Regulation of purine nucleotide biosynthesis: in yeast and beyond.

Purine nucleotides are critically important for the normal functioning of cells due to their myriad of activities. It is important for cells to maintain a balance in the pool sizes of the adenine-containing and guanine-containing nucleotides, which occurs by a combination of de novo synthesis and salvage pathways that interconvert the purine nucleotides. This review describes the mechanism for regulation of the biosynthetic genes in the yeast Saccharomyces cerevisiae and compares this mechanism with that described in several microbial species.

ade9 is an allele of SER1 and plays an indirect role in purine biosynthesis.

In this study we demonstrate that ade9 plays an indirect role in purine biosynthesis as a non-functional allele of SER1 in Saccharomyces cerevisiae. The SER1 locus, encoding 3-phosphoserine aminotransferase required for serine biosynthesis, is located on chromosome XV and resides approximately where ade9 had previously been mapped genetically. A minimal functional construct of SER1 is necessary and sufficient to complement both the adenine- and serine-requiring phenotypes of ade9 strains. In addition, adequate exogenous serine levels mask the adenine phenotype of ade9. A disruption of SER1 behaves in the same manner phenotypically as the original ade9 strain. Therefore, ade9 can be more accurately described as an allele of SER1.

Interaction between the retinoid X receptor and transcription factor IIB is ligand-dependent in vivo.

The retinoid X receptor (RXR) influences gene activation through heterodimeric and homodimeric association with DNA and associates with TATA binding protein, TAF110, and cAMP response element-binding protein-binding protein; yet the molecular mechanisms responsible for gene activation by RXRs remain incompletely defined. Since the general transcription factor IIB (TFIIB) is a common target of sequence-specific transcriptional activators, we suspected that RXR might regulate target genes via an interaction with TFIIB. Coimmunoprecipitation, far Western analysis, and glutathione S-transferase binding studies indicated that murine RXR beta (mRXR beta) was capable of binding to human TFIIB in vitro. Functional analysis with a dual-hybrid yeast system and cotransfection assays revealed the interaction of mRXR beta with TFIIB to be ligand-dependent in vivo. Truncation experiments mapped the essential binding regions to the carboxyl region of mRXR beta (amino acids (aa) 254-389) and two regions in the carboxyl region of TFIIB (aa 178-201 and aa 238-271). Furthermore, the delta 390-410 mRXR beta mutant bound to TFIIB in vitro but was not active in the dual-hybrid yeast system, suggesting that the extreme carboxyl region of RXR was required for in vivo interaction with TFIIB. These data indicate that interaction of mRXR beta with TFIIB is specific, direct, and ligand-dependent in vivo and suggest that gene activation by RXR involves TFIIB.

Evidence that complex formation by Bas1p and Bas2p (Pho2p) unmasks the activation function of Bas1p in an adenine-repressible step of ADE gene transcription.

Bas1p and Bas2p (Pho2p) are Myb-related and homeodomain DNA binding proteins, respectively, required for transcription of adenine biosynthetic genes in Saccharomyces cerevisiae. The repression of ADE genes in adenine-replete cells involves down-regulation of the functions of one or both of these activator proteins. A LexA-Bas2p fusion protein was found to activate transcription from a lexAop-lacZ reporter independently of both BAS1 function and the adenine levels in the medium. In contrast, a LexA-Bas1p fusion activated the lexAop reporter in a BAS2-dependent and adenine-regulated fashion. The DNA binding activity of Bas2p was not needed for its ability to support activation of the lexAop reporter by LexA-Bas1p, indicating that LexA-Bas1p recruits Bas2p to this promoter. The activation functions of both authentic Bas1p and LexA-Bas1p were stimulated under adenine-repressing conditions by overexpression of Bas2p, suggesting that complex formation by these proteins is inhibited in adenine-replete cells. Replacement of Asp-617 with Asn in Bas1p or LexA-Bas1p allowed either protein to activate transcription under repressing conditions in a manner fully dependent on Bas2p, suggesting that this mutation reduces the negative effect of adenine on complex formation by Bas1p and Bas2p. Deletions of N-terminal and C-terminal segments from the Bas1p moiety of LexA-Bas1p allowed high-level activation by the truncated proteins independently of Bas2p and adenine levels in the medium. From these results we propose that complex formation between Bas1p and Bas2p unmasks a latent activation function in Bas1p as a critical adenine-regulated step in transcription of the ADE genes.

