An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')H (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')ll enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus-and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.
Oncogenes carried by the transferred DNA (T‐DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants. Other T‐DNA genes regulate the tumorous growth in ways that are not yet understood. To determine the function of T‐DNA gene 5, its coding region was expressed in Escherichia coli. Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28‐fold increase in conversion of tryptophan to indole‐3‐lactate (ILA), an auxin analogue. Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells. cis‐regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax‐1. ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole‐3‐acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins. It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants.
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