Presence of a pertussis toxin-sensitive G protein α subunit inSporothrix schenckii

Abstract

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.

Chromosomal Polymorphism in the Sporothrix schenckii Complex

“…PCR was used to amplify gene fragments from β-tubulin (β-tub) [10], calmodulin (Calm) [22], catalase (Cata) [23], chitin synthase 1 (CHS1) [24], Internal Transcribed Spacer (ITS) [25], Pho85 cyclin-dependent kinase (Pho85) [26], protein kinase C Ss-2 (PKC) [27], G protein α subunit (GProt) [28], and topoisomerase II (Topo) [29] with primers designed to target specific gene sequences (Table S1). The DNA sample from S. schenckii s. str.…”

Characterization and ligand identification of a membrane progesterone receptor in fungi: existence of a novel PAQR in Sporothrix schenckii

“…DNA and RNA were obtained from S. schenckii yeast cells as described previously [54]. Poly A + RNA was obtained from total RNA using the mRNA Purification Kit from Amersham Biosciences (Piscataway, NJ, USA) and used as template for cDNA synthesis.…”

“…Nested primers were designed to improve the original amplification reactions. Bands from the 5′ nested PCR were excised from the gel and cloned as described previously [54]. Primers for RACE were designed based on the sequence obtained from the yeast two-hybrid assay.…”

“…For RTPCR, RNA was extracted as described previously [54]. The cDNA was obtained using the RETROscript ™ First Strand Synthesis kit (Ambion, Applied Biosystems, Foster City, CA, USA) and used as template.…”

“…The following PCR parameters were used: an initial denaturation step at 94°C for 30 sec, followed by 25 cycles of denaturation at 94°C for 5 sec, annealing at 40°C for 10 sec, and extension at 72°C for 2 min. The RTPCR products were cloned as described previously [54] and the inserts sequenced using commercial sequencing services from Davis Sequencing (Davis, CA, USA).…”