Promoter fusions to the Activator transposase gene cause distinct patterns of Dissociation excision in tobacco cotyledons

Abstract

To explore the effects of altering the level of Activator (Ac) transposase (TPase) expression, a series of plasmids was construced in which heterologous promoters were fused to the TPase gene. Promoters for the cauliflower mosaic virus (CaMV) 35S transcript and the octopine synthase (ocs) and nopaline synthase (nos) genes were tested. These fusions, and constructs expressing TPase from the wild-type Ac promoter, were introduced into tobacco, and their activity was monitored by crossing to a line carrying Dissociation (Ds) in a streptomycin phosphotransferase gene (Ds::SPT). The SPT marker provides a record of somatic excisions of Ds that occur during embryo development. The patterns of somatic variegation that resulted from transactivation by each fusion were distinct and strikingly different from the pattern triggered by the wild-type Ac constructs. Unlike wild-type Ac, which caused transposition throughout embryo development, each fusion gave rise to sectors of discrete size. Sectors triggered by the CaMV 35S fusion were largest, ocs sectors were intermediate, and nos were smallest. These patterns appear to indicate differential timing of the activation of these promoters during embryogeny. Measurement of transcript abundance for each transformant indicated that the CaMV 35S-transformed plants accumulated approximately 1000-fold more TPase mRNA than plants containing wild-type Ac, whereas ocs- and nos-transformed lines accumulated about 100-fold and 20-fold higher levels, respectively. Measurements of germinal excision frequencies driven by the chimeric TPase fusions, however, indicated that increasing transcription does not necessarily result in an increase in germinal excision. These measurements showed that the ocs and nos fusions have very low rates of germinal excision. Only the CaMV 35S fusion transformants were found to have higher rates than the Ac constructs, although significant pod-to-pod variation was observed. Gel blot analysis of DNA from progeny carrying germinal excision events resulting from the CaMV 35S fusion showed that excision is associated with reinsertion and that siblings sometimes carry the same transposition events. These findings suggest that in tobacco there is no direct proportionality between TPase expression and Ac-Ds transposition activity. This possibility has important implications for understanding the regulation of Ac transposition and for designing efficient gene tagging systems.