UMP Synthase Research Articles

Identification of a Nonsense Mutation in CWC15 Associated with Decreased Reproductive Efficiency in Jersey Cattle

With the recent advent of genomic tools for cattle, several recessive conditions affecting fertility have been identified and selected against, such as deficiency of uridine monophosphate synthase, complex vertebral malformation, and brachyspina. The current report refines the location of a recessive haplotype affecting fertility in Jersey cattle using crossover haplotypes, discovers the causative mutation using whole genome sequencing, and examines the gene’s role in embryo loss. In an attempt to identify unknown recessive lethal alleles in the current dairy population, a search using deep Mendelian sampling of 5,288 Jersey cattle was conducted for high-frequency haplotypes that have a deficit of homozygotes at the population level. This search led to the discovery of a putative recessive lethal in Jersey cattle on Bos taurus autosome 15. The haplotype, denoted JH1, was associated with reduced fertility, and further investigation identified one highly-influential Jersey bull as the putative source ancestor. By combining SNP analysis of whole-genome sequences aligned to the JH1 interval and subsequent SNP validation a nonsense mutation in CWC15 was identified as the likely causative mutation underlying the fertility phenotype. No homozygous recessive individuals were found in 749 genotyped animals, whereas all known carriers and carrier haplotypes possessed one copy of the mutant allele. This newly identified lethal has been responsible for a substantial number of spontaneous abortions in Jersey dairy cattle throughout the past half-century. With the mutation identified, selection against the deleterious allele in breeding schemes will aid in reducing the incidence of this defect in the population. These results also show that carrier status can be imputed with high accuracy. Whole-genome resequencing proved to be a powerful strategy to rapidly identify a previously mapped deleterious mutation in a known carrier of a recessive lethal allele.

Introduction of a Fluorine Atom at C3 of 3-Deazauridine Shifts Its Antimetabolic Activity from Inhibition of CTP Synthetase to Inhibition of Orotidylate Decarboxylase, an Early Event in the de Novo Pyrimidine Nucleotide Biosynthesis Pathway

The antimetabolite prodrug 3-deazauridine (3DUrd) inhibits CTP synthetase upon intracellular conversion to its triphosphate, which selectively depletes the intracellular CTP pools. Introduction of a fluorine atom at C3 of 3DUrd shifts its antimetabolic action to inhibition of the orotidylate decarboxylase (ODC) activity of the UMP synthase enzyme complex that catalyzes an early event in pyrimidine nucleotide biosynthesis. This results in concomitant depletion of the intracellular UTP and CTP pools. The new prodrug (designated 3F-3DUrd) exerts its inhibitory activity because its monophosphate is not further converted intracellularly to its triphosphate derivative to a detectable extent. Combinations with hypoxanthine and adenine markedly potentiate the cytostatic activity of 3F-3DUrd. This is likely because of depletion of 5-phosphoribosyl-1-pyrophosphate (consumed in the hypoxanthine phosphoribosyl transferase/adenine phosphoribosyl transferase reaction) and subsequent slowing of the 5-phosphoribosyl-1-pyrophosphate-dependent orotate phosphoribosyl transferase reaction, which depletes orotidylate, the substrate for ODC. Further efficient anabolism by nucleotide kinases is compromised apparently because of the decrease in pK(a) brought about by the fluorine atom, which affects the ionization state of the new prodrug. The 3F-3DUrd monophosphate exhibits new inhibitory properties against a different enzyme of the pyrimidine nucleotide metabolism, namely the ODC activity of UMP synthase.

Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.

