Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium...

Sequences in the 3'-untranslated region of two different octopine T-DNA genes were analyzed with regard to their significance in polyadenylation. Poly(A) addition sites were localized precisely by S1 nuclease mapping with T-DNA-derived mRNAs isolated from tobacco. The gene encoding transcript 7' contains two AATAAA hexanucleotides, respectively 119 bp and 170 bp downstream of the TAA stop codon. A single poly(A) site was mapped 24-25 bp downstream of the first AATAAA. Further, we show that a mutant octopine synthase gene, which has lost part of its 3'-untranslated region by deletion, is still active. This mutant gene terminates 19 bp upstream from the major wild-type polyadenylation site. The deletion also removes the AATAAT signal preceding this site. The mutant octopine synthase gene contains a minimum of four different poly(A) sites. The most prominent of these sites is identical to the minor poly(A) site of the wild-type gene, and is preceded by a sequence AATGAATATA. Three other sites are located within the adjacent plant DNA, giving rise to hybrid T-DNA/plant DNA transcripts. The two most distal sites are probably dependent on a motif AATAAATAAA, found 29 bp away from the T-DNA/plant DNA junction.