Different 3' end regions strongly influence the level of gene expression in plant cells.

We have investigated the functional role of a 3' end region on the expression of a reporter gene in plant cells. In stably transformed plants, expression of the reporter gene without a plant gene 3' end is variable and depends on the fortuitous presence of polyadenylation signals in the downstream sequences. When the reporter gene is flanked by pBR322 DNA, 3'-processing and polyadenylation occurs at (a) cryptic site(s) within these vector sequences. Using a transient gene expression system, we present a deletion analysis of the 3' end of the octopine synthase gene showing that the most proximal polyadenylation signal per se is not sufficient to ensure expression but that a downstream (G)T-rich sequence is also required. Optimal expression of the fusion gene requires more than 98 base pairs and at most 142 base pairs downstream from the most distal polyadenylation site. We analyzed the expression of chimeric genes with 3' end sequences originating from different plant genes. In the transient expression assay, all constructs direct similar neomycin phosphotransferase II activities. However, in stably transformed tissue, the gene constructs displayed characteristic expression levels which varied as much as 60-fold. This result suggests a role for 3' end sequences in post-transcriptional processes such as efficiency of 3'-processing and/or mRNA stability.

A negative selection scheme for tobacco protoplast-derived cells expressing the T-DNA gene 2.

The amido hydrolase encoded by the T-DNA gene 2 catalyzes the conversion of indole-acetamide, α-naphthalene acetamide, and other substrate analogues into the corresponding auxins. As a result, only gene 2-expressing protoplast-derived tobacco cells can grow in medium containing low concentrations (0.2-1 µM) of α-naphthalene acetamide as auxin precursor. However, in a mixture of SR1 and SR1, gene 2 (+) protoplast-derived cells, cross-feeding occurs and consequently no positive selection for gene 2 is obtained. A 100-times higher concentration of α-naphthalene acetamide (between 30 and 300 µM) provides a negative selection scheme. Only the tobacco cells expressing gene 2 are sensitive to the high naphthalene acetamide concentration and cannot grow to colonies, while cells lacking the gene 2 product regenerate calli even in mixed gene 2 (+) and gene 2 (-) cell populations. Thus, gene 2 might provide a unique biochemically defined marker to investigate mutations and gene inactivation.

Isolation of tobacco DNA segments with plant promoter activity.

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.