Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

Studies on Deoxyribonucleotide Synthesis in Vicia faba

The incorporation of uniformly labelled [¹⁴C] cytidine into the nucleic acids was studied in root tips of Vicia faba. Cytidine was found to be incorporated into RNA and DNA and the specific activities of the individual mononucleotides were deter- mined. The pyrimidine nucleotides were degrade and the ratio between the specific activity of the pentose and the specific activity of the base was determined for each nucleotide. CMP of RNA and deoxy CMP of DNA bad almost the same pentose: base ratios as The cytidine added to the incubation medium. It was concluded that the administered cytidine or a derivative of it was reduced to the corresponding deoxycytidine compound without breakage of the bond between pentose and base. [¹⁴C)-cytidine was transformed to UMP of RNA with some loss of radioactivity from the pentose and had almost the same pentose: base ratio as deoxy TMP of DNA. This indicates that the formation of thymidine phosphates involved The reduction of a uridine compound. Furthermore the incorporation of ¹⁴C-labelled thymidine, deoxyadenosine and deoxyguanosine into DNA was studied. Deoxyguanosine was found to be incorporated only to a slight extent. This finding has been discussed in relation to previous results.     

Methacrylate polymerization by AzoRNA: potential usefulness for chromosomal localization of genes

An azo pyrimidine nucleotide has been prepared and enzymatically attached to oligo(A) primers. The nucleotide's azo pyrimidine group has previously been shown to initiate polymerization of methacrylate esters designed to bind marker groups for visualization by microscopy. When attached to RNA molecules complementary to a chromosomal DNA segment, these nucleotides may allow localization of the DNA segment following in situ hybridization of the probe, methacrylate polymerization, and marker attachment. Since mRNA molecules of potential interest as probes bear a 3′-poly(A) tail, the modified nucleotides were added to oligo(A) primers as models. First, N⁴-ureidocytosine nucleotides were enzymatically added to ApApA, (Ap)₉A, or [5′-³²P]-(pA)₁₀, using the modified cytidine 5′-diphosphate and “primer-dependent” polynucleotide phosphorylase (M. luteus). In the case of the ApApA-primed reaction, the N⁴-ureidocytosine nucleotides in the product polynucleotide were converted into azo nucleotides by oxidation with N-bromosuccinimide. The other two primers were employed to study the time course of polynucleotide formation and to verify that primer was indeed being utilized by the enzyme. The suitability of the modified nucleotide for in situ hybridization studies was examined. Poly(N⁴-ureidocytidylic acid) was prepared from poly(C) and semicarbazide by the bisulfite-catalyzed transamination reaction. It was found that 95% of the N⁴-ureidocytosine nucleotides in this polynucleotide survive the elevated temperatures typically required for DNA:DNA denaturation and RNA:DNA annealing. When poly(N⁴-ureidocytidylic acid) was mixed with poly(I) in buffered aqueous salt solutions, no evidence for hybridization was found, so binding of the probe RNA to the denatured chromosomal DNA molecule via the modified nucleotides is not expected. Upon oxidation of poly(N⁴-ureidocytidylic acid) with N-bromosuccinimide, the azo nucleotides were formed, as judged by the appearance of a characteristic peak at approximately 350 nm in the uv-absorption spectrum of the yellow-orange product, azoRNA. The azo nucleotides in azoRNA exhibited the expected acid lability, which is known to be accompanied by 1-glyceryl methacrylate polymerization in the case of the simple azo pyrimidine. Because 1-glyceryl metharcylate bears substituent glycol groups for attaching heavy atoms or fluorescent markers, it is possible that probe RNA molecules bearing azo nucleotides may be useful for localizing low-multiplicity genes along eukaryotic chromosomes.     

