RESUMO
The yeast-to-mycelium transition in Sporothrix schenckii has been shown to respond to protein kinase C (PKC) effectors, indicating the involvement of PKC in this regulation. In this study, we identified the presence of two pkcl-like genes in S. schenckii. Using fungal genomic DNA as template and primers targeted to conserved sequences in the Saccharomyces cerevisiae pkc1 gene, two partially overlapping extra long polymerase chain reaction (XL-PCR) products were obtained. These XL-PCR products were sequenced and found to encode part of the C3/C4 domains of two different PKC-like proteins. The presence of two different genes was confirmed by Southern blot analysis. These two genes were named pkcSs-1 and pkcSs-2. The sequence of the pkcSs-2 gene was completed and revealed an open reading frame of 3942 nucleotides interrupted by five introns. A transcript of 8.7 kb was detected in northern blot analysis of poly A+ RNA. The pkcSs-2 gene encodes a protein of 1194 amino acids and 132.84 kDa that contains the characteristic structure and domains of other fungal PKCs reported to date. Using reverse transcription-PCR (RT-PCR), the pkcSs-2 gene was found to be expressed at all intervals tested during the yeast-to-mycelium transition.
Assuntos
Proteína Quinase C/genética , Sporothrix/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Sequência Conservada , DNA Fúngico/análise , Expressão Gênica , Genoma Fúngico , Dados de Sequência Molecular , Morfogênese/genética , Micélio/enzimologia , Filogenia , Reação em Cadeia da Polimerase , Proteína Quinase C/isolamento & purificação , Homologia de Sequência de Aminoácidos , Sporothrix/genética , Sporothrix/crescimento & desenvolvimento , Transcrição GênicaRESUMO
Protein kinase C (PKC) plays an important role in the control of proliferation and differentiation of a wide range of cell types, and fungi are no exception. Previous results reported by us on the effects of the phorbol ester, 12-myristate-13-acetate phorbol (PMA) and other PKC effector molecules, on dimorphism in Sporothrix schenckii suggested the presence of this enzyme in the fungus and its involvement in the control of morphogenetic transitions. The work summarized here confirms the presence of PKC in yeast and mycelium extracts of S. schenckii. Different isoforms of this enzyme were found to be present in the yeast and mycelium forms of the fungus and were identified by Western blot analysis using affinity purified anti-PKC isoforms specific antibodies: the gamma and zeta isoforms were detected in both the yeast and mycelium forms of the fungus, while the beta isoform was only detected in the yeast form. The presence of PKC was confirmed biochemically by measuring total enzyme activity in both forms of the fungus. No significant differences were observed for the PKC activity level recorded for both the mycelium and yeast forms of the fungus (p < or = 0.05). These data confirm the presence of PKC activity in Sporothrix schenckii and constitutes the first evidence concerning the differential expression of PKC isoforms in the mycelium and yeast forms of a dimorphic fungus, supporting the possible involvement of this important signal transduction enzyme in the control of morphogenesis in this fungus.
