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Analysis of prokaryotic antisense mechanisms in eukaryotic cells

Details zum Projekt
Projektlaufzeit: 01/199912/2002

In prokaryotes, gene regulation by antisense transcripts is a well documented mechanism. Surprisingly, molecular interactions of completely complementary RNAs is controlled by complex secondary structures in the molecules and disruption of these structures results in a failure of antisense mediated gene regulation even though complementarity of the molecules is maintained. In contrast, antisense mechanisms in eukaryotes are barely understood and rather suggest that flexible structures rather than distinct folding enhances intermolecular interaction. We have designed an assay system to elucidate the the ability of prokaryotic antisense systems to promote anisense mediated gene silencing in the eukaryote Dictyostelium.. Fusion of the E. coli copT (target) sequence to the ß-galactosidase gene and regulated expression of copA (antisense) from a high copy number gene construct allows for modifying ß-galactosidase activity and thus proves that the copA/T system is functional in Dictyostelium. Modified copA/T structures which are still completely complementary but known to be far less efficient in E. coli due to structural changes, have been itroduced into Dictyostelium. These structures are also less efficient in the eukaryote, suggesting that structural features play an important role in the interaction kinetics of sense and antisense molecules and/or the formation of a functional complex which prevents translation or promotes RNA degradation. Constructs expressing a double stranded copA/copT RNA in the form of a fold-back hairpin (similar to RNAi), are as efficient as expressed antisense RNAs and thus suggest that these mechanisms of gene silen


Zuletzt aktualisiert 2017-11-07 um 14:50