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Bidirectionalization of polar promoters in plants

Abstract

A typical eukaryotic promoter consists of a minimal promoter and other upstream cis elements1,2,3. The minimal promoter is essentially a TATA box region where RNA polymerase II, TATA-binding protein (TBP), and TBP-associated factors (TAFs) bind to initiate transcription4, but minimal promoters alone have no transcriptional activity1. The cis elements, to which tissue-specific or development-specific transcription factors bind, individually or in combination, determine the spatio-temporal expression pattern of a promoter at the transcriptional level1. The arrangement of upstream cis elements followed by a minimal promoter sets the polarity of the promoter. Promoters in plants that have been cloned and widely used for both basic research and biotechnological application are generally unidirectional, directing only one gene that has been fused at its 3′ end (downstream). It is often necessary to introduce multiple genes into plants for metabolic engineering and trait stacking5. It is also desirable to minimize or avoid repeated use of a single promoter that may cause transcriptional gene silencing6. Here we describe a strategy to make polar promoters bidirectional so that one promoter can direct the expression of two genes, one on each end of the promoter.

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Acknowledgements

We thank Dr. Richard Amasino (University of Wisconsin, Madison), Drs. George Wagner, Maelor Davies, Arthur Hunt, and Deane Falcone (University of Kentucky, Lexington) for critically reading this manuscript, Dr. Indu Maiti (University of Kentucky, Lexington) for the PClSV promoter, and Dr. Pal Maliga (State University of New Jersey, Piscataway) for pPZP family vectors. The work was supported by the US Department of Energy Basic Energy Sciences (Grant no. DE-FG02-99ER20330 to S.G.), the US Department of Agriculture National Research Initiative (Grant no. 2001-35304-09994 to S.G.), and the Tobacco and Health Research Institute at the University of Kentucky (grant to S.G.). Y.H. was supported in part by the University of Kentucky's Research Challenge Trust Fund (Plant Sciences).

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Correspondence to Susheng Gan.

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Figure 1: Diagram of promoter bidirectionalization strategy and some genetic constructs.
Figure 2: Patterns of GUS expression in various transgenic Arabidopsis seedlings.
Figure 3: Comparison of gene expression levels directed by bidirectionalized promoters versus related conventional promoters.