RESUMEN

UNLABELLED: Percutaneous transluminal angioplasties of coronary and peripheral vessels are frequently used and replace open surgery in a certain percentage. Hemostasis in most of these patients is reduced or inhibited and often leads to hemorrhage from the puncture track. Due to this fact, hospitalization is not only mandatory, but also surgical revision of the puncture site is often required. We designed and produced a coaxial delivery system, which is mounted on the indwelling guide-wire after withdrawal of the instrumentation for angioplasty. The delivery system is advanced down to the outer wall of the punctured vessel and 1 cc of human two-compound fibrin glue is released. Based on our experience with laboratory and animal research, which we already presented at the 7th International Symposium on Pediatric Surgical Research in Heidelberg, May 27-28, 1994, we conducted the first trials in interventional cardiology. In 1996, a first group of 10 patients, aged 49 to 80 years, underwent sealing of the right femoral artery after diagnostic evaluation (n = 3) of coronary balloon dilatation (n = 7). In patients, the local manual compression time was less than 5 minutes and 1 patient needed 10 minutes of digital compression. In one case, bleeding continued and a compression bandage was successful, whereas in another case the local hematoma formation needed surgical revision with suture of the ruptured vessel wall. CONCLUSION: Puncture-track sealing with locally applied fibrin glue seems to be an efficient tool to avoid bleeding after interventions of coronary and peripheral vessels. In the meantime, the device has been improved by a target system to optimize the delivery of the glue exactly at the outer wall of the vessel.

Asunto(s)

Angioplastia Coronaria con Balón , Angioplastia de Balón , Adhesivo de Tejido de Fibrina/administración & dosificación , Hemostasis Quirúrgica/instrumentación , Hemostáticos/administración & dosificación , Punciones , Anciano , Anciano de 80 o más Años , Angioplastia de Balón/métodos , Angioplastia Coronaria con Balón/métodos , Animales , Femenino , Arteria Femoral , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto

RESUMEN

We have cloned proteins that interact with the nuclear orphan receptor RZR beta using the yeast two-hybrid system. We identified, amongst a number of other genes, the nucleoside diphosphate kinase (NDPK)-2 also known as Nm23-2, c-myc regulatory factor PuF and differentiation inhibitory factor, RZR beta specifically interacts with Nm23-2 but not with the closely related tumor metastasis suppressor candidate gene product Nm23-1. In contrast ROR alpha interacts with both Nm23 proteins. These findings were corroborated by in vitro interaction assays based on GST-pulldown experiments. With-n-myc we propose a candidate gene regulated by ROR alpha/RZR beta and Nm23, based on the finding that the respective DNA binding sites in the first intron are conserved in several mammalian species.

Asunto(s)

Proteínas de Unión al GTP Monoméricas , Nucleósido-Difosfato Quinasa , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Genes myc , Humanos , Ratones , Datos de Secuencia Molecular , Nucleósido Difosfato Quinasas NM23 , Unión Proteica , Ratas , Homología de Secuencia de Ácido Nucleico , Especificidad por Sustrato , Factores de Transcripción/genética

RESUMEN

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with the Saccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in the PKC1-mediated signalling pathway. Subsequently, Pkc1p and the other PKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of the PKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Fúngicas/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteína Quinasa C/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Clonación Molecular/métodos , Proteínas Fúngicas/genética , Genes Fúngicos/genética , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética

RESUMEN

Using a DNA fragment derived from the Saccharomyces cerevisiae protein kinase C gene (PKC1) as a probe to screen an ordered array library of genomic DNA from the dimorphic pathogenic fungus Candida albicans, the C. albicans PKC1 gene (CaPKC1) was isolated. The CaPKC1 gene is predicted to encode a protein of 1079 amino acids with 51% sequence identity over the entire length with the S. cerevisiae Pkc1 protein and is capable of functionally complementing the growth defects of a S. cerevisiae pkc1 delta mutant strain on hypo-osmotic medium. Deletion of both endogenous copies of the CaPKC1 gene in diploid C. albicans cells resulted in an osmotically remedial cell lysis defect of both the budding and the hyphal growth form and morphologically aberrant cells of the budding form. Despite these abnormalities, the transition between the two growth forms of C. albicans occurred normally in pkc1/pkc1 double disruptants. Capkc1p was modified at its C-terminus with two repeats of the Staphylococcus aureus protein A IgG-binding fragment (ZZ-sequence tag) and partially purified by chromatography on DEAE-Sepharose and IgG-Sepharose. In vitro, Capkc1p preferably phosphorylated the S. cerevisiae Pkc1p pseudosubstrate peptide and myelin basic protein, but not histones, protamine or dephosphorylated casein, and failed to respond to cofactors known to activate several mammalian PKC isozymes.

