Incorporation Of Adenine Research Articles

Energy requirement for biosynthesis of DNA in Escherichia coli: Role of membrane-bound energy-transducing ATPase (coupling factor)

A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T. H., Mével-Ninio, M. and Valentine, R. C. (1973) Biochim. Biophys. Acta 314, 267–275; Thipayathasana, P. and Valentine, R. C. (1974) Biochim. Biophys. Acta 347, 464–468; Mével-Ninio, M. and Yamamoto, T. (1974) Biochim. Biophys. Acta 357, 63–66) has been found to be blocked for anaerobic DNA synthesis. The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1 6 the rate of DNA synthesis in the wild type culture under similar conditions. Addition of NO 3 - or O 2 restores DNA biosynthesis in the mutant. The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis. It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.

Testosterone stimulation of a rapidly labeled, low-molecular-weight RNA fraction in human hepatic erythroid cells in culture.

The addition of erythropoietin to cell cultures of erythroid cells of human fetal liver resulted in an increased incorporation of thymidine, adenine, and uridine into trichloroacetic acid-insoluble cell fractions and in an increased uptake of adenine and uridine into the cell. Although the effects of testosterone and erythropoietin on heme synthesis in these cells are known to be very similar, there was no effect of testosterone on the total incorporation of radioactive precursors into DNA or RNA. The RNA synthesized after short pulses of radioactive uridine, when analyzed on sucrose gradients containing 1% sodium dodecyl sulfate, consisted of a homogeneous peak sedimenting at 10 plus or minus 2 S, which is quite different from the heterogeneous, high-molecular-weight RNA synthesized under identical conditions in primary cultures of human fetal lung, kidney, or liver parenchymal cells. Addition of testosterone to liver erythroid cells in cultures for 5 hr followed by a 1-hr uridine pulse resulted in a 3-fold increase of RNA species with an average sedimentation coefficient of 14 plus or minus 3 S. The similarity with the sedimentation coefficient of the globin mRNA described in other systems and the high degree of specialization of the erythroid cells suggest that this RNA may be a stable intermediate involved in the synthesis of hemoglobin.

A Comparison of the Radiation Sensitivities of Thymic and Thoracic Duct Lymphocytes

The dose-response curve for adenine incorporation, an index of cell death when measured 24 hr after irradiation, was biphasic for both thymic and thoracic duct lymphocyte populations X-irradiated in vitro. However, the magnitude of reduced adenine incorporation was greater for thymic lymphocytes than for thoracic duct lymphocytes at each dose examined. Measurement of the rate of injury development in the two cell populations indicates that the difference between the two dose-response curves is the result of a slower rate of injury development in thoracic duct lymphocytes. Correction for differences in rate of injury development indicates that the radiosensitivities of the two types of lymphocyte populations are identical.

Normal Activity of Metabolic Pathways Involved in the Formation and Utilization of Phosphoribosylpyrophosphate in Erythrocytes of Patients with Primary Metabolic Gout

The activity of metabolic pathways involved in the formation and utilization of phosphoribosylpyrophosphate (PRPP) was studied in. The erythrocytes of 34 patients with idiopathic metabolic gout. The activities of the oxidative pentose shunt, of the hypoxanthine-guanine and adenine phosphoribosyltransferases (HGPRT, APRT) and of PRPP synthetase, as well as the rates of PRPP generation and of adenine incorporation into nucleotides were found to be normal in the erythrocytes of all these patients. Four patients with metabolic gout due to enzymatic abnormalities, two relatives with partial deficiency of HGPRT and two relatives with mutant feedback-resistant PRPP synthetase, were studied for comparison. The significance of the results is discussed in relation to postulated mechanisms for purine overproduction in metabolic gout.

Nucleic acid precursor incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and jejunal villus epithelium.

SYNOPSISAutoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [3H]adenine primarily into RNA while that from [3H]adenosine and [3H]guanosine was present mainly in DNA. Label from [3H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [3H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [3H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [3H]adenosine. Incorporation of label from [3H]guanosine was similar for parasitized and control cells. A small quantity of label from each [3H]precursor was incorporated into DNA of villus epithelial cell nuclei.

Studies on the biochemical basis of spontaneous mutation. II. The incorporation of a base and its analogue into DNA by wild-type, mutator and antimutator DNA polymerases.

The incorporation and turnover of adenine and its analogue 2-aminopurine into DNA by purified wild-type, mutator, and antimutator T4 DNA polymerase have been measured. Antimutators incorporate less 2-aminopurine into DNA than does wild type, and imitators incorporate more than wild type. Analysis of these data is consistent with the idea that the incorporation of 2-aminopurine is influenced primarily by the ratio of 3′-exonuclease to polymerase activities of the different enzymes. The experimental results conform to a model equation, which expresses the base incorporation frequency as a function of the polymerase insertion and removal activities. Some of the implications of the model equation are examined in this and the accompanying paper.