Biosynthesis of pyrimidine nucleotides in cultured root callus ofCucurbita pepo

Summary Callus cultures derived from roots of summer squash (Cucurbita pepo L. c.v. Early Prolific Straightneck) grown in the dark at 27° C on Murashige and Skoog medium supplemented per liter with 30 g sucrose, 100 mg myo-inositol, 10 mg indole-butyric acid, 2 mg glycine, 1 mg thiamin, 0.5 mg nicotinic acid, 0.5 mg pyridoxine, and 2 g Gelrite were capable of synthesizing pyrimidine nucleotides both de novo and through salvage of existing pyrimidine nucleotides and bases. Evidence that the de novo biosynthesis of pyrimidine nucleotides proceeded via the orotate pathway in this tissue included: (a) demonstration of the incorporation of NaH¹⁴CO₃ and [¹⁴C₆]orotic acid into uridine nucleotides (ΣUMP), and (b) demonstration that the addition of 6-azauridine blocked the incorporation of these two precursors into ΣUMP. The synthesis of pyrimidine nucleotides through the salvage of existing pyrimidine bases and ribosides was demonstrated by measuring the incorporation of [¹⁴C₂]uracil and [¹⁴C₂]uridine into ΣUMP. Salvage of both [¹⁴C₂]uracil and [¹⁴C₂]uridine was sensitive to inhibition by 6-azauridine or one of its metabolites. The orotic acid pathway for the de novo biosynthesis of pyrimidine nucleotides was demonstrated to be sensitive to end-product inhibition. Uridine, or one of its metabolites, inhibited the incorporation of NaH¹⁴CO₃, but not [¹⁴C₆]orotic acid, into ΣUMP. Evidence is presented suggesting that Aspartate carbomoyltransferase is the site of feedback control. This work was supported by the Citrus Research Center and Agricultural Experiment Station of the University of California, Riverside, CA. Submitted in partial fulfillment of the requirements of the University of California for the Master of Science degree in botany (F-F.L.)     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Effects of nucleic acid compounds on viability and cell composition of Bdellovibrio bacteriovorus during starvation

The effects of various exogenous nucleic acid compounds on the viability and cell composition of Bdellovibrio bacteriovorus starved in buffer were measured. In decreasing order of effectiveness, these compounds were found to decrease the rate of loss of viability and the loss of cell carbon, cell ribonculeic acid, and cell protein: glutamate > ribonucleoside monophosphates > ribonucleosides > deoxyribonucleoside monophosphates. Similar sparing effects were not observed with nucleic acid bases, deoxyribonucleosides, ribose, ribose-5-phosphate, deoxyribose, and deoxyribose-5-phosphate. Appreciable increases in the respiration rate over the endogenous rate did not occur when cell suspensions were incubated with individual or mixtures of nucleic acid compounds. Formation of ¹⁴CO₂ by cell suspensions incubated with carbon 14-labeled nucleic acid compounds indicated ribonucleosides and ribonucleoside monophosphates were respired and to a small extent, were incorporated into cell material of non-growing cells. The respired ¹⁴CO₂ was derived mainly from the ribose portion of these molecules. No respired ¹⁴CO₂ or incorporated carbon 14 was found with bdellovibrios incubated with other nucleic acid compounds tested, including free ribose. During growth of B. bacteriovorus on Escherichia coli in the presence of exogenous UL-¹⁴C-ribonucleoside monophosphates, 10–16% of the radioactivity was in the respired CO₂ and of the radioactivity incorporated into the bdellovibrios, only 40 to 50% resided in the cell nucleic acids. However, during growth on ¹⁴C-adenine,-uracil, or-thymidine labeled E. coli, only trace amounts of ¹⁴CO₂ were found and 90% or more of the incorporated radioactivity was in the bdellovibrio nucleic acids. It is concluded that bdellovibrio can use ribonucleoside monophosphates during growth and starvation as biosynthetic precursors for synthesis of both nucleic acids and other cell materials as well as catabolizing the ribose portion for energy purposes.Abbreviations HM buffer 5 mM N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (pH 7.6) containing 0.1 mM CaCl₂ and MgCl₂ - DNA deoxyribonucleic acid - RNA ribonucleic acid - Ar, Cr, Gr, Ur ribonucleosides of adenine, cytosine, guanine, uracil, respectively - dTr deoxythymidine - AMP, CMP, GMP, UMP ribonucleoside monophosphates of adenine, cytosine, guanine, and uracil, respectively - dTMP deoxythymidine monophosphate - ATP adenosine triphosphate - PFU plaque-forming units     

Improved method to prepare RNA-free DNA from mammalian cells

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-³H]uridine-labeled nucleic acid with 50 μg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 μg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3′ to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 μg/ml RNase T1, which preferentially cleaves RNA 3′ to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-³H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.     

