RACE and RAGE Cloning in Parasitic Microbial Eukaryotes

Abstract

Many gene-cloning strategies and gene survey often provide partial sequence data. To exploit the information from these partial sequences numerous PCR-based approaches have been developed to clone full-length open reading frames. These approaches can be successful using small quantities of cDNA or genomic DNA as starting material and avoid the need to go through the complex and tedious process of constructing and screening gene libraries. Here we present two of these approaches, called RACE and RAGE, we used to successfully clone partial and full-length ORFs from amitochondriate parasitic microbial eukaryotes. The RACE approach uses cDNA as template for PCR cloning whereas RAGE uses genomic DNA. These two approaches were used to complement each other to provide full-length genes. The amitochondriate microbial eukaryotes we are investigating are of interest from both evolutionary and biomedical perspectives. We have investigated genes of mitochondrial origins in the obligate intracellular parasite called microsporidia. In these organisms spores are the only source of material that can be isolated from host cells and typically yield small amount of mRNA and genomic DNA for cloning. A full-length mitochondrial Hsp70 could be cloned and sequenced and specific antibody raised against a fusion protein. The highly specific antibody allowed us to demonstrate for the first time the presence of mitochondrial-like organelles in microsporidia.