The transcriptional activators BAS1, BAS2, and ABF1 bind positive regulatory sites as the critical elements for adenine regulation of ADE5,7.

Adenine repression of the purine nucleotide biosynthetic genes in Saccharomyces cerevisiae involves down-regulation of the activator protein BAS1 or BAS2 by an unknown mechanism. To determine the minimal cis-acting requirements for adenine regulation, hybrid promoter constructs were made between ADE5,7 promoter fragments and a CYC1-lacZ reporter. A 139-nucleotide fragment containing two BAS1 binding sites was sufficient to confer adenine regulation on the CYC1-lacZ reporter. Analysis of deletion and substitution mutations led to the conclusion that the proximal BAS1 binding site is both necessary and sufficient for regulation, whereas the distal site augments the function of the proximal site. By performing saturation mutagenesis, we found two essential regions that flank the proximal site. An ABF1 consensus sequence is within one of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and appears to bind BAS2. No substitution mutations led to high level constitutive promoter activity as would be expected from removal of an upstream repression sequence. Our results indicate that ABF1, BAS1, and BAS2 are required for ADE5,7 promoter function and that adenine repression most likely involves activator modification or a negative regulator that does not itself bind DNA.

Effects of myocardial viability assessment with positron emission tomography on clinical management and patient outcomes.

OBJECTIVE: To evaluate the effects of myocardial viability assessment with positron emission tomography on cardiac revascularization decision-making and consequential outcomes of patients with multivessel coronary artery disease. METHODS: Thirty-three patients with multivessel coronary disease and heart failure were studied in this series, using 13NH for myocardial perfusion and F-18-deoxy-glucose for myocardial metabolism. Viable myocardium (mis-matched perfusion-metabolism) was visually and quantitatively analyzed in anterior, apical, septal, inferior, and lateral segments of the left ventricle. Left ventricular ejection fraction (LVEF) was also measured with first-pass radionuclide angiocardiography. RESULTS: Based on the assessment of myocardial viability, 19 patients (group A) with sufficient viable myocardium underwent revascularization (coronary bypass graft and/or angioplasty), and 14 patients (group B) without sufficient viable myocardium received conservative medical treatment. During an average of 17-month follow-up, there were 2 (10.5%) deaths in group A and 2 in group B (14.3%) deaths (P > 0.5). Patients with revascularization showed significantly improved average LVEFs post-revascularization, without revascularization procedure-related mortality; patients with medical treatment had an initial average LVEF of 25% and class II-III (NYHA) average cardiac function with a survival rate of 86% in average, which was better than that reported in literature. CONCLUSION: Positron emission tomography is useful in myocardial viability assessment for cardiac revascularization decision-making through precisely selecting suitable patients for revascularization and avoiding operations on those who will not benefit, which results in promising effects on outcomes of patients with multivessel coronary disease and severe left ventricular dysfunction.

Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2.

The transcriptional activator protein GCN4 is responsible for increased transcription of more than 30 different amino acid biosynthetic genes in response to starvation for a single amino acid. This induction depends on increased expression of GCN4 at the translational level. We show that starvation for purines also stimulates GCN4 translation by the same mechanism that operates in amino acid-starved cells, being dependent on short upstream open reading frames in the GCN4 mRNA leader, the phosphorylation site in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha), the protein kinase GCN2, and translational activators of GCN4 encoded by GCN1 and GCN3. Biochemical experiments show that eIF-2 alpha is phosphorylated in response to purine starvation and that this reaction is completely dependent on GCN2. As expected, derepression of GCN4 in purine-starved cells leads to a substantial increase in HIS4 expression, one of the targets of GCN4 transcriptional activation. gcn mutants that are defective for derepression of amino acid biosynthetic enzymes also exhibit sensitivity to inhibitors of purine biosynthesis, suggesting that derepression of GCN4 is required for maximal expression of one or more purine biosynthetic genes under conditions of purine limitation. Analysis of mRNAs produced from the ADE4, ADE5,7, ADE8, and ADE1 genes indicates that GCN4 stimulates the expression of these genes under conditions of histidine starvation, and it appeared that ADE8 mRNA was also derepressed by GCN4 in purine-starved cells. Our results indicate that the general control response is more global than was previously imagined in terms of the type of nutrient starvation that elicits derepression of GCN4 as well as the range of target genes that depend on GCN4 for transcriptional activation.

