RESUMEN
We have developed a multiplex assay covering 16 microsatellite loci, amplified in four polymerase chain reaction (PCR) assays, and loaded on the ABI DNA Analyzer in two separate panels. The assay was tested on 603 individuals originating from wild populations and hatchery stocks of Atlantic sturgeon. The assay was also tested on 12 individuals of European sturgeon and appeared to be almost equally useful. The multiplex assay designed in this study can be successfully applied in studies requiring high genetic resolution, such as relatedness analysis, selective breeding programs, and stock identification of Atlantic sturgeon.
Asunto(s)
Bioensayo/métodos , Peces/genética , Variación Genética/genética , Genética de Población , Repeticiones de Microsatélite/genética , Reacción en Cadena de la Polimerasa/métodos , Animales , Técnicas de GenotipajeRESUMEN
We have cloned and analysed the arcA gene which encodes a transcriptional activator necessary for the high-level expression of two genes for enzymes of the arginine catabolic pathway in Aspergillus nidulans: agaA (for arginase) and otaA (for ornithine transaminase, OTAse). Here we present complete genomic and cDNA sequences for, and describe the pattern of expression of, the arcA gene. This gene contains one intron and encodes a polypeptide of 600 amino acids. The deduced protein belongs to the family of Zn(2)Cys(6) fungal regulatory proteins. ARCA is the first known protein of this family that has glycine instead of the conserved proline at the fifth position in the second, six-residue, loop of the Zn cluster domain. We have established that transcription of the arcA gene is not self-regulated and does not depend on arginine. Two mutations in arcA, one gain-of-function and one loss-of-function, have been sequenced and the effects of these mutations on the expression of the agaA gene at the transcriptional level are reported.
Asunto(s)
Arginina/metabolismo , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reguladores , Transactivadores/genética , Secuencia de Aminoácidos , Arginasa/biosíntesis , Arginasa/genética , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Mutación , Ornitina-Oxo-Ácido Transaminasa/biosíntesis , Ornitina-Oxo-Ácido Transaminasa/genética , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Dedos de ZincRESUMEN
The arginase structural gene (agaA) from Aspergillus nidulans has been cloned and characterised. Depending on the growth conditions of the mycelium, transcripts of this gene have different 5'ends. These differences could result either from the presence of multiple transcription initiation sites or from differential processing of mRNA. The agaA mRNA has a long 5'UTR with a potentially complex secondary structure. Putative arginine binding aptamers were found in this UTR suggesting interesting possibilities for regulation of the agaA expression.
Asunto(s)
Arginasa/genética , Aspergillus nidulans/genética , ARN Mensajero/genética , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Genes Fúngicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN Mensajero/química , Transcripción GenéticaRESUMEN
The ornithine transaminase (otaA) gene of Aspergillus nidulans has been cloned by transformation of the A. nidulans pro-ota- mutant strain with a cosmid gene library. The otaA gene contains two introns and potentially codes for a 453-aa-long protein. The deduced amino-acid sequence is homologous to known ornithine transaminases from Saccharomyces cerevisiae, Plasmodium falciparum, Vigna aconitifolia, rat, mouse and man, particularly in the pyridoxal phosphate-binding domain. The expression of the otaA gene is specifically induced by arginine, and is also under the control of nitrogen-metabolite and carbon-catabolite repression. Regulation of the gene occurs at both transcriptional and post-transcriptional levels. The promoter region of otaA contains putative AREA and CREA binding-sites. Fusion proteins containing AREA or CREA DNA-binding domains bind some of these sites. CREA binding-sites correspond very well to the CREA-binding consensus sequence which is SYGGRG. AREA binding-sites are composed of GATT sequences which are not typical binding sites for the GATA - binding family of transcription factors.
Asunto(s)
Aspergillus nidulans/genética , Ornitina-Oxo-Ácido Transaminasa/genética , Secuencia de Aminoácidos , Aspergillus nidulans/enzimología , Aspergillus nidulans/crecimiento & desarrollo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/genética , Datos de Secuencia Molecular , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Arginase (EC 3.5.3.1) of Aspergillus nidulans, the enzyme which enables the fungus to use arginine as the sole nitrogen source was purified to homogeneity. Molecular mass of the purified arginase subunit is 40 kDa and is similar to that reported for the Neurospora crassa (38.3 kDa) and Saccharomyces cerevisiae (39 kDa) enzymes. The native molecular mass of arginase is 125 kDa. The subunit/native molecular mass ratio suggests a trimeric form of the protein. The arginase protein was cleaved and partially sequenced. Two out of the six polypeptides sequenced show a high degree of homology to conserved domains in arginases from other species.
