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PubMed1991

Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon.

A 12.5-kilobase segment of Bacillus subtilis chromosomal DNA containing the entire pyrimidine biosynthetic (pyr) gene cluster has been cloned and sequenced. The sequenced DNA has seven cistrons encoding the six enzymes of de novo pyrimidine nucleotide biosynthesis and two open reading frames of unknown function. Based on the sequence and mapping of transcripts, the genes in this cluster appear to be transcribed on one large polycistronic message in the order ORF1, pyrB, pyrC, pyrAA, pyrAB, ORF2, pyrD, pyrF, pyrE. The deduced amino acid sequences for six pyrimidine biosynthetic enzymes from B. subtilis and comparisons with the corresponding sequences from numerous other species are presented. The 3' ends of the reading frames overlap the 5' ends of the downstream open reading frames for all cistrons in the cluster except ORF1 and pyrB, which are separated by a 145-base pair intercistronic region. The start of transcription was mapped by primer extension to a G residue 158 nucleotides upstream from the translation initiation codon of ORF1. This site is preceded by a typical B. subtilis sigma A-dependent promoter. A promoter indicator plasmid was used to show that this region carried a promoter from which transcription was regulated by pyrimidines. Transcription from this promoter was not detected in primer extension experiments using mRNA prepared from B. subtilis cells grown in the presence of excess uracil. No transcripts initiating from the intercistronic space between ORF1 and pyrB were detected with S1 nuclease mapping; however, a transcription terminator was detected in this region that reduced but did not fully block transcriptional readthrough. This terminator was not regulated by pyrimidines in the growth medium under the conditions tested. The region immediately following the promoter and 5' to ORF1 has a potential transcription terminator sequence. The roles, if any, of these transcription terminators in the regulation of pyr operon expression are yet to be determined. However, deletion of the terminator immediately following the promoter abolished pyrimidine regulation of transcription in the indicator plasmid.

Amino Acid Sequence↗

PubMed1994

Origin of the N-1, C-2 and C-2' atoms of the pyrimidine moiety of thiamin in Saccharomyces cerevisiae.

The precursor of the pyrimidine moiety of thiamin in S. cerevisiae was studied. The tracers [15N]- and [2'-13C]-pyridoxine were chemically synthesized, and incorporated efficiently into the pyrimidine. The mass fragmentation pattern showed that the nitrogen atom of pyridoxine was incorporated into the N-1 of the pyrimidine. In addition, the C-2' atom of pyridoxine was incorporated into the C-2' atom of the pyrimidine. These results and the structural resemblance between the pyrimidine and pyridoxine revealed that the C-2, C-2' and N-1 unit of the pyrimidine are derived from the C-2, C-2' and N-1 unit of pyridoxine.

Carbon↗

PubMed1993

Heat-shock treatment selectively affects induction and repair of cyclobutane pyrimidine dimers in transcriptionally active genes in ultraviolet-irradiated human fibroblasts.

The effect of hyperthermia on induction and repair of UV-radiation-induced cyclobutane pyrimidine dimers was investigated in the genome overall and in transcriptionally active and inactive genes in confluent human fibroblasts. Hyperthermia treatment (30 min, 45 degrees C) of human fibroblasts resulted in an increase in the protein content of isolated nuclei (protein aggregation) similar to that observed for HeLa S3 cells. The faster rate of disaggregation of nuclear proteins and the higher survival rate of heated fibroblasts in comparison with those for HeLa cells provide further evidence for a possible role of protein aggregation in heat-induced cell killing. Determination of the frequencies of cyclobutane pyrimidine dimers in the genome overall and in restriction fragments of the active adenosine deaminase (ADA) gene and inactive 754 locus revealed that hyperthermia selectively inhibits the induction of cyclobutane pyrimidine dimers in transcriptionally active DNA. Removal of cyclobutane pyrimidine dimers from the ADA gene was strongly delayed during the first 8 h in 10 J/m2 UV-irradiated fibroblasts. Such inhibition of repair of cyclobutane pyrimidine dimers was not observed for the 754 gene, indicating that inhibition of repair by hyperthermia is generally not mediated by inactivation of repair enzymes. It is proposed that the inhibition of induction and repair of cyclobutane pyrimidine dimers in active genes by hyperthermia is related to the heat-induced aggregation of proteins with the nuclear matrix, proximal to which active genes are located. Our results are consistent with a functional compartmentalization of DNA repair at the nuclear matrix.