Enteric MRI contrast agents: comparative study of five potential agents in humans.

We compared the effectiveness of 1 mM Geritol, 12% corn oil emulsion, Kaolin-pectin, single contrast oral barium sulfate, and effervescent granules as enteric magnetic resonance imaging (MRI) contrast agents. Five volunteers were recruited. Each volunteer ingested for examinations, separated by at least one week, either 500 ml of each of the liquid preparations or two packets of the CO2 granules (producing 400 ml of CO2 per packet). Abdominal MR images were then obtained using a 1.5 T Magnetom imager and SE 550/22, SE 2000/45/90 and FISP 40/18/40 degrees pulse sequences. The oil emulsions were best tolerated. Barium sulfate caused the greatest amount of nausea, followed by Geritol and Kaolin-pectin. With FISP 40/18/40 degrees, 60%-80% of the small bowel was well delineated using oil emulsion, Kaolin-pectin, or barium sulfate. We conclude that oil emulsion was by far the best enteric MR contrast agent in our study. Good delineation of the small bowel and pancreas can be achieved using oil emulsion and gradient echo pulse sequences. The lack of side-effects and the excellent taste make it highly acceptable to human subjects.

Barium sulfate suspension as a negative oral MRI contrast agent: in vitro and human optimization studies.

In vitro proton spectroscopy with line-width measurements and MR imaging were performed on various concentrations of commercially available single contrast (SC), double contrast, oral and rectal barium sulfate suspensions, as well as potassium sulfate, barium chloride, barium hydroxide, and 97% pure barium sulfate suspensions. Approximately 500 ml of 20%, 40%, 60%, and 70% w/w suspensions of SC oral barium sulfate suspensions were administered to four normal volunteers, respectively, and MR images were obtained at both 1.5 T and 0.15 T. Subsequently, 500 ml of 60% w/w suspensions of SC oral barium sulfate suspensions were administered to five normal volunteers and imaged at 1.5 T. All of the inert suspensions produced line-width broadening but the SC oral barium sulfate suspension at 50% and 70% stayed in suspension even after hours of standing undisturbed. As much as 80% of the small bowel and the entire colon were well visualized using the combination of 60% or 70% w/w SC barium sulfate suspensions with SE 550/22 and FISP pulse sequences. The effect was less at 0.15 T and also with the SE 2000/45/90 pulse sequences. We conclude that barium sulfate suspensions are useful as oral MRI contrast agents.

Purification of the Escherichia coli purine regulon repressor and identification of corepressors.

The Escherichia coli pur regulon repressor protein was overproduced in a phage T7 expression system. The overexpressed repressor constituted approximately 35% of the soluble cellular protein. Pur repressor was purified to near homogeneity by two chromatographic steps. Hypoxanthine or guanine was required for binding of purified repressor to purF operator DNA. Apparent dissociation constants of 3.4 nM were determined for binding of holorepressor to purF operator and of 1.7 and 7.1 microM were determined for aporepressor interaction with guanine and hypoxanthine, respectively. A requirement for hypoxanthine or guanine for conversion of aporepressor to holorepressor in vitro supports the earlier report (U. Houlberg and K.F. Jensen, J. Bacteriol. 153:837-845, 1983) that these purine bases are involved in regulation of pur gene expression in Salmonella typhimurium and confirms that hypoxanthine and guanine are corepressors.

Autoregulation of Escherichia coli purR requires two control sites downstream of the promoter.

