Plant Physiol.
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Premature Polyadenylation at Multiple Sites within a Bacillus thuringiensis Toxin Gene-Coding Region1

Scott H. Diehn2, Wan-Ling Chiu3, E. Jay De Rocher, and Pamela J. Green*

Michigan State University-Department of Energy Plant Research Laboratory (S.H.D., W.-L.C., E.J.D.R., P.J.G.), Department of Botany and Plant Pathology (S.H.D.), and Department of Biochemistry (P.J.G.), Michigan State University, East Lansing, Michigan 48824

Some foreign genes introduced into plants are poorly expressed, even when transcription is controlled by a strong promoter. Perhaps the best examples of this problem are the cry genes of Bacillus thuringiensis (B.t.), which encode the insecticidal proteins commonly referred to as B.t. toxins. As a step toward overcoming such problems most effectively, we sought to elucidate the mechanisms limiting the expression of a typical B.t.-toxin gene, cryIA(c), which accumulates very little mRNA in tobacco (Nicotiana tabacum) cells. Most cell lines transformed with the cryIA(c) B.t.-toxin gene accumulate short, polyadenylated transcripts. The abundance of these transcripts can be increased by treating the cells with cycloheximide, a translation inhibitor that can stabilize many unstable transcripts. Using a series of hybridizations, reverse-transcriptase polymerase chain reactions, and RNase-H-digestion experiments, poly(A+) addition sites were identified in the B.t.-toxin-coding region corresponding to the short transcripts. A fourth polyadenylation site was identified using a chimeric gene. These results demonstrate for the first time to our knowledge that premature polyadenylation can limit the expression of a foreign gene in plants. Moreover, this work emphasizes that further study of the fundamental principles governing polyadenylation in plants will have basic as well as applied significance.

1   This work was supported by grants from the Department of Energy, the U.S. Department of Agriculture, Project Green, Michigan State University Research Excellence Funds, Midwest Plant Biotechnology Consortium, Consortium for Plant Biotechnology Research, and matching funds from Ciba-Geigy, Sandoz, Pioneer, Anheuser-Busch, ICI, Agrigenetics, and DowElanco. S.H.D. and E.J.D.R. were supported in part by a National Institutes of Health predoctoral traineeship and a U.S. Department of Agriculture postdoctoral fellowship, respectively.
2   Present address: Pioneer Hi-Bred International, Inc., Traits and Technology Development, 7300 NW 62nd Avenue, P.O. Box 1004, Johnston, IA 50131-1004.
3   Present address: Department of Biology, University of Richmond, Richmond, VA 23173.
*   Corresponding author; e-mail green{at}pilot.msu.edu; fax 1-517-355-9298.

Plant Physiol. (1998) 117: 1433-1443
Copyright Clearance Center:   0032-0889/98/117//11
© 1998 American Society of Plant Physiologists




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