Monitoring of BLAD, DUMPS, CVM, BC and FXID in Turkish native cattle breeds

Bu çalışmada, Türkiye yerli sığır ırkları Sığır Lökosit Bağlanma Yetersizliği (BLAD), Üridin Monofosfat Sentezi Yetersizliği (DUMPS), Kompleks Omurga Deformasyonu (CVM), Sığır Sitrülin Birikimi (BC) ve Faktör XI Eksikliği (FXID) genetik kusurları bakımından tanımlanmıştır. Otozamal kromozomlar üzerinde bulunan resesif genlerle belirlenmekte olan bu genetik kusurlar büyük ekonomik kayıplara neden olmaktadır. Araştırmada, Yerli Kara (200 baş), Doğu Anadolu Kırmızısı (100 baş), Boz ırk (100 baş), Güneydoğu Anadolu Sarısı (50 baş), Güneydoğu Anadolu Kırmızısı (50 baş) ve Zavot (9 baş) ırklarına ait toplam 509 baş sığır materyal olarak kullanılmıştır. Genomik DNA kandan elde edilmiş ve polimeraz zincir reaksiyonu (PCR) yöntemi ile üzerinde durulan genetik kusurlara ait gen bölgeleri çoğaltılmıştır. PCR ürünleri TaqI (BLAD), AvaI (DUMPS), EcoT22I (CVM) ve AvaII (BC) restriksiyon enzimleri ile muamele edilerek sığırların genotipleri agaroz jel elektroforez yöntemiyle belirlenmiştir. FXID genetik kusuru bakımından sığırların genotipleri ise sadece PCR ürünlerinin agaroz jel elektroforez yöntemindeki bant modelleri kullanılarak tespit edilmiştir. Araştırmada, incelenen 509 baş sığırdan hiçbirinin BLAD, DUMPS, CVM, BC ve FXID genetik kusurlarının taşıyıcısı olmadığı saptanmıştır. Bu araştırmanın sonuçlarına göre, BLAD, DUMPS, CVM, BC ve FXID mutant allellerinin Türkiye yerli sığır ırklarında bulunmadığı söylenebilir.

The PRPP Synthetase Spectrum: What Does it Demonstrate About Nucleotide Syndromes?

Defects in X-linked phosphoribosylpyrophosphate synthetase 1 (PRPS1) manifest as follows: (1) PRS-I enzyme “superactivity” (gain-of-function mutations affecting allosteric regions); (2) PRS-I overexpression (which may be linked to miRNA mutation); (3) severe PRS-I deficiency/Arts syndrome (missense mutations producing loss-of-function); (4) moderate PRS-I deficiency/Charcot–Marie–Tooth disease-5 (less severe loss-of-function mutations); and (5) mild PRS-I deficiency/Deafness-2 (mutations producing slight destabilization). Similar to Lesch–Nyhan disease, PRPS1-related disorders arise from phosphoribosyl-pyrophosphate (PRPP)-dependent nucleotide “depletion” of purine nucleotides (e.g., ATP, GTP). S-adenosylmethionine (SAMe) appears to partially alleviate purine depletion via a PRPP-independent path. Synthesis of pyrimidine nucleotides is PRPP dependent, with uridine monophosphate synthase deficiency producing pyrimidine nucleotide depletion. But pyrimidine salvage from uridine does not require PRPP, and this nucleoside is transported freely to pyrimidine-depleted tissues. Regulation of nicotinamide nucleotides is less clear; synthesis from pyridine nucleobases is PRPP dependent. Nucleotide “depletion” contrasts with nucleotide “toxicity,” exemplified by the purine disorders adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies or by pyrimidine nucleotidase deficiency. These are characterized by the accumulation of one or more abnormal nucleotides such as succinyl- or deoxy-nucleotides or their metabolites, which interrupt other nucleotide or related pathways or are toxic to specific cell types. Theoretically, purine toxicity disorders would not be ameliorated by SAMe therapy, and this was confirmed for one adenylosuccinate lyase-deficient child. Nucleotide defects may also be seen as an aspect of mitochondrial disease, with SAMe-based mitochondrial therapy perhaps meriting further investigation.