Asunto(s)
Arginasa/aislamiento & purificación , Aspergillus nidulans/enzimología , Secuencia de Aminoácidos , Secuencia Conservada , Datos de Secuencia Molecular , Peso Molecular , Alineación de SecuenciaRESUMEN
The protein nitrogen regulator I (NRI)-phosphate is known to activate the initiation of transcription of the Escherichia coli glnA gene. This activation is facilitated by the binding of the protein to NRI-specific sites located upstream of the sigma 54-dependent glnA promoter. To determine whether binding of NRI-phosphate to upstream sites is sufficient for activation, we placed several promoters not normally activated by NRI-phosphate downstream of NRI binding sites and measured activation in intact cells and in an in vitro transcription system. We found that the sigma 70-dependent lac promoter was not activated, that the sigma 54-dependent Klebsiella pneumoniae nifH promoter was weakly activated, and that a nifH promoter altered in the RNA polymerase binding site was almost as well activated as the glnA promoter. We conclude that the sensitivity of the susceptible promoter depends on the presence of NRI binding sites, but that the presence of bound NRI-phosphate upstream of a promoter is not sufficient for activation of transcription by RNA polymerase. This activation is determined by the structure of the RNA polymerase binding site. We suggest that sigma 54-but not sigma 70-dependent promoters are susceptible to activation by NRI-phosphate and that the nucleotide sequence of the sigma 54-RNA polymerase binding site is an important determinant of the efficiency of activation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Fijación del Nitrógeno/genética , Regiones Promotoras Genéticas , Transactivadores , Factores de Transcripción , Transcripción Genética , Secuencia de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Genotipo , Datos de Secuencia Molecular , Proteínas PII Reguladoras del Nitrógeno , Plásmidos , Factor sigma/metabolismoRESUMEN
Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I (NRI), the product of glnG, were obtained by two different selection procedures. The mutant proteins were purified and characterized. The concentrations of mutant proteins needed to activate transcription at the glnAp2 promoter were three to four times lower than that of the wild-type NRI. The rate of phosphorylation of these proteins and the stability of mutant NRI phosphate were found to be similar to those of the wild-type NRI. In one of the mutants, the site of the mutation was localized in the DNA region specifying the central domain of NRI.
Asunto(s)
Proteínas Bacterianas , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Genes Bacterianos , Mutación , Transactivadores , Factores de Transcripción , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Regulación de la Expresión Génica , Genotipo , Proteínas PII Reguladoras del Nitrógeno , Fosforilación , Plásmidos , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
We have shown that the Aspergillus nidulans ans1 sequence enhances the efficiency of transformation when introduced into vectors containing argB or trpC genes. Increased efficiency of transformation is also observed when ans1 is present on a second cotransforming plasmid. In an attempt to explains the ans1 transactivity we have performed analysis of some cotransformants.
Asunto(s)
Aspergillus nidulans/genética , Genes Bacterianos/genética , Transformación Bacteriana/genética , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Hibridación de Ácido Nucleico , Plásmidos/genéticaRESUMEN
We have constructed a series of deletion plasmids which contain the Aspergillus nidulans argB gene for ornithine carbamoyltransferase (OTC). These deletions comprise the 5' upstream sequence of the argB gene. The pro- arg- strain of A. nidulans was transformed with the above plasmids. Several arg+ transformants of integration types I and II, obtained using each of the deletion plasmids, were studied, and their ability to de-repress OTC level by proline starvation was compared. It was concluded that nucleotides located between -150 and -50 bp upstream of the argB gene are significant for its cross-pathway regulation. This regulatory region contains three copies of the TGACTC hexanucleotide which is a cis-acting regulatory sequence of general amino acid control in yeast.
Asunto(s)
Aspergillus nidulans/genética , Genes Fúngicos , Ornitina Carbamoiltransferasa/genética , Secuencias Reguladoras de Ácidos Nucleicos , Aspergillus nidulans/enzimología , Secuencia de Bases , ADN de Hongos , Escherichia coli/genética , Datos de Secuencia Molecular , Ornitina Carbamoiltransferasa/metabolismo , Plásmidos , Prolina/metabolismo , Mapeo Restrictivo , Transformación GenéticaRESUMEN
Aspergillus nidulans argB mutant was transformed with the plasmid DNA containing the argB gene. Analysis of transformants revealed that transformation was due to integration of either argB gene alone or the whole plasmid DNA into the A. nidulans genome. In 5 out of 23 transformants studied, integration took place in the locus different than the original argB locus. The amplification of integrated sequences was often observed. Integrated DNA was found to be mitotically stable, while the meiotic stability depends on the mode of integration. The activity of the ornithine carbamoyltransferase (the argB gene product) was measured and in some transformants bearing the amplified argB sequence was found to be strongly elevated.
