Related Experiment Videos
Artificial ribozyme and antisense gene expression in Saccharomyces cerevisiae
1CSIRO Division of Plant Industry, Canberra, Australia.
Antisense Research and Development
|January 1, 1994
Summary
Hammerhead ribozymes and antisense genes effectively reduced chloramphenicol acetyltransferase (CAT) gene expression by up to 90% when acting in cis within Saccharomyces cerevisiae. However, trans-acting versions showed no significant effect, suggesting a need for co-localization.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- Hammerhead ribozymes and antisense oligonucleotides are tools for gene silencing.
- Understanding their in vivo efficacy in model organisms like Saccharomyces cerevisiae is crucial for therapeutic applications.
- Reporter gene systems are vital for quantifying gene function and regulation.
Purpose of the Study:
- To develop and validate a reporter gene system for analyzing hammerhead ribozyme and antisense gene function in vivo.
- To compare the gene silencing efficacy of cis- and trans-acting ribozymes and antisense sequences targeting the chloramphenicol acetyltransferase (CAT) gene.
- To investigate the mechanisms underlying ribozyme and antisense activity in Saccharomyces cerevisiae.
Main Methods:
- Construction of a quantitative reporter gene system in Saccharomyces cerevisiae.
- Utilizing a chloramphenicol acetyltransferase (CAT) reporter gene.
- Testing cis- and trans-acting configurations of hammerhead ribozyme and antisense genes.
- Measuring CAT gene expression levels via reporter gene activity.
- Analyzing RNA levels of chimeric and target transcripts.
Main Results:
- Cis-acting ribozymes and antisense genes reduced CAT gene expression by up to 90%.
- In vitro cleavage by cis-ribozymes did not correlate with reduced steady-state RNA levels, suggesting alternative silencing mechanisms.
- Trans-acting ribozymes and antisense genes showed no significant effect on chromosomal CAT gene expression.
- Efficacy appears dependent on co-localization of ribozyme/antisense and target RNAs, favoring cis-acting configurations.
Conclusions:
- A novel reporter system enables sensitive, quantitative analysis of ribozyme and antisense gene function in vivo.
- Cis-acting hammerhead ribozymes and antisense RNAs are highly effective gene silencers in Saccharomyces cerevisiae.
- Trans-acting ribozymes and antisense RNAs exhibit limited efficacy, likely due to the lack of RNA co-localization.
- Findings highlight the importance of RNA localization for effective gene silencing strategies in yeast.