Short communication: Distribution of recessive genetic defect carriers in Chinese Holstein

In dairy cattle, 4 important recessive hereditary diseases exist: complex vertebral malformation (CVM), bovine leukocyte adhesion deficiency (BLAD), citrullinemia (CTLN), and deficiency of uridine monophosphate synthase (DUMPS). Holstein Associations in developed countries have established monitoring systems for such disorders in Holstein bulls for decades. Over the past decades, China has continuously imported Holstein semen and embryos, mainly from North America but also from Europe. The dissemination of such genetic defects was undetermined until now, although efforts were taken to develop molecular techniques and detect carriers for CVM and BLAD in small populations of Chinese dairy cattle. Thus, herein we extensively screened 732 proven bulls participating in artificial insemination programs and 136 young bulls entering progeny test from 15 bull stations in China for CVM, BLAD, CTLN, and DUMPS. The proportion of carriers of the defects was found to be 7.72, 1.38, 0.23, and 0.12%, respectively. Given our findings, early diagnostic and monitoring systems on recessive inherited disorders among proven and young bulls entering the national genetic improvement programs for dairy cattle of China should be established immediately, in which a series of measures will be taken to prevent further spreading of such disorders and gradually eliminate them in the dairy cattle population in China.

The Leishmania donovani UMP Synthase Is Essential for Promastigote Viability and Has an Unusual Tetrameric Structure That Exhibits Substrate-controlled Oligomerization

The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.

Frequency of Uridine Monophosphate Synthase Gly213Ala Polymorphism in Caucasian Gastrointestinal Cancer Patients and Healthy Subjects, Investigated by Means of New, Rapid Genotyping Assays

Uridine monophosphate synthase (UMPS) is a fundamental enzyme in pyrimidine synthesis. A single-nucleotide polymorphism, a G-C transversion at the 638th nucleotide, was demonstrated to increase UMPS activity and suggested to have clinical effects. The aims of this study were to set up simple genotyping methods and investigate the UMPS 638G>C polymorphism in the Caucasian population. Two hundred forty-one patients with gastrointestinal cancers and 189 healthy subjects were enrolled. Genomic DNA was extracted from peripheral blood. A polymerase chain reaction-restriction fragment length polymorphism (RFLP) method was implemented using a forward primer incorporating a mismatched base to produce an artificial restriction site and BsrI restriction enzyme digestion; a denaturing high performance liquid chromatography (DHPLC) method was developed to further speed up UMPS genotyping. A 153 bp UMPS gene fragment was successfully amplified and analyzed in all samples. RFLP and DHPLC results showed a 100% match and where confirmed by direct sequencing. UMPS genotype distribution was similar in patients with cancer and control subjects. Although no association was detected between UMPS variants and gastrointestinal cancer risk in Caucasians, polymerase chain reaction-RFLP with BsrI digestion and DHPLC set up at 59°C are reliable and cost-effective methods to genotype UMPS.

Identification of the UMP Synthase Gene by Establishment of Uracil Auxotrophic Mutants and the Phenotypic Complementation System in the Marine DiatomPhaeodactylum tricornutum

Uridine-5'-monophosphate synthase (UMPS), the critical step of the de novo pyrimidine biosynthesis pathway, which is a housekeeping plastid process in higher plants, was investigated in a marine diatom, the most crucial primary producer in the marine environment. A mutagenesis using an alkylation agent, N-ethyl-N-nitrosourea, was carried out to the marine diatom Phaeodactylum tricornutum. Cells were treated with 1.0 mg mL(-1) N-ethyl-N-nitrosourea and were screened on agar plates containing 100 to 300 mg L(-1) 5-fluoroorotidic acid (5-FOA). Two clones survived the selection and were designated as Requiring Uracil and Resistant to FOA (RURF) 1 and 2. The 50% effective concentration of 5-FOA on growth of RURF1 was about 5 mm, whereas that in wild-type cells was 30 μm. The ability to grow in the absence of uracil was restored by a P. tricornutum gene that potentially encoded UMPS or the human umps gene, HUMPS. Because the P. tricornutum gene was able to restore growth in the absence of uracil, it was designated as ptumps, encoding a major functional UMPS in P. tricornutum. RNA interference to the ptumps targeting the 5' region of ptumps resulted in the occurrence of a clear RURF phenotype in P. tricornutum. This RNA interference phenotype was reverted to the wild type by the insertion of HUMPS, confirming that the ptumps encodes UMPS. These results showed direct evidence of the occurrence of novel-type UMPS in a marine diatom and also revealed the potential usage of this gene silencing and complementation system for molecular tools for this organism.

