Elimination of transcriptional interference between tandem genes in plant cells

Plant cells are commonly transformed with two or more tandemly arranged gen es, but how orientation affects their expression is not well understood. We investigated the amount of transcriptional interference occurring between two adjacent genes by cloning luciferase and green fluorescent protein (GFP ) genes (promoter - coding sequence - terminator) in all possible orientati ons and expressing the genes in tobacco protoplasts. When two genes are ori ented head-to-tail (--> -->), the expression of the downstream gene was red uced 80% by the upstream gene. When two genes are oriented tail-to-tail (-- > <--), the expression of the upstream gene was reduced 53% by the expressi on of the downstream gene. There was no interference when the orientation w as head-to-head (<-- -->). Using a chemically inducible gene expression sys tem, we showed that the downstream gene expression was reduced 71% by the i nduction of an upstream gene. Inserting a mammalian transcription blocker s equence eliminated the interference between the genes in tail-to-tail orien tation. The interference in the head-to-tail orientation was eliminated by inserting a 2322-bp lambda phage DNA fragment. The terminators in gene cons tructs did not prevent the transcriptional interference, and the interferen ce was eliminated by designing the orientation of genes and by placing a tr anscription blocker or a lambda phage sequence between genes.