Deoxycytidine Triphosphate Deaminase: Identification and Function in Salmonella typhimurium

The biosynthesis of 2'-deoxyuridine monophosphate (dUMP) has been studied in a cytidine- and uracil-requiring mutant of Salmonella typhimurium (DP-55). The dUMP pool and the thymidine monophosphate (dTMP) pool of DP-55, grown in the presence of (3)H-uracil and unlabeled cytidine, are found to have the same specific activities. However, only 30% of the dUMP and the dTMP is synthesized from a uridine nucleotide. Seventy per cent is derived directly from a cytosine compound. The identification and partial purification of a Mg(2+)-dependent 2'-deoxycytidine triphosphate (dCTP) deaminase from S. typhimurium suggests that the combined action of dCTP deaminase and 2'-deoxyuridine triphosphate pyrophosphatase accounts for 70% of the dUMP, and therefore the dTMP, synthesized in vivo. The introduction of a thymine requirement (i.e., a block in thymidylate synthetase) into DP-55 results in a 100-fold increase in the size of the dUMP pool. However, the relative contribution of the uridine and cytidine pathways to dUMP synthesis is unaltered. The high dUMP pool is accompanied by extensive catabolism of dUMP to uracil. Partial thymine starvation of the cells results in a significant increase in the dUMP and dCTP pools. Moreover, an increase in the contribution of the dCTP pathway to dUMP synthesis is observed. As a result of these changes the catabolism of dUMP to uracil is augmented.     

Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells.

Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.     

Repression of cytidine triphosphate synthetase in Salmonella typhimurium by pyrimidines during uridine nucleotide depletion

Regulation of the synthesis of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2) was investigated in Salmonella typhimurium. CTP synthetase appeared to be repressed only when intracellular concentrations of uridine nucleotides were significantly lowered. Under such nucleotide pool conditions, a cytidine compound and, to a lesser degree, a thymidine compound appeared as putative repressing metabolites of enzyme synthesis.     

Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis.

Long-term, 32-P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3-H]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3-H to 32-P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14-C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.     

Effect of D-galactosamine on nucleotides in the rat heart. Effects of cytidine and uridine administration

The treatment of rats by galactosamine (2 mmol/kg i.p.), which dramatically alters the metabolism of pyrimidine nucleotides in the liver, has been used to investigate the dynamics of pyrimidine nucleotides in the rat heart. Six hours after administration of the drug, the UTP and UDPG myocardial contents were decreased by respectively 40 and 52% while the sum of uracil nucleotides was increased by 66% and that of cytosine nucleotides by 15%. When administered 5 h after galactosamine treatment, cytidine (750 nmol/rat i.v.) induced a further increase in cytosine nucleotides (46% above control value 1 h later) without however effect on uracil nucleotides. On the other hand, the administration of uridine (250 nmol/rat, i.v. 5 h after galactosamine), while restoring UTP, UDPG and the pool of uracil nucleotides, provoked a decrease in cytosine nucleotide level (-17%). In the absence of galactosamine treatment, the administration of uridine and cytidine did not induce changes in nucleotide levels despite a rise in blood cytidine concentration. All these observations support the hypothesis that: 1. the pathway for cytosine nucleotide synthesis predominant in the heart is that utilizing preformed exogenous cytidine and 2. this pathway is mainly controlled by the intracellular concentration of UTP rather than that of CTP.     

Regulation of PRPP and nucleoside tri and tetraphosphate pools in Escherichia coli under conditions of nitrogen starvation.

The ribonucleoside triphosphate, deoxyribonucleoside triphosphate, 3' -diphosphate guanosine 5' -diphosphate (ppGpp), and 5-phosphoribosyl 1-pyrophosphate (PRPP) pools in Escherichia coli B were determined by thin-layer chromatography during changing conditions to ammonium starvation. The intracellular concentrations of all nucleotides were found to change in a well-defined order several minutes before andy observed change in the optical density of the culture. The levels of purine nucleoside triphosphates (adenosine 5' -triphosphate [CTP], dCTP) and uridine nucleotides (uridine 5' -triphosphate, deoxythymidine 5'-triphosphate). The deoxyribonucleotides thus behaved as the ribonucleotides. The levels of ppGpp increased 11-fold after the decrease in uridine nucleotides, when the accumulation of stable ribonucleic acid (RNA) stopped. The level of the nucleotide pool did not stabilize until 30 min after the change in optical density. The pool of dGTP dropped concomitantly with the pool of CTP. The nucleotide precursor PRPP exhibited a transient increase, wtih maximum value of four times the exponential levels at the onset of starvation. Apparently the cell adjusts early to starvation by reducing either the phosphorylating activity or the nucleotide biosynthetic activity. As in other downshift systems, the accumulation of stable RNA stopped before the break in optical density and before the stop in protein accumulation. Cell divisions were quite insensitive to the control mechanisms operating on RNA and protein accumulation under ammonium starvation, since the cells continued to divide for 21 min without any net accumulation of RNA.     

