Molecular cloning, characterization and expression of PmRsr1, a Ras-related gene from yeast form of Penicillium marneffei

Abstract

GTP-binding proteins such as Ras act as molecular switches in a large number of signal pathways. In this report, we isolated and characterized a novel Ras small monomeric GTPase Rsr1 gene, designated PmRsr1, from yeast-form Penicillium marneffei. The full-length PmRsr1 cDNA sequence is 1,866 bp in size, and contains an open reading frame of 642 bp encoding 213 amino acids. The predicted molecular mass of PmRsr1 is 24.41 kDa with an estimated theoretical isoelectric point of 9.21. The deduced amino acid sequence of PmRsr1 shows 87% identity with that of Aspergillus fumigatus and A. clavatus. Eight exons and seven introns are identified within the 2,102 bp PmRsr1 genomic DNA sequence of P. marneffei. The open reading frame was subcloned into the pcDNA6-myc-His B expression vector, and the recombinant plasmid was transfected into Vero cell line. The expressed fusion protein was analyzed by SDS-PAGE and western blotting. Differential expression of the PmRsr1 was demonstrated by real-time RT-PCR. The expression of PmRsr1 was the highest in the yeast phase comparing with that in the mycelia and conidia phases.