Mutants of Escherichia coli unable to metabolize cytidine: isolation and characterization

Summary Strains of Escherichia coli have been selected, which contain mutations in the udk gene, encoding uridine kinase. The gene has been located on the chromosome as cotransducible with the his gene and shown to be responsible for both uridine and cytidine kinase activities in the cell.An additional mutation in the cdd gene (encoding cytidine deaminase) has been introduced, thus rendering the cells unable to metabolize cytidine. In these mutants exogenously added cytidine acts as inducer of nucleoside catabolizing enzymes indicating that cytidine per se is the actual inducer.When the udk, cdd mutants are grown on minimal medium the enzyme levels are considerably higher than in wild type cells. Evidence is presented indicating that the high levels are due to intracellular accumulation of cytidine, which acts as endogenous inducer.Abbreviations and Symbols FU 5-fluorouracil - FUR 5-fluorouridine - FUdR 5-fluoro-2'deoxyuridine - FCR 5-fluorocytidine - FCdR 5-fluorodeoxycytidine - THUR 3, 4, 5, 6-tetrahydrouridine - UMP uridine monophosphate - CMP cytidine monophosphate - dUMP deoxyuridine monophosphate. Genes coding for: cytidine deaminase - edd uridine phosphorylase - udp thymidine phosphorylase - tpp purmnucleoside phosphorylase - pup uridine kinase (=cytidine kinase) - udk UMP-pyrophosphorylase - upp. CytR regulatory gene for cdd, udp, dra, tpp, drm and pup Enzymes EC 2.4.2.1 Purine nucleoside phosphorylase or purine nucleoside: orthophosphate (deoxy)-ribosyltransferase - EC 2.4.2.4 thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - EC 2.4.2.3 uridine phosphorylase or uridine: orthophosphate ribosyltransferase - EC 3.5.4.5 cytidine deaminase or (deoxy)cytidine aminohydrolase - EC 4.1.2.4 deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - EC 2.4.2.9 UMP-pyrophosphorylase or UMP: pyrophosphate phosphoribosyltransferase - EC 2.7.1.48 uridine kinase or ATP: uridine 5-phosphotransferase     

Pathways of Pyrimidine Salvage in Streptomyces

Using 5-fluoropyrimidine analogues, high-performance liquid chromatography (HPLC), and the feeding of pyrimidine compounds to pyrimidine auxotrophs, the pathways for salvage of exogenous pyrimidine nucleosides and bases in Streptomyces were established. Selection for resistance to the analogues resulted in the isolation of strains of S. griseus lacking the following enzyme activities: uracil phosphoribosyltransferase (upp) and cytidine deaminase (cdd). The conversion of substrates in the pathway was followed using reverse-phase HPLC. The strains deficient in salvage enzymes were also verified by this method. In addition, feeding of exogenous pyrimidines to strains lacking the biosynthetic pathway confirmed the salvage pathway. Data from the analogue, HPLC, and feeding experiments showed that Streptomyces recycles the pyrimidine base uracil, as well as the nucleosides uridine and cytidine. Cytosine is not recycled due to a lack of cytosine deaminase.     

Fusion of the lac genes to the promotor for the cytidine deaminase gene of Escherichia coli K-12

Summary Phage Mu has been inserted into the structural gene for cytidine deaminase (cdd). By the use of phage (lac, Mu) the promoter for the cdd gene has been fused to lacZ. In these strains lacZ expression is regulated by the cytR repressor protein and is therefore induced by cytidine. The fusion strains were used for the isolation of cddo mutants. Plaque forming phages carrying the different cdd-lacZ fusions were isolated. Studies of the cdd-Mu strains showed that the cdd gene is transcribed clockwise with respect to the Escherichia coli map.     

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

Summary The Bacillus subtilis cdd gene encoding cytidine/2-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis 43 and 37 RNA polymerase holoenzymes during growth and stationary phase.Abbreviations SDS sodium dodecyl sulphate - Ap ampicillin resistance - Tet^r tetracycline resistance - Km^r kanamycin resistance     

关键基因的修饰对枯草芽孢杆菌尿苷合成的影响

【目的】探究磷酸核糖焦磷酸(PRPP)合成酶(prs)和氨甲酰磷酸合成酶(pyr AA/pyr AB)的点突变,以及异源5′-核苷酸酶(sdt1)的过表达,对枯草芽孢杆菌尿苷生物合成的影响。【方法】依据推断的变构位点,分别在prs基因和pyr AB基因编码序列中引入点突变;将点突变的prs基因在染色体xyl R位点整合表达,pyr AB基因则在染色体原位被修饰;sdt1基因在染色体sac B位点整合过表达。通过对重组菌摇瓶发酵液中尿苷、胞苷和尿嘧啶的分析,表征相关基因修饰对尿苷合成的影响。【结果】在PRPP合成酶中引入Asn120Ser、Leu135Ile和Glu52Gly或Val312Ala点突变,分别导致尿苷积累量提高67%和96%。进一步在氨甲酰磷酸合成酶中引入Ser948Phe、Thr977Ala和Lys993Ile点突变,导致尿苷积累量又增加了182%,达到6.97 g/L。在此基础上,过表达异源5′-核苷酸酶,导致尿苷产量增加17%,达到8.16 g/L。【结论】PRPP合成酶和氨甲酰磷酸合成酶的酶活或反馈抑制调节机制,是限制尿苷过量合成的重要因素。PRPP合成酶的Asn120Ser和Leu135Ile点突变,以及氨甲酰磷酸合成酶的Ser948Phe、Thr977Ala和Lys993Ile点突变,能够显著促进尿苷合成。PRPP合成酶附加的Glu52Gly或Val312Ala点突变,有利于尿苷合成。异源的嘧啶专一性5′-核苷酸酶的引入,也对尿苷的合成有明显的促进作用。     

ENZYMIC CONVERSION OF URIDINE NUCLEOTIDE TO CYTIDINE NUCLEOTIDE BY RAT BRAIN

—After in vivo administration of [¹⁴C]uridine monophosphate to rats, radioactivity appears in cytidine nucleotides at comparable rates in the brain and the liver. An in vitro assay, on the soluble fraction of rat brain showed that the enzyme required for amination of uridine nucleotide to cytidine nucleotide occurs in the brain.     

Metabolism of cytidine and uridine in bean leaves

The metabolism of cytidine-2-¹⁴C and uridine-2-¹⁴C was studied in discs cut from leaflets of bean plants (Phaseolus vulgaris L.). Cytidine was degraded to carbon dioxide and incorporated into RNA at about the same rates as was uridine. Both nucleosides were converted into the same soluble nucleotides, principally uridine diphosphate glucose, suggesting that cytidine was rapidly deaminated to uridine and then metabolized along the same pathways. However, cytidine was converted to cytidine diphosphate and cytidine triphosphate more effectively than was uridine. Cytidine also was converted into cytidylic acid of RNA much more extensively and into RNA uridylic acid less extensively than was uridine. Azaserine, an antagonist of reactions involving glutamine (including the conversion of uridine triphosphate to cytidine triphosphate), inhibited the conversion of cytidine into RNA uridylic acid with less effect on its incorporation into cytidylic acid. On the other hand, it inhibited the conversion of orotic acid into RNA cytidylic acid much more than into uridylic acid. The results suggest that cytidine is in part metabolized by direct conversion to uridine and in part by conversion to cytidine triphosphate through reactions not involving uridine nucleotides.     

N4-hydroxycytidine-a new mutagen of a base analogue type

N⁴-hydroxycytidine (N⁴-OHcyd)¹ is incorporated into nucleic acids of a cytidine-requiring strain of S.typhimurium 1045 and can act mutagenically. The reversion frequency of pyrG^? → pyrG⁺ is 10–20 fold higher than the spontaneous background. N⁴OHcyd-induced revertants show a strong inhibitory effect in the presence of N⁴OHcyd. The influence of N⁴OHcyd on cytidine metabolism is discussed.     

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5'' cyclic AMP and CRP protein.

Summary The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.Abbreviations and Symbols cAMP cyclic adenosine 3:5-monophosphate - CRP cAMP receptor protein. Genes coding for: adenyl cyclase - cya cAMP receptor protein - crp cytidine deaminase - cdd uridine phosphorylase - udp thymidine phosphorylase - tpp purine nucleoside phosphorylase - pup; cytR regulatory gene for cdd, udp, dra, tpp, drm, and pup - deoR regulatory gene for dra, tpp, drm, and pup     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes.

Rat liver microsomes showed very active uridine diphosphate-galactose pyrophosphatase activity leading to the hydrolysis of uridine diphosphate-galactose into galactose1-phosphate and finally into galactose. The activity was observed in presence of buffers with wide ranges of pH. Different concentrations of divalent cations, such as Mn²⁺, Mg²⁺, and Ca²⁺ had no significant effect on the enzyme activity. A number of nucleotides and their derivatives inhibited the pyrophosphatase activity. Of these, different concentrations of uridine monophosphate, cytidine 5′-phosphate and cytidine 5′-diphosphate have slight or no effect; cytidine 5′-triphosphate, adenosine 5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-diphosphate-glucose and guanosine 5′-diphosphate-glucose showed strong inhibitory effect whereas cytidine 5′-diphosphate-choline showed a moderate effect on the pyrophosphatase. All these nucleotides also showed variable stimulatory effects on uridine diphosphate-galactose:glycoprotein galactosyltransferase activity in the microsomes which could be partly related to their inhibitory effects on uridine diphosphate-galactose pyrophosphatase. Among them uridine monophosphate, cytidine 5′-phosphate, and cytidine 5′-diphosphate stimulated galactosyltransferase activity without showing appreciable inhibition of pyrophosphatase, cytidine 5′-diphosphate-choline, although did not inhibit pyrophosphatase as effectively as cytidine 5′-triphosphate, guanosine 5′-triphosphate, adenosine 5′-triphosphate, cytidine 5′-diphosphate-glucose, and guanosine 5′-diphosphate-glucose but stimulated galactosyltransferase activity as well as those. The fact that cytidine 5′-diphosphate-choline stimulated galactosyltransferase more effectively than cytidine 5′-phosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate suggested an additional role of the choline moiety in the system. It has been also shown that cytidine 5′-diphosphate-choline can affect the saturation of galactosyltransferase enzyme at a much lower concentration of uridine diphosphate-galactose. Most of the pyrophosphatase and galactosyltransferase activities were solubilized by deoxycholate and the membrane pellets remaining after solubilization still retained some galactosyltransferase activity which was stimulated by cytidine 5′-diphosphate-choline. In different membrane fractions a concerted effect of both uridine diphosphate-galactose pyrophosphatase and glycoprotein:galactosyltransferase enzymes on the substrate uridine diphosphate-galactose is indicated and their eventual controlling effects on the glycopolymer synthesis in vitro or in vivo need careful evaluation.     

Metabolism of 4-N-Hydroxy-Cytidine in Escherichia coli

4-N-hydroxy-cytidine was found to substitute for uridine as a pyrimidine supplement for the growth of Escherichia coli Bu⁻. Measurement of the incorporation of 4-N-hydroxy-cytidine-2-¹⁴C into ribonucleic acid and deoxyribonucleic acid revealed that this compound was converted to cytidine or uridine before utilization. Two pathways for metabolism were considered: (i) the reduction of 4-N-hydroxy-cytidine to cytidine followed by deamination, (ii) the direct hydrolysis of hydroxylamine from 4-N-hydroxy-cytidine to yield uridine. A threefold increase in cytidine (deoxycytidine) deaminase (EC 3.5.4.5) activity, when the cells were grown on 4-N-hydroxy-cytidine, suggested the involvement of this enzyme. More direct proof was obtained by purifying the deaminase 185-fold and finding that it released hydroxylamine from 4-N-hydroxy-cytidine at one-fiftieth the rate at which ammonia was removed from cytidine. This result is consistent with the slower rate of growth of the Bu⁻ cells on 4-N-hydroxy-cytidine than cytidine and suggests that the second pathway is the major route for utilization of this compound.     

Crystal Structure of 04-Methyl Uridine: Stacking Induced Changes in the Geometry of the Pyrimidine Ring and Its Mutagenic Role

Abstract

The geometric properties of the pyrimidine ring of O⁴-methyl uridine more closely resemble those of cytidine than diketo uridine. Differences between the independent molecules of O⁴-methyl uridine are observed in the C(7)-O(4)-C(4)-C(5)-C(6) bond orders and the planarity of the pyrimidine rings. These differences are attributed to the monopole-induced dipole interactions between the ribose ring oxygen atom and a neighboring base of molecule A. A survey of the literature reveals that similar stacking-induced effects occur in other structures, involving both pyrimidine and purines. Finally, two base pairing schemes between O⁴-methyl uridine and guanosine, in which two hydrogen bonds can form, have been presented. Of these two the mispair with Watson-Crick geometry is favored.     

Some kinetic studies on ribonucleosides degradation by extracts of penicillium citrinum

Cell-free extracts of 3–4 days old mats of nitrate-grown Penicillium citrinum catalyze the hydrolytic cleavage of the N-glycosidic bonds of inosine, guanosine and adenosine optimally at pH 4, 0.1 M citrate buffer. The same extracts catalyze the hydrolytic deamination of cytidine at a maximum rate in 0.08 M Tris-acetate buffer pH 6.5, 40°C and 50°C were the most suitable degrees for purine nucleoside hydrolysis and cytidine deamination, respectively. The incubation of the extracts at 60°C, in the absence of cytidine caused a loss in the deaminating activity, while freezing and thawing had no effect on both activities. The deaminating activity seems to be cytidine specific as neither cytosine, adenine, adenosine nor guanosine could be deaminated. Uridine competively inhibited this activity, while ammonia had no effect. The apparent K_m value of this enzyme for cytidine was 1.57×10^?3M and its K_i value for uridine was 7.8×10^?3M. The apparent K_m values of the N-glycosidic bond cleaving enzyme for inosine, guanosine and adenosine were 13.3, 14.2 and 20×10^?3 M, respectively.     

Isolation by tritium suicide of uridine-cytidine kinase-defective mutants in Chinese hamster V79 cells

Tritium suicide was shown to be a highly effective method for isolating mutants defective in uridine-cytidine kinase in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of three kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [³H]uridine for 10 min, followed by storage of ³H-labelled cells at −70 °C for 4–7 days. Nine clones that survived the third kill cycle were tested for incorporation of [³H]uridine and for uridine kinase activity in extracts. Eight of these clones were defective in whole-cell uridine incorporation and in uridine kinase activity. A kinetic study was made on the uridine-cytidine kinase activity of three of the mutants. The apparent V_max of the mutants was reduced approx. 10-fold when either uridine or cytidine was used as substrate. In contrast, the apparent K_m of uridine was reduced approx. 12-fold in the mutants with only a 2-fold (probably insignificant) reduction in K_m's for cytidine or for ATP.     

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5′-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

Biosynthesis of cytidine nucleotides in rat liver after administration of D-galactosamine

Following the administration of D-galactosamine the utilization of [2-¹⁴C]orotic acid for the synthesis of the cytidine components of the acidsoluble extract and liver RNA cytosine is markedly decreased. The depression of the specific activity of the cytidine components takes place after application of low doses of the drug which do not interfere with the specific activity of the uridine components of the acid-soluble extract or of liver RNA uracil. Simultaneously the administration of [U-¹⁴C]cytidine paralleled by its enhanced liver uptake. The total amount of uridine as well as cytidine components of the acid-soluble extract following the administration of D-galactosamine increases; however, the molar ratio of both pyrimidines does not change. The alterations of the cytidine metabolism after the administration of the drug are accompanied by the increased level of microsomal cytochrome P-450.     

Cytidine and Uridine Increase Striatal CDP-Choline Levels Without Decreasing Acetylcholine Synthesis or Release

Summary Aims: Treatments that increase acetylcholine release from brain slices decrease the synthesis of phosphatidylcholine by, and its levels in, the slices. We examined whether adding cytidine or uridine to the slice medium, which increases the utilization of choline to form phospholipids, also decreases acetylcholine levels and release. Methods: We incubated rat brain slices with or without cytidine or uridine (both 25–400 μM), and with or without choline (20–40 μM), and measured the spontaneous and potassium-evoked release of acetylcholine. Results: Striatal slices stimulated for 2 h released 2650±365 pmol of acetylcholine per mg protein when incubated without choline, or 4600±450 pmol/mg protein acetylcholine when incubated with choline (20 μM). Adding cytidine or uridine (both 25–400 μM) to the media failed to affect acetylcholine release whether or not choline was also added, even though the pyrimidines (400 μM) did enhance choline`s utilization to form CDP-choline by 89 or 61%, respectively. The pyrimidines also had no effect on acetylcholine release from hippocampal and cortical slices. Cytidine or uridine also failed to affect acetylcholine levels in striatal slices, nor choline transport into striatal synaptosomes. Conclusion: These data show that cytidine and uridine can stimulate brain phosphatide synthesis without diminishing acetylcholine synthesis or release.