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THE PLANT CELL, Vol 4, Issue 5 573-582, Copyright © 1992 by American Society of Plant Biologists
Promoter Fusions to the Activator Transposase Gene Cause Distinct Patterns of Dissociation Excision in Tobacco Cotyledons
S. R. Scofield, K. Harrison, S. J. Nurrish and JDG. Jones
Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom
To explore the effects of altering the level of Activator (Ac) transposase
(TPase) expression, a series of plasmids was constructed 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.
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