Screening for bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, complex vertebral malformation, bovine citrullinaemia, and factor XI deficiency in Holstein cows reared in Turkey

BackgroundBovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins.MethodsGenomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not.ResultsFourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID).ConclusionThis study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.

ACaenorhabditis elegansmodel of orotic aciduria reveals enlarged lysosome‐related organelles in embryos lackingumps‐1function

Gut granules are cell type-specific lysosome-related organelles found within the intestinal cells of Caenorhabditis elegans. To investigate the regulation of lysosome-related organelle size, we screened for C. elegans mutants with substantially enlarged gut granules, identifying alleles of the vacuolar-type H(+)-ATPase and uridine-5'-monophosphate synthase (UMPS)-1. UMPS-1 catalyzes the conversion of orotic acid to UMP; this comprises the two terminal steps in de novo pyrimidine biosynthesis. Mutations in the orthologous human gene UMPS result in the rare genetic disease orotic aciduria. The umps-1(-) mutation promoted the enlargement of gut granules to 250 times their normal size, whereas other endolysosomal organelles were not similarly affected. UMPS-1::green fluorescent protein was expressed in embryonic and adult intestinal cells, where it was cytoplasmically localized and not obviously associated with gut granules. Whereas the umps-1(-) mutant is viable, combination of umps-1(-) with mutations disrupting gut granule biogenesis resulted in synthetic lethality. The effects of mutations in pyr-1, which encodes the enzyme catalyzing the first three steps of de novo pyrimidine biosynthesis, did not phenotypically resemble those of umps-1(-); instead, the synthetic lethality and enlargement of gut granules exhibited by the umps-1(-) mutant was suppressed by pyr-1(-). In a search for factors that mediate the enlargement of gut granules in the umps-1(-) mutant, we identified WHT-2, an ABCG transporter previously implicated in gut granule function. Our data suggest that umps-1(-) leads to enlargement of gut granules through a build-up of orotic acid. WHT-2 possibly facilitates the increase in gut granule size of the umps-1(-) mutant by transporting orotic acid into the gut granule and promoting osmotically induced swelling of the compartment.

English

The aim of this study was to identify the deficiency of uridine monophosphate synthase (DUMPS) and the complex vertebral malformation (CVM) in Iranian Holstein bulls. A total of 144 blood samples were prepared of Holstein bulls in Abbas Abad Animal Breeding Center and Ferdowsi University of Mashhad's Dairy Farm in Khorasan state of Iran. Genomic PCR-RFLP protocol was performed to amplify the polymorphic region of the bovine uridine monophosphate synthase UMPS gene. Also, genomic PCR-SSCP method was performed for CVM to amplify the polymorphic region of the bovine solute carrier family 35 member 3 (SLC35A3) genes. The results of this study demonstrated that there was no carrier of DUMPS and CVM in Iranian bulls in these centers.

Functional analysis of pyrimidine biosynthesis enzymes using the anticancer drug 5‐fluorouracil inCaenorhabditis elegans

Pyrimidine biosynthesis enzymes function in many cellular processes and are closely associated with pyrimidine antagonists used in cancer chemotherapy. These enzymes are well characterized from bacteria to mammals, but not in a simple metazoan. To study the pyrimidine biosynthesis pathway in Caenorhabditis elegans, we screened for mutants exhibiting resistance to the anticancer drug 5-fluorouracil (5-FU). In several strains, mutations were identified in ZK783.2, the worm homolog of human uridine phosphorylase (UP). UP is a member of the pyrimidine biosynthesis family of enzymes and is a key regulator of uridine homeostasis. C. elegans UP homologous protein (UPP-1) exhibited both uridine and thymidine phosphorylase activity in vitro. Knockdown of other pyrimidine biosynthesis enzyme homologs, such as uridine monophosphate kinase and uridine monophosphate synthetase, also resulted in 5-FU resistance. Uridine monophosphate kinase and uridine monophosphate synthetase proteins are redundant, and show different, tissue-specific expression patterns in C. elegans. Whereas pyrimidine biosynthesis pathways are highly conserved between worms and humans, no human thymidine phosphorylase homolog has been identified in C. elegans. UPP-1 functions as a key regulator of the pyrimidine salvage pathway in C. elegans, as mutation of upp-1 results in strong 5-FU resistance.

