Presence of a pertussis toxin-sensitive G protein alpha subunit in Sporothrix schenckii.
As an initial step in the study of the role of G proteins in signal transduction in Sporothrix schenckii, we identified a Galphai subunit using different experimental approaches. Western blots of fungal membrane preparations using anti-Galphacommon and anti-Galphai1-Galphai2 antibodies identified a band of approximately 41 kDa. Pertussis toxin-catalyzed 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 Galphai sequences, was used as a probe to isolate a clone from an S. schenckii genomic library. A partial sequence for a Galphai 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 Galphai 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 Galphai homolog in a pathogenic dimorphic fungus.
Delgado
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Department of Microbiology and Medical Zoology, University of Puerto Rico, San Juan, USA.
Rodríguez-del Valle
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D037342:Pertussis Toxin; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D013175:Sporothrix / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D018076:DNA, Complementary; D005656:Fungal Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins