RESUMO
Nitrogen metabolite repression (NMR) in filamentous fungi is controlled by the GATA transcription factors AreA and AreB. While AreA mainly acts as a positive regulator of NMR-sensitive genes, the role of AreB is not well understood. We report the characterization of AreB and its interplay with AreA in the gibberellin-producing fungus Fusarium fujikuroi. The areB locus produces three different transcripts that each code for functional proteins fully complementing the areB deletion mutant that influence growth and secondary metabolism. However, under nitrogen repression, the AreB isoforms differ in subcellular localization indicating distinct functions under these conditions. In addition, AreA and two isoforms of AreB colocalize in the nucleus under low nitrogen, but their nuclear localization disappears under conditions of high nitrogen. Using a bimolecular fluorescence complementation (BiFC) approach we showed for the first time that one of the AreB isoforms interacts with AreA when starved of nitrogen. Cross-species complementation revealed that some AreB functions are retained between F. fujikuroi and Aspergillus nidulans while others have diverged. By comparison to other fungi where AreB was postulated to function as a negative counterpart of AreA, AreB can act as both repressor and activator of transcription in F. fujikuroi.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica , Nitrogênio/metabolismo , Mapeamento de Interação de Proteínas , Núcleo Celular/química , Citoplasma/química , Fusarium/crescimento & desenvolvimento , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação GenéticaRESUMO
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.
Assuntos
Arginase/genética , Aspergillus nidulans/genética , RNA Mensageiro/genética , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Fúngico , Genes Fúngicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Mensageiro/química , Transcrição GênicaRESUMO
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.
Assuntos
Aspergillus nidulans/genética , Ornitina-Oxo-Ácido Transaminase/genética , Sequência de Aminoácidos , Aspergillus nidulans/enzimologia , Aspergillus nidulans/crescimento & desenvolvimento , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Dados de Sequência Molecular , Ornitina-Oxo-Ácido Transaminase/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Arginase/isolamento & purificação , Aspergillus nidulans/enzimologia , Sequência de Aminoácidos , Sequência Conservada , Dados de Sequência Molecular , Peso Molecular , Alinhamento de SequênciaRESUMO
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.