RESUMEN
In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker.
Asunto(s)
Aspergillus oryzae/genética , Sitios de Ligazón Microbiológica/genética , Eliminación de Gen , Integrasas/metabolismo , Mutagénesis Insercional/métodos , Mutagénesis Sitio-Dirigida/métodos , Aspergillus nidulans/genética , Escherichia coli/genética , Genes Fúngicos/genética , Marcadores Genéticos/genética , Integrasas/genética , Plásmidos/genética , Selección GenéticaAsunto(s)
Criptococosis/diagnóstico , Cryptococcus neoformans , Dermatomicosis/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva , Neoplasias Hepáticas , Anfotericina B/uso terapéutico , Antifúngicos/uso terapéutico , Criptococosis/tratamiento farmacológico , Criptococosis/patología , Dermatomicosis/tratamiento farmacológico , Dermatomicosis/patología , Diagnóstico Diferencial , Humanos , Huésped Inmunocomprometido , Masculino , Persona de Mediana Edad , MusloRESUMEN
A 47-year-old woman presented with multiple bluish subcutaneous nodules on the trunk and upper extremities. The histological diagnosis of a subcutaneous nodule was cavernous hemangioma. Multiple cavernous hemangiomas were also found in her cerebrum, cerebellum, and medulla oblongata on magnetic resonance imaging examination. We did not detect any mutations in the two loci of the TIE2 gene that have been reported in familial venous malformations.
