The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

Studies of the orotidine 5′-monophosphate decarboxylase activity of crude extracts of human cells

The specific activity of orotidine 5-monophosphate (OMP) decarboxylase in cultured human fibroblasts is an exponential function of the concentration of cell protein in the extract. Low concentrations of uridylic or cytidylic acid augment the catalytic activity of dilute solutions of cell extract but not of concentrated ones. In the presence of urea, specific activity becomes independent of protein concentration, and uridylic or cytidylic acid augments activity over all concentrations of cell extract. These results, as well as other observations, suggest that the decarboxylase may be composed of subunits which are in dynamic equilibrium with an aggregate. At least one of the subunits is likely to have catalytic activity for the reaction, though less activity than the aggregate. The effect of the mutant gene for orotic aciduria on OMP decarboxylase is easily demonstrated when cell extracts are assayed in either the presence or the absence of urea.Supported by Program Project Grants 1-PO1-GM 15419 and GM 18153-1, National Institutes of Health, United States Public Health Service.     

Cloning and Sequence Analysis of cDNA 3′-end in Wheat DREB Gene

Adverse environmental conditions, such as drought and low temperature, greatly affect the plant‘s growth, development, survival and geographical distribution. They have become the critical factors which limit crop quality and productivity. To improve crop tolerance to these abiotic stresses,breeding researches have been focused on     

Ls-RRNA 5′ Nucleotide Sequence of Rohdea japonica (Liliaceae) and its Evolutionary Significance

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Transformation system for a wastewater treatment yeast, Hansenula fabianii J640: isolation of the orotidine-5′-phosphate decarboxylase gene (URA3 ) and uracil auxotrophic mutants

A transformation system for Hansenula fabianii J640, a commonly used wastewater treatment yeast, was constructed. As a host cell, a uracil auxotrophic mutant designated as H. fabianii J640 u-1, which was confirmed to have a mutation at the locus of the gene for orotidine-5′-phosphate (OMP) decarboxylase (URA3), was obtained by positive selection using 5-fluoroorotic acid. A plasmid named pHFura3, which includes a 795-bp open-reading frame of the OMP decarboxylase H. fabianii, was obtained by complementation of the Escherichia colipyrF mutant. pHFura3 could transform H. fabianii J640 u-1 by a non-homologous and frequently multicopy integration into the host genomic DNA. Received: 25 March 1997 / Received revision: 12 July 1997 / Accepted: 1 August 1997     

The 5′ Nucleotide Sequence of Vesicular Stomatitis Viral RNA

The 5' nucleotide sequence of vesicular stomatitis viral RNA is pppApCpGp....     

Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5′-monophosphate decarboxylase

Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having a COOH-terminal orotate phosphoribosyltransferase (OPRT) and an NH₂-terminal orotidine 5′-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, were stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (k_cat/K_m). The k_cat was selectively enhanced up to three orders of magnitude, while the K_m was not much affected and remained at low μM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a “super-enzyme” with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivities and metabolic functions on molecular evolution.     

Sequence simplicity and evolution of the 3′ untranslated region of the histone H1° Gene

The H1° gene has a long 3′ untranslated region (3′UTR) of 1,125 nucleotides in the rat and 1,310 in humans. Analysis of the sequences shows that they have features of simple DNA that suggest involvement of replication slippage in their evolution. These features include the length imbalance between the rat and human sequences; the abundance of single-base repeats, two-base runs and other simple motifs clustered along the sequence; and the presence of single-base repeat length polymorphisms in the rat and mouse sequences. Pairwise comparisons show numerous short insertions/deletions, often flanked by direct repeats. In addition, a proportion of short insertions/deletions results from length differences in conserved single-base repeats. Quantification of the sequence simplicity shows that simple sequences have been more actively incorporated in the human lineage than in the rodent lineage. The combination of insertions/deletions and nucleotide substitutions along the sequence gives rise to three main regions of homology: a highly variable central region flanked by more conserved regions nearest the coding region and the polyA addition site. Correspondence to: P. Suau     

Isolation and Sequence Analysis of Bovine αsl-Casein cDNA Clone

Nucleoside oxidase, a novel nucleoside oxidizing enzyme has been purified from a crude extract of Pseudomonas maltophilia LB-86 by a ammonium sulfate fractionation, heat treatment, column chromatography on DEAE-Toyopearl, and gel filtration twice on Sephacryl S-200. The overall purification was approximately 60-fold with a yield of 35 %. The purified enzyme gave a single protein band on acrylamide gel electrophoresis.

