THE ADE6 GENE OF THE FISSION YEAST AS A TARGET FOR ANTISENSE AND RIBOZYME RNA-MEDIATED SUPPRESSION

A genetic system for the analysis of antisense and ribozyme mechanisms is a much needed experimental tool, and yeast represent a favorable o rganism on which to base such a system, We have shown previously that the fission yeast Schizosaccharomyces pombe has potential to satisfy t he requirements of such a system, This report describes experiments de signed to determine if antisense and ribozyme RNA-mediated gene suppre ssion will be generally applicable to other genes in S. pombe. Antisen se and ribozyme RNAs designed to suppress the ade6 gene were expressed at high levels from episomal expression vectors, The ade6 gene was ch osen as a target as mutations within the gene confer adenine auxotroph y and a red colony phenotype, and it was expected that antisense or ri bozyme RNA-mediated mutant phenocopies would exhibit the same readily detectable phenotype. No phenotypic indication of ade6 suppression was detected in transformed yeast, and ade6 target mRNA was analyzed by p rimer extension and Northern analysis. Initially, conflicting results were obtained from these techniques, which were determined to be due t o duplex formation between antisense and target RNA in vitro. No detec table reduction in the ade6 mRNA levels was found, and it was conclude d that the gene was not suppressed by the antisense or ribozyme RNAs t ested. These results confirm that in S. pombe as with other organisms, the susceptibility of genes to RNA-mediated suppression may be gene s pecific and that design of antisense and ribozyme genes will be an emp irical process.