Browsing by Author "Avila, M"

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    Differential regulation of genes encoding manganese peroxidase (MnP) in the basidiomycete Ceriporiopsis subvermispora
    (2003) Manubens, A; Avila, M; Canessa, P; Vicuña, R
    We previously identified and characterized three mnp genes coding for manganese peroxidase (MnP) in the white rot fungus Ceriporiopsis subvermispora. In this work, we assessed transcript levels of mnp genes in liquid cultures of this fungus grown under various conditions. In the absence of Mn2+, mnp1 and mnp2 mRNA were detected by Northern hybridization, irrespective of the lack of extracellular MnP activity. Addition of Mn2+ to the cultures led to a marked increase in both transcripts, the highest titers being observed at 10 muM Mn2+ mnp1 mRNA was not detected at Mn2+ concentrations above 80 muM, whereas mnp2 mRNA was still observed at 320 muM Mn2+. Differential regulation of these genes was confirmed by the addition of Cu2+, Zn2+, Ag+ and Cd2+. These metal ions dramatically elevated both transcripts and also allowed the detection of the mnp3 transcript. In most cases, the increase in mRNA levels was partially abolished by the simultaneous presence of Mn2+, although the latter was strictly required to detect extracellular MnP activity. However, the lignin-related compound syringic acid specifically increased the mnp1 transcript, although only in the absence of Mn2+. These results indicate that there is no clear correlation between mnp mRNA levels and MnP activity. In addition, they strongly suggest that Mn2+ plays a post-transcriptional role which is essential for the presence of active MnP in the extracellular fluid.
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    Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns
    (2003) Larrondo, LF; Avila, M; Salas, L; Cullen, D; Vicuña, R
    Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described Ics-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active laccase. Relative to the isozymic forms of the native C. subvermispora enzyme, the A. niger-produced laccase had a higher molecular mass and gave a single band on IEF gels. In contrast, A. nidulans transformants secreted several isoforms remarkably similar to those of the native system. Considered together with previously reported Southern blots and protein sequencing, expression in A. nidulans supports the view that C. subvermispora has a single laccase gene and that multiple isoforms result from post-translational processes. In addition, several lines of evidence strongly suggest that under copper limitation, A. nidulans secretes apoprotein which can be reconstituted by a short incubation with Cu(I) and to a lesser extent with Cu(II).