DNA Repair↗

PubMed1977

Selective inhibition of pyrimidine biosynthesis and effect on proliferative growth of colonic cancer cells.

A highly selective inhibition of de novo pyrimidine synthesis in the intact cell has been demonstrated by the action of N-(phosphonacetyl)-L-aspartate (PALA), a transition-state analog inhibitor of the reaction catalyzed by asparate transcarbamylase. The effect of pyrimidine deprivation induced by this agent on the viability and survival of human normal (WI-38) and colonic cancer cells (HT-29) was examined. The PALA-treated, pyrimidine-deprived cells failed to grow but demonstrated a normal rate of glucose utilization with impaired glycogen synthesis. Pyrimidine deprivation and lack of cell growth were maintained long after PALA removal. Growth inhibition of HT-29 cells by PALA was found to reflect an apparent steady state between newly formed and dying cells induced by limited pyrimidine availability. The highly selective nature of PALA action was confirmed by the ability of an exogenous source of pyrimidine to restore the normal growth pattern of the cell. Significant antitumor activity of PALA was found against a transplantable colonic tumor (line 26) carried in mice.

Animals↗

PubMed1974

Hereditary hemolytic anemia with human erythrocyte pyrimidine 5'-nucleotidase deficiency.

A severe deficiency of a red cell pyrimidine 5'-nucleotidase was found to be associated with hereditary hemolytic anemia in four members of three kindreds. The syndrome was characterized by marked increases above normal in red cell basophilic stippling, total nucleotides, and GSH and by a fairly severe deficiency of ribosephosphate pyrophosphokinase (EC 2.7.6.1.). Patient erythrocytes uniquely contained large amounts of pyrimidine 5'-ribonucleotides. In earlier studies, these were erroneously considered to be adenosine phosphates, since all previous investigations of the nucleotides of human red cells and reticulocytes have shown 97% or more to contain adenine. Total nucleotides in patient cells were present in amounts 3-6 times greater than normal, and approximately 80% contained pyrimidine. The ultraviolet spectral curves of deproteinized red cell extracts exhibited a shift in maximum absorbance from the usual 256-257 nm to approximately 266-270 nm, and absorbance at 250, 270, 280, and 290 nm, expressed as a ratio of that at 260 nm, differed greatly from normal. The spectral characteristics of extracts provide the basis of a readily performed screening procedure, which does not require enzyme assay. The nucleotidase activity in deficient red cells assayed less than 14%, and usually less than 10%, of normal and much less in terms of reticulocyte-rich blood, where it was consistently found to be increased. The enzyme has a pH optimum of 7.5-8.0, is inhibited by EDTA, and does not utilize purine 5'-ribonucleotides or beta-glycerophosphate as substrates. While comparatively few family members have been available thus far for study, initial data are compatible with an autosomal, recessive mode of transmission of the deficiency. The pyrimidine 5'-ribonucleotides are presumably derived from RNA degradation and, not being diffusible, accumulate when the enzyme catalyzing their dephosphorylation is deficient. It is postulated that the prominent basophilic stippling results from retarded ribosomal RNA degradation secondary to accumulation of degradation products, namely pyrimidine 5'-ribonucleotides. Ribosephosphate pyrophosphokinase deficiency is considered to be an epiphenomenon. The mechanism responsible for increased red cell GSH is unknown.

Adenosine Triphosphate↗

PubMed1986

Age-specific prevalence of antibodies to pyrimidine kinase enzymes of herpes simplex virus types 1 and 2, and of varicella zoster virus.

The age-specific prevalence of antibodies to pyrimidine kinase enzymes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) and of varicella zoster virus (VZV) was measured in serum specimens from 360 persons. Specific inhibition of viral enzyme activity by the serum was used as an indication of the presence of antibodies to the enzyme. For HSV-1 and HSV-2 together the overall prevalence of positive sera increased with age and reached about 50% in the older age groups, the major part of the positive sera being HSV-1 positive. Sera inhibitory to the VZV pyrimidine kinase enzyme were detected only sporadically. Six percent of the tested sera repeatedly inhibited the cytosolar corresponding enzyme of uninfected host cells by an unidentified mechanism. The results of the pyrimidine kinase antibody-assay test were compared to those obtained by complement fixation (CF) and enzyme immunoassay (EIA) in a separate set of 50 sera. The correlation between the CF and the EIA tests was good in this selected serum set with CF titres from less than 8 to 64. Twenty-four percent of the CF positive sera were negative by the pyrimidine kinase antibody assay, while all pyrimidine-kinase-antibody-positive sera also had antibodies detectable by the two other methods. These results confirm and extend earlier observations on the occurrence of antibodies in human sera to HSV and VZV pyrimidine kinase enzymes.