Asunto(s)
Aspergillus nidulans/genética , ADN de Hongos/análisis , Genes Fúngicos , Ornitina Carbamoiltransferasa/genética , Transformación Genética , Aspergillus nidulans/enzimología , Enzimas de Restricción del ADN , Vectores Genéticos , Metilación , Hibridación de Ácido Nucleico , Ornitina Carbamoiltransferasa/metabolismo , PlásmidosRESUMEN
The transcriptional organization and sequence of the Aspergillus nidulans argB gene, encoding ornithine carbamoyl transferase (OCTase; E.C. 2.1.3.3.), was determined. Transcription of the gene begins within a methionine-initiated open translation reading frame, indicating that a second methionine codon of the open reading frame is used for translation initiation. The predicted length of the OCTase precursor peptide is 359 amino acids, and it contains a highly basic amino terminus that is probably involved in mitochondrial targeting. There is extensive homology between Aspergillus OCTase and mammalian and bacterial OCTases and weaker homology between the Aspergillus polypeptide and bacterial arginine carbamoyl transferase.
Asunto(s)
Aspergillus nidulans/genética , Genes Bacterianos , Genes , Ornitina Carbamoiltransferasa/genética , Secuencia de Aminoácidos , Aspergillus nidulans/enzimología , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , Especificidad de la Especie , Transcripción GenéticaRESUMEN
The Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OTCase) is not expressed in Escherichia coli. However, E. coli OTCase-deficient strains transformed with plasmids carrying the argB gene from A. nidulans reverted to prototrophy at a high frequency. In these derivatives the argB gene became functional due to DNA rearrangements upstream of the coding sequence. Two types of rearrangement were characterized. One was identified as an insertion of IS2. The second was a deletion that resulted in transcription of the argB gene from the TcR gene promoter and translation from a newly created ribosome-binding site formed at the junction between the A. nidulans and vector DNA sequences.
Asunto(s)
Aspergillus nidulans/genética , Regulación de la Expresión Génica , Genes Bacterianos , Genes Fúngicos , Ornitina Carbamoiltransferasa/genética , Secuencia de Bases , Elementos Transponibles de ADN , ADN Bacteriano , ADN de Hongos , Escherichia coli/genética , Plásmidos , Biosíntesis de Proteínas , Transcripción GenéticaRESUMEN
Differential centrifugation of the Aspergillus nidulans cell lysate shows that ornithine carbamoyltransferase (EC 2.1.3.3) appears mainly in the particulate (organellar) fraction. The enzyme was located to the mitochondria by co-sedimentation with cytochrome oxidase in isopycnic density gradient and by cytochemical-electron microscopic means. Arginase (EC 3.5.3.1) and ornithine delta-aminotransferase (E.C. 2.6.1.13) were found to reside in cytosol. The release of ornithine carbamoyltransferase from the organellar fraction by various agents indicates that the enzyme resides in the mitochondrial matrix. In Saccharomyces cerevisiae the plasmid pSAL43, carrying cloned Aspergillus nidulans ornithine carbamoyltransferase gene, directs the synthesis of the enzyme partially associated with yeast mitochondria even though the homologous yeast enzyme is exclusively cytosolic. The implications of these findings are discussed.
Asunto(s)
Aspergillus nidulans/enzimología , ADN Recombinante , Ornitina Carbamoiltransferasa/análisis , Saccharomyces cerevisiae/enzimología , Arginasa/análisis , Aspergillus nidulans/ultraestructura , Centrifugación por Gradiente de Densidad , Citosol/enzimología , Histocitoquímica , Microscopía Electrónica , Mitocondrias/enzimología , Organoides/enzimología , Ornitina Carbamoiltransferasa/genética , Ornitina-Oxo-Ácido Transaminasa/análisis , Plásmidos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , SolubilidadRESUMEN
An Aspergillus nidulans DNA fragment composed of two adjacent SalI subfragments (1.8 and 0.85 kb) that carries an argB gene complementing the yeast arg3 mutation has been isolated from two different gene libraries. Hybridization results and immunological tests indicate that the cloned fragment contains the A. nidulans structural gene coding for ornithine carbamoyltransferase (OTCase). Using the cloned gene as a probe, the specific mRNA was identified. The level of this RNA observed in A. nidulans strains grown under various conditions correlated with the level of the OTCase activity, suggesting transcriptional control of OTCase synthesis. Expression of the cloned gene in Saccharomyces cerevisiae does not depend on its orientation in the vector. In Escherichia coli, the cloned gene does not function; however arg- transformants revert to prototrophy with high frequency possibly due to DNA rearrangements within the recombinant plasmid.
