Elimination of transcriptional interference between tandem genes in plant cells

Plant cells are commonly transformed with two or more tandemly arranged genes, 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 orientations and expressing the genes in tobacco protoplasts. When two genes are oriented head-to-tail (-->-->), the expression of the downstream gene was reduced 80% by the upstream gene. When two genes are oriented tail-to-tail (--><--), the expression of the upstream gene was reduced 53% by the expression of the downstream gene. There was no interference when the orientation was head-to-head (<---->). Using a chemically inducible gene expression system, we showed that the downstream gene expression was reduced 71% by the induction of an upstream gene. Inserting a mammalian transcription blocker sequence eliminated the interference between the genes in tail-to-tail orientation. The interference in the head-to-tail orientation was eliminated by inserting a 2322-bp lambda phage DNA fragment. The terminators in gene constructs did not prevent the transcriptional interference, and the interference was eliminated by designing the orientation of genes and by placing a transcription blocker or a lambda phage sequence between genes.