Adolescent↗

PubMed1991

Different intrastrand and interstrand contributions to stacking account for roll variations at the alternating purine-pyrimidine sequences in A-DNA and A-RNA.

An explanation is suggested for the roll alternation between low and high values in A-type nucleic acid duplexes containing alternating sequences of purine and pyrimidine residues. The explanation combines two points. (1) Roll inevitably occurs in A-type duplexes due to geometrical reasons. (2) Intrastrand base stacking is much more impaired by roll than interstrand base stacking in A-type duplexes. Therefore purine-pyrimidine steps, whose bases mainly exhibit an intrastrand stacking, resist roll and decrease it. By contrast, bases at pyrimidine-purine steps exhibit a significant interstrand stacking that is tolerant to roll in A-type nucleic acid duplexes. In consequence, it is favourable if the purine-pyrimidine and pyrimidine-purine steps adopt low and high rolls, respectively in A-conformations of DNA and RNA molecules containing alternating purine-pyrimidine sequences. This is actually observed in the relevant molecular crystal structures.

Base Composition↗

PubMed1981

Control of Pyrimidine Biosynthesis in Synchronously Dividing Cells of Helianthus tuberosus.

Factors with potential for regulating pyrimidine biosynthesis in plant tissue have been explored in quiescent cells of Helianthus tuberosus induced to divide by auxin addition. Investigations confined to the first highly synchronous cell cycle of the tuber explants revealed that the relative activity of asparate carbamoyltransferase (ACTase) to ornithinecarbamoyltransferase (OCTase) (enzymes competing for carbamoyl phosphate for the pyrimidine and arginine pathways, respectively) changes from 0.5 in quiescent cells to 3.0 by the end of the first cell cycle. This was interpreted as a change in the state of cell function from accumulation of storage arginine to cell division with a concomitant demand for pyrimidine nucleotides for nucleic acid synthesis. The rise in ACTase activity began at the same time as the initiation of DNA synthesis and was dependent on continued DNA synthesis. OCTase activity declined whether or not auxin was added to the medium, whereas ACTase activity was observed to decline only in the absence of DNA synthesis.The low cellular concentration of the shared substrate, carbamoyl phosphate (2 micromolar), favored utilization of this substrate by the pyrimidine pathway over the arginine pathway because of the low K(m) (0.08 80 micromolar) for this substrate by ACTase compared to that for OCTase (9.0 millimolar). Unexpectedly, the total concentration of the feedback inhibitor for the pyrimidine pathway, UMP, was found to have more than doubled in dividing tissue at a time when pyrimidine nucleotide demand had increased. It is concluded that compartmentation decreased UMP in the vicinity of ACTase and/or that the extra UMP stabilizes newly synthesized ACTase in preparation for an even greater demand for nucleic acid synthesis in the second and subsequent cell cycles.

Journal Article↗

PubMed1976

Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.

Cumulative repression of Escherichia coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.

Arginine↗

PubMed1984

Identification of cytidine diphosphodiesters in erythrocytes from a patient with pyrimidine nucleotidase deficiency.

Pyrimidine 5'-nucleotidase deficient (PND) erythrocytes contain elevated levels of pyrimidine nucleotides and relatively normal purine nucleotide levels. The composition of this nucleotide pool has been examined by others, but not all of the abnormal red cell metabolites in this disorder were identified. We have isolated and positively confirmed the identity of cytidine diphosphate (CDP)-choline and CDP-ethanolamine from PND red cells using methods including proton FT-NMR, spectroscopy, and comparative mass spectrometry. The concentrations of these and other pyrimidine nucleotidase-deficient erythrocyte nucleotides were determined using anion-exchange high performance liquid chromatography and ultraviolet (u.v.) detection. The pyrimidine diphosphodiesters appear to be the most prominent abnormal pyrimidine nucleotides in PND red cells, accounting for 55% of the total red cell pyrimidine nucleotides in this disorder. It is proposed that these abnormal phosphodiesters may be related to the accelerated hemolysis in PND.

