RESUMEN
Mutations truncating as many as 143 C-terminal residues from the transcriptional activator encoded by the areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, do not significantly reduce the ability of the areA product to activate expression of most genes under areA control. Such mutations can even have a gain-of-function, derepressed phenotype, consistent with a critical role for this region in modulating the activity of the areA protein. However, expression of a few genes under areA control is substantially impaired by such C-terminal truncations, indicating that regions of an activator protein can play differing roles in the control of different structural genes. This underlines the advantages of being able to monitor effects of areA mutations on expression of large numbers of structural genes. Additionally, it is shown that truncation of as many as 153 C-terminal residues, virtually all amino acids C-terminal to the DNA-binding region, is compatible with retention of some areA function.
Asunto(s)
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Factores de Transcripción/genética , Transcripción Genética , Activación Transcripcional , Secuencia de Aminoácidos , Aspergillus nidulans/metabolismo , Secuencia de Bases , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Fenotipo , Eliminación de Secuencia , Factores de Transcripción/metabolismoRESUMEN
Of five amidohydrolase activities subject to nitrogen metabolite repression in Aspergillus nidulans, L-asparaginase shows clearest evidence of also being subject to repression by atmospheric oxygen. Such oxygen repressibility is only evident under nitrogen metabolite derepressed conditions. Asparaginase levels are also considerably elevated by areA300, an altered function allele of the positive acting wide domain regulatory gene areA mediating nitrogen metabolite repression and are drastically reduced by loss of function mutations in areA. A. nidulans has two L-asparaginase enzymes and it has been shown by the use of appropriate mutants that these regulatory effects are exerted on the expression of that specified by the ahrA gene but probably not that specified by the apnA gene.
Asunto(s)
Asparaginasa/genética , Aspergillus nidulans/enzimología , Aerobiosis , Anaerobiosis , Asparaginasa/biosíntesis , Aspergillus nidulans/genética , Represión Enzimática/efectos de los fármacos , Mutación , Oxígeno/farmacologíaRESUMEN
The synthesis of thymine 7-hydroxylase, an alpha-ketoglutarate dependent dioxygenase, is subject both to nitrogen metabolite repression and to oxygen repression, while synthesis of the other pyrimidine salvage pathway dioxygenase, pyrimidine deoxyribonucleoside 2'-hydroxylase, is subject to neither. are A300, an allele of the positive acting regulatory gene are A mediating nitrogen metabolite repression in Aspergillus nidulans, considerably elevates levels of thymine 7-hydroxylase, probably alleviating at least partly both nitrogen metabolite repression and oxygen repression. are A300 has little or no effect on levels of pyrimidine deoxy-ribonucleoside 2'-hydroxylase but does elevate net uptake capacities for thymine, thymidine and deoxyuridine two-fold. are A300 was selected as allowing thymine to supplement a pyrimidine auxotrophy and was found to allow supplementation by thymidine, other pyrimidine nucleosides and pyrimidine salvage intermediates as well. This is the first reported evidence for are A control over an activity(-ies) not directly concerned with nitrogen source utilization.
Asunto(s)
Aspergillus nidulans/metabolismo , Genes Fúngicos , Genes Reguladores , Pirimidinas/metabolismo , Alelos , Aspergillus nidulans/genética , Oxigenasas de Función Mixta/metabolismo , Nitrógeno/metabolismo , Consumo de OxígenoRESUMEN
Partially purified preparations from Aspergillus nidulans were shown to catalyze two alpha-ketoglutarate dependent dioxygenase reactions: the pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3) and the thymine 7-hydroxylase (EC 1.14.11.6) reactions. These reactions showed an absolute requirement for alpha-ketoglutarate and molecular oxygen and were stimulated by Fe(II), ascorbate and catalase. Both reactions demonstrated a stoichiometry such that for each mole of substrate (deoxyribonucleoside or pyrimidine) hydroxylated one mole of CO2 was produced from alpha-ketoglutarate. These two activities were separated using DEAE-Sephacel chromatography.
Asunto(s)
Aspergillus nidulans/enzimología , Oxigenasas de Función Mixta/aislamiento & purificación , Ácidos Cetoglutáricos/farmacología , Oxigenasas de Función Mixta/metabolismo , Oxígeno/farmacologíaRESUMEN
The utilization of thymidine by Neurospora crassa is initiated by the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and the consequent formation of thymine and ribose. Thymine must then be oxidatively demethylated by the thymine 7-hydroxylase and uracil-5-carboxylic acid decarboxylase reactions. This article shows that the 2'-hydroxylase reaction can be regulated differently than the oxidative demethylation process and suggests that the 2'-hydroxylase has, in addition to the role of salvaging the pyrimidine ring, the role of providing ribose not only for the utilization of the demethylated pyrimidine but also for other metabolic processes. One way that this difference in regulation was observed was with the uc-1 mutation developed by Williams and Mitchell. The present communication shows that this mutation increases the activities of the 7-hydroxylase and the decarboxylase but has no comparable effect on the 2'-hydroxylase. Qualitatively similar effects on these enzymes were bought about by growth of wild-type Neurospora in media lacking ammonium ion, such as the Westergaard-Mitchell medium. The 2'-hydroxylase and 7-hydroxylase are also differently affected by the carbon dioxide content of the atmosphere above the growing culture and the growth temperature. Studies with inhibitors indicated that the carbon dioxide effect is dependent on protein synthesis.
Asunto(s)
Neurospora crassa/metabolismo , Neurospora/metabolismo , Timidina/metabolismo , Dióxido de Carbono/farmacología , Carboxiliasas/metabolismo , Sistema Libre de Células , Dactinomicina/farmacología , Inducción Enzimática/efectos de los fármacos , Oxigenasas de Función Mixta/metabolismo , Mutación , Neurospora crassa/enzimología , Proflavina/farmacología , Compuestos de Amonio Cuaternario/farmacología , Ribosa/biosíntesis , Temperatura , Timina/biosíntesisRESUMEN
The experiments in this report involve the following series of reactions which were previously demonstrated with purified enzyme preparations from Neurospora crassa: thymidine a yields thymine ribonucleoside b yields thymine c yields 5-hydroxymethyluracil d yields 5-formyluracil e yields uracil-5-carboxylic acid f yields uracil. The evidence for some of the reactions occurring in vivo has been incomplete and for others totally lacking. In this paper intact cells of Neurospora are shown to be capable of converting the substrates of each of the reactions to the corresponding products. Studies are described which were carried out in vivo and in vitro with the pyrimidineless strains pyr-4,uc-1,uc-2 and pyr-4,uc-1,uc-3, developed by Williams and Mitchell. The results reported in the present paper indicate that (reaction a) and the uc-3 mutation affects thymine 7-hydroxylase (reactions c,d, and e). Evidence is presented for the 2'-hydroxylase reaction being the major, if not only, way by which Neurospora can initiate the conversion of thymidine to the pyrimidines of nucleic acids and for the 2'-hydroxylation of thymidine and deoxyuridine being catalyzed by the same enzyme. Deoxycytidine was shown not to be hydroxylated in intact cells but instead deaminated to deoxyuridine, which in turn was converted to uridine. Further studies with the uc-3-carrying strain showed that an enzyme other than thymine 7-hydroxylase can also convert 5-formyluracil to uracil-5-carboxylic acid.