Molecular Cloning and DNA Analysis of the Orotidine-5′-Phosphate Decarboxylase Gene from the Yeast Saccharomyces exiguus Yp74L-3

The orotidine-5′-phosphate decarboxylase gene of Saccharomyces exiguus Yp74L-3 was cloned as a DNA fragment complementing a ura4 mutation of this yeast. The coding region of the gene is 807 bp in length, and represents 68.7% similarity to the corresponding gene of S. cerevisiae (URA3). The cloned URA4 gene was shown to be located on the 790-kbp Chromosome (chr) VIII of S. exiguus Yp74L-3. The neighbor-joining phylogenetic tree based on the orotidine-5′-phosphate decarboxylase coding sequences indicates that S. exiguus Yp74L-3 is closely related to Kluyveromyces yeasts, as well as to a S. cerevisiae laboratory strain. Received: 4 February 2000 / Accepted: 3 July 2000     

Orotidine-5′-Phosphate Decarboxylase and Pyrophosphorylase of Bean Leaves

This report includes results demonstrating the existence of orotidine-5'-phosphate decarboxylase and orotidine-5'-phosphate pyrophosphorylase in plant leaves. The decarboxylase enzyme, purified 8 fold from leaves of etiolated pinto beans (Phaseolus vulgaris L.), had a pH optimum of 6.3. It was strongly inhibited by 6-azauridine-5'-phosphate; a concentration of 12 mum decreased the reaction rate 60%. The enzyme was not dependent upon magnesium ions or inhibited by p-chloromercuribenzoate. It was present in other parts of the bean plant and was found in young leaves of tomato (Lycopersicon esculentum Mill.) and Canada thistle (Cirsium arvense L.)The enzyme orotidine-5'-phosphate pyrophosphorylase, which catalyzes the formation of orotidine-5'-phosphate from orotic acid and 5-phosphoribosyl-1-pyrophosphate, was found in the etiolated bean leaves, and was also present in the leaves of tomato and Canada thistle. It was stimulated by manganous or magnesium ions and had a pH optimum of 7.2. The K(m) value obtained by varying the concentrations of 5-phosphoribosyl-1-pyrophosphate was 75 mum, and when orotic acid was varied the resulting K(m) was 3.5 mum.The presence of these 2 enzymes in higher plants, combined with previous results with inhibitors and labeled metabolites, indicates that the normal pathway of pyrimidine nucleotide synthesis in higher plants proceeds through orotic acid and OMP.     

Transformation and Functional Expression of the rFCA-RRM2 Gene in Rice

The primary aim of the present study was to investigate the overexpression of the rice (Oryza sativa L. subsp, japonica var. Zhonghua 11) flowering control gene (rFCA-RRM2) in monocotyledonous model rice. Constitutive expression of rFCA-RRM2 from the Actl-5 rice promoter caused late flowering in transgenic rice and increased grain weight that was more than 50% higher than that of control plants, which is the first demonstration of rFCA-RRM2 being able to increase rice production. Late flowering was accompanied by strong phenotype and some morphological modifications. These observations suggest that rFCA-RRM2 is a useful tool for phenotype improvement and yield enhancement in cereal crops.     

Purification and Properties of Orotidine-5′-phosphate Pyrophosphorylase in Micrococcus glutamicus.

A coupled enzyme system of orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase has been purified approximately 30-fold from cell-free extract of Micrococcus glutamicus 534 Co–147 by means of acid treatment and fractionations with ammonium sulfate and ethanol addition, and properties of the enzyme system have been studied.

Optima of pH, temperature and substrate concentrations for the activity of the purified enzyme system have been investigated, and compared with those of the same enzyme system from dried brewer’s yeast. Furthermore, effects of various inhibitors on the enzyme activity have been examined and it has become evident that the enzyme system is completely inactivated by addition of chelating agent such as EDTA, and regenerated by further addition of magnesium ion.     

