Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae: combinatorial expression of the five PRS genes in Escherichia coli

The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no detectable PRPP synthase activity. In contrast PRPP could be detected in growing cells containing PRS1 PRS2, PRS1 PRS3, PRS5 PRS2, or PRS5 PRS4. These apparent conflicting results indicate that, apart from the PRS1 PRS3-specified enzyme, PRS-specified enzyme is functional in vivo but unstable when released from the cell. Certain combinations of three PRS genes appeared to produce an enzyme that is stable in vitro. Thus, extracts of strains harboring PRS1 PRS2 PRS5, PRS1 PRS4 PRS5, or PRS2 PRS4 PRS5 as well as extracts of strains harboring combinations with PRS1 PRS3 contained readily assayable PRPP synthase activity. The data indicate that although certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. The activity of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5 was found to be dependent on Pi, to be temperature-sensitive, and inhibited by ADP.     

Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in Saccharomyces cerevisiae

The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es).     

PRS5, the Fifth Member of the Phosphoribosyl Pyrophosphate Synthetase Gene Family in Saccharomyces cerevisiae, Is Essential for Cell Viability in the Absence of either PRS1 or PRS3

In Saccharomyces cerevisiae, an open reading frame, YOL061w, encodes a polypeptide with sequence similarity to the four known 5-phosphoribosyl-1(α)-pyrophosphate synthetase (PRS) genes since it contains a divalent cation binding site and a phosphoribosyl pyrophosphate binding site. We regard YOL061w as the fifth member of the PRS gene family, PRS5. Loss of Prs5p has a significant impact on PRS enzyme activity, causing it to be reduced by 84%. On the other hand, Δprs5 strains are not affected in growth or in the size of their nucleotide pools. However, simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 rendered the strains inviable, which implies that PRS5 plays an important role in the maintenance of PRS function in S. cerevisiae.     

Cloning of cDNAs for human phosphoribosylpyrophosphate synthetases 1 and 2 and X chromosome localization of PRPS1 and PRPS2 genes

Cloned cDNAs representing the entire, homologous (80%) translated sequences of human phosphoribosylpyrophosphate synthetase (PRS) 1 and PRS 2 cDNAs were utilized as probes to localize the corresponding human PRPS1 and PRPS2 genes, previously reported to be X chromosome linked. PRPS1 and PRPS2 loci mapped to the intervals Xq22-q24 and Xp22.2-p22.3, respectively, using a combination of in situ chromosomal hybridization and human x rodent somatic cell panel genomic DNA hybridization analyses. A PRPS1-related gene or pseudogene (PRPS1L2) was also identified using in situ chromosomal hybridization at 9q33-q34. Human HPRT and PRPS1 loci are not closely linked. Despite marked cDNA and deduced amino acid sequence homology, human PRS 1 and PRS 2 isoforms are encoded by genes widely separated on the X chromosome.     

Proteomic characterization in the hippocampus of prenatally stressed rats

Rats exposed to early life stress are considered as a valuable model for the study of epigenetic programming leading to mood disorders and anxiety in the adult life. Rats submitted to prenatal restraint stress (PRS) are characterized by an anxious/depressive phenotype associated with neuroadaptive changes in the hippocampus. We used the model of PRS to identify proteins that are specifically affected by early life stress. We therefore performed a proteomic analysis in the hippocampus of adult male PRS rats. We found that PRS induced changes in the expression profile of a number of proteins, involved in the regulation of signal transduction, synaptic vesicles, protein synthesis, cytoskeleton dynamics, and energetic metabolism. Immunoblot analysis showed significant changes in the expression of proteins, such as LASP-1, fascin, and prohibitin, which may lie at the core of the developmental programming triggered by early life stress.     

Primary structure of E. coli alanine transfer RNA: relation to the yeast phenylalanyl tRNA synthetase recognition site

Nucleotide sequence comparison of tRNAs aminoacylated by yeast phenylalanyl tRNA synthetase (PRS) have lead to the proposal that the specific nucleotides of the dihydrouridine (diHU) stem region and adenosine at the fourth position from the 3′ end are involved in the PRS recognition site. Kinetic analysis and enzymatic methylation have shown that the size of the diHU loop and the methylation of guanine at position 10 from the 5′ end both directly affect the PRS aminoacylation kinetics. $\text{E. coli}$ tRNA₁^A1a, which is aminoacylated by PRS, should therefore have 1- the specific nucleotides of the diHU stem region and, 2- adenosine at position 4 from the 3′ end. The PRS aminoacylation kinetics of this tRNA indicates that this molecule 3- has a diHU loop of 8 nucleotides and 4- has an unmethylated guanine at position 10 from the 5′ end. We report here the complete sequence of $\text{E. coli}$ tRNA₁^A1a and confirmation of each of these four predictions.     