Orotic aciduria and uridine monophosphate synthase: A reappraisal

Three subtypes of hereditary orotic aciduria are described in the literature, all related to deficiencies in uridine monophosphate synthase, the multifunctional enzyme that contains both orotate: pyrophosphoryl transferase and orotidine monophosphate decarboxylase activities. The type of enzyme defect present in the subtypes has been re-examined by steady-state modelling of the relative outputs of the three enzymic products, uridine monophosphate, urinary orotic acid and urinary orotidine. It is shown that the ratio of urinary outputs of orotidine to orotate provides a means of testing for particular forms of enzyme defect. It is confirmed that the type I defect is caused by loss of uridine monophosphate synthase activity. Cells and tissue of type I cases have a residual amount of activity that is qualitatively unchanged: the relative rates of the transferase and decarboxylase do not differ from those of wild-type enzyme. The single claimed case of type II, thought to be due to specific inactivation of orotidine monophosphate decarboxylase, is shown to have a product spectrum inconsistent with that claim. It is proposed that this type II form does not differ sufficiently to be accepted as separate from type I. The third subtype, hereditary orotic aciduria without megaloblastic anaemia, occurs in two cases. It has the product spectrum expected of a defect in orotidine monophosphate decarboxylase. This form is the only one that appears to have a qualitatively different uridine monophosphate synthase. The possibility that orotidine monophosphate may control flux through the pyrimidine biosynthesis pathway in hereditary orotic aciduria is discussed.

Identification of Alleles for Factor XI (FXID) and Uridine Monophosphate Synthase (DUMPS) Deficiencies in Holstein Cows Reared in Antalya

Summary Defi ciencies of Factor XI (FXID) and Uridine Monophosphate Synthase (DUMPS) are autosomal recessive inherited diseases in Holstein cattle breed. The aim of this study was to investigate whether FXI defi ciency and DUMPS alleles exist in Holstein cattle reared in Antalya region Turkey or not. 504 Holstein cows were used for this study. PCR (Polymerase Chain Reaction) was used for determination of FXI genotypes and, PCR-RFLP (PCR- Restriction Fragment Length Polymorphism) was used for determination of DUMPS genotypes. As a result, DUMPS allele was not detected while the prevalence of FXID carriers was found as 0.4%.

Structures of the Human Orotidine-5′-Monophosphate Decarboxylase Support a Covalent Mechanism and Provide a Framework for Drug Design

UMP synthase (UMPS) catalyzes the last two steps of de novo pyrimidine nucleotide synthesis and is a potential cancer drug target. The C-terminal domain of UMPS is orotidine-5'-monophosphate decarboxylase (OMPD), a cofactor-less yet extremely efficient enzyme. Studies of OMPDs from micro-organisms led to the proposal of several noncovalent decarboxylation mechanisms via high-energy intermediates. We describe nine crystal structures of human OMPD in complex with substrate, product, and nucleotide inhibitors. Unexpectedly, simple compounds can replace the natural nucleotides and induce a closed conformation of OMPD, defining a tripartite catalytic site. The structures outline the requirements drugs must meet to maximize therapeutic effects and minimize cross-species activity. Chemical mimicry by iodide identified a CO(2) product binding site. Plasticity of catalytic residues and a covalent OMPD-UMP complex prompt a reevaluation of the prevailing decarboxylation mechanism in favor of covalent intermediates. This mechanism can also explain the observed catalytic promiscuity of OMPD.