Pleiotropic mutants of Chinese hamster cells with altered cytidine 5′-triphosphate synthetase

Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5-triphosphate (CTP) and deoxycytidine 5-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.This work was supported by Grant GM 30608 from the U.S. Public Health Service.     

Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis

Tribby, Ilse I. E. (University of Chicago, Chicago, Ill.), and James W. Moulder. Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis. J. Bacteriol. 91:2362-2367. 1966.-Uninfected L cells and the meningopneumonitis agent propagated in L cells utilized exogenous adenine, guanine, and their ribonucleosides and deoxyribonucleosides for synthesis of both deoxyribonucleic acid (DNA) and ribonucleic acid. Cytosine, cytidine, and uridine were also incorporated into the nucleic acids of both host and parasite. L cells and the meningopneumonitis agent incorporated uracil, thymine, and deoxyuridine very poorly. L cells utilized thymidine and deoxycytidine almost exclusively for DNA synthesis, but the meningopneumonitis agent did not incorporate these nucleosides at all. Since the L cell had previously been shown to convert added thymidine to its nucleotides, mainly the triphosphate, it was concluded that the meningopneumonitis agent can utilize neither the thymidine nor the thymidine nucleotides of the L-cell pool, and that it can probably synthesize the thymidine triphosphate needed for DNA synthesis from the uridine of the L-cell pool.     

In situ regulation of mammalian CTP synthetase by allosteric inhibition

The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.     

Pyrimidine Salvage Pathways In Toxoplasma Gondii

ABSTRACT. Pyrimidine salvage enzyme activities in cell-free extracts of Toxoplasma gondii were assayed in order to determine which of these enzyme activities are present in these parasites. Enzyme activities that were detected included phosphoribosyltransferase activity towards uracil (but not cytosine or thymine), nucleoside phosphorylase activity towards uridine, deoxyuridine and thymidine (but not cytidine or deoxycytidine), deaminase activity towards cytidine and deoxycytidine (but not cytosine, cytidine 5'-monophosphate or deoxycytidine 5'-monophosphate), and nucleoside 5'-monophosphate phosphohydrolase activity towards all nucleotides tested. No nucleoside kinase or phosphotransferase activity was detected, indicating that T. gondii lack the ability to directly phosphorylate nucleosides. Toxoplasma gondii appear to have a single non-specific uridine phosphorylase enzyme which can catalyze the reversible phosphorolysis of uridine, deoxyuridine and thymidine, and a single cytidine deaminase activity which can deaminate both cytidine and deoxycytidine. These results indicate that pyrimidine salvage in T. gondii probably occurs via the following reactions: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively; uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase; and uracil is metabolized to uridine 5'-monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5'-monophosphate is the end-product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii.     

Selective high metabolic lability of uridine, guanosine and cytosine triphosphates in response to glucose deprivation and refeeding of untransformed and polyoma virus-transformed hamster fibroblasts.

Sugar deprivation of hamster fibroblasts (NIL) affected the steady state levels (pool sizes) of cellular acid soluble nucleotides in the folloing fashion: the pools of UTP, GTP and CTP decreased to a much greater extent than the cellular ATP pools, with the UTP pools undergoing the most dramatic reduction. Sugar deprivation of polyoma-transformed NIL cells (PyNIL) yielded even sharper decreases in the nucleoside triphosphate pools with relative changes similar to those of the untransformed cells. Inhibition of protein synthesis by cycloheximide, initiated at the onset of (and continued during) sugar deprivation, prevented the reduction in pool sizes and yielded values slightly higher than those observed for pool sizes in cells cultured in sugar-supplemented medium.Refeeding glucose to sugar-depleted hamster fibroblasts led to rapid increases (within 1 hour) in the UTP and CTP pools to levels well above the pool sizes observed in cells which were continuously cultured (16 hours) in sugar supplemented medium. Feeding NIL or PyNIL cells with fructose instead of glucose as the only hexose source did not appreciably affect any of the ribonucleoside triphosphate pool sizes. Measurements of hexose uptake by NIL and PyNIL cells under a variety of conditions suggest that hexose transport is not regulated by the total cellular pools of ATP or any of the other ribonucleoside triphosphates.     

Profiles of pyrimidine biosynthesis,salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.     

