Laboratory Calibrations of the [3H]Adenine Technique for Measuring Rates of RNA and DNA Synthesis in Marine Microorganisms

The nucleic acid synthesis rates of several marine phytoplankton and bacteria grown in chemostat and batch cultures were measured by using [³H]adenine. The [³H]adenine synthesis rates showed excellent agreement with the known rates of synthesis estimated from chemical RNA and DNA data. Under certain conditions, RNA turnover and ATP pool compartmentalization produce inaccuracies in synthesis measurements made with [³H]adenine. However, accurate measurements of the rates of microbial RNA and DNA synthesis can be made in any environmental situation provided a few simple precautions are observed. First, time course experiments are recommended. Second, experiments should be conducted for periods long enough to avoid problems arising from disequilibria of internal ATP pools. Finally, exogenous [³H]adenine should remain in the medium over the length of the time course.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. IV. Effect on methylase-mediated methylation of DNA.

The possibility that carcinogens may affect methylase-mediated methylation of replicating DNA was investigated. A system eminently suitable for this purpose is liver regenerating after partial hepatectomy, as one injection of dimethylnitrosamine (DMN) given during the ensuing period of increased DNA synthesis induces hepatocellular carcinoma. Methylation of DNA by DNA methylase normally occurs only in proportion to DNA synthesis. Therefore simultaneous measurements were made of synthesis (incorporation of [14C]adenine into DNA adenine, or of d[5-3H]cytidine into DNA cytosine), and of methylation (incorporation of [methyl-3H]methionine into 5-methylcytosine of DNA) in liver regenerating after partial hepatectomy. After treatment with DMN, the ratio of methylation: synthesis remained within the normal range. Methyl methanesulphonate (MMS), a compound which damages DNA in regenerating liver in a similar but not identical way to DMN and which does not induce tumors in liver even when given after partial hepatectomy, caused an increase in methylation in relation to synthesis. These experiments therefore do not support the view that altered DNA methylase activity is involved in carcinogenesis.     

Ribonucleic acid synthesis in rabbit erythroid cells.

Kinetic studies on the synthesis of RNA in mature bone-marrow erythroid cells from rabbits were made by measuring the incorporation of [2-3H]adenosine into the ATP pool and RNA over periods up to 8h. By use of equations to fit the pool specific radioactivity and an equation using the same type of pool to generate the rate of linear DNA synthesis, good agreement between the pool parameters is found, provided that the ATP pool is measured in whole cell extracts, and assuming that the dATP and ATP pools equilibrate rapidly. RNA-synthesis rates were measured by using curve fits to equations developed by using the pool specific-radioactivity curves. The rate of synthesis of poly(A)-containing RNA varied in three experiments from 90 to 220mol/min per cell, with half-life of nuclear processing of 12-22 min with a mean of 16 min. Ribosomal RNA is synthesized at a rate of 70-200 mol/min per cell with an average half-life of nuclear processing of 37 min for the 18S RNA and 214 min for the 28S RNA. When the stable rRNA components are subtracted from the nRNA synthesis, the rate of nRNA synthesis is between 2 and 6fg/min per cell with an average half-life of degradation of 27 min. The rate of synthesis of poly(A)-containing RNA is 1.5-3.5% of the RNA-synthesis rates. These rates are compared with the RNA-synthesis rates found in L cells and concentrations of globin mRNA found in various erythroid-cell preparations.     

Estimates of bacterial growth from changes in uptake rates and biomass.

Rates of nucleic acid synthesis have been used to examine microbiol growth in natural waters. These rates are calculated from the incorporation of [3H]adenine and [3H]thymidine for RNA and DNA syntheses, respectively. Several additional biochemical parameters must be measured or taken from the literature to estimate growth rates from the incorporation of the tritiated compounds. We propose a simple method of estimating a conversion factor which obviates measuring these biochemical parameters. The change in bacterial abundance and incorporation rates of [3H]thymidine was measured in samples from three environments. The incorporation of exogenous [3H]thymidine was closely coupled with growth and cell division as estimated from the increase in bacterial biomass. Analysis of the changes in incorporation rates and initial bacterial abundance yielded a conversion factor for calculating bacterial production rates from incorporation rates. Furthermore, the growth rate of only those bacteria incorporating the compound can be estimated. The data analysis and experimental design can be used to estimate the proportion of nondividing cells and to examine changes in cell volumes.     