The expression of Escherichia coli purR, which encodes the pur regulon repressor protein, is autoregulated. Autoregulation at the level of transcription requires two operator sites, designated purRo1 and purRo2 (O1 and O2). Operator O1 is in the region of DNA between the transcription start site and the site for translation initiation, and O2 is in the protein-coding region. The repressor protein binds noncooperatively to O1 with a sixfold-higher affinity than to O2, and saturation of O1 by the repressor precedes saturation of O2. Both O1 and O2 function in the two- to threefold autoregulation in vivo, as determined by measurement of beta-galactosidase and mRNA from purR-lacZ translational fusions. Of all the genes thus far known to be regulated by the Pur repressor, only purR employs a two-operator mechanism.

Regulation of the Escherichia coli glyA gene by the purR gene product.

The purine regulon repressor protein, PurR, was shown to be a purine component involved in glyA regulation in Escherichia coli. Expression of glyA, encoding serine hydroxymethyltransferase activity, was elevated in a purR mutant compared with a wild-type strain. When the purR mutant was transformed with a plasmid carrying the purR gene, the serine hydroxymethyltransferase levels returned to the wild-type level. The PurR protein bound specifically to a DNA fragment carrying the glyA control region, as determined by gel retardation. In a DNase I protection assay, a 24-base-pair region was protected from DNase I digestion by PurR. The glyA operator sequence for PurR binding is similar to that reported for several pur regulon genes.

The spleen: an integrated imaging approach.

In this review we attempted to demonstrate the imaging appearance of the spectrum of entities that may involve the spleen. The systematic approach reviewing the radiologic findings by modality (plain films, ultrasound, CT, scintigraphy, angiography, and MRI) has been used. We have opted to present the information following pathologic categories (congenital, infections, cysts, benign and malignant tumors, vascular, trauma, and miscellaneous) rather than a pattern approach. A pattern approach may not be practical in spleen since patterns are few and nonspecific. Although a specific diagnosis can be obtained rarely, the information provided by imaging coupled with clinical and laboratory data is capable in many cases of significantly altering patient management. Surgery, percutaneous intervention, medical therapy, or simple observation can be pursued based on the radiological findings.

Regulation of Escherichia coli purF. Mutations that define the promoter, operator, and purine repressor gene.

Mutations were constructed in vitro which identify the -35 promoter element and the operator site of the Escherichia coli purF operon as well as confirm the -10 promoter sequence. The operator was localized by a two-base change at positions -26 and -27, relative to the start of transcription. This mutation abolished repression of a purF-lacZ fusion. In the wild-type, repression of single copy and multicopy purF-lacZ constructs was equally effective. This indicates that cells contain a greater than 100-fold excess of purR-encoded repressor than is needed to regulate the chromosomal purF operon. Thus, cells contain sufficient repressor to regulate the other pur regulon genes. Two independent purR mutations were isolated which abolished repression of purF and purF-lacZ. We conclude that there is a single repressor protein-operator regulatory system to sense purine or purine nucleotide pools.

Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator.

The Escherichia coli gene purR, encoding a repressor protein, was cloned by complementation of a purR mutation. Gene purR on a multicopy plasmid repressed expression of purF and purF-lacZ and reduced the growth rate of host cells by limiting the rate of de novo purine nucleotide synthesis. The level of a 1.3-kilobase purR mRNA was higher in cells grown with excess adenine, suggesting that synthesis of the repressor may be regulated. The chromosomal locus of purR was mapped to coordinate 1755-kb on the E. coli restriction map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508). Pur repressor bound specifically to purF operator DNA as determined by gel retardation and DNase I footprinting assays. The amino acid sequence of Pur repressor was derived from the nucleotide sequence. Pur repressor subunit contains 341 amino acids and has a calculated Mr of 38,179. Pur repressor is 31-35% identical with the galR and cytR repressors and 26% identical with the lacI repressor. These four repressors are likely homologous. Amino acid sequence similarity is greatest in an amino-terminal region presumed to contain a DNA-binding domain. A similarity is also noted in the operator sites for these repressors.