PREMATURE POLYADENYLATION AT MULTIPLE SITES WITHIN A BACILLUS-THURINGIENSIS TOXIN GENE-CODING REGION

Some foreign genes introduced into plants are poorly expressed, even w hen 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 effective ly, we sought to elucidate the mechanisms limiting the expression of a typical B.t.-toxin gene, crylA(c), which accumulates very little mRNA in tobacco (Nicotiana tabacom) cells. Most cell lines transformed wit h the crylA(c) B.t.-toxin gene accumulate short, polyadenylated transc ripts. The abundance of these transcripts can be increased by treating the cells with cycloheximide, a translation inhibitor that can stabil ize many unstable transcripts. Using a series of hybridizations, rever se-transcriptase polymerase chain reactions, and RNase-H-digestion exp eriments, poly(A(+)) addition sites were identified in the B.t.-toxin- coding region corresponding to the short transcripts. A fourth polyade nylation site was identified using a chimeric gene. These results demo nstrate for the first time to our knowledge that premature polyadenyla tion can limit the expression of a foreign gene in plants. Moreover, t his work emphasizes that further study of the fundamental principles g overning polyadenylation in plants will have basic as well as applied significance.