Identification of guanosine 3′:5′-monophosphate in the fruit of Zizyphus jujuba

Evidence is presented here confirming the identification of guanosine 3′: 5′-monophosphate (c GMP) in the tissue of higher plants. The c GMP activity detected in fruits of Zizyphus jujuba was separated from the c AMP activity also present. The separated sample was extensively purified by Bio-Rad AG 1 × 4 and aluminium oxide CC, and by TLC. The purified sample showed the same physicochemical properties as authentic c GMP by TLC using different solvents and by UV spectroscopy, and was decomposable by cyclic nucleotide-specific phosphodiesterase. The identification was further supported by HPLC. The amount of c GMP present increases 90-fold during fruit ripening.     

a/α-Control of DNA Repair in the Yeast Saccharomyces Cerevisiae: Genetic and Physiological Aspects

It has long been known that diploid strains of yeast are more resistant to γ-rays than haploid cells, and that this is in part due to heterozygosity at the mating type (MAT) locus. It is shown here that the genetic control exerted by the MAT genes on DNA repair involves the a1 and α2 genes, in a RME1-independent way. In rad18 diploids, affected in the error-prone repair, the a/α effects are of a very large amplitude, after both UV and γ-rays, and also depends on a1 and α2. The coexpression of a and α in rad18 haploids suppresses the sensitivity of a subpopulation corresponding to the G(2) phase cells. Related to this, the coexpression of a and α in RAD(+) haploids depresses UV-induced mutagenesis in G(2) cells. For srs2 null diploids, also affected in the error-prone repair pathway, we show that their G(1) UV sensitivity, likely due to lethal recombination events, is partly suppressed by MAT homozygosity. Taken together, these results led to the proposal that a1-α2 promotes a channeling of some DNA structures from the mutagenic into the recombinational repair process.     

Separation and Characterization of Two Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidases from Baker’s Yeast

We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against β-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the β-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.     

Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidase from Baker’s Yeast

Pyridoxamine (pyridoxine) 5′-phosphate oxidase purified from baker’s yeast was found to have a molecular weight of ca, 55,000 daltons based on polyacrylamide gel electrophoresis. The size of the enzyme subunit was analyzed by gel electrophoresis in the presence of sodium dodecylsulfate. This showed that the enzyme was composed of two nonidentical subunits with a molecular weight of 27,000 and 25,000 daltons. Fluorescence titration of the apoenzyme with FMN suggested that the holoenzyme contained one mol of FMN per mol of the enzyme. The K_m value of FMN for apoenzyme was calculated to be ca. 16 nm on both activities of pyridoxamine 5′-phosphate oxidase and pyridoxine 5′-phosphate oxidase.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

The Saccharomyces cerevisiae gene YPR011c encodes a mitochondrial transporter of adenosine 5′-phosphosulfate and 3′-phospho-adenosine 5′-phosphosulfate

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier family, nearly all of which have been functionally characterized. In this study, the identification of the mitochondrial carrier for adenosine 5′-phosphosulfate (APS) is described. The corresponding gene (YPR011c) was overexpressed in bacteria. The purified protein was reconstituted into phospholipid vesicles and its transport properties and kinetic parameters were characterized. It transported APS, 3′-phospho-adenosine 5′-phosphosulfate, sulfate and phosphate almost exclusively by a counter-exchange mechanism. Transport was saturable and inhibited by bongkrekic acid and other inhibitors. To investigate the physiological significance of this carrier in S. cerevisiae, mutants were subjected to thermal shock at 45 °C in the presence of sulfate and in the absence of methionine. At 45 °C cells lacking YPR011c, engineered cells (in which APS is produced only in mitochondria) and more so the latter cells, in which the exit of mitochondrial APS is prevented by the absence of YPR011cp, were less thermotolerant. Moreover, at the same temperature all these cells contained less methionine and total glutathione than wild-type cells. Our results show that S. cerevisiae mitochondria are equipped with a transporter for APS and that YPR011cp-mediated mitochondrial transport of APS occurs in S. cerevisiae under thermal stress conditions.     

