ARTIFICIAL RIBOZYME AND ANTISENSE GENE-EXPRESSION IN SACCHAROMYCES-CEREVISIAE

A sensitive, quantitative reporter gene-based experimental system for the in vivo analysis of hammerhead ribozyme and antisense gene functio n in Saccharomyces cerevisiae is described. The system was constructed to test the activity of ribozyme and antisense genes targeting the ch loramphenicol acetyltransferase gene (CAT) in both a cis and trans con figuration relative to the target. When both target and ribozyme or an tisense genes were transcribed in the same mRNA from an expression vec tor, CAT expression was reduced by up to 90%. Although the cis-positio ned ribozyme molecule cleaved the target RNA in vitro, the steady stat e RNA levels of these chimeric transcripts were increased several fold relative to control mRNAs. This observation indicates a mechanism of suppression of CAT gene expression other than duplex-dependent degrada tion of mRNA. When the ribozyme or antisense genes were transcribed in trans from a plasmid-based expression vector, expression of a CAT gen e integrated into a chromosome was unaffected. The effect of the cis-l ocated RNA molecules may be dependent on an interaction requiring co-l ocalization of ribozyme or antisense and target mRNAs during or immedi ately after target gene transcription. The failure of such a co-locali zation of these RNAs when synthesized in trans may contribute to the l ack of efficacy seen in the trans-acting ribozymes or antisense RNAs. These observations are consistent with other studies reporting ineffic ient trans-acting ribozyme and antisense activity in S. cerevisiae.