Asunto(s)

Candida albicans/enzimología , Candida albicans/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteína Quinasa C/genética , Secuencia de Aminoácidos , Animales , Candida albicans/crecimiento & desarrollo , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C/metabolismo , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

Silver ions and silver-containing compounds have been used as topical antimicrobial agents in a variety of clinical situations. We have previously shown that the enzyme phosphomannose isomerase (PMI) is essential for the biosynthesis of Candida albicans cell walls. In this study, we find that PMI can be inhibited by silver ions. This process is shown to be irreversible, and is a two-step process, involving an intermediate complex with a dissociation constant, Ki, of 59 +/- 8 microM, and a maximum rate of inactivation of 0.25 +/- 0.04 min-1 in 50 mM Hepes buffer, pH 8.0 at 37 degrees C. The enzyme can be protected against this inactivation by the substrate mannose 6-phosphate, with a dissociation constant of 0.31 +/- 0.04 mM, close to its Km value. Flamazine (silver sulfadiazine) is a silver-containing antibiotic which is used clinically as a topical antimicrobial and antifungal agent. We compared the ability of silver sulfadiazine and two other silver-containing compounds to irreversibly inactivate C. albicans PMI. The addition of the organic moiety increased the affinity of the compounds, with silver sulfadiazine showing a Ki of 190 +/- 30 nM. In all cases, the maximum inhibition rate was similar, implying a similar rate-determining step. Silver sulfadiazine does not inhibit Escherichia coli PMI, and this suggests a role of the only free cysteine, Cys-150, in the inactivation process. To confirm this, we mutated this residue to alanine in C. albicans PMI. The resultant Cys150 --> Ala mutant protein showed similar Vm and Km values to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Candida albicans/enzimología , Manosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Compuestos Organometálicos/farmacología , Sulfadiazina de Plata/farmacología , Plata/farmacología , Alanina/genética , Antifúngicos/farmacología , Secuencia de Bases , Candida albicans/genética , Carbazoles/farmacología , Cationes Monovalentes/farmacología , Cisteína/genética , Relación Dosis-Respuesta a Droga , Indoles/farmacología , Manosa-6-Fosfato Isomerasa/genética , Cloruro de Mercurio/farmacología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida

RESUMEN

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta-glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.

Asunto(s)

Matriz Extracelular/metabolismo , Proteínas Fúngicas/genética , Glucanos/biosíntesis , Proteínas de la Membrana/genética , Proteína Quinasa C/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , beta-Glucanos , Secuencia de Aminoácidos , Secuencia de Bases , Pared Celular/metabolismo , Pared Celular/ultraestructura , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Aparato de Golgi/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteína Quinasa 1 Activada por Mitógenos , Datos de Secuencia Molecular , Fenotipo , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Homología de Secuencia de Aminoácido

RESUMEN

The Saccharomyces cerevisiae PKC1 gene encodes a homolog of mammalian protein kinase C (Levin, D. E., Fields, F.O., Kunisawa, R., Bishop, J.M., and Thorner, J. (1990) Cell 62, 213-224). A protein of 150 kDa is recognized by a polyclonal antiserum raised against a trpE-Pkc1 fusion protein. In subcellular fractionations, Pkc1p associates with the 100,000 x g particulate fraction. This association is resistant to extraction with high salt concentrations, alkali buffer, or nonionic detergents, suggesting that Pkc1p may be associated with a large protein complex. Pkc1p modified at its COOH terminus with two repeats of the Staphylococcus aureus protein A IgG-binding fragment (ZZ sequence tag) was able to fully restore the growth defects of a pkc1ts strain at restrictive temperature. ZZ-tagged Pkc1p was partially purified by chromatography on DEAE-Sepharose, followed by IgG-Sepharose. In vitro, Pkc1p phosphorylates the pseudosubstrate peptide and myelin basic protein, but not histones. Replacing an isoleucine with an arginine 2 amino acids COOH-terminal of the acceptor serine in the substrate peptide resulted in a 10-fold decrease of Km. Pkc1p activity was independent of cofactors such as phospholipids, diacylglycerol, and Ca2+, known to activate several mammalian protein kinase C isoenzymes, making it a rather distantly related member of the protein kinase C superfamily.