Differential Changes in Pyridoxine 5′-Phosphate Oxidase Immunoreactivity and Protein Levels in the Somatosensory Cortex and Striatum of the Ischemic Gerbil Brain

Pyridoxal 5'-phosphate (PLP) is an important cofactor in a wide range of biochemical reactions, such as the metabolism of various amino acids, including GABA. PLP is synthesized by the oxidation of pyridoxine 5'-phosphate (PNP), which is catalyzed by PNP oxidase (PNPO). We observed the changes in cresyl violet-positive neurons, PNPO immunoreactivity and PNPO protein levels in the somatosensory cortex and striatum in gerbils after 5 min of transient forebrain ischemia. Cresyl violet-positive neurons showed condensed cytoplasm in the somatosensory cortex and lateral part of the striatum at 2 days after ischemia/reperfusion. PNPO immunoreactivity began to increase in neurons in layers III and V at 3 h after ischemia/reperfusion and this immunoreactivity was significantly increased at 12 h after ischemia/reperfusion. Thereafter, PNPO immunoreactivity decreased with time after ischemia/reperfusion. PNPO-immunoreactive neurons were only slightly detected in the lateral part of the striatum at 12 h after ischemia/reperfusion. In addition, the PNPO protein levels in both the somatosensory cortex and striatum homogenates peaked at 12 h after ischemia/reperfusion. These results indicate that PNPO is significantly increased in the ischemic somatosensory cortex and lateral part of the striatum, and this change in the level of PNPO may be associated with the neuronal damage induced by ischemia.     

Cloning, Functional Identification and Sequence Analysis of Flavonoid 3′-hydroxylase and Flavonoid 3′,5′-hydroxylase cDNAs Reveals Independent Evolution of Flavonoid 3′,5′-hydroxylase in the Asteraceae Family

Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Phylogenetic analysis of known sequences of F3′H and F3′5′H indicated that F3′5′H was recruited from F3′H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3′Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3′H activity but surprisingly, three recombinant proteins showed F3′5′H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3′5′H. Furthermore, sequence analysis of these unusual F3′5′H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3′5′H is necessary for the synthesis of 3′,4′,5′-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3′5′H probably reflects the adaptive value of efficient attraction of insect pollinators.     

Functional Identification of a Putative β-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus acidophilus

The putative β-galactosidase gene (lacZ) of Lactobacillus acidophilus has a very low degree of homology to the Escherichia coli β-galactosidase gene (lacZ) and locates in a special lac gene cluster which contains two β-galactosidase genes. No functional characteristic of the putative β-galactosidase has been described so far. In this study, the lacZ gene of L. acidophilus was hetero-expressed in E. coli and the recombinant protein was purified by a three-step procedure. The product of the lacZ gene was also extracted from L. acidophilus ATCC 4356 and active staining was carried out. The enzymatic properties of the purified recombinant LacZ were assayed. The results of hetero-expression showed the recombinant LacZ without tag had β-galactosidase activity. The purified recombinant LacZ had a specific activity of 43.2 U/mg protein. The result of active staining showed that the functional product of the lacZ gene did exist in L. acidophilus. The L. acidophilus β-galactosidase (LacZ) had an optimal pH of 6, an optimal temperature of 37°C and could hydrolyze 73% of lactose in milk in 30 h at 10°C. The L. acidophilus β-galactosidase (LacZ) was identified as cold-adapted β-galactosidase in this study for the first time, and may be useful for lactose removal from dairy products at low temperatures.     

Identification of the 5′‐Nucleotidase Activity Altered in Neurological Syndromes

5′‐nucleotidases comprise a family of enzymes involved in the regulation of intracellular and extracellular nucleotide concentration. There is increasing knowledge about an involvement of these activities in the aetiology of neurological disorders. In this paper we present a protocol for the identification of the altered enzyme in fibroblasts primary culture from patients and controls.