Comparative genomic analysis reveals a critical role of de novo nucleotide biosynthesis for Saccharomyces cerevisiae virulence.

In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings.

To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.

Effect of 5-amino-4-imidazolecarboxamide riboside (AICA-riboside) on the purine nucleotide synthesis and growth of rat kidney cells in culture: study with [15N]aspartate.

The present investigation evaluates the effect of AICA-Riboside on the synthesis of purine nucleotides and the growth of normal rat kidney cells in culture. Experiments in the presence and absence of various concentrations of AICA-Riboside were conducted with Dulbecco's Modified Eagle's Medium supplemented with either 1 mM [15N]aspartate or [14N]aspartate. Addition of 50 microM AICA-Riboside to the incubation medium significantly stimulated intracellular adenine nucleotide concentrations following incubation for 48 hours. This stimulation was associated with augmented cell growth and DNA concentration. In contrast, with concentrations above 100 microM of AICA-Riboside in the incubation medium, there was a remarkable inhibition of cell growth and a significant depletion of intracellular pools of adenine nucleotides and DNA. Experiments with [15N]aspartate showed that the initial rate (0-24 hours) of [6-15NH2]adenine nucleotide formation from 1 mM [15N]aspartate was 38.8 +/- 9.6, 67.9 +/- 12.5, and 20.1 +/- 3.8 pmol h-1/10(6) cells in the presence of 0 (control), 50 microM and 500 microM AICA-Riboside, respectively. These observations indicate that the main effect of AICA-Riboside is on the formation of AMP from aspartate and IMP via the sequential action of adenylosuccinate synthetase and adenylosuccinate lyase. The current studies suggest that AICA-Riboside could be used as a factor mediating renal cell mitosis in culture. AICA-Riboside has a biphasic effect on the growth of renal epithelial cells in culture and on their intracellular purine nucleotides and DNA concentration.

Polypyrimidine segments in Drosophila melanogaster DNA: II. Chromosome location and nucleotide sequence.

Long pyrimidine tracts, purified from Drosophila melanogaster DNA after treatment with formic acid-diphenylamine, were used as template for E. coli RNA polymerase to produce a polynucleotide containing only purines. This polypurine RNA hybridized specifically to D. melanogaster DNA with high efficiency at low Cot values. The resulting hybrid showed high thermal stability. When polypurine RNA was subjected to complete hydrolysis with ribonuclease T1, over 90% of the nucleotide products were ApGp and ApApGp. Partial hydrolysis yielded a distinct additional component, ApApGpApGp + ApGpApApGp. We conclude that the major sequence in the polypurine transcript is (ApGpApApGp)n. In situ hybridization to salivary gland polytene chromosomes and to metaphase chromosomes from neural ganglia indicated that polypyrimidines complementary to polypurine RNA are located in heterochromatin. In femal cells, the predominant labeling was on centromeric heterochromatin of the 2nd chromosome. We have verified the location of polypyrimidines in neural ganglion cells, by using a cytological marker of chromosomes 2. In male cells, hybrid was also found on the Y chromosome.

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17 .

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.