Crystallization and preliminary X-ray crystallographic analysis of orotate phosphoribosyltransferase from Helicobacter pylori.
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Orotic acid phosphoribosyltransferase (PyrE) (EC 2.4.2.10) is a key enzyme in de novo uridine monophosphate (UMP) biosynthesis. It catalyzes the reaction between orotic acid and 5-phosphoribosyl-1-pyrophosphate (PRPP) to yield orotidine monophosphate (OMP), which is transformed to uridine monophosphate by decarboxylation. H. pylori PyrE was crystallized at 294 +/- 1 K by the hanging drop vapor-diffusion method. The crystals belong to the space group P2(1)2(1)2(1) with unit-cell dimensions a = 95.8, b = 104.9, c = 281.1 A, alpha = beta = gamma = 90 degrees. A set of diffraction data was collected to 3.29 A resolution using synchrotron X-ray radiation. (+info)
Comparative gene genealogical analyses of strains of serotype AD identify recombination in populations of serotypes A and D in the human pathogenic yeast Cryptococcus neoformans.
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Cryptococcus neoformans is a major pathogen of humans throughout the world. Using commercial monoclonal antibodies to capsular epitopes, strains of C. neoformans manifest five serotypes: A, B, C, D and AD. Previous studies demonstrated significant divergence among serotypes A, B, C and D, which are typically haploid. In contrast, most strains of serotype AD are diploid or aneuploid and result from recent hybridization between strains of serotypes A and D. Whether serotypes A, B, C and D represent strictly asexual lineages is not known. Using comparative genealogical analyses of two genes, the authors investigated whether recombination occurred among strains within serotypes A and D. For each of 14 serotype AD strains, a portion (642 bp) of the orotidine monophosphate pyrophosphorylase (URA5) gene was cloned and sequenced. Each of these 14 strains contained two different alleles and sequences for both alleles were obtained. The URA5 gene genealogy was compared to that derived from the laccase (LAC) gene, which was reported recently for the same 14 strains. For both genes, each of the 14 serotype AD strains contained two phylogenetically distinct alleles: one allele was highly similar to those from serotype A strains and the other to alleles from serotype D strains. However, within both the serotype A allelic group and the serotype D allelic group, there was significant incongruence between genealogies derived from URA5 and LAC. The results suggest recombination in natural populations of both serotypes A and D. (+info)
Effect of carbon source on pyrimidine biosynthesis in Pseudomonas alcaligenes ATCC 14909.
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The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in Pseudomonas alcaligenes ATCC 14909 was investigated. The de novo pyrimidine biosynthetic enzymes were measured in extracts of P. alcaligenes ATCC 14909 cells and of cells from an auxotroph deficient for orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic enzyme activities in ATCC 14909 were influenced by pyrimidine supplementation to the culture medium but not by the carbon source present. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be controlled at the transcriptional level by a pyrimidine-related compound. Its regulation seemed to be subject to less transcriptional control by a pyrimidine-related compound than what was observed in the closely related species Pseudomonas pseudoalcaligenes. (+info)
Extensive allelic variation in Cryptococcus neoformans.
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The orotidine monophosphate pyrophosphorylase (OMPPase) gene locus of the DNA of 13 Cryptococcus neoformans var. neoformans strains, including 10 recent clinical isolates, was studied by using restriction fragment length polymorphisms and nucleotide sequence analysis. The OMPPase locus (URA5) is highly polymorphic, and at least six alleles were identified. The nucleotide sequences of some alleles differed by up to 5%. The majority of the nucleotide polymorphisms in the protein-coding region occurred at the third codon position and were silent. The low frequency of replacement nucleotide substitutions relative to silent nucleotide substitutions implied that there is strong selection against amino acid changes in OMPPase. The allelic variation suggested that there is extensive genomic diversity among C. neoformans clinical isolates from one geographic area. The various alleles are potentially useful markers in the study of the population structure, epidemiology, and pathogenesis of C. neoformans strains. (+info)
Attenuation in the rph-pyrE operon of Escherichia coli and processing of the dicistronic mRNA.
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We have substituted on a plasmid the native promoter of the Escherichia coli rph-pyrE operon with an inducible transcription-initiation signal. The plasmid was used to study the mRNA chains derived from the operon at different intracellular concentrations of UTP and as a function of time following induction of transcription. The results showed that dicistronic rph-pyrE mRNA was formed when the UTP pool was low, and that a monocistronic rph mRNa was the major transcription product in high-UTP pools, thus supporting an UTP-controlled attenuation mechanism for regulation of pyrE gene expression. However, the dicistronic rph-pyrE transcript was rapidly processed into two monocistronic mRNA units, and a cleavage site was mapped near the attenuator in the intercistronic region, close to the site where transcription was terminated in high-UTP pools. Furthermore, the major 3' end of the pyrE mRNA was mapped near a palindromic structure of similarity to the family of repetitive extragenic palindromic sequences, 35 nucleotide residues after stop codon of the pryE gene. (+info)
Both gene expression for orotate phosphoribosyltransferase and its ratio to dihydropyrimidine dehydrogenase influence outcome following fluoropyrimidine-based chemotherapy for metastatic colorectal cancer.