Effect of short-term salt stress on the metabolic profiles of pyrimidine, purine and pyridine nucleotides in cultured cells of the mangrove tree, Bruguiera sexangula

To investigate the short‐term (3 h) effect of salt on the metabolism of purine, pyrimidine and pyridine nucleotides in mangrove (Bruguiera sexangula) cells, we examined the uptake and overall metabolism of radiolabelled intermediates involved in the de novo pathways and substrates of salvage pathways for nucleotide biosynthesis in the presence and absence of 100 mM NaCl. Uptake by the cells of substrates for the salvage pathways was much faster than uptake of intermediates of the de novo pathways. The activity of the de novo pyrimidine biosynthesis estimated by [2‐¹⁴C]orotate metabolism was not significantly affected by the salt. About 20–30% of [2‐¹⁴C]uridine, [2‐¹⁴C]uracil and more than 50% of [2‐¹⁴C]cytidine were salvaged for pyrimidine nucleotide biosynthesis. However, substantial quantities of these compounds were degraded to ¹⁴CO₂ via β‐ureidopropionate (β‐UP), and degradation of β‐UP was increased by the salt. The activities of the de novo pathway, estimated by [2‐¹⁴C] 5‐aminoimidazole‐4‐carboxamide ribonucleoside, and the salvage pathways from [8‐¹⁴C]adenosine and [8‐¹⁴C]guanosine for the purine nucleotide biosynthesis were not influenced by the salt. Most [8‐¹⁴C]hypoxanthine was catabolised to ¹⁴CO₂, and other purine compounds are also catabolised via xanthine. Purine catabolism was stimulated by the salt. [³H]Quinolinate, [carbonyl‐¹⁴C]nicotinamide and [carboxyl‐¹⁴C]nicotinic acid were utilised for the biosynthesis of pyridine nucleotides. The salvage pathways for pyridine nucleotides were significantly stimulated by the salt. Trigonelline was synthesised from all pyridine precursors that were examined; its synthesis was also stimulated by the salt. We discuss the physiological role of the salt‐stimulated reactions of nucleotide metabolism.     

Interrelationships between water and cellular metabolism in Artemia cysts. VI. RNA and protein synthesis

Using ¹⁴CO₂ as a labelled precursor the relationship between the initiation of protein and RNA synthesis, and water concentration, has been examined in cysts (encysted embryos) of the brine shrimp, Artemia salina. Although incorporation of radioactivity into amino acids and nucleotides occurred in cysts at hydrations as low as 0.3 g H₂O/g dried cysts, incorporation into proteins and RNA was not measurable until the cysts had achieved a hydration in the range of 0.6–0.6 g/g. In no case was radioactivity detected in DNA of unemerged cysts. Fully hydrated cysts (about 1.3 g/g) that were actively synthesizing proteins and RNA, stopped doing so when dehydrated to levels below the same hydration range: thus, the hydration dependence does not involve appreciable hysteresis. The hydration range required to initiate synthesis of these macromolecules is essentially the same as that previously shown to initiate embryonic development.     

The effect of some platinum compounds on the biosynthesis of RNA and its precursors.

