Molecular cloning, characterization and differential expression of Cdc42 in Fonsecaea monophora

The cell divisions cycle 42 (Cdc42) gene has been characterized in the fungi, such as Candida albicans, Penicillium marneffei, and Wangiella (Exophiala) dermatitidis, which plays important roles during growth and development. The partial cDNA sequence of Cdc42 of Fonsecaea monophora (F. monophora), designated FmCdc42, was obtained using degenerate primers based on the conserved domain of the other fungi Cdc42. Then the complete cDNA sequence of FmCdc42 was obtained by 5′ and 3′ RACE. The full-length cDNA is 1,510 bp in size which had an open reading frame (ORF) of 582 bp, encoding 193 amino acid residues. The predicted molecular mass of FmCdc42 is 21.5 kDa with an estimated theoretical isoelectric point of 5.67. The deduced amino acid sequence of FmCdc42 shows 99% identity to that of Wangiella (Exophiala) dermatitidis. 5 exons and 4 introns are identified within the 1,617 bp FmCdc42 genomic DNA sequence of F. monophora. The ORF could be subcloned into the pCDNA6/myc-His B expression vector. The recombinant protein about 27.5 kD infusion protein had high expression level in Vero cells with SDS-PAGE and Western blot analysis. Quantitative real time RT-PCR revealed that FmCdc42 was the highest expression in the sclerotic bodies’ stage compared with that in the mycelia and conidia stages, which indicated that the FmCdc42 may be involved in formation of F. monophora sclerotic bodies.