Underestimation of DNA synthesis by [h]thymidine incorporation in marine bacteria

A direct comparison of [H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [H]thymidine incorporation and isotope dilution assays.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Errors from using 3H-labeled ribonucleoside triphosphates to monitor nuclear RNA synthesis in vitro

The [3H]XTPs are used widely to monitor RNA synthesis in vitro. Recently, we discovered that they reflected only 40-45% of the true rate of nuclear RNA synthesis. Thus, when [8-14C]GTP was used, 1466 pmol [8-14C]GMP was incorporated per mg DNA/10 min. On the other hand, when [8-3H]GTP was used, only 564 pmol [8-3H]GMP was incorporated per mg DNA/10 min. There are three obvious factors that could have contributed to this greater than 2-fold difference in the apparent incorporation rate: commercial [8-3H]GTP sample was contaminated with substances causing the assay medium to be less efficient in RNA synthesis; 3H exchange occurred during acid washing of the [3H]RNA; and there was a greater quenching effect on [3H]RNA. Experiments were designed to test each of these alternatives. We are able to conclude that none of the above three are contributing factors. Our data also show that the 3H label was removed after it was incorporated into RNA. Similar differences were observed when 3H and 14C labeled pairs of ATP, UTP and CTP were compared. Furthermore, when nuclei were fractionated into nucleolar and nucleoplasmic fractions and carried out RNA synthesis, the loss of 3H label was observed mainly from the nucleoplasmic fraction.     

Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology

The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-³H]adenine, [methyl-³H]thymidine, and [5-³H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [³H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [³H]thymidine and [³H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented.     

Role of the intramitochondrial adenine nucleotides as intermediates in the uncoupler-induced hydrolysis of extramitochondrial ATP.

1. A formula is given that describes the appearance of [14C]ATPADP outside the mitochondria after the addition of [14C] 1atp during the steady-state uncoupler-induced hydrolysis of extramitochondrial ATP. If the transported adenine nucleotides equilibrate with the intramitochondrial pool, [14C]ADP0 would be expected to appear with a lag phase that corresponds with the time needed for the radioactive labelling of the intramitochondrial adenine nucleotide pool. 2. The rates of formation of [14C]ADP outside the mitochondria after addition of [14C]ATP during the steady-state uncoupler-induced ATP hydrolysis catalysed by rat-liver mitochondria at 0 degree C were measured. 3. In the presence of carbonyl cyanide m-chlorophenylhydrazone the time course of the [14]ADPo formation was the same as that predicted on the basis of the above assumption. 4. In the presence of the less effective uncoupler, 2,4-dinitrophenol, the time course of [14C]ADPo formation was not consistent with the theoretical predictions: no lag phase was present and the measured rate was higher than the maximal calculated rate. These results can be explained by assuming a functional interaction between the adenine nucleotide translocator and the mitochondrial ATPase (F1). 5. It is concluded that under phosphorylating as well as dephosphorylating conditions, the adenine nucleotide translocator and the mitochondrial ATPase can be functionally linked to catalyse phosphorylation or dephosphorylation of extramitochondrial ADP or ATP, without participation of the intramitochondrial adenine nucleotides.     