The effect of cis-DDP (cis-diamminedichloroplatinum(II)), trans-DDP (trans-diamminedichloroplatinum(II)), SPC (spermine-platinum(II) complex), and K2PtCl4 on the ribomononucleotide and RNA metabolism was studied. When Ehrlich ascites tumor cells were preincubated with the aforementioned compounds and then labeled with [C14]uridine a clear-cut suppression of the radioactive labeling of RNA was observed. As radioactivity incorporated into the pool of the free uridine nucleotides in the cells treated with platinum compounds was even higher in comparison with that of the non-treated cells a conclusion may be drawn with certainty that the platinum compounds studied inhibit RNA biosynthesis. It was also found that under the effect of these compounds in the in vivo-assessed rate of the conversion of uridine nucleotides into cytidine nucleotides was considerably diminished. Using NaH14CO3 as a radioactive precursor it was shown that platinum compounds also inhibited purine biosynthesis de novo, in particular the conversion of IMP into GMP and AMP. The pronounced inhibitory effect of the platinum compounds on essential steps of the pyrimidine and purine biosynthesis de novo may be at least partly responsible for the firmly established inhibition in the present study of RNA biosynthesis by platinum compounds. The inhibition of the synthesis of the mononucleotides and RNA by the platinum compounds may be closely related to their cytostatic and cytotoxic activities.     

Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo

The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash (Cucurbita pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides proceeds via the orotate pathway in C. pepo included: (a) demonstration of the incorporation of [¹⁴C]NaHCO₃, [¹⁴C]carbamylaspartate, and [¹⁴C]orotic acid into uridine nucleotides; (b) the isolation of [¹⁴C]orotic acid when [¹⁴C]NaHCO₃ and [¹⁴C]carbamylaspartate were used as precursors; (c) the observation that 6-azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and (d) demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts.     

Dietary nucleotide requirements of the mosquito, Culex pipiens

Dietary nucleic acid, or certain constituents thereof, is essential for full development of the mosquito Culex pipiens. Yeast RNA, whole or hydrolysed, is fully effective. Sperm DNA is totally ineffective on its own, but becomes fully effective when supplemented with uridylic acid. RNA can be replaced with only partial success by a mixture of its component nucleotides (adenylic, guanylic, cytidylic, and uridylic acids), but with the addition of thymidylic acid (characteristic of DNA but present in RNA only as a minor component of transfer RNA), the resulting five-nucleotide mixture is a fully adequate replacement for yeast RNA. Single and multiple deletions from this five-nucleotide mixture showed a minimal requirement for three nucleotides: adenylic acid (a purine ribonucleotide) and thymidylic acid (a pyrimidine deoxyribonucleotide) were both specifically required; as the third nucleotide, either cytidylic or uridylic acid (both pyrimidine ribonucleotides) was equally satisfactory. Mixtures of the five nucleosides or five deoxynucleotides corresponding in base composition to the effective five-nucleotide mixture were only partially effective substitutes.     

Cytidine nucleotide biosynthesis and the level of cytochrome P-450 in rat liver microsomes after administration of colchicine.

Colchicine displays a biphasic effect on the biosynthesis of cytidine nucleotides in rat liver; an initial depression is followed by activation of synthesis. Depending on the duration of exposure to colchicine, the changes of the values of the ratio of specific radioactivity of cytosine to uracil in the acid-soluble pool and 3′-CMP to 3′-UMP of cytoplasmic ribosomal RNA are inversely proportional to changes of the levels of cytochrome P-450 in liver microsomes. The utilization of [2-¹⁴C]orotic acid for the biosynthesis of DNA cytosine as affected by the exposure to colchicine reflects the changes in the specific radioactivity of the cytidine components of the acid-soluble pool. However, the maximal radioactivity in DNA thymine is reached under these conditions only at longer time intervals.     

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.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

Absorption,Translocation and Metabolism of Nicotinamide in Some Plants

The seedlings of rice, eggplant and tomato at the 5th leaf stage of growth readily absorbed exogenous ¹⁴C-nicotinamide through the root and the foliage in water culture. Within the 24 hr period after the bigining of cultivation, the radioactivity gradually translocated from the part treated with ¹⁴C-nicotinamide to the whole plant body. This compound was rapidly metabolised in the plants to at least six metabolites, in which three compounds were identified as nicotinic acid, NAD and NADP. ¹⁴C-Nicotinic acid was also taken up quickly through the root of rice and its metabolism showed a similar pattern to that of ¹⁴C-nicotinamide. The incorporation of radioactivity into NAD and NADP from ¹⁴C-nicotinamide added to cultivating solution at a concentration of 0.21 ppm was decreased to 10~20% by the simultaneous addition of unlabeled nicotinic acid at a concentration about 1000 times higher than that of the labeled one. It was concluded that the biosynthesis of these pyridine nucleotides from nicotinamide was chiefly via nicotinic acid. The formation of ¹⁴C-nicotinamide in the ¹⁴C-nicotinic acid metabolism suggested a breakdown of NAD. Three unknown compounds observed in both the metabolisms described above were not intermediates in the pyridine nucleotide biosynthesis.     

Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [³H]Thd, [¹⁴C]orotate or with inorganic ³²P_i. Expression of incorporated radioactivity as log₁₀% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [³H]Thd was asymmetrically distributed among the isostichs, i.e., ³H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., ratios for the first 10 isostichs averaged 1.43 ± 0.08 and 1.28 ± 0.05, depending on the method of analysis; furthermore, the ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [³H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [³H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecuar weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [¹⁴C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [³H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of ¹⁴C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from ³²P_i, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since ³²P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of ³²P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the ‘replitase’ concept for channeling dNTPs to replicating forks.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Metabolic fate of guanosine in higher plants

The aim of the present study was to investigate the metabolic fate of guanine nucleotides in higher plants. The rate of uptake of [8-¹⁴C]guanosine by suspension-cultured Catharanthus roseus cells was more than 20 times higher than that of [8-¹⁴C]guanine. The rate of uptake of [8-¹⁴C]guanosine increased with the age of the culture. Pulse-chase experiments with [8-¹⁴C]guanosine revealed that some of the guanosine that had been taken up by the cells was converted to guanine nucleotides and incorporated into nucleic acids. A significant amount of [8-¹⁴C]guanosine was degraded directly to xanthine, allantoin and allantoic acid, with the generation of ¹⁴CO₂ as the final product. The rate of salvage of [8-¹⁴C]guanosine for the synthesis of nucleic acids was highest in young cells, while the rate of degradation increased with the age of the cells. In segments of roots from Vigna mungo seedlings, nearly 50% of the [8-¹⁴C]guanosine that had been absorbed over the course of 15 min was recovered in guanine nucleotides. A significant amount of the radioactivity in nucleotides became associated with nucleic acids and ureides during ‘chase’ periods. In segments of young leaves of Camellia sinensis, [8-¹⁴C]guanosine was initially incorporated into guanine nucleotides, nucleic acids, theobromine and ureides, and the radioactivity in these compounds was transferred to caffeine and CO₂ during a 24-h incubation. Our results suggest that guanosine is an intermediate in the catabolism of guanine nucleotides and that it is re-utilised for nucleotide synthesis by ‘salvage’ reactions. Guanosine was catabolised by the conventional degradation pathway via xanthine and allantoin. In some plants, guanosine is also utilised for the formation of ureide or the biosynthesis of caffeine.     

Asparate transcarbamylase activity in etiolated cowpea hypocotyls treated with 2,4-dichlorophenoxyacetic Acid

Treating etiolated cowpea (Vigna unguiculata) seedlings with 2,4-dichlorophenoxyacetic acid resulted in 2.5-, 3.9-, and 6.5-fold increases in DNA, soluble protein, and RNA, respectively, over untreated controls 84 hours after treatment. Aspartate transcarbamylase activity increased within 12 hours after treatment, and by 84 hours it was almost 12-fold greater than that in the untreated controls. During that time, activity in untreated controls dropped 60%. The assay used ¹⁴C-aspartate, which was then separated from the ¹⁴C-ureidosuccinate product by Dowex 50 (H⁺ form) column chromatography. Thin layer chromatography of the reaction product indicated that most of the carbamyl-phosphate-dependent radioactivity co-chromatographed with ureidosuccinate. The reaction has a pH optimum near 10.0 and is inhibited by uridine 5′-phosphate and succinate. The data suggest that aspartate transcarbamylase is important in pyrimidine biosynthesis in 2,4-dichlorophenoxyacetic acid-treated seedlings.