Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a systemic mycosis restricted geographically to Central and South America. It is a dimorphic fungus which undergoes a mycelial (M) to yeast (Y) transition in a temperature-dependent way. Rho GTPases are key regulatory proteins during fungal morphogenesis. In Saccharomyces cerevisiae, Candida albicans and Aspergillus fumigatus, β -1,3-glucan synthase is regulated by Rho1 [1-3]. In Schizosaccharomyces pombe, Rho1 and Rho2 regulate the syntheses of β -1,3-glucan and α -1,3-glucan, respectively . We identified and cloned two different RHO genes from P. brasiliensis by PCR and colony hybridization . Intron confirmation was performed by RT-PCR (Life Technologies, Rockville, USA). Sequence analysis revealed that translations of both putative P. brasiliensis DNA fragments resembled either Rho1 or Rho2 proteins, by comparison to other fungal Rho GTPases . Therefore, the putative P. brasiliensis RHO genes were designated PbrRHO1 and PbrRHO2. PbrRHO1 presents a size of 950 bp, with an open reading frame (ORF) of 573 bp, interrupted by four introns. PbrRHO2 is 957 bp long, showing a discontinuous ORF of 612 bp with four introns. The deduced sequences of these genes show 191 and 204 amino acids for Rho1 and Rho2, respectively. Expression studies and complementation in S. cerevisiae rho null mutants are under progress. The sequences of the P. brasiliensis RHO1 (accession number AY392528) and RHO2 (accession number AY496954) have been deposited in GenBank. ACKNOWLEDGEMENT: To FONACIT (Caracas, Venezuela) for Grant G97-0000615 1.- Cabib, E. et al. J. Biol. Chem. 2001; 276: 19679-19682. 2.- Kondoh, O. et al. J. Bacteriol. 1997; 179: 7734-7741. 3.- Beauvais, A. et al. J. Bacteriol. 2001; 183: 2273-2279. 4.- Calonge, T. M. et al. Mol. Cell Biol. 2000; 11: 4393-4401. 5.- Sambrook, J. et al. (Eds). 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 6.- Altschul, S. F. et al. Nucleic Acids Res. 1997; 25: 3389-3402.
Full conference title:
14th Annual Focus on Fungal Infections
- FFI 14th (2004)