Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry.

Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.

Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase.

1,3-beta-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p. The residual glucan synthase activity present in strains with deletions of fks1 is (i) immunodepleted by antibodies prepared against FKS2 peptides, demonstrating that Fks2p is also a component of the enzyme, and (ii) more sensitive to the echinocandin L-733,560, explaining the increased sensitivity of fks1 null mutants to this drug. Simultaneous disruption of FKS1 and FKS2 is lethal, suggesting that Fks1p and Fks2p are alternative subunits with essential overlapping function. Analysis of FKS1 and FKS2 expression reveals that transcription of FKS1 is regulated in the cell cycle and predominates during growth on glucose, while FKS2 is expressed in the absence of glucose. FKS2 is essential for sporulation, a process which occurs during nutritional starvation. FKS2 is induced by the addition of Ca2+ to the growth medium, and this induction is completely dependent on the Ca2+/calmodulin-dependent phosphoprotein phosphatase calcineurin. We have previously shown that growth of fks1 null mutants is highly sensitive to the calcineurin inhibitors FK506 and cyclosporin A. Expression of FKS2 from the heterologous ADH1 promoter results in FK506-resistant growth. Thus, the sensitivity of fks1 mutants to these drugs can be explained by the calcineurin-dependent transcription of FKS2. Moreover, FKS2 is also highly induced in response to pheromone in a calcineurin-dependent manner, suggesting that FKS2 may also play a role in the remodeling of the cell wall during the mating process.

Colocalization of somatostatin receptor sst5 and insulin in rat pancreatic beta-cells.

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is secreted by pancreatic delta-cells and inhibits the secretion of both insulin and glucagon. SRIF initiates its actions by binding to a family of six G protein-coupled receptors (sst1, -2A, -2B, -3, -4, and -5) encoded by five genes. Messenger RNA for both sst2 and sst5 have been reported in the rat pancreas, and the sst2A receptor protein has been localized to rat pancreatic alpha and pancreatic polypeptide-secreting cells in the islets as well as to pancreatic acinar cells. In this study we have used double immunostaining to show that the sst5 protein is expressed exclusively in the beta-cells of rat pancreatic islets and localizes with insulin-secreting alpha-cells. The sst5 receptor is not colocalized with sst2A. Thus, in the rat SRIF inhibits pancreatic insulin and glucagon secretion via different sst receptor subtypes.

Construction of a shuttle vector consisting of the Escherichia coli plasmid pACYC177 inserted into the Streptomyces cattleya phage TG1.

The Escherichia coli plasmid, pACYC177, was inserted into the single PstI site of a deletion derivative of the Streptomyces cattleya phage, TG1. The hybrid molecule can be propagated as a phage in S. cattleya and as a plasmid in E. coli and is readily transferred between the two species by transfection and transformation. The kanamycin-resistance-encoding gene derived from pACYC177 is not expressed in lysogens of the hybrid phage. Analysis of deletion mutants of the hybrid phage indicated that at least 7.5 kb of phage DNA is dispensable. Some of the deletion mutants fail to lysogenize S. cattleya (Lyg- phenotype). The locations of these deletions are consistent with the location of the phage att site as previously established by Southern hybridization analysis. The thiostrepton-resistance-encoding gene derived from Streptomyces azureus was inserted into Lyg+ and Lyg- deletion derivatives and is expressed in S. cattleya.

Vectors for generating nested deletions and facilitating subcloning G+C-rich DNA between Escherichia coli and Streptomyces sp.

New multiple cloning sites (MCS), which facilitate the subcloning of G+C-rich DNA, were added to pUC18, M13mp18, pVE616 (a pBR322-derived insertion vector), and the low-copy-number Streptomyces vector, pIJ922. The MCS in these vectors contain sites found infrequently in Streptomyces DNA, facilitating the exchange of subclones between the vectors. The MCS added to M13mp18 and pUC18 was also designed to generate nested deletions within subcloned fragments.

Isolation and characterization of the Streptomyces cattleya temperate phage TG1.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli.

An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E. coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A. W., and Brown, G. M. (1968) J. Biol. Chem. 243, 2349-2358). Some of the properties of GTP cyclohydrolase II are: (a) divalent cations are required for activity (Mg2+ is most effective); (b) its molecular weight, estimated by filtration on Sephadex G-200, is 44,000; (c) the K-m for GTP is 41 mum; (d) its pH optimum is 8.5; and (e) its activity is inhibited by inorganic pyrophosphate, one of the products of the reaction. Compounds not used as substrate are: GDP, GMP, guanosine, dGTP, ATP, ITP, and XTP. Properties a, b, c, and e (above), as well as the nature of the products, distinguish this enzyme from GTP cyclohydrolase I. Since GTP cyclohydrolase II apparently is not concerned with the biosynthesis of folic acid, the possible physiological role of this enzyme in the biosynthesis of riboflavin is considered in the light of the present investigations and the previously published work on riboflavin biosynthesis by other investigators.

Regulation of the synthesis of glutamine synthetase by the PII protein in Klebsiella aerogenes.

