Characterization of a protein kinase C gene in Sporothrix schenckii and its expression during the yeast-to-mycelium transition.

Abstract

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.