5'-Nucleotidase↗

PubMed1978

[Distribution of 5-methylcytosine in pyrimidine oligonucleotides of higher plant DNA].

The level of pyrimidine clusters (isopliths) and distribution of 5-methylcytosine in DNAs of some archegonial (ferns, ginkgo) and flowering (cordyline, tulip, wheat) plants has been studied. DNA from Cordyline australis (Liliaceae) is one of the GC-type (GC = 57.5 mol.%), has very low methylation level (amount of m5C is about 1 mol.%) and significantly differs from other species DNAs studied in terms of pyrimidine distribution patterns, i.e. amount of dipyrimidine fragments is higher than that of monopyrimidine ones. All other plant DNAs under study are of the AT-type, have similar pyrimidine distribution patterns and are characterized by high degree of pyrimidine blocking; specific differences in the level (approximately 10-fold) and type of genome methylation are found. In all DNAs studied 5-methylcytosine is unevenly distributed in pyrimidine isopliths: the degree of cytosine methylation [m5C(C+m5C)] is decreased with an increase in the length of isopliths, irrespective of the ratio of thymine and cytosine residues. 5-methylcytosine is mainly accumulated in mono- and dipyrimidine fragments (60--68%). Specific differences in 5-methylcytosine content in trinucleotides and long-chain pyrimidine oligonucleotides are revealed.

Cytosine↗

PubMed2002

Capillary electrophoresis for detection of inherited disorders of purine and pyrimidine metabolism: a selective approach.

We developed a capillary electrophoresis method as a diagnostic tool for purine and pyrimidine metabolic disorders. Optimal conditions allowed the separation of the major diagnostic metabolites in urine samples within an analysis time of 10 min and with a separation efficiency of about 350,000 theoretical plates/m. The diagnostically important metabolites (adenine, adenosine, 2-deoxyadenosine, 2-deoxyguanosine, 2,8-dihydroxyadenine, guanosine, hypoxanthine, orotidine, orotic acid, and creatinine) were detectable at concentrations of 1.0-5.7 micromol/L. The method gives a linear calibration curve for tested purine and pyrimidine derivatives within the range of 5-500 micromol/L (r > 0.996) The coefficients of variation for the within- and between-day imprecisions were less than 3.2 and 5.8%, respectively. Characteristic abnormalities were detected in the electropherograms of urine samples from patients with purine and pyrimidine enzyme deficiencies. We provide electrophoretic and spectral characteristics of intermediates in purine and pyrimidine metabolism and possible artifacts from medication and their UV-absorbing compounds. Our method allows the detection of the majority of inborn errors of purine and pyrimidine metabolism.

Adolescent↗

PubMed1998

Purines and pyrimidines determination in urine using high-performance liquid chromatography.

Single purine and pyrimidine bases are involved in two fundamental metabolic pathways that lead to formation of the building stones of DNA and RNA. Purine and pyrimidine nucleotides are also critically important metabolites in many cellular functions. The main breakdown of purines and pyrimidines produces uric acid and B-minoacids, respectively. Therefore, the study of purine and pyrimidine compounds in body fluid has high clinical relevance. We report, in this work, our experience in purines and pyrimidines determination in urine from children presenting with a clinical picture suggesting an inborn these pathways.

Biomarkers↗

PubMed1984

Salvage of pyrimidine nucleosides by Trichomonas vaginalis.

Trichomonas vaginalis is incapable of de novo pyrimidine biosynthesis because it cannot incorporate bicarbonate, aspartate or orotate into its pyrimidine nucleotides or nucleic acids. The organism can salvage exogenous cytidine greater than uridine greater than uracil and thymidine, and incorporate them into the nucleotide pool. A portion of cytidine is converted to CMP, CDP and CTP by cytidine phosphotransferase and nucleotide kinases. Some cytidine and most of uracil are, however, converted first to uridine by cytidine deaminase and uridine phosphorylase respectively; uridine is then incorporated into UMP, UDP and UTP by uridine phosphotransferase and nucleotide kinases. The two phosphotransferases, found mainly in the non-sedimentable fraction of T. vaginalis, provide the main avenue of pyrimidine salvage. No significant levels of pyrimidine phosphoribosyl transferase or nucleoside kinases can be detected in the extract. T. vaginalis has no appreciable dihydrofolate reductase or thymidylate synthetase; it grows normally in millimolar concentrations of methotrexate, pyrimethamine, or trimethoprim, and cannot incorporate labels from exogenous uracil or uridine into DNA. It has an enzyme thymidine phosphotransferase in the sedimentable fraction which converts thymidine to TMP. Thymidine salvage in T. vaginalis is thus totally isolated from the rest of the pyrimidine salvage.