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Activation of 5-fluorouracil into its nucleotides requires phosphorylation by three pathways involving orotate phosphoribosyl-transferase (OPRT), uridine phosphorylase (UP), or thymidine phosphorylase (TP). In this study, we investigated the association between gene expressions of these three enzymes and antitumour effect. Gene expressions in primary colorectal tumours were analysed by a real-time reverse transcriptional-polymerase chain reaction method in 37 patients receiving oral treatment of tegafur-uracil and leucovorin for metastatic diseases. The median values of OPRT mRNA expressions were 1.39 and 0.85 for responding tumours and nonresponding tumours, respectively, showing a statistically significant difference (P=0.0008). Responding tumours had statistically lower expressions of TP mRNA than nonresponding tumours (P=0.006). However, there was no difference in UP mRNA expression between responding and nonresponding tumours. Patients with high OPRT (>/=1.0) gene expression survived longer than those with low OPRT (<1.0) expression. Dihydropyrimidine dehydrogenase (DPD) gene expressions were measured. Responding tumours had a statistically higher OPRT/DPD ratio than the nonresponding ones (P=0.003). When the median value of the OPRT/DPD ratio was selected as the cutoff value, patients with a high OPRT/DPD ratio survived statistically longer than those with a low ratio (P=0.0014). In conclusion, both the expression of OPRT gene and the OPRT/DPD ratio might be useful as predictive parameters for the efficacy of fluoropyrimidine-based chemotherapy for metastatic colorectal cancer. (+info)
Construction of a complete URA5 deletion strain of a human pathogenic yeast Cryptococcus neoformans.
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Cryptococcus neoformans is an opportunistic human pathogen, which infects the central nervous system causing the fatal disease, meningitis. In order to understand the genetic background of this human pathogen, the basic molecular manipulation techniques of deletion, overexpression, and so on have been developed. URA5, a gene encoding orotate phosphoribosyltransferase, has frequently been used to introduce foreign gene fragments by complementing ura5 mutant strains, which are not, however, stable; reversion to uracil prototroph is thus frequently observed on selective condition. The high possibility of reversion makes it inconvenient to use this mutation to identify appropriate transformants and thus, manipulation in molecular genetics. We report here the isolation of a stable ura5 mutant of C. neoformans, designated as TAD1, by eliminating the URA5 gene by homologous recombination using the biolistic DNA delivery system. The availability of the stable ura5 mutant offers the advantage that no spontaneous reversion occurs so that a satisfactory rate of homologous recombination can be achieved. The strain will allow efficient genomic analysis in C. neoformans. (+info)
Prognostic significance of orotate phosphoribosyltransferase activity in bladder carcinoma.
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BACKGROUND: 5-Fluorouracil (5-FU), an antitumor agent, is used clinically against a variety of malignancies, including bladder carcinoma. 5-FU is a prodrug, and orotate phosphoribosyltransferase (OPRT) is the principal enzyme that converts 5-FU directly into an active antitumor metabolite, 5-fluoro-2'-deoxyuridine 5'-monophosphate. In addition, OPRT is the key enzyme in the de novo DNA and RNA synthetic process. To the authors' knowledge, little is known regarding the significance of OPRT in various malignancies, including bladder carcinoma. The authors analyzed the activity levels of OPRT in 60 bladder carcinomas and evaluated the association between the level of OPRT activity and the stage and grade status of bladder carcinoma. They also examined the prognostic significance of OPRT activity in patients with bladder carcinoma and the correlation between OPRT activity levels in bladder carcinoma cells and the sensitivity of those cells to 5-FU. METHODS: OPRT activity levels in nonfixed, fresh-frozen specimens of bladder carcinoma and normal bladder were determined enzymatically using a 5-FU phosphorylation assay. The sensitivity of bladder cells to 5-FU was assessed using a microculture tetrazolium dye assay. RESULTS: The activity levels of OPRT were approximately 7.5-fold higher in bladder carcinoma specimens compared with the activity levels in normal bladder specimens. OPRT activity in muscle-invasive bladder carcinoma was 2-fold higher compared with the activity in superficial bladder carcinoma (classified as Ta and T1). In addition, the activity of OPRT in T1 bladder carcinoma was 2-fold higher compared with the activity in Ta bladder carcinoma. The level of OPRT activity in Grade 3 bladder carcinoma was 6-fold and 2-fold higher compared with the activity in Grade 1 and Grade 2 bladder carcinoma, respectively. Patients who had Ta and T1 bladder carcinoma with low OPRT activity had a longer postoperative tumor free period compared with patients who had bladder carcinoma with high OPRT activity in the 3-year follow-up. There was a positive association between the activity levels of OPRT and thymidylate synthase/thymidine kinase, which are the key enzymes in the de novo/salvage DNA synthetic process. OPRT activity in bladder carcinoma cells was correlated positively with their sensitivity to 5-FU. CONCLUSIONS: to the authors' knowledge, the current study is the first to demonstrate that OPRT activity levels in bladder carcinoma were higher compared with its activity in the normal bladder tissues and that OPRT activity levels were correlated positively with the stage and grade of bladder carcinoma. In addition, high OPRT activity levels in patients with superficial bladder carcinoma predicted early recurrence and high sensitivity to 5-FU. These results suggest that the level of OPRT activity may be used both as a prognostic parameter and as a predictive indicator for 5-FU efficacy in patients with bladder carcinoma and that OPRT may be a molecular therapeutic target in bladder carcinoma. (+info)