Two PCR products were obtained when using fungal DNA as template and the GESGKST/KWIHCF primer pair one belonging to ssg-1 and the other to ssg-2 of approximately 620 and 645 bp, respectively. The ssg-2 PCR product (645 bp) established the presence of a new gene encoding another Gα subunit in S. schenckii. Figure 1A shows the sequencing strategy used for the identification of this new G protein α subunit gene. Once the coding sequence was completed, it was confirmed using yeast cDNA as template and the
MGACMS/KDSGIL primer pair. A 1,065 bp ORF was obtained, containing the coding region of the ssg-2 cDNA as shown in Figure 1B. Using the same primer pair and genomic DNA as template a 1,333 bp PCR product
SBI-0206965 molecular weight was obtained. Sequencing of this PCR product confirmed the sequences obtained previously and showed the presence and position of Ferrostatin-1 cell line 4 introns. These introns had the consensus GT/AG junction splice site and interrupted the respective codons after the second nucleotide. The first intron interrupted the codon for G42 and consisted of 82 bp, the second intron interrupted the codon for Y157 and consisted of 60 bp, the third intron interrupted the codon for H200 and consisted of 60 bp, the fourth intron starts interrupted the codon H323 and consisted of 67 bp. With the exception of the regions where introns were present in the genomic sequence of the ssg-2 gene, the cDNA sequence and genomic sequence were identical. The overlapping of these two sequences
confirmed the presence of the introns in the genomic sequence. The cDNA and genomic sequence of ssg-2 have GenBank accession numbers AF454862 and AY078408, respectively. Figure 1 cDNA and derived amino acid sequences of the S. schenckii ssg-2 gene. Figure 1A shows the sequencing strategy used for ssg-2. The size and location in the gene of the various fragments obtained from PCR and RACE are shown. The black boxes indicate the size and relative position of the introns. Figure 1B shows the cDNA and derived amino acid sequence of the ssg-2 gene. PF-01367338 supplier Non-coding regions are given in lower case letters, coding regions and amino acids are given in upper case letters. The sequences that make up the GTPase over domain are shaded in gray, the five residues that identify the adenylate cyclase interaction site are given in red and the putative receptor binding site is shown in blue. Bioinformatic characterization of SSG-2 The derived amino acid sequence (GenBank accession number AAL57853) revealed a Gα subunit of 355 amino acids as shown in Figure 1B. The calculated molecular weight of the ssg-2 gene product was 40.90 kDa. Blocks analysis of the amino acid sequence of SSG-2 revealed a G-protein alpha subunit signature from amino acids 37 to 276 with an E value of 5.2e-67 and a fungal G-protein alpha subunit signature from amino acids 61 to 341 with an E value of 3.3e-28 .