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell. Received: 15 July 1996 / Accepted: 24 October 1996     

Effect of the catecholestrogen 2-hydroxyestradiol on the renin-angiotensin system in the rat

The effects of the catecholestrogen 2-hydroxyestradiol (250 and 500 micrograms/day, each for 7 days) on plasma renin substrate (PRS), activity (PRA) and concentration (PRC) were studied in male rats as compared with those of estradiol (250 micrograms/day, for 7 days) and vehicle alone (for 7 days). Pre-treatment levels of PRS, PRA, PRC and the PRA/PRC ratio were similar in four groups. After vehicle treatment, PRS, PRA, PRC and the PRA/PRC ratio remained unchanged. Estradiol treatment, however, produced an increase in PRS, an increase in PRA but no change in PRC. The PRA/PRC ratio after estradiol treatment was high. On the other hand, 2-hydroxyestradiol treatment caused no increase in PRS at a daily dose of 250 micrograms and a slight but significant increase in PRS at a daily dose of 500 micrograms. This treatment also produced increases in PRA as well as PRC at the two daily doses. These increases in PRA and PRC tended to be higher at a daily dose of 500 micrograms than at a daily dose of 250 micrograms. The PRA/PRC ratios after 2-hydroxyestradiol treatment were unaltered at the two daily doses. It is concluded that, while 2-hydroxyestradiol is less active in increasing PRS than estradiol, the compound is capable of increasing PRC.     

In situ characterization of mercury-resistant growth-promoting fluorescent pseudomonads

Pseudomonas fluorescens strains PRS9 and GRS1 (wild type) were made mercury resistant PRS9Hg(r) (147 microM HgCl2) and GRS1Hg(r) (55 microM HgCl2), respectively, in King's medium by enrichment selection and their in situ root colonization studies were carried out. Mercury resistant mutant of PRS9 was stable and resulted in significant increase in root and shoot fresh weight (P < 0.05). Both the mutants are positive for indoleacetic acid (IAA), 'P' solubilization and siderophore production. PRS9, potent 'P' solubilizer, exhibited higher 'P' solubilization as compared to GRS1. After 2 weeks of inoculation, the population level of wild type PRS9 and its mercury resistant mutants has increased (50 fold). Mercury resistance has no adverse effect on the growth promoting properties of mutants besides being comparable in its morphological and physiological properties with their wild type counterpart. Furthermore, mercury resistant character facilitates rhizospheric competition and thus helpful for establishment of growth promoting strains where metal ions are either limiting and/or present at toxic level.     

Complete nucleotide sequence of human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) cDNA and a comparison with human and rat PRPS gene families.

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) were isolated from a glioblastoma cell line MGC 1 cDNA library. The longest clone contained 2,075 base pairs (bp) almost covering the 2.3-kb mRNA and the base sequence of the coding region (954 bp) had a 92.0% sequence homology with that of rat PRS I cDNA. The deduced amino acid sequences were identical between human and rat PRS I. This perfect conservation has heretofore not been reported for other enzymes involved in nucleotide metabolism and glycolysis. A comparison with other isoforms of this enzyme, PRS II and PRS III, showed that the human PRS I was 79.9 and 92.2% homologous in the coding sequence and 95.3 and 94.0% in the deduced amino acid sequence to human PRS II and PRS III, respectively. The high value of the synonymous difference between PRS I and PRS II cDNAs places their time of divergence long before that of the radiation of mammals. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago.     

Molecular cloning of the yeast proteasome PRS2 gene identical to the suppressor gene scl1+

The YC7-alpha gene encoding a subunit of yeast proteasomes named PRS2 has been isolated and sequenced. Southern blot analysis after electrophoretic separation of yeast chromosomal DNAs showed that the PRS2 gene is located in chromosome VII, unlike the PRS1 gene for subunit YC1, is located on chromosome XV. Surprisingly, the overall structure of the PRS2 gene was found to be identical to that of the suppressor scl1+ gene restoring the SCL1-1 mutation that suppresses crl3 cycloheximide-resistant, temperature-sensitive lethality. The identity of these two genes indicates that the suppressive role of the scl1+ gene product on crl3 mutation could be attributed to proteasomal function.     