Animals↗

PubMed1997

Inborn errors of pyrimidine degradation: clinical, biochemical and molecular aspects.

The pyrimidines, uracil and thymine, are degraded in four steps. The first three steps of pyrimidine catabolism, controlled by enzyme shared by both pathways, result in the production of the neurotransmitter amino acid beta-alanine from uracil and the nonfunctional (R)-(-)-beta-aminoisobutyrate from thymine. The fourth step is controlled by several aminotransferases, which have different affinities for beta-alanine, beta-aminoisobutyrate and GABA. Defects concerning the first three steps all lead to a reduced production of beta-alanine; defects of the transaminases involving the metabolism of beta-alanine and GABA lead to accumulation of these neurotransmitter substances. In addition, other metabolites will accumulate or be reduced depending on the specific enzyme defect. Analysis of the abnormal concentrations of these metabolites in the body fluids is essential for the detection of patients with pyrimidine degradation defects. Clinically these disorders are often overlooked because symptomatology is highly aspecific. The growth in our knowledge concerning inborn errors of pyrimidine degradation has emphasized the importance of the clinical awareness of these defects as a possible cause of neurological disease and a contraindication for treatment of cancer patients with certain pyrimidine analogues. The various defects are discussed and attention is paid to clinical genetic and diagnostic aspects.

Amidohydrolases↗

PubMed1998

Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1.

Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.

Binding Sites↗

PubMed1985

Pyrimidine salvage in Giardia lamblia.

We have found that the anaerobic protozoan parasite Giardia lamblia is incapable of de novo pyrimidine metabolism, as shown by its inability to incorporate orotate, bicarbonate, and aspartate into the pyrimidine nucleotide pool. Results from high performance liquid chromatography of pyrimidine and pyrimidine nucleoside pulse-labeled nucleotide pools and enzyme assays suggest that the parasite satisfies its pyrimidine nucleotide needs predominantly through salvage of uracil by a cytoplasmic uracil phosphoribosyltransferase. Exogenous uridine and cytidine are primarily converted to uracil by the action of uridine hydrolase and cytidine deaminase before incorporation into nucleotide pools. Direct salvage of cytosine occurs to a relatively limited extent via cytosine phosphoribosyltransferase. G. lamblia relies on salvage of exogenous thymidine for ribosylthymine monophosphate (TMP) synthesis, accomplished primarily through the action of a 100,000 g-pelletable thymidine phosphotransferase.

Animals↗

PubMed1978

Pyrimidine metabolism in microplasmodia of Physarum polycephalum.

If microplasmodia of Physarum polycephalum are exposed to 14C-labelled pyrimidine nucleosides or bases, an unusual pattern of metabolism is found. Only the nucleosides are taken up. Analysis of the distribution of the radioactivity in the cells revealed that ribonucleosides and deoxyribonucleosides are incorporated into nucleotides; however, a substantial catabolism takes place. Thus incubation with [2-14C]pyrimidine nucleosides readily gives rise to [14C]O2, particularly in the case of [2-14C]thymidine. Due to this a significant part of the trichloroacetic-acid-insoluble radioactivity from exogenously supplied [2-14C]thymidine is not associated with DNA. The pattern of labelling of nucleoside triphosphates from exogenously supplied nucleosides indicated that the de novo synthesis of nucleotides was only partly repressed. An unusual conversion of deoxycytidine into cytidine was noted. Enzyme analysis on cell-free extracts revealed that pyrimidine nucleosides can be salvaged by kinases and that their initial catabolism is initiated by hydrolases. Incubation of microplasmodia with pyrimidine analogues showed that only nucleoside analogues are toxic. The experimental results have led us to propose a scheme for the metabolism of pyrimidine nucleosides and bases in Physarum polycephalum.

Biological Transport↗