Certain mutations at the glaB locus result in the failure to fully derepress glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] and to convert it to the active nonadenylylated form in response to nitrogen limitation. In these mutants the PII regulatory protein is altered such that it cannot be converted by uridylyltransferase to the form stimulating deadenylylation of glutamine synthetase by adenylyltransferase. Additional mutations as well as insertions of transposon Tn5 at the glnB site result in the loss of PII. The loss of PII does not prevent adenylylation and deadenylylation of glutamine synthetase but reduces the rates of these reactions. Cells lacking PII have a high level of glutamine synthetase even when they are grown with an excess of ammonia and the enzyme is highly adenylylated. The results suggest that the PII protein plays a role, independent of its effect on adenylylation, in the regulation of the level of glutamine synthetase.

Regulation of glutamine synthetase by regulatory protein PII in Klebsiella aerogenes mutants lacking adenylyltransferase.

A mutation of Klebsiella aerogenes causing production of an altered PII regulatory protein which stimulates overadenylylation of glutamine synthetase and also prevents its derepression was combined with mutations abolishing the activity of adenylyltransferase. The results support the idea that PII plays a role in the regulation of the level of glutamine synthetase which is independent of its interaction with adenylyltransferase.

Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola.

The gene (tutA) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed. The enzyme was expressed at high levels in E. coli in the presence of isopropyl-beta-D-thiogalactopyranoside or lactose as an inducer. L-Dihydroxyphenylalanine was synthesized in high yield from catechol, pyruvate, and ammonia by induced cells.

Regulation of synthesis of glutamine synthetase by adenylylated glutamine synthetase.

We have examined three mutants of Klebsiella aerogenes whose genetic lesions (glnB, glnD, and glnE) are in loci unlinked to the structural gene for glutamine sythetase (glnA) and in which the control of both the level and state of adenylylation of glutamine synthetase is altered. Each mutation alters a different component of the adenylylation system of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. Inability of the cell to deadenylylate glutamine synthetase (glnB and glnD) greatly decreases its production, while inability to adenylylate glutamine sythetase (glnE) results in its constitutively high production. These results together with our previous results indicate that adenylylated glutamine synthetase inhibits the transcription of glnA.

Role of glnA-linked genes in regulation of glutamine synthetase and histidase formation in Klebsiella aerogenes.

We isolated mutants of Klebsiella aerogenes with insertions of transposon Tn5 linked to the structural gene for glutamine synthetase, glnA. We found that K. aerogenes, like Escherichia coli and Salmonella typhimurium, contains a regulatory gene, glnG, which is closely linked to but distinct from glnA. The product of glnG is essential for normal regulation of the synthesis of glutamine synthetase and histidase. We analyzed two mutations which affected the regulation of the formation of these enzymes and appeared to be outside glnG. The results of our studies suggest the presence in K. aerogenes of at least two regulatory genes located in the glnA region, namely, glnG and another gene, the transcription of which can be initiated at the promoter of glnA.

Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase.

The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA.

Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase.

A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.

Cloning of a Streptomyces clavuligerus DNA fragment encoding the cephalosporin 7 alpha-hydroxylase and its expression in Streptomyces lividans.

A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7 alpha-hydroxylase (CH) of Streptomyces clavuligerus NRRL 3585 and used to screen a DNA library from this organism. The library was constructed in the lambda GEM-11 phage system. After plaque purification and reprobing, positive recombinant phages were chosen for further analysis. Characterization of the cloned DNA by restriction mapping and Southern hybridization showed that a 1.5-kb SalI fragment hybridized to the probe. Polymerase chain reaction assays using this fragment as a template and the probe as a primer indicated that the fragment carries the entire putative CH gene (cmcI). This was confirmed through the expression of CH enzymatic activity when the fragment was introduced into Streptomyces lividans. A putative beta-lactamase activity was detected in S. lividans.

Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast.

The structurally unrelated immunosuppressants FK506 and cyclosporin A (CsA) act similarly, inhibiting a Ca(2+)-dependent signal required for interleukin-2 transcription and T-cell activation. Each drug binds to its cytosolic receptor, FKBP-12 and cyclophilin, respectively, and the drug-receptor complexes inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In yeast, calcineurin has been implicated in recovery from alpha-mating factor arrest. Here we show that FK506 bound to yeast FKBP-12 appears to form a complex with yeast calcineurin. Moreover, recovery from mating factor arrest is highly sensitive to FK506 or CsA, and this sensitivity requires the presence of FKBP-12 or cyclophilin, respectively. These results define a key physiological target of an FK506- and CsA-sensitive signal pathway in yeast, suggest a high degree of mechanistic conservation with mammalian cells, and indicate that further examination of the yeast system should provide insight into the same process in T cells.

The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.

In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounced slow-growth phenotype, (ii) hypersensitivity to FK506 and cyclosporin A, (iii) a slight increase in sensitivity to echinocandin, and (iv) a significant reduction in 1,3-beta-D-glucan synthase activity in vitro. The nucleotide sequence encodes a 215-kDa polypeptide predicted to be an integral membrane protein with 16 transmembrane helices, consistent with previous observations that the etg1-1 mutation results in echinocandin-resistant glucan synthase activity associated with the nonextractable membrane fraction of the enzyme. These results suggest that FKS1 encodes a subunit of 1,3-beta-D-glucan synthase. The residual activity present in the disruption mutant, the nonessential nature of the gene, and results of Southern blot hybridization analysis point to the existence of a glucan synthase isozyme.