Carbon-specific phytoplankton growth rates: a comparison of methods

Measurements of biomass and growth rate of two axenic algalcultures were carried out using three different methodologicalapproaches: the specific ¹⁴C-labelling of chlorophyll a, [³H]adenineincorporation into DNA and net organic carbon assimilation.Time-course experiments revealed that the specific activitiesof chlorophyll a were significantly higher than the specificactivity of total algal carbon in six of seven experiments.When the specific activity of chlorophyll a is used to calculatethe carbon biomass and growth rate, the carbon biomass of thealgae will thus be underestimated and the specific growth ratewill be too high. Determination of growth rates from incorporationof [³H]adenine gave lower values than those obtained from netorganic carbon assimilation and from ¹⁴C incorporation intochlorophyll a. Problems with adenine saturation are suggested.When [³H]adenine is used to measure growth rates in dense algalcultures, additions of >1 µM [³H]adenine are oftenrequired to maximally label the extracellular and intracellularadenine pools and hence DNA.     

Stimulation by abscisic acid of RNA synthesis in discs from healthy and tobacco mosaic virus-infected tobacco leaves

Uptake of abscisic acid from the culture medium by discs of healthy and tobacco mosaic virus-infected tobacco leaves was measured. Small (two to five-fold) increases in abscisic acid concentration in discs caused increases in rates of [³H]uridine and [³H]adenine incorporation into total nucleic acid, virus RNA and host ribosomal RNA. Net accumulation of virus RNA was also enhanced by abscisic acid. This evidence for stimulation of RNA synthesis is compared with previous reports showing inhibition of RNA synthesis in other tissues. It is suggested that the increase in endogenous abscisic acid caused by tobacco mosaic virus infection may be at least partly responsible for observed increases in rates of RNA synthesis after infection.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.     

The effect of chemical mutagens on purine and pyrimidine nucleotide biosynthesis

Nucleotide biosynthesis in Novikoff hepatoma cells is markedly altered by a variety of chemical mutagens, whether the mechanism of mutagenesis is by base substitution, covalent binding (adduct formation), intercalation, or cross-linking of DNA. The compounds investigated (N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide, 9-aminoacridine, and mitomycin C), at concentrations that cause some inhibition of RNA and DNA synthesis, bring about a large increase in the pool levels of all four nucleoside triphosphates. At the same time, reactions leading to the synthesis of CTP from exogenous uridine and GTP and ATP from exogenous hypoxanthine are severely inhibited. The formation of UTP from uridine and ATP from adenosine, by more direct phosphorylation reactions, appears relatively unaffected. The increase in nucleotide pool size cannot be accounted for by a corresponding increase in de novo purine and pyrimidine nucleotide synthesis, as experiments with labeled formate and aspartate show similar inhibitions by the mutagens. With the salvage precursors, [3H]uridine and [3H]hypoxanthine, the mutagens can produce a widely divergent reduction in the labeling of RNA-CMP versus RNA-UMP and of RNA-GMP versus RNA-AMP, mostly a result of these agents causing large differences in the specific activities of the respective triphosphate precursors. These observations suggest that, in addition to the reactions with DNA, nucleotide biosynthesis could be another important biochemical target of chemical mutagens.     

The pathway of adenylate catabolism in Azotobacter vinelandii. Evidence for adenosine monophosphate nucleosidase as the regulatory enzyme.

Cell-free, dialyzed extracts from Azotobacter vinelandii rapidly dephosphorylate [U-14C]ATP to labeled ADP and AMP, which is then degraded to hypoxanthine, the end product of AMP catabolism under the experimental conditions which were used. The intermediates of the pathway from ATP to hypoxanthine have been identified by thin layer chromatography and quantitated by the 14-C content. The concentrations of intermediates present during the production of hypoxanthine are consistent with AMP nucleosidase being responsible for AMP degradation in these extracts. This result was confirmed in experiments which utilized rabbit antibody prepared against purified AMP nucleosidase. The antibody inhibited AMP nucleosidase activity in cell-free extracts but did not inhibit adenine demanase or adenosine deaminase from the same extracts. In the presence of antibody prepared against purified AMP nucleosidase, the dialyzed extracts showed a marked reduction in the production of hypoxanthine from ATP. Other enzymes which could be responsible theoretically for the conversion of AMP to hypoxanthine were not detected by standard assay procedures. These results are consistent with AMP degradation proceeding by way of AMP nucleosidase to yield adenine and ribose 5-phosphate. The adenine is then converted to hypoxanthine by adenine deaminase. Both of these enzymes were present in sufficient quantities to account for the observed rates of hypoxanthine formation. The rate of hypoxanthine formation decreases during the time course of the [U-14-C]ATP degradation experiments, even though the concentration of AMP remains high. This decrease in the rate of hypoxanthine formation as a function of time is attributed to the decreasing ATP and increasing P0-4 concentrations, since ATP is an activator of AMP nucleosidase and P0-4 is an inhibitor. These observations suggest that the in vivo activity of AMP nucleosidase could also be regulated by changes in the relative ratios of ATP:AMP:P0-4.     