Asunto(s)

Genes Fúngicos , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Prueba de Complementación Genética , Cinética , Mamíferos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Fosfopéptidos/química , Fosfopéptidos/aislamiento & purificación , Fosfopéptidos/metabolismo , Plásmidos , Proteína Quinasa C/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/enzimología , Especificidad por Sustrato

RESUMEN

The Saccharomyces cerevisiae HIS7 gene was cloned by its location immediately downstream of the previously isolated and characterized ARO4 gene. The two genes have the same orientation with a distance of only 416 bp between the two open reading frames. The yeast HIS7 gene represents the first isolated eukaryotic gene encoding the enzymatic activities which catalyze the fifth and sixth step in histidine biosynthesis. The open reading frame of the HIS7 gene has a length of 1,656 bp resulting in a gene product of 552 amino acids with a calculated molecular weight of 61,082. Two findings implicate a bifunctional nature of the HIS7 gene product. First, the N-terminal and C-terminal segments of the deduced HIS7 amino acid sequence show significant homology to prokaryotic monofunctional glutamine amidotransferases and cyclases, respectively, involved in histidine biosynthesis. Second, the yeast HIS7 gene is able to suppress His auxotrophy of corresponding Escherichia coli hisH and hisF mutants. HIS7 gene expression is regulated by the general control system of amino acid biosynthesis. GCN4-dependent and GCN4-independent (basal) transcription use different initiator elements in the HIS7 promoter.

Asunto(s)

Complejos Multienzimáticos/genética , Saccharomyces cerevisiae/genética , Transferasas/genética , Secuencia de Aminoácidos , Aminohidrolasas , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Transferasas/metabolismo

RESUMEN

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.

Asunto(s)

Proteína Quinasa C/genética , Saccharomyces cerevisiae/genética , Medios de Cultivo , Citometría de Flujo , Eliminación de Gen , Prueba de Complementación Genética , Ligamiento Genético , Microscopía Electrónica , Mutación , Proteína Quinasa C/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Transformación Genética , Equilibrio Hidroelectrolítico

RESUMEN

Sequencing of an open reading frame 450 bp downstream from the yeast VPS35 gene revealed a putative peptide of 452 amino acids and 52.7 kDa. The predicted amino acid sequence has 45% identity with the 55-kDa subunit of the 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (EC 2.7.1.105/EC 3.1.3.46) from rat liver and 42% identity with 480 amino acids in the center of the recently reported 93.5-kDa subunit of yeast 6-phosphofructo-2-kinase (EC 2.7.1.105). The product of the new yeast gene is similar to the entire sequence of the bifunctional rat liver enzyme and, unlike yeast 6-phosphofructo-2-kinase, has the histidine residue essential for fructose-2,6-bisphosphatase activity. Extracts from a chromosomal null mutant strain, fbp26::HIS3, incubated in the presence of [2-32P]fructose 2,6-P2, lacked in autoradiograms the characteristic 56-kDa labeled band observed in wild-type. The same band was intensified 3-fold over wild-type level with the FBP26 gene introduced on multicopy in the fbp26::HIS3 background. A similar increase was found for fructose-2,6-bisphosphatase activity in the same extracts. The FBP26 gene did not cause detectable increase in 6-phosphofructo-2-kinase activity when introduced on multicopy in a pfk26::LEU2 mutant, indicating that its gene product is predominantly a fructose-2,6-bisphosphatase. Growth on glucose, fructose, galactose, pyruvate, and glycerol/lactate was not impaired in strains carrying the fbp26::HIS3 allele.

Asunto(s)

Genes Fúngicos , Monoéster Fosfórico Hidrolasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Hígado/enzimología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfofructoquinasa-2 , Fosfotransferasas/genética , Ratas , Alineación de Secuencia

RESUMEN

We have previously described a temperature-sensitive pmi40-1 mutant of Saccharomyces cerevisiae which is defective in glycosylation and secretion because of a thermolabile phosphomannose isomerase (PMI) activity. Inactivation of PMI at the restrictive temperature of 37 degrees C prevents synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death. Here, we report the isolation of the PMI40 gene by complementation of the corresponding mutation. The PMI40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S. cerevisiae in that it is short and the branch-forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC. The 48.2-kDa protein predicted to be encoded by PMI40 contains amino acid sequences corresponding to those of internal peptides derived from purified S. cerevisiae PMI. Deletion of the PMI40 coding sequence results in a strain requiring D-mannose for growth. The PMI40 gene is located on chromosome V, and its transcription is increased 12-fold when cells are grown on D-mannose as sole carbon source instead of D-glucose. PMI enzyme activity, however, is not increased in D-mannose-grown cells, and PMI protein levels remain constant, suggesting that the PMI40 gene is subject to additional levels of regulation.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Manosa-6-Fosfato Isomerasa/genética , Manosa/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Fúngicos , Análisis Mutacional de ADN , Monofosfato de Dolicol Manosa/metabolismo , Prueba de Complementación Genética , Glicosilación , Guanosina Difosfato Manosa/biosíntesis , Intrones/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Mapeo Restrictivo , Transcripción Genética

RESUMEN

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.

