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 organism on which to base such a system. We have shown previously that the fission yeast Schizosaccharomyces pombe has potential to satisfy the requirements of such a system. This report describes experiments designed to determine if antisense and ribozyme RNA-mediated gene suppression will be generally applicable to other genes in S. pombe. Antisense and ribozyme RNAs designed to suppress the ade6 gene were expressed at high levels from episomal expression vectors. The ade6 gene was chosen as a target as mutations within the gene confer adenine auxotrophy and a red colony phenotype, and it was expected that antisense or ribozyme 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 primer extension and Northern analysis. Initially, conflicting results were obtained from these techniques, which were determined to be due to duplex formation between antisense and target RNA in vitro. No detectable reduction in the ade6 mRNA levels was found, and it was concluded that the gene was not suppressed by the antisense or ribozyme RNAs tested. These results confirm that in S. pombe as with other organisms, the susceptibility of genes to RNA-mediated suppression may be gene specific and that design of antisense and ribozyme genes will be an empirical process.
Atkins
D
D
R.W. Johnson Pharmaceutical Research Institute, Sydney NSW, Australia.
Patrikakis
M
M
Izant
J G
JG
D000225:Adenine / Q000096:biosynthesis; D001483:Base Sequence; D018390:Gene Targeting; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016372:RNA, Antisense / Q000494:pharmacology*; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D012568:Schizosaccharomyces / Q000235:genetics*; D013489:Suppression, Genetic
D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D000225:Adenine