Orotic Acid Excretion and Arginine Metabolism

The urinary excretion of orotic acid, an intermediate in the pyrimidine biosynthetic pathway, is markedly increased in many inborn errors of the urea cycle and in a number of other disorders involving arginine metabolism. Carbamoyl phosphate, which accumulates within hepatic mitochondria in patients with ornithine transcarbamoylase deficiency, can diffuse to the cytosol and enter the pyrimidine pathway, resulting in greatly increased orotic acid production and excretion. This orotic aciduria also occurs in inborn errors of the mitochondrial ornithine/citrulline transporter, arginase, argininosuccinate synthetase, and argininosuccinate lyase. Increased orotic acid excretion is also found in a number of hypoargininemic states, such as lysinuric protein intolerance. However, orotic aciduria should not be used uncritically as an index of arginine deficiency because it is found in patients with arginase deficiency who exhibit hyperargininemia. Increased orotic acid excretion can also arise as a result of impairments of pyrimidine synthesis, whether brought about by a genetic defect (e.g., in UMP synthase) or by drugs that inhibit the terminal part of the pathway (e.g., allopurinol or 6-azauridine). When used appropriately, measurement of urinary orotic acid is a valuable tool for the study of many derangements of arginine metabolism, including arginine depletion, and to assess the efficacy of therapies used to replete this amino acid.

Pseudo-merohedral twinning in monoclinic crystals of human orotidine-5′-monophosphate decarboxylase

Human UMP synthase is a bifunctional enzyme that catalyzes the penultimate and last steps in the de novo biosynthesis of UMP. In contrast to prokaryotes, UMP synthase from higher eukaryotes combines the orotate phosphoribosyltransferase and the orotidine-5'-monophosphate (OMP) decarboxylase activities on a single polypeptide chain. The decarboxylase activity is unusual in that it represents the fastest rate acceleration of any enzyme studied to date. Although several crystal structures of OMP decarboxylases have been described, the precise decarboxylation mechanism remains elusive. The crystal structure of the OMP decarboxylase domain from human UMP synthase was determined by molecular replacement using data from a highly twinned monoclinic crystal. The space group is P2(1), with unit-cell parameters a = 69.18, b = 61.70, c = 69.17 A, beta = 113.06 degrees. Self-rotation function analysis and various intensity statistics revealed the presence of pseudo-merohedral twinning, but these tests underestimated the true twin fraction of alpha approximately = 0.44. Data analysis, the origin of the twinning and structure determination are discussed.

Japanese Black Cattle with Orotic Aciduria Detected by Gas-Chromatography/Mass-Spectrometry

A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.

A fluoroorotic acid-resistant mutant of Arabidopsis defective in the uptake of uracil

A fluoroorotic acid (FOA)-resistant mutant of Arabidopsis thaliana was isolated by screening M2 populations of ethyl methane sulphonate (EMS)-mutagenized Columbia seed. FOA resistance was due to a nuclear recessive gene, for1-1, which locates to a 519 kb region in chromosome 5. Assays of key regulatory enzymes in de novo pyrimidine synthesis (uridine monophosphate synthase) and salvage biochemistry (thymidine kinase) confirmed that FOA resistance in for1-1/for1-1 plants was not due to altered enzymatic activities. Uptake studies using radiolabelled purines, pyrimidines, and [14C]FOA reveal that for1-1/for1-1 plants were specifically defective in the uptake of uracil or uracil-like bases. To confirm such specificity, genetic crosses show that FOR1 is a distinct locus from FUR1 which encodes a deoxyuridine nucleoside transporter. In addition, for1-1/for1-1 plants were restored to FOA sensitivity by transformation with the Escherichia coli uracil transporter gene uraA driven by the cauliflower mosaic virus (CaMV) 35S promoter. Molecular mapping studies reveal that FOR1 does not correspond to loci belonging to any of the six known nucleobase transporter families identified in the Arabidopsis genome. Moreover, FOR1 does not appear to regulate the transcript levels of either uracil transporter-encoding loci At2g03590 or At2g03530. The above results strongly suggest that the for1-1 mutant allele affects a transport mechanism that is specific for the uptake of uracil.