Asunto(s)

Proteínas Portadoras , Proteínas Fúngicas/genética , Hidrolasas/biosíntesis , Proteínas de la Membrana/genética , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Fosfatasa Alcalina/biosíntesis , Secuencia de Aminoácidos , Ácido Aspártico Endopeptidasas/biosíntesis , Secuencia de Bases , Carboxipeptidasas/biosíntesis , Catepsina A , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Precursores de Proteínas/metabolismo , Serina Endopeptidasas/biosíntesis , Fracciones Subcelulares/química

RESUMEN

In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway. Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells. The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene. Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine. The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli. ARO4 gene expression is regulated by the general control system of aa biosynthesis. As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions. However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions.

Asunto(s)

3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , 3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Tirosina/farmacología

RESUMEN

The phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonate-7-phosphate (dHp1P) synthase from Saccharomyces cerevisiae has been purified to apparent homogeneity by a 1250-fold enrichment of the enzyme activity present in wild-type crude extracts, employing an overproducing strain. The estimated molecular mass of 42 kDa corresponds to the calculated molecular mass of 42.13 kDa deduced from the previously determined primary sequence. Gel filtration indicates that the active enzyme is a monomer. The enzyme is an Fe protein and is inactivated by EDTA in a reaction which is reversible by several bivalent metal ions. The Michaelis constant of the enzyme is 18 microM for phosphoenolpyruvate (P-pyruvate) and 130 microM for erythrose 4-phosphate (Ery4P) and the rate constant was calculated as 10 s-1. Inhibition by phenylalanine is competitive with respect to erythrose 4-phosphate and non-competitive to phosphoenolpyruvate, with a Ki of 10 microM.

Asunto(s)

3-Desoxi-7-Fosfoheptulonato Sintasa/aislamiento & purificación , Aldehído-Liasas/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , 3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , 3-Desoxi-7-Fosfoheptulonato Sintasa/fisiología , Cromatografía , Cromatografía por Intercambio Iónico , Ácido Edético/farmacología , Concentración de Iones de Hidrógeno , Iones , Peso Molecular , Fenilalanina/farmacología , Especificidad por Sustrato

RESUMEN

The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine. The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment. Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein 256 amino acids. In addition, three mutant alleles of ARO7 were cloned and sequenced. These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity. A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains. In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.

Asunto(s)

Corismato Mutasa/metabolismo , Genes Fúngicos , Genes , Isomerasas/metabolismo , Mutación , Saccharomyces cerevisiae/enzimología , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Corismato Mutasa/genética , Clonación Molecular , Retroalimentación , Genotipo , Cinética , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Tirosina/farmacología

RESUMEN

The ARO3 gene encodes one of two 3-deoxy-D-arabino-heptulosonate-7-phosphate isoenzymes in Saccharomyces cerevisiae catalyzing the first step in the biosynthesis of aromatic amino acids. The ARO3-encoded 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) is feedback inhibited by phenylalanine; its isoenzyme, the ARO4 gene product, is inhibited by tyrosine. Both genes ARO3 and ARO4 are strongly regulated under the general control regulatory system. Cells carrying only one intact isogene are phenotypically indistinguishable from a wild-type strain when grown on minimal medium. The complete functional ARO3 promoter comprises 231 base pairs and contains only one TGACTA binding site for the general control activator protein GCN4. Mutating this element to TTACTA inhibits binding of GCN4 and results in a decreased basal level of ARO3 gene product and slow growth of a strain defective in its isogene ARO4. In addition, ARO3 gene expression cannot be elevated under amino acid starvation conditions. An ARO3 aro4 strain with gcn4 genetic background has the same phenotype of low ARO3 gene expression and slow growth. The amount of GCN4 protein present in repressed wild-type cells therefore seems to contribute to a basal level of ARO3 gene expression. The general control activator GCN4 has thus two functions: (i) to maintain a basal level of ARO3 transcription (basal control) in the presence of amino acids and (ii) to derepress the ARO3 gene to a higher transcription rate under amino acid starvation (general control).

