Artificial Ribozyme and Antisense Gene Expression in Saccharomyces cerevisiae

Abstract

A sensitive, quantitative reporter gene-based experimental system for the in vivo analysis of hammerhead ribozyme and antisense gene function in Saccharomyces cerevisiae is described. The system was constructed to test the activity of ribozyme and antisense genes targeting the chloramphenicol acetyltransferase gene (CAT) in both a cis and trans configuration relative to the target. When both target and ribozyme or antisense genes were transcribed in the same mRNA from an expression vector, CAT expression was reduced by up to 90%. Although the cis-positioned ribozyme molecule cleaved the target RNA in vitro, the steady state 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 degradation of mRNA. When the ribozyme or antisense genes were transcribed in trans from a plasmid-based expression vector, expression of a CAT gene integrated into a chromosome was unaffected. The effect of the cis-located RNA molecules may be dependent on an interaction requiring co-localization of ribozyme or antisense and target mRNAs during or immediately after target gene transcription. The failure of such a co-localization of these RNAs when synthesized in trans may contribute to the lack of efficacy seen in the trans-acting ribozymes or antisense RNAs. These observations are consistent with other studies reporting inefficient trans-acting ribozyme and antisense activity in S. cerevisiae.