RESUMO
The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from Aspergillus, yeast, and mammals, indicating an ancient origin.
Assuntos
Aspergillus nidulans/genética , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/genética , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Aspergillus nidulans/metabolismo , Sequência Conservada , Proteínas Fúngicas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Mutação , Nitrogênio/metabolismo , Sinais de Localização Nuclear , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The use of native promoters to drive transgene expression has facilitated overexpression studies in Drosophila and other insects. We identified 12 Tubulin family members from the genome sequence of the red flour beetle, Tribolium castaneum, and used the promoter from one of these to drive constitutive expression of a transgene. The activity of the T. castaneum alpha-Tubulin1 (TcalphaTub1) putative promoter was pre-tested in conjunction with an eye-color gene, T. castaneum vermilion (Tcv), by transient expression in Tcv-deficient embryos. Such embryos showed complete rescue of larval eyespot pigmentation. We also examined the TcalphaTub1 expression pattern in germline transformants using the enhanced green fluorescent protein (EGFP) reporter. Beetles transformed with this piggyBac-based reporter ubiquitously expressed EGFP at all stages.