Asunto(s)

3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Aldehído-Liasas/biosíntesis , ADN Polimerasa I/biosíntesis , Proteínas Fúngicas/fisiología , Genes Fúngicos , Genes Reguladores , Regiones Promotoras Genéticas , Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/fisiología , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , ADN/análisis , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Mutación , Plásmidos , Saccharomyces cerevisiae/enzimología , Factores de Transcripción/genética , Transcripción Genética

RESUMEN

We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites. A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene. In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene. In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.

Asunto(s)

Clonación Molecular , Genes Fúngicos , Recombinación Genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Southern Blotting , Escherichia coli/genética , Leucina/biosíntesis , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Plásmidos , Mapeo Restrictivo

RESUMEN

In Saccharomyces cerevisiae, the genes ARO3 and ARO4 encode isoenzymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. Both genes are derepressed seven-fold under the general control of amino acid biosynthesis. A previously isolated 1.7 kb fragment containing the ARO3 gene and the 5'- and 3'-flanking regions was sequenced. The endpoints of the ARO3 transcript coding for a 370 amino acid protein were mapped by primer extension experiments and S1 nuclease digestion. Promoter elements involved in transcription initiation and responsible for the strong general control derepression response are discussed.

Asunto(s)

3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Aldehído-Liasas/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Electroforesis en Gel de Poliacrilamida , Endonucleasas , Genes , Isoenzimas/genética , Datos de Secuencia Molecular , Fenilalanina/fisiología , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Endonucleasas Específicas del ADN y ARN con un Solo Filamento

RESUMEN

The TRP1 gene of Saccharomyces cerevisiae is the only TRP gene which is not derepressible by the general control regulatory system. In the TRP1 promoter transcription starts at five initiation sites, organized in two clusters. The two transcripts of the first, more upstream cluster include a long leader sequence of approximately 200 bp. A transcriptional terminator element located in the 5' region of the TRP1 gene is essential for accurate gene expression. In partial TRP1 promoters lacking the terminator, like the original EcoRI TRP1 fragment used in numerous vectors, plasmid-encoded transcription is initiated predominantly in adjacent vector regions, resulting mainly in large, poorly translated transcripts. This poor translation is not due to mRNA instability. The effect can be suppressed by introducing artificial transcription barriers between vector sequences and the truncated EcoRI TRP1 fragment.

Asunto(s)

Isomerasas Aldosa-Cetosa , Regulación de la Expresión Génica , Genes Fúngicos , Genes Reguladores , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Regiones Terminadoras Genéticas , Transcripción Genética , Secuencia de Bases , Carbohidrato Epimerasas/metabolismo , Mapeo Cromosómico , Clonación Molecular , Hibridación de Ácido Nucleico , Plásmidos , Transformación Genética

RESUMEN

Transcription of the gene for phosphoribosyl-anthranilate isomerase (TRP1) from the TRP1 promoter is initiated only approximately half as frequently as, for example, from the TRP3 promoter, but TRP1 mRNA is approximately twice as stable as TRP3 mRNA. Therefore, the steady state amount of TRP1 mRNA in yeast cells, grown without amino acid limitation, is similar to the steady-state amount of TRP3 mRNA. The protein concentration of both enzymes in yeast cells is about the same, but the basal specific enzyme activity in permeabilized cells of the TRP1 gene product N-(5'-phosphoribosyl-1)-anthranilate isomerase is about 2-3 times higher than that of any of the other TRP enzymes. According to the kinetic parameters of the purified isomerase protein, the enzyme is more active than, for example, the purified TRP3 enzyme indoleglycerol-phosphate synthase. It is suggested that the TRP1 gene of Saccharomyces cerevisiae might be the result of a rearrangement event, separating the N-(5'-phosphoribosyl-1)-anthranilate isomerase domain from the indoleglycerol-phosphate synthase domain and putting the catalytically more active isomerase domain behind a weak and nonregulated constitutive promoter.

Asunto(s)

Isomerasas Aldosa-Cetosa , Carbohidrato Epimerasas/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Triptófano/genética , Permeabilidad de la Membrana Celular , Regulación de la Expresión Génica , Semivida , Indol-3-Glicerolfosfato Sintasa/genética , Cinética , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Triptófano/biosíntesis