increase in single-strand breaks.     

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.     

Nucleic acid synthesis and ribonucleic acid polymerase specificity in germinating and outgrowing spores of Bacillus subtilis.

Nucleic acid synthesis was studied during germination and outgrowth of normal spores of Bacillus subtilis, as well as of spores carrying the genome of phage phie. In a system in which development was restricted to the spore-darkening phase, synthesis of ribonucleic acid (RNA), but not deoxyribonucleic acid (DNA), was detected. The extent of RNA synthesis and turnover, during this phase was similar for the two types of spores. In a partially darkened population of spores of either type, there was little RNA degradation, whereas there was considerable turnover in a fully darkened population. The DNA-dependent RNA polymerase of dormant or dark spores was not active in vitro with phi DNA as template, although a sigma-like factor could be separated from the polymerizing activity by zone centrifugation. Within 40 min after resuspension of dark spores in a medium that allows outgrowth, the enzyme acquired the ability to transcribe the phage DNA efficiently. During outgrowth, both normal and carrier spores synthesized DNA, but in carrier spores this DNA was almost entirely phage specific. The pattern of RNA accumulation in normal spores was in two distinct phase (0 to 60 min and 90 to 180 min). The second phase was absent in outgrowing carrier spores. The burst of phage in carrier spores occurred at 160 to 180 min.     

Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis.

A rapid micromethod is described for the preparation of nucleic acid-free extracts from Escherichia coli that involves precipitation with polyethylene glycol. Extracts can be prepared from growing cells in 75 min by three short, low-speed centrifugations. The extract did not inhibit added purified ribonucleic acid (RNA) polymerase, suggesting that major inhibitors of RNA synthesis had been removed. This extract should be ideal for assessing the properties of mutant RNA polymerases. The rapid chromatography of the extracts with step elution from deoxyribonucleic acid- and diethylaminoethyl-cellulose columns resulted in high yields of substantially pure RNA polymerase. We used this technique to purify 35S-labeled RNA polymerase. This system should find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.     

Effect of protein synthesis on plasmid maintenance in Streptococcus faecalis.

Plasmid-to-chromosome ratios in Enterobacteriaceae, upon interruption of protein synthesis by chloramphenicol, are either conserved or increased when measured by dye buoyant density centrifugation. We have found, on the other hand, that the effect of inhibition of protein synthesis on the amount of covalently closed circular deoxyribonucleic acid visualized by this method in two strains of Streptococcus faecalis appears to differ from these established systems. A three- to sixfold decrease in covalently closed circular deoxyribonucleic acid was observed when lysates of chloramphenicol-treated cultures were submitted to dye buoyant density centrifugation. A loss of covalently closed circular deoxyribonucleic acid was also evident from electrophoretic profiles of these lysates. Several conditions which could account for the apparent loss of covalently closed circular deoxyribonucleic acid upon inhibition of protein synthesis are discussed.     

Pyrophosphate-condensing activity linked to nucleic acid synthesis.

In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation.     

Isolation and properties of a testicular ribonucleic acid polymerase

A procedure is described for isolation in a soluble form of a ribonucleic acid polymerase from rat testes. Evidence is presented that this enzyme catalyzes three distinct reactions: (a) deoxyribonucleic acid-directed synthesis of RNA in the presence of all four major ribonucleoside triphosphates; (b) DNA-primed formation of polyadenylic acid and other ribohomopolymers in the presence of single ribonucleoside triphosphate substrates; (c) synthesis of complementary polyribonucleotides in the presence of various ribohomopolymer primers. The properties of these reactions are discussed with respect to metal ion requirements, affinities for ribonucleoside triphosphates and primer polynucleotides, heat denaturation of DNA primers, and the effects of ionic strength, beta-mercaptoethanol, polyamines, temperature, and inhibitors on the rates and extent of the reactions. The testicular ribonucleic acid polymerase is a very unstable enzyme that can be stabilized by high concentrations of glycerol.     

Effect of aflatoxin B1 on chromatin-bound ribonucleic acid polymerase and nucleic acid and protein synthesis in germinating maize seeds

The treatment of germinating maize seeds (cv. Ganga 2) with aflatoxin B1 resulted in suppression of ribonucleic acid (RNA), protein, and deoxyribonucleic acid (DNA) synthesis at 3, 4, and 5 h, respectively. At or below the concentrations inhibitory for these in vivo syntheses, the toxin inhibited chromatin-bound DNA-dependent RNA polymerase activity. The synthesis of both polyadenylated and non-polyadenylated RNA was inhibited, but the effect on the former was more pronounced. Equilibrium dialysis and difference spectral and viscometric analyses showed a binding of aflatoxin B1 to DNA isolated from the seeds. It is proposed that the inhibition of RNA synthesis in maize seeds by the toxin is due to the interference with the RNA polymerase activity, which seems, at least partially, due to the impairment of DNA template functions.     

Effect of aflatoxin B1 on chromatin-bound ribonucleic acid polymerase and nucleic acid and protein synthesis in germinating maize seeds.

The treatment of germinating maize seeds (cv. Ganga 2) with aflatoxin B1 resulted in suppression of ribonucleic acid (RNA), protein, and deoxyribonucleic acid (DNA) synthesis at 3, 4, and 5 h, respectively. At or below the concentrations inhibitory for these in vivo syntheses, the toxin inhibited chromatin-bound DNA-dependent RNA polymerase activity. The synthesis of both polyadenylated and non-polyadenylated RNA was inhibited, but the effect on the former was more pronounced. Equilibrium dialysis and difference spectral and viscometric analyses showed a binding of aflatoxin B1 to DNA isolated from the seeds. It is proposed that the inhibition of RNA synthesis in maize seeds by the toxin is due to the interference with the RNA polymerase activity, which seems, at least partially, due to the impairment of DNA template functions.     

Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid.

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serum-stimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the G0 leads to G1 leads to S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.     

Characterization of deoxyribonucleases induced by poxviruses

Increases in deoxyribonuclease activity assayed at alkaline pH can be observed in poxvirus-infected cells when native or denatured deoxyribonucleic acid (DNA) is used as substrate. The deoxyribonuclease assayable with native DNA as substrate, induced in HeLa cells by cowpoxvirus or vaccinia virus WR, can be separated from the corresponding enzyme present in normal cells by chromatography on diethylaminoethyl cellulose. In addition, the two enzymes induced in the virus-infected cells differ from each other in their chromatographic properties. The two induced enzymes have been further characterized with respect to properties of enzymatic reaction.     

Partial amino acid sequence of two forms of human post-gamma-globulin.

We have purified two different electrophoretic forms of Post-γ-globulin defined by electrophoresis as “native slow” form and “fast” form respectively. Amino acid sequences of the first fifty-two residues of the “native slow” form and twenty-nine of the “fast” form were determined. The sequence shows that the “fast” form lacks the first nine amino acids of the “native slow” form.This observation is consistent with the existence of a “native slow” form that is degraded into other more acidic forms of Post-γ-globulin by loss of basic amino acids.