Measurement of cyclic 3',5'-adenosine monophosphate synthesis in growing Escherichia coli.

The net synthesis of cAMP by an adenine auxotroph of $\text{Escherichia coli}$ was measured by assaying the incorporation of tritium from [³H]-adenine into cyclic [³H] AMP during exponential growth. Synthesis of cAMP ceased abruptly when glucose was added to cells growing in glycerol and then recovered to an intermediate rate of synthesis after 0.5–1.0 generation. Cyclic AMP appeared to be synthesized from a precursor pool that turned over more rapidly than total cellular ATP. The rates of cAMP synthesis measured by this technique are compatible with the cellular levels of cAMP previously measured in this strain(3).     

Mode of enhancement in ribonucleic acid synthesis directed by chromium (III)-bound deoxyribonucleic acid

By forming a complex with calf thymus DNA, Cr(III), i.e., CrCl3 and Cr(NO3)3, significantly enhanced its template activity for in vitro RNA synthesis as assayed by 3H incorporation from [5-3H]uridine triphosphate (UTP). The extent of the augmentation in RNA synthesis was proportional to the binding ratio of Cr(III) to the template DNA. K2CrO4, on the other hand, neither bound to DNA nor enhanced its template activity. Experiments using rifampicin and heparin suggested that incorrect and nonviable initiation sites for RNA synthesis became functional in Cr(III)-bound DNA. The incorporation of [gamma-32P]adenosine triphosphate (ATP) into RNA synthesized on Cr(III)-bound DNA was 8 to 9 times greater than that on control DNA. This value was much higher than that of the 3H incorporation form [5-3H]UTP, i.e., the incorporation of 32P on Cr(III) bound DNA was 8 to 9 times greater that of 3H and less than twice that on control DNA. These results suggest that Cr(III) possibly induces the abnormal synthesis of RNA of a very low molecular weight, for most if not all the molecules, by binding to the template DNA.     

Synthesis and turnover of hdRNA and mRNA in the salivary gland of the blowfly Calliphora erythrocephala.

We have measured the absolute molar rates of synthesis, accumulation, and turnover of blowfly salivary gland heterodisperse RNA. Twelve- and 84-hr-stage third-instar Calliphora erythrocephala larvae were injected with [³H]adenosine, and its flow into glandular ATP, heterodisperse RNA, and polyadenylated RNA was each quantitated over a 360-min time course. The results of these experiments indicate that at least 80% of the newly synthesized heterodisperse RNA mass is a >28 S nuclear species whose average first-order half-life is approximately 20 min. The remaining 20% of the heterodisperse RNA has a 6–28 S size distribution, accumulates in the cytoplasm, and is associated with functional polysomes. The average first-order half-life of this more stable species is 20–25 hr. In addition, we have independently quantitated by optical methods the developmental change in the content of polysome-associated mRNA. The mRNA in these studies also has an average first-order half-life of 25 hr and accounts for 25–55% of the mRNA mass predicted by the incorporation-kinetic analysis of the pulse-labeled heterodisperse RNA. Despite the increased polyteny of the older stage glands, the rates of synthesis and accumulation of each of the individual heterodisperse RNA classes are the same at the 12- and 84-hr stages. Collectively, these results demonstrate that salivary gland functional specialization results from the accumulation of long-lived mRNA and not from changes in the overall rate of mRNA synthesis.     

Mechanism of inhibition of Escherichia coli RNA polymerase by captan.

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.