Abstract
As an initial step in the study of the role of G proteins in signal transduction in Sporothrix schenckii, we identified a Gαi subunit using different experimental approaches. Western blots of fungal membrane preparations using anti-Gαcommon and anti-Gαi1-Gαi2 antibodies identified a band of approximately 41 kDa. Pertussis toxincatalyzed adenosine diphosphate (ADP)-ribosylation of these membrane fractions confirmed the presence of a protein substrate of 41 kDa. A 357 bp polymerase chain reaction (PCR) product obtained using fungal DNA as template and primers targeted to conserved Gαi sequences, was used as a probe to isolate a clone from an S. schenckii genomic library. A partial sequence for a Gαi subunit was obtained from this clone. The sequence was completed using the rapid amplification of cDNA ends (RACE) technique with mycelium and yeast cDNA. The cDNA sequence revealed a 1059 bp open reading frame encoding a 353 amino acid Gαi subunit of 41 kDa, more than 90% identical to the CPG-1 of Cryphonectria parasitica, and GNA-1 of Neurospora crassa. The genomic sequence was obtained by PCR using fungal DNA, and revealed a 1250 bp sequence and the presence of three introns. These results provide evidence for the first time of the presence and expression of a Gαi homolog in a pathogenic dimorphic fungus.