Transactivation of Ds by Ac-transposase gene fusions in tobacco

Abstract

To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A2 promoter CHI A2. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5' regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.

Transposition-Based Plant Transformation

“…EcoRV digests were run on gel, blotted, and hybridized with radioactively labeled probes as described previously (Rommens et al, 1992). Primers for amplification of fragments that were used to generate probes to visualize the presence of the nptII (Ds) and Ac-Tpa genes (remaining T-DNA) were Pr13-Pr14 and Pr15-Pr16, respectively.…”

“…Separation of the two functions of Ac creates a twocomponent transposition system. An expression cassette for the Tpa gene represents the first component, whereas the second component consists of a nonautonomous Dissociation (Ds) element that contains the terminal sequences required for transposition (Rommens et al, 1992). Such two-component systems have been introduced into a variety of plant species by transforming plants with T-DNAs carrying both Ds and the Ac-Tpa gene (Hehl and Baker, 1989).…”

Transposition pattern of a modified Ds element in tomato

“…The Ds-r element contains both the GUS gene driven by a 0.8 kb fragment of the CaMV 35S promoter and parts of plasmid pA-CYC 184 (origin of replication and chloramphenicol resistance gene) between the 586 bp 5' Ac terminal sequence and the 448 bp 3' Ac terminal sequence [46]. The construction of the modified Ac element present between the borders of the T-DNA of SLJ1515 was initiated from a clone of the maize bronze allele bz-m2 (Ac) [17].…”

“…To examine whether Ds-r was either still present between the borders of this T-DNA or had transposed from the T-DNA to positions in the plant DNA, hybridizations were performed with the 35S promoter of cauliflower mosaic virus as a probe. DNA isolation, restriction digests, Southern blotting, hybridizations and autoradiography were performed as described previously [46]. Fragments indicative of an untransposed Ds-r have sizes of 8.2 kb and 1.9 kb.…”

“…Recently, we reported the construction of a Ds element containing bacterial plasmid sequences between the ends of Ac [46]. This element has the origin of replication of plasmid pACYC184 and the chloramphenicol resistance gene (functional in Escherichia coli) inserted between 0.5 kb Ac terminal sequences.…”