A 1-kb Bacteriophage Lambda Fragment Functions as an Insulator to Effectively Block Enhancer-Promoter Interactions in Arabidopsis thaliana

Enhancers are known to be capable of overriding the specificity of nearby promoters in a distance-dependent manner, which is problematic when multiple promoters coexist in a single transgene unit. In an attempt to determine whether enhancer activation function is inversely related to its distance from the target promoter, we inserted 1-, 2-, and 4-kb bacteriophage lambda fragments, respectively, between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP) fused to the beta-glucuronidase (GUS) coding region. In the absence of an insert sequence, the 35S enhancer activates AGIP-driven GUS expression in vegetative tissues of transgenic Arabidopsis thaliana lines. Moreover, neither the 2-kb nor the 4-kb lambda fragment was able to block GUS expression in transgenic leaves, implying that the 35S enhancer can override a distance barrier of at least 4 kb in our system. Unexpectedly, insertion of the 1- kb lambda insert into the same site resulted in diminished GUS expression in transgenic leaves. Our data indicate that this fragment functions as a true enhancer-blocking insulator that could potentially be utilized to minimize enhancer-promoter interference between multiple transcriptional units within a plasmid vector during plant transformation experiments.