Isolation of fatty acids from the mycelium of Penicillium stipitatum Thom and their effect on Ehrlich ascites carcinoma cells.

A total of 5 fractions were isolated from the mycelium ofPenicillium stipitatum Thom obtained by submerged cultivation. Three of them inhibited the incorporation of14C-labelled precursors into Ehrlich ascitic cells (EAC) at concentrations lower than 100 μg/ml. The fraction M-72-3, inhibiting mainly adenine incorporation, was further separated into 6 fractions. The highest effect on EAC cells was exerted by subfraction IV which consisted of free fatty acids; its main effective components were oleic linoleic and linolenic acid. Their cyto-inhibitory effect on EAC cells was confirmed by their application in a pure form.

Regulation of nuclear DNA replication by thechloroplast in Chlamydomonas.

The experiments described in this paper implicate chloroplast protein synthesis in the regulation of nuclear DNA replication. The inhibition of nuclear DNA replication in the lower eukaryote, Chlamydomonas reinhardi strain 21gr, was examined after growth of cells with a series of antibiotics (streptomycin, neamine, spectinomycin, cleocin, chloramphenicol, and rifampicin) each of which has a known effect upon chloroplast RNA or protein synthesis in this organism. Each antibiotic inhibited nuclear DNA replication at drug concentrations at which there was little or no inhibition of adenine incorporation into chloroplast DNA. That chloroplast DNA was replicating under these conditions rather than merely being repaired, was shown first by the high incorporation rates and second by a (14)N-(15)N density transfer experiment in which chloroplast DNA doubled in the presence of streptomycin, while no incorporation into nuclear DNA was detected. A small DNA peak, Component III, located between nuclear and chloroplast DNA's in CsCl gradients, possibly mitochondrial, was more pronounced in DNA from antibiotic-inhibited cultures than from controls.

The accumulation of adenosine 3′,5′-monophosphate in cerebral cortical slices of the quaking mouse, a neurologic mutant

Norepinephrine, adenosine, veratridine, and adenosine-biogenic amine combinations elicit an accumulation of cyclic AMP in cerebral cortical slices from C57B1/6J control (+/+), quaking (qk/qk) and heterozygous (qk/+) mice. The percent conversion of radioactive adenine nucleotides to cyclic AMP in slices previously incubated with radioactive adenine or adenosine is markedly lower in slices from quaking and heterozygous mice compared to controls with all stimulatory agents except an adenosine-norepinephrine combination. Total incorporation of radioactive adenine or adenosine into adenine nucleotides is, however, significantly higher with slices of quaking and heterozygous mice. The absolute amount of radioactive cyclic AMP and the levels of endogenous cyclic AMP are nearly identical in the 3 groups of mice following incubation with all stimulatory agents except adenosine. The absolute accumulation of both radioactive and endogenous cyclic AMP was significantly lower in quaking and heterozygous mice after incubation with adenosine. The ratio of [ 14C]- to [ 3H]cyclic AMP in slices previously incubated with [ 14C]adenine and [ 3H]adenosine is dependent upon the stimulatory agent. The ratio with each agent is consistently lower in quaking mice compared to controls. The data provide evidence for biochemical alterations in nucleotide metabolism in cortical slices of both quaking and heterozygous mice. These effects would not appear to be directly associated with hypomyelination and tremor of the quaking mouse, since the heterozygous mouse, with one quaking gene does not show the latter gross abnormalities.

The effect of long-term in vitro incubation on the intermediates of red cell metabolism.

Whole human blood was incubated for periods of up to 70 hours at 37 C under conditions that would cause minimal modifications in normal metabolic controls. Insofar as possible, the conditions utilized were similar to those found in vivo. Control of pH within the limits of 7.2 to 7.5 was achieved by the addition of isotonic Tris buffer or isotonic TES buffer. Glucose, inorganic phosphate, adenine, and pyruvate were added as needed throughout the incubation period. The ADP/ATP ratio and levels of ATP, total adenine nucleotides, 2,3‐diphosphoglycerate and other metabolic intermediates were maintained near those of fresh erythrocytes. The maintenance and control of red blood cell adenine nucleotide levels are discussed, with particular emphasis on factors affecting the rate of incorporation of adenine into adenine nucleotides.

Effect of 5-bromodeoxyuridine on chromatin transcription in confluent fibroblasts.

