Accelerated transcription of PRPS1 in X-linked overactivity of normal human phosphoribosylpyrophosphate synthetase. (1/125)

Phosphoribosylpyrophosphate (PRPP) synthetase (PRS) superactivity is an X-linked disorder characterized by gout with overproduction of purine nucleotides and uric acid. Study of the two X-linked PRS isoforms (PRS1 and PRS2) in cells from certain affected individuals has shown selectively increased concentrations of structurally normal PRS1 transcript and isoform, suggesting that this form of the disorder involves pretranslational dysregulation of PRPS1 expression and might be more appropriately termed overactivity of normal PRS. We applied Southern and Northern blot analyses and slot blotting of nuclear runoffs to delineate the process underlying aberrant PRPS1 expression in fibroblasts and lymphoblasts from patients with overactivity of normal PRS. Neither PRPS1 amplification nor altered stability or processing of PRS1 mRNA was identified, but PRPS1 transcription was increased relative to GAPDH (3- to 4-fold normal in fibroblasts; 1.9- to 2.4-fold in lymphoblasts) and PRPS2. Nearly coordinate relative increases in each process mediating transfer of genetic information from PRPS1 transcription to maximal PRS1 isoform expression in patient fibroblasts further supported the idea that accelerated PRPS1 transcription is the major aberration leading to PRS1 overexpression. In addition, modulated relative increases in PRS activities at suboptimal Pi concentration and in rates of PRPP and purine nucleotide synthesis in intact patient fibroblasts indicate that despite an intact allosteric mechanism of regulation of PRS activity, PRPS1 transcription is a major determinant of PRPP and purine synthesis. The genetic basis of disordered PRPS1 transcription remains unresolved; normal- and patient-derived PRPS1s share nucleotide sequence identity at least 850 base pairs 5' to the consensus transcription initiation site.  (+info)

Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana. (2/125)

cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison and phylogenetic analysis indicate that these two isozymes belong to a novel class of phosphoribosyl diphosphate synthases.  (+info)

Effects of X chromosome number and parental origin on X-linked gene expression in preimplantation mouse embryos. (3/125)

Diploid androgenetic mouse embryos, possessing two sets of paternally inherited chromosomes, and control fertilized embryos were used to examine the relative effects of X chromosome number and parental chromosome origin on androgenone viability and X-linked gene expression. A significant difference in efficiency of blastocyst formation was observed between XX and XY androgenones in some experiments, but this difference was not uniformly observed. Significant effects of both X chromosome number and parental origin on X-linked gene expression were observed. Male and female control embryos expressed the XIST: RNA initially. This expression was followed by a preferential reduction in XIST: RNA abundance in male embryos, indicating that dosage compensation for the X chromosome may normally require the downregulation of XIST: RNA expression in male embryos, in conjunction with the production of stable XIST: transcripts in female embryos. By the late blastocyst stage, XX control embryos expressed significantly more XIST: RNA than did XY embryos. Unlike their normal counterparts, XX androgenones did not express significantly more XIST: RNA than did XY androgenones at the late blastocyst stage. Androgenones exhibited severe repression of the Pgk1 gene, but during development to the late blastocyst stage Pgk1 mRNA expression increased in XX androgenones and decreased in XY androgenones. Thus, the initial repression of the Pgk1 gene in XX androgenones was lost as the XIST: RNA declined in abundance, and this loss was correlated with a failure of XX androgenones to express significantly more XIST: RNA than did XY androgenones. These results indicate that androgenones may lack a factor that is expressed from the maternal genome and required for dosage compensation in preimplantation embryos. The results also indicate that early dosage compensation in preimplantation embryos may normally be reversible, thus providing flexibility to meet different developmental requirements of the embryonic and extraembryonic lineages.  (+info)

Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase. (4/125)

A steady state kinetic investigation of the P(i) activation of 5-phospho-d-ribosyl alpha-1-diphosphate synthase from Escherichia coli suggests that P(i) can bind randomly to the enzyme either before or after an ordered addition of free Mg(2+) and substrates. Unsaturation with ribose 5-phosphate increased the apparent cooperativity of P(i) activation. At unsaturating P(i) concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with P(i) directs the subsequent ordered binding of Mg(2+) and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg(2+) and substrates via a slow pathway where P(i) binds to the enzyme last. The random mechanism for P(i) binding was further supported by studies with competitive inhibitors of Mg(2+), MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying P(i) at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing P(i) concentrations. Results from ADP inhibition of P(i) activation suggest that these effectors compete for binding to a common regulatory site.  (+info)

The importance of the five phosphoribosyl-pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p. (5/125)

Phosphoribosyl-pyrophosphate synthetase (Prs) catalyses the synthesis of phosphoribosyl pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Deltaprs1 and Deltaprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.  (+info)

Characterization of the promoter of PRS1 in Saccharomyces cerevisiae identifies three regions potentially involved in control of expression. (6/125)

The transcription initiation site of the Saccharomyces cerevisiae PRS1 gene was mapped at -179 bp. Measurement of beta-galactosidase activity of the successively deleted PRS1 promoter linked to lacZ and integrated at the ura3 locus defined three DNA regions involved in the control of PRS1 expression. Gel shift analysis confirmed the data.  (+info)

Protein factors associated with the SsrA.SmpB tagging and ribosome rescue complex. (7/125)

SsrA RNA acts as a tRNA and mRNA to modify proteins whose synthesis on ribosomes has stalled. Such proteins are marked for degradation by addition of peptide tags to their C termini in a reaction mediated by SsrA RNA and SmpB, a specific SsrA-RNA binding protein. Evidence is presented here for the existence of a larger ribonucleoprotein complex that contains ribosomal protein S1, phosphoribosyl pyrophosphate synthase, RNase R, and YfbG in addition to SsrA RNA and SmpB. Biochemical, genetic, and phylogenetic results suggest potential roles for some of these factors in various stages of the ribosome rescue and tagging process and/or the presence of functional interactions between one or more of these proteins and SsrA.  (+info)

Class II recombinant phosphoribosyl diphosphate synthase from spinach. Phosphate independence and diphosphoryl donor specificity. (8/125)

A recombinant form of spinach (Spinacia oleracea) phosphoribosyl diphosphate (PRPP) synthase isozyme 3 resembling the presumed mature enzyme has been synthesized in an Escherichia coli strain in which the endogenous PRPP synthase gene was deleted, and has been purified to near homogeneity. Contrary to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6.  (+info)