Asunto(s)
Aspergillus nidulans/genética , Ornitina Carbamoiltransferasa/genética , Aspergillus nidulans/enzimología , Precipitación Química , Clonación Molecular , ADN de Hongos/biosíntesis , Escherichia coli/metabolismo , Genes Fúngicos , Inmunoquímica , Hibridación de Ácido Nucleico , Ornitina Carbamoiltransferasa/biosíntesis , Plásmidos , ARN de Hongos/biosíntesis , Transformación GenéticaRESUMEN
Fragments of Aspergillus nidulans DNA obtained after digestion with EcoRI, BamHI and HindIII endonucleases were cloned in Escherichia coli in plasmid pBR322. These gene banks were used for transformation of 15 E. coli auxotrophic mutants and in 5 cases prototrophic clones containing recombinant plasmids were selected. Three different recombinant plasmids conferring prototrophy to pyrF, proAB and argIF mutants were analyzed. Hybridization experiments indicated that in two of these plasmids the inserted fragment of DNA hybridized not only to the A. nidulans but also to the E. coli DNA.
Asunto(s)
Aspergillus nidulans/genética , ADN de Hongos/aislamiento & purificación , Escherichia coli/genética , Genes Fúngicos , Plásmidos/aislamiento & purificación , Clonación Molecular , ADN de Hongos/metabolismo , Desoxirribonucleasa BamHI/metabolismo , Desoxirribonucleasa EcoRI/metabolismo , Desoxirribonucleasa HindIII/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Hibridación de Ácido NucleicoRESUMEN
Using N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, ethyl methanesulphonate or 4-nitroquinoline-1-oxide mutagenesis and an enrichment method for the isolation of auxotrophs, 25 mutants with defects in the adA locus were obtained after screening 41,376 colonies. One of these, adA24, did not complement with any of the other adA mutants, had a very high reversion rate and had some other properties which usually characterize strains carrying nonsense mutations. All revertants of adA24 carried dominant suppressor mutations. A group of adA24 suppressors was tested for allele and locus specificity. They were found to suppress only some adA alleles, and at the same time, some mutations in the methG, methH, argB and proA loci. It is proposed that the allele specific and locus non-specific adenine suppressors are suppressors of nonsense mutations.
Asunto(s)
Aspergillus nidulans/genética , Supresión Genética , Adenina/biosíntesis , Adenosina Monofosfato/biosíntesis , Alelos , Mapeo Cromosómico , Genes , Genes Dominantes , Ligamiento Genético , MutaciónRESUMEN
We examined wild-type and mutant strains of Klebsiella aerogenes for the relative amounts of ribonucleic acid (RNA) hybridizing specifically to deoxyribonucleic acid from a transducing phage carrying glnAK, the structural gene for glutamine synthetase. Our data showed a positive correlation between the intracellular level of glutamine synthetase and the level of glnA messenger RNA; we were unable to detect glnA messinger RNA in strains devoid of glutamine synthetase protein. Therefore, it is possible that transcription of glnA is not regulated simply by repression mediated through the glutamine synthetase protein; rather, autogenous control in this system may involve activation of transcription. Our experiments also suggest that the promotor of the glnA gene is located at the rha proximal end of the gene.
Asunto(s)
Glutamato-Amoníaco Ligasa/biosíntesis , Klebsiella pneumoniae/metabolismo , Operón , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Genes , Klebsiella pneumoniae/enzimología , Modelos Biológicos , Mutación , Transcripción GenéticaRESUMEN
The areA gene which is known to be involved in ammonium repression in Aspergillus nidulans was found to participate in regulation of arginine catabolism. Mutations in this gene are hypostatic to mutations in arcA, suDpro and suEpro genes which are responsible for regulation of synthesis of arginine catabolic enzymes.