Robust expression in yeast cells of a reporter gene driven by rumen protozoal promoter sequences

The objectives of this study were the identification of genes that show relatively strong levels of expression in the rumen protozoan, Isotricha intestinalis, and the demonstration that promoters from such genes can be used in the construction of recombinant expression vectors. In order to identify highly expressed genes, a cDNA library was constructed for I. intestinalis, and RNA expression analysis conducted on 62 clones using a filter array hybridization assay. Expression levels for individual clones ranged from easily detectable to below the detection threshold of the technique. Eleven cDNAs showed relatively intense hybridization signals, and the gene for one of these clones, I87, was characterized in detail. The ability of the I87 promoter to drive the expression of recombinant genes was tested by linking it to the luciferase reporter gene in a yeast shuttle vector and transforming Saccharomyces cerevisiae cells for expression analysis. The results showed that a rumen protozoal gene promoter is capable of directing the expression of a reporter gene in S. cerevisiae. Accession numbers: I87 gene, AY247961, Isotricha sp. BBF-2003 ESTs, CB305319–CB305329