Pyrimidine deoxyribonucleotide metabolism during maturation and germination of white spruce (Picea glauca) somatic embryos: metabolic fate of C-labelled cytidine, deoxycytidine and thymidine

Pyrimidine deoxyribonucleotide metabolism was investigated during maturation and germination of white spruce somatic embryos by following the metabolic fate of [2‐¹⁴C]cytidine, [2‐¹⁴C]deoxycytidine and [2‐¹⁴C]thymidine. The de-novo pathway of deoxyribonucleotides was estimated indirectly, by the ability of the tissue to incorporate cytidine into DNA after conversion to dCTP. The salvage pathway was estimated by the utilization of labelled cytidine, deoxycytidine and thymidine for synthesis of deoxyribonucleotides and nucleic acids. Utilization of cytidine for DNA synthesis, via the de novo pathway, was always lower than that observed for RNA throughout the course of the experiment. Incorporation of cytidine into RNA was found to occur either directly, after conversion to CTP, mediated by the enzymes cytidine kinase, nucleoside monophosphate kinase and nucleoside diphosphate kinase, or indirectly, after conversion to UTP via uridine and UMP. Active incorporation of uridine into RNA of white spruce-cultured cells was demonstrated previously. Salvage of deoxycytidine and thymidine was operative in maturing and germinating white spruce somatic embryos, as label from both compounds was recovered in nucleotides and DNA. However, the utilization of these precursors by the cells was different. Salvage of deoxycytidine was always higher than that observed for thymidine, which was extensively catabolized to CO₂ at all stages of embryo development.     

Pyrimidine metabolism by intracellular Chlamydia psittaci.

Pyrimidine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined mutations affecting pyrimidine metabolism. C. psittaci AA Mp cannot synthesize pyrimidines de novo, as assessed by its inability to incorporate aspartic acid into nucleic acid pyrimidines. In addition, the parasite cannot take UTP, CTP, or dCTP from the host cell, nor can it salvage exogenously supplied uridine, cytidine, or deoxycytidine. The primary source of pyrimidine nucleotides is via the salvage of uracil by a uracil phosphoribosyltransferase. Uracil phosphoribosyltransferase activity was detected in crude extracts prepared from highly purified C. psittaci AA Mp reticulate bodies. The presence of CTP synthetase and ribonucleotide reductase is implicated from the incorporation of uracil into nucleic acid cytosine and deoxycytidine. Deoxyuridine was used by the parasite only after cleavage to uracil. C. psittaci AA Mp grew poorly in mutant host cell lines auxotrophic for thymidine. Furthermore, the parasite could not synthesize thymidine nucleotides de novo. C. psittaci AA Mp could take TTP directly from the host cell. In addition, the parasite could incorporate exogenous thymidine and thymine into DNA. Thymidine kinase activity and thymidine-cleaving activity were detected in C. psittaci AA Mp reticulate body extract. Thus, thymidine salvage was totally independent of other pyrimidine salvage.     

Fatty acid synthesis by isolated chromoplasts from the daffodil. Energy sources and distribution patterns of the acids

1. Fatty acid synthesis in isolated intact chromoplasts from [1-¹⁴C]acetate was made possible by using ATP, ADP (via adenylate kinase), and, with decreasing efficiency, UTP, CTP, and GTP as energy sources. 2. The glycolytic path from dihydroxyacetone phosphate to acetyl-CoA operates within the chromoplasts. The glycolytic intermediates, especially 2-phosphoglycerate and phosphoenolpyruvate, served as very effective energy donors for fatty acid synthesis by phosphorylating the endogenous adenine nucleotide pool. 3. In the presence of exogenous ATP or ADP, appreciable amounts of in vitro formed fatty acids were found as acyl-CoA and subsequent products, mainly phosphatidylcholine. When other energy sources were used most of the acids formed were in the free form, and to a minor extent, in the phosphatidic acid and diacylglycerol fractions. Similar results have recently been reported for spinach chloroplasts (Kleinig and Liedvogel 1979, FEBS Lett.101, 339–342).Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - UTP uridine triphosphate - CTP cytidine triphosphate - GTP gnanosine triphosphate     

Two nucleoside uptake systems in Lactococcus lactis: competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of (33)P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H(3)BO(3)-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K(m) for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.     

Characterization of Sertoli cell RNA synthetic activities in vitro at selected times during sexual maturation

In this study, Sertoli cell RNA synthetic activity in vitro was characterized at selected times during sexual maturation. Sertoli cells, isolated from rat testes undergoing the first wave of spermatogenesis and placed in culture for 4 days, exhibited 2-fold increases in soluble ribonucleotide pools and in total RNA concentrations over the age span of 18-35 days. High performance liquid chromatographic analysis of the ribonucleotide pools in Sertoli cells cultured from 18- and 33- to 34-day-old rats revealed that, in addition to the overall age-related doubling of concentrations, uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools were disproportionately increased 4- and 6-fold, respectively. In general, Sertoli cell contained relatively small amounts of UTP in comparison to several other cell types, but exhibited a high ADP:ATP ratio. A uniform 2-fold increase in the base composition of Sertoli cell RNA per mg DNA was observed over the age span of 18-35 days, with no preferential increase in any one specific nucleotide. There was no change in [3H]uridine incorporation (2 h) into RNA per cell (pmol/mg DNA), but decreased specific activity of the RNA (pmol/mg RNA) in Sertoli cells cultured from 35-day-old rats as compared to those from 18- to 19-day-old rats. Similar differences were noted in the specific activity of label incorporated into specific RNA bases. In contrast, the specific activity of the UTP-CTP soluble pool/mg DNA was only slightly increased. These data indicate that processes related to RNA synthesis in the Sertoli cell undergo a number of changes during the period of sexual maturation.     

Compartmentation of dCTP pools. Evidence from deoxyliponucleotide synthesis

The nucleotide fraction of cultured 3T6 and 3T3 mouse fibroblasts contains deoxy-CDP choline and deoxy-CDP ethanolamine as well as the corresponding riboliponucleotides. In permeabilized cells both deoxyliponucleotides were formed from dCTP. In intact cells they could be labeled from [5-3H] deoxycytidine or cytidine via transformation of the nucleosides to dCTP. Their turnover was slow compared to that of dCTP. When rapidly growing 3T3 cells were labeled during 90 min from deoxycytidine the specific activity of dCDP choline was 2.4 times higher than that of dCTP while after labeling from cytidine both nucleotides (and CTP) reached the same specific activity under steady state conditions. Also dCDP ethanolamine was labeled more rapidly from deoxycytidine than from cytidine. Our results suggest that the deoxyliponucleotides were synthesized from a dCTP pool that was labeled preferentially from deoxycytidine. Earlier work (Nicander, B., and Reichard, P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1347-1351) had demonstrated synthesis of DNA from a dCTP pool labeled preferentially from cytidine. Taken together our results suggest that deoxyliponucleotides and DNA are synthesized from separate dCTP pools.     

Pyrimidine Nucleotide-Stimulated Thromboxane A2 Release from Cultured GLIA

1. Uridine triphosphate (UTP), uridine diphosphate (UDP), cytidine triphosphate (CTP), and deoxythymidine triphosphate (TTP) caused concentration-dependent increases in the release of thromboxane A₂ (TXA₂) from cultured glia prepared from the newborn rat cerebral cortex. Although each of the pyrimidine nucleotides displayed similar potencies, CTP and TTP were considerably less effective than either UTP or UDP. The purine nucleotide ATP was equally as potent as the pyrimidine nucleotides but was marginally less effective than either UTP or UDP.2. The ability of UTP, UDP, TTP, and CTP to promote TXA₂ release from cultured glia was inhibited in a concentration-dependent manner by suramin and was markedly reduced when incubations were performed either in Ca²⁺-free medium or on cultures which had been maintained in serum-free growth medium for 4 days prior to experimentation.3. Challenges with UTP and UDP in combination were found to elicit a response which was no different from the effects of these nucleotides alone; in addition, their effects were reversed by the phospholipase A₂ inhibitor ONO-RS-082. A slight reduction in UTP-and UDP-stimulated TXA₂ release was observed in cultures grown in the presence of leucine methyl ester, a treatment reported to limit microglial survival.4. These results suggest that glia are targets for extracellular pyrimidine nucleotides and that their ability to release eicosanoids from these cells may be important in the brain's response to damage.     

Re-utilization of pyrimidine nucleotides during rat liver regeneration.

The changes in the specific radioactivities of the pool of total acid-soluble uridine nucleotides and of uridine and cytidine components of total cellular and nuclear RNA were monitored in regenerating rat liver for 12 days after partial hepatectomy. Evidence is presented for the re-utilization of pyrimidine nucleotides derived from cytoplasmic RNA degradation for the synthesis of new RNA. The extent of recycling was assessed and the true rate of rRNA turnover determined more accurately. The reutilization of the uridine components of RNA was 7.0%/day during the proliferative and 3.2%/day during the post-proliferative phase, whereas that of the cytidine nucleotides was more pronounced (9.6%/day and 18.1%/day respectively). The results reveal the existence of partial compartmentalization of pyrimidine ribonucleoside triphosphate pools in the nucleus and cytoplasm of rat liver cells.