3T6 mouse fibroblasts were grown in 5-bromodeoxyuridine (BrdU) so that approximately 20% of the thymine residues in DNA were replaced by BrdU. BrdU replacement caused an alteration in the relative incorporation of labeled nucleotide precursors into RNA. The RNA synthesized by cells grown in BrdU has a lower proportion of adenine and a higher guanine complement. This was shown for (a) nascent RNA made in vivo by confluent monolayers of cells in culture; (b) RNA synthesized in vitro on a chromatin template with either homologous or heterologous RNA polymerase; and (c) RNA synthesized in vitro on a DNA template with a highly purified RNA polymerase. The product was completely digested by RNase. The relative decrease in the incorporation of adenine into RNA was reserved when BrdU-treated cells were allowed to proliferate in BrdU-free medium.

Biosynthesis of Riboflavin: THE STRUCTURE OF THE PURINE PRECURSOR

Abstract A mutant of Aerobacter aerogenes was isolated which is unable to synthesize purines de novo and to convert xanthylic acid to guanylic acid. Under suitable conditions, this mutant excretes xanthosine and riboflavin into the culture medium. The incorporation of 14C-labeled adenine, hypoxanthine, xanthine, and guanine into riboflavin and xanthosince was studied with nonproliferating and proliferating cells of the mutant. No radioactivity from 14C-labeled adenine, hypoxanthine, or xanthine is incorporated into riboflavin, whereas all of these compounds are efficiently converted to xanthosine. On the other hand, [2-14C]guanine is incorporated into riboflavin without significant dilution, whereas only traces of radioactive label are incorporated into xanthosine. These results document with a high degree of certainty that the biosynthesis of riboflavin starts with guanine or a guanine compound.

Metabolism, Macromolecular Synthesis, and Nuclear Behavior of Cryptococcus albidus at 37 C

The temperature-sensitive events which prevent Cryptococcus albidus from growing at 37 C were investigated. Cultures incubated at 37 C immediately after inoculation did not increase in optical density nor in cell numbers, and by 24 h 90% of cells in such cultures were deformed and dead. When cultures in log phase were shifted from 23 to 37 C the optical density increased but the cell numbers did not. Morphological observations revealed that the increase in turbidity at 37 C represented enlargement and distortion of cells without appreciable replication. Uptake and incorporation of (14)C-leucine were similar at 23 and 37 C. There was no difference in (14)CO(2) evolution from cells at either temperature. Uptake and incorporation of adenine-8-(14)C into RNA was slightly lower in cells incubated at 37 C. There was, however, a 60% reduction in incorporation of adenine-8-(14)C into DNA after 3 hr at 37 C. Nuclear staining revealed that nuclear migration did not occur in cells incubated at 37 C. Thus the data indicate that both adenine incorporation into DNA and nuclear migration prior to nuclear division by C. albidus are temperature sensitive.

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation. The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone. It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.

Stimulation of cyclic AMP production by the rat testis during incubation with hCG in vitro.

The response of the isolated rat testis to hCG in vitro was evaluated in terms of cyclic AMP synthesis and release into the incubation medium. Simultaneous measurement of 14C adenine incorporation into cyclic AMP and total release of immunoreactive cyclic AMP showed a constant ratio between incorporation and mass of nucleotide, indicating that proportionate synthesis and release of cyclic AMP occurred during stimulation with hCG. The maximum levels of cyclic AMP in tissue and medium were observed after incubation for 2 hr, with a drop at the fourth hr. The extent of the cyclic AMP response to hCG increased with gonadotropin dose over the range 1–500 ng/ml hCG, with a tendency to fall at extremely high hCG levels. As little as 1 ng/ml hCG produced detectable release of cyclic AMP into the incubation medium; at this and higher hCG doses, testosterone release was maximal and remained constant despite increasing cyclic AMP production and release with high levels of hCG. The release of cyclic AMP during hCG st...

The relationship between rates of [ 3H]uridine and [ 3H]adenine incorporation into RNA and the measured rates of RNA synthesis during the cell cycle

The rates of [5- 3H]uridine uptake into Chinese hamster V79 cells and its incorporation into RNA increase 10-fold during the cell cycle. In contrast, the rates of [ 3H]adenine uptake and incorporation increase only 2-fold over the same time period. The pattern of [ 3H]thymidine uptake into cells is unlike that of either [5- 3H]uridine or [ 3H]adenine, suggesting that transport of each of these nucleic acid precursors behaves differently during the cell cycle. The rates of RNA synthesis during the cell cycle were measured by relating the amount of [ 3H]adenine incorporated into RNA to the specific radioactivity of the [ 3H]ATP pool at 6 different time intervals. The rate of RNA synthesis doubles during the cell cycle. The cellular ATP pool undergoes a 2-fold increase, and the specific radioactivity of the [ 3H]ATP pool remains constant. [ 3H]adenine incorporation in this system provides a better measure of RNA synthetic rate than does [ 3H]uridine.

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17 .

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.