Reaction products from inosine were identified as inosine-5′-aldehyde and inosine-5′-carboxylic acid. The enzyme catalyzed the oxidation of inosine to inosine-5′-carboxylic acid via inosine-5′-aldehyde using molecular oxygen as a primary electron acceptor with no formation of hydrogen peroxide.     

Investigation into mechanism of orotidine 5′-monophosphate decarboxylase enzyme by MM-PBSA/MM-GBSA and molecular docking

In this work, we studied the binding affinity of orotidine 5′-monophosphate (OMP) and 6-hydroxy-UMP (BMP) for Saccharomyces cerevisiae orotidine 5′-monophosphate decarboxylase (OMPDC) enzyme by using Molecular Mechanics-Poisson–Boltzmann Surface Area (MM-PBSA) and the Molecular Mechanics-Generalised Born Surface Area (MM-GBSA) calculations. In all simulations, Asp91, which is an important residue in the enzyme active site, was considered in both anionic (present in the native form of the enzyme) and neutral states. A series of 10-ns molecular dynamics simulations were performed for the four OMPDC–ligand complexes, two ligand-free enzymes and two free ligands, followed by MM-PBSA/MM-GBSA calculations on the collected snapshots, and molecular docking calculations using the free enzymes and ligands. The results of MM-PBSA/MM-GBSA calculations indicate that all of the OMPDC–ligand complexes form favourable systems in water, which is in agreement with corresponding experimental data. The results of the MM-PBSA and molecular docking methods also showed that OMPDC–BMP complexes, transition state analogue and inhibitor of the OMPDC enzyme have the highest binding affinities. The fact that in the native anionic state BMP shows a higher binding affinity compared with the substrate suggests the contribution of a transition state stabilisation mechanism in the debatable catalytic mechanism of the OMPDC enzyme.     

Molecular analysis of the Aureobasidium pullulans ura3 gene encoding orotidine-5′-phosphate decarboxylase and isolation of mutants defective in this gene

Orotidine-5′-phosphate decarboxylase (OMP decarboxylase) catalyses the final step in the pyrimidine biosynthesis, the conversion of orotidine-5′-phosphate (OMP) to uridine-5′-phosphate. The ura3 gene of Aureobasidium pullulans, encoding OMP decarboxylase, was isolated from an Aureobasidium genomic library constructed in the plasmid pBlueskriptSK⁻. The ura3 gene of A. pullulans has an open reading frame of 271 amino acid residues. Analysis of the sequence revealed the presence of two introns. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Aspergillus niger, Neurospora crassa, Phycomyces blakesleeanus and Homo sapiens. The ura3 gene is the third Aureobasidium gene that has been cloned and analysed. We have also isolated ura3 mutants by selection of ethyl methanesulphonate mutagenised cells on 5-fluoroorotic acid. Transformation of these A. pullulans mutant strains to prototrophy showed the functionality of the cloned gene. Received: 16 July 1999 / Revision received: 20 September 1999 / Accepted: 24 September 1999     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Template-directed oligomerization of 3-isoadenosine 5′-phosphate

Summary Template-directed oligomerization of an activated derivative of 3-isoadenosine 5-phosphate (piA) on polyuridylic acid [poly(U)] was studied. The reaction of ImpiA is more efficient than the corresponding reaction of ImpA, and produces 3–5-linked oligomers while the reaction of ImpA gives only 2–5-linked oligomers. The base pairing between piA and poly(U) in this system is probably of the Hoogsteen type (involving the 6-amino group and N7 of 3-isoadenosine) rather than of the Watson-Crick type.