Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models.

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Abstract
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.