The suppression of nonsense mutations in plants

In plants very little is known concerning the regulation of tRNA gene transcription, and the development of in vitro transcription systems has proven to be quite difficult. We have studied tRNA gene expression in plants using an approach which involves the conversion of normal cellular tRNAs to nonsense suppressors, whose expression can be monitored by their ability to suppress nonsense mutations in the gene encoding chloramphenicol acetyltransferase (CAT). Four tRNA{dollar}sp{lcub}rm Trp{rcub}{dollar} suppressor gene constructs were utilized in the experiments described here. Two of these constructs were synthesized de novo based upon the sequence of a wheat germ tRNA{dollar}sb{lcub}rm UGG{rcub}sp{lcub}rm Trp{rcub}{dollar} and hence contain no wild type flanking sequences. The other two tRNA{dollar}sp{lcub}rm Trp{rcub}{dollar} suppressors were generated by site directed mutagenesis of a cloned tRNA{dollar}sb{lcub}rm UGG{rcub}sp{lcub}rm Trp{rcub}{dollar} from Arabidopsis thaliana. This work was carried out both in a transient assay system which employed carrot protoplasts as well as in transgenic tobacco plants. Transient assays revealed that the cloned tRNA{dollar}sp{lcub}rm Trp{rcub}{dollar} genes are able to function as nonsense suppressors, generating readthrough products at a level 0.014% that of wild type constructs, or approximately 10 fold above background levels. The synthetic genes, however, are not expressed at sufficiently high enough levels to permit their detection by this assay. Heterologous in vitro transcription data indicate that this difference is probably due to the almost twenty fold higher transcription of the cloned tRNA{dollar}sp{lcub}rm Trp{rcub}{dollar} genes versus the synthetic genes. In transgenic tobacco plants the synthetic genes were again unable to suppress the nonsense mutations in the CAT gene. Work with the cloned Arabidopsis tRNAs is still underway. We went on to utilize these transgenic plants to look at the effects of viral infection on translational suppression. There is some evidence in mammalian systems that the level of specific suppressor tRNAs required for translational readthrough of viral RNA, is elevated in response to viral infection. Our transgenic tobacco plants afforded us with a functional assay for suppressor tRNA activity in tobacco mosaic virus (TMV) infected plants. Our work indicates that there is no virally mediated increase in translational suppression when transgenic tobacco plants containing CAT genes bearing nonsense mutations are infected with TMV.