Effect of an AM fungal consortium and Pseudomonas on the growth and nutrient uptake of Eucalyptus hybrid

 Eucalyptus is an important tree species used for afforestation of large tracts of marginal and wastelands. Eucalyptus-arbuscular mycorrhizal fungal (AMF) interactions in seedling establishment and growth promotion have been inadequately dealt with. Efforts were made to assess the role of AMF-pseudomonad (PRS9, plant growth promotory fluorescent Pseudomonas) interactions in growth promotion and nursery establishment of E. hybrid. Seedlings were subjected to six different treatments: (i) uninoculated control, (ii) 400 AM spores, (iii) 800 AMF spores, (iv) PRS9 (v) 400 AMF spores + PRS9, (vi) 800 AMF spores + PRS9, with the different P regimes of 10, 20 and 30 ppm. Root length, shoot length, root and shoot fresh and dry weights were maximal at 400 AMF spores and 20 ppm soil P. Shoot P content was maximal at 800 AMF spores followed by 400 AMF spores and 400 AMF spores + PRS9. In general, plant growth was greater at 20 ppm P. Root P content increased significantly with 400 AMF spores followed by 800 at 20 ppm P. Independent of soil P levels, the quality index of mycorrhizal treatments without PRS9 was significantly higher than the treatments including PRS9 or PRS9 alone. Mycorrhizal inoculation efficiency was superior at 10 ppm P irrespective of the treatment. AM alone (400 spores) significantly improved the inoculation efficiency. PRS9 in association with AM fungi inhibited growth promotion and nutrient uptake Accepted: 8 September 1999     

Prenatal restraint stress generates two distinct behavioral and neurochemical profiles in male and female rats

Prenatal Restraint Stress (PRS) in rats is a validated model of early stress resulting in permanent behavioral and neurobiological outcomes. Although sexual dimorphism in the effects of PRS has been hypothesized for more than 30 years, few studies in this long period have directly addressed the issue. Our group has uncovered a pronounced gender difference in the effects of PRS (stress delivered to the mothers 3 times per day during the last 10 days of pregnancy) on anxiety, spatial learning, and a series of neurobiological parameters classically associated with hippocampus-dependent behaviors. Adult male rats subjected to PRS ("PRS rats") showed increased anxiety-like behavior in the elevated plus maze (EPM), a reduction in the survival of newborn cells in the dentate gyrus, a reduction in the activity of mGlu1/5 metabotropic glutamate receptors in the ventral hippocampus, and an increase in the levels of brain-derived neurotrophic factor (BDNF) and pro-BDNF in the hippocampus. In contrast, female PRS rats displayed reduced anxiety in the EPM, improved learning in the Morris water maze, an increase in the activity of mGlu1/5 receptors in the ventral and dorsal hippocampus, and no changes in hippocampal neurogenesis or BDNF levels. The direction of the changes in neurogenesis, BDNF levels and mGlu receptor function in PRS animals was not consistent with the behavioral changes, suggesting that PRS perturbs the interdependency of these particular parameters and their relation to hippocampus-dependent behavior. Our data suggest that the epigenetic changes in hippocampal neuroplasticity induced by early environmental challenges are critically sex-dependent and that the behavioral outcome may diverge in males and females.     

PRS3 encoding an essential subunit of yeast proteasomes homologous to mammalian proteasome subunit C5.

We found by computer analysis that a putative yeast proteasome subunit gene named PRS3 that encodes a protein very similar to subunit C5 of rat and human proteasomes is located immediately 3' to the ERD2 gene of Saccharomyces cerevisiae. The similarity of the primary structures of the two suggests that this subunit may have a common function in proteasomes of all eukaryotes. The protein, deduced from the open reading frame of PRS3, consists of 242 amino acid residues with a calculated molecular weight of 27,077. Chromosomal disruption of the PRS3 gene created a recessive lethal mutation. Physical mapping by hybridization to intact S. cerevisiae chromosomal DNA showed that the PRS3 gene is located on chromosome II, unlike two other subunit genes, PRS1 and PRS2, which are located on chromosomes XV and VII, respectively. These findings indicate that the PRS3 protein is a subunit of yeast proteasomes that is essential for cell viability.     

Efficient detection of DNA polymorphism in cabbage and rice cultivars by PCR-RF-SSCP (PRS)

PCR (polymerase chain reaction)-RF(restriction fragment)-SSCP (single-strand conformation polymorphism) - designated here as PRS - is a combined method of SSCP and PCR-RFLP (restriction fragment length polymorphism) - designated as CAPS (cleaved amplified polymorphic sequence) - and was efficient in detecting intraspecific variation of the SLR1 gene in Brassica oleracea. One to six nucleotide changes in restriction fragments of the SLR1 gene were detected as different bands in PRS. In an analysis of randomly chosen DNA fragments in cabbage, PRS detected DNA polymorphism between different cultivars with more than 60% of the primer pairs used except for a combination of two cultivars having highly similar characteristics. In rice, no DNA polymorphism was found between two Japonica cultivars, while more than 80% of the primer pairs showed DNA polymorphism between Japonica cultivars and Indica cultivars. PRS had a 1.5- to twofold greater ability to detect DNA polymorphism in these cabbage and rice cultivars than CAPS. The present study indicated that PRS is potentially useful for the identification of crop cultivars and genetic mapping of DNA fragments including genes of interest.