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

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Abstract

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 λ fragments, respectively, between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP) fused to the β-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 λ 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 λ 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.

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Acknowledgments

We thank Mr. Dennis Bennett for technical assistance. This study was funded by the United States Department of Agriculture (USDA)-Agricultural Research Service Headquarter 2005 and 2007 classes of postdoctoral grants and a USDA Cooperative State Research, Education, and Extension Service Biotechnology Risk Assessment Research grant (2006-03701).

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  1. Stacy D. Singer & Jean-Michel Hily

    Present address: Department of Plant Pathology, New York State Agricultural Experiment Station, Cornell University, 630 West North Street, Geneva, NY, 14456, USA

Authors and Affiliations

  1. USDA-ARS Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, WV, 25430, USA

    Stacy D. Singer, Jean-Michel Hily & Zongrang Liu

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  1. Stacy D. Singer
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  2. Jean-Michel Hily
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Correspondence to Zongrang Liu.

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Singer, S.D., Hily, JM. & Liu, Z. A 1-kb Bacteriophage Lambda Fragment Functions as an Insulator to Effectively Block Enhancer–Promoter Interactions in Arabidopsis thaliana . Plant Mol Biol Rep 28, 69–76 (2010). https://doi.org/10.1007/s11105-009-0122-3

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  • DOI: https://doi.org/10.1007/s11105-009-0122-3

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