Density-dependent fluctuations in membrane permeability in logarithmically growing cell cultures

The effect of population density on cellular uptake of adenine, leucine, thymidine, and uridine was studied in monolayer cultures of a mammalian cell line not subject to density-dependent inhibition of growth. Experiments were conducted during the logarithmic phase of the growth cycle using cultures of equal age but at different cell densities to avoid effects of aging or of the transition to stationary-phase growth. Results indicate that uptake of leucine is independent of cell density, whereas uptake of adenine, thymidine, and uridine vary inversely with density. At higher densities incorporation of radioactive adenine, thymidine, and uridine into cellular macromolecules is reduced to approximately the same extent as uptake suggesting that utilization is limited by entry into the cell.

Metabolism of Rickettsia typhi and Rickettsia akari in irradiated L cells.

L cells that had been exposed to 3,000 r of (60)Co the previous day were used to study the growth and metabolism of Rickettsia typhi and R. akari. Viable (unirradiated) L cells were used to study the effect of rickettsial infection on host-cell metabolism. Monolayers were infected with a rickettsial multiplicity of 1.2 and given Eagle's minimal essential medium containing 25 mmN-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid buffer and 10% calf serum. At various intervals, cycloheximide (2 mug/ml) was added to one set of cultures, to inhibit eukaryotic protein and deoxyribonucleic acid (DNA) metabolism; phosphate-buffered saline (PBS) was added to another set. After 1 hr, the cultures received a mixture of 15 (14)C-labeled amino acids or adenine-8-(14)C. The cultures were harvested 16 hr later and were tested for incorporation of labeled carbon into the fraction precipitated by cold trichloroacetic acid. Viable cells were exposed to thymidine-2-(14)C for 2-hr periods. Infectivity of R. typhi increased to a peak of 150 to 400 hemolytic units/culture on day 4; the titer remained approximately the same on days 5 and 6, and declined rapidly on day 7. Total amino acid incorporation was about the same in infected and uninfected cultures up to day 6, but metabolic activity was reduced to a negligible level on day 7 in infected cells. Cycloheximide-resistant activity was higher in the infected cultures, with a peak equivalent to one-half the total activity at day 4 to 5. Total as well as cycloheximide-resistant adenine incorporation was higher in the infected cells between days 3 and 5 after infection, with a peak at day 3 to 4. Somewhat similar results were obtained with R. akari, except that the cycle of infection and of cycloheximide-resistant activity proceeded and was completed more rapidly. (14)C-DNA of both rickettsiae was isolated from infected cultures that had received labeled adenine. With labeled thymidine, which was not incorporated by the rickettsiae, it was shown that R. typhi and R. akari differ considerably in their effects on the host cell. R. typhi elicited moderate inhibition, whereas R. akari infection led to a complete inhibition of thymidine incorporation by the third day, at the time of highest rickettsial activity. It is concluded that rickettsiae have the necessary enzymes for protein and nucleic acid synthesis, but, thus far, these enzymes have been activated or induced only in an intracellular environment.

Effects of cerebral stimuli on adenine incorporation into nucleotides and RNA in brain slices from the rat.

Comparisons have been made of the rates of incorporation of [8-14C]adenine into RNA and of the specific activities of labelled ATP, in rat brain cortex slices incubated under conditions affecting nucleotide and RNA synthesis.The presence of 50 mM KCl in the incubation medium causes a considerable reduction of the rate of [8-14C]-adenine incorporation into RNA, with no diminution of the specific activity of the cerebral ATP or of the rate of nucleotide formation from adenine. It is inferred that cerebral RNA synthesis is suppressed by 50 mM KCl in the incubation medium.When K+ is omitted from the incubation medium or in the presence of 198 mM NaCl or 5 mM sodium L-glutamate, both the rate of [8-14C]adenine incorporation into RNA and the specific activity of cerebral ATP are diminished to approximately the same extent. This suggests that the process of RNA synthesis in the brain tissue is but little affected either by the increased ceil concentration of Na+ or by the diminished ATP concentration that obtain under these incubation conditions. The process, however, of [8-14C]adenine incorporation into cell nucleotides is markedly suppressed.The presence of protoveratrine (10 μM) causes at least 40% reduction in the rates of [8-14C]adenine incorporation into both RNA and nucleotides with little reduction in the specific activity of the cerebral ATP. The effects of protoveratrine are abolished by tetrodotoxin, indicating that the effects of protoveratrine are confined to the neurons.It is concluded that reductions of the specific activity of cerebral ATP derived from labelled adenine are due to the diminished rates of nucleotide formation from adenine that occur under specific incubation conditions. Such reductions may give rise to the observed diminutions in the rates of incorporation of labelled adenine into RNA. The relatively small fail in the specific activity of isolated ATP after incubation of brain tissue in the presence of protoveratrine is attributed to the localization of the effects of this drug to the neurons, in which the content and specific activity of ATP are suppressed, while those in the glia are undiminished.