A chimeric transactivator allows tetracycline-responsive gene expression in whole plants

Abstract

The chimeric transcriptional activator tTA, a fusion between the Tn10 encoded Tet repressor and the activation domain of the Herpes simplex virion protein VP16, was stably expressed in transgenic tobacco plants. It stimulates transcription of the beta-glucuronidase (gus) gene from an artificial promoter consisting of 7 tet operators and a TATA-box. Tetracycline, which interferes with binding of tTA to operator DNA, reduces gus expression over several orders of magnitude. This stringency of regulation suggests that the system can be used to construct transgenic plants encoding a potentially lethal gene product. Furthermore, the specific and fast inactivation of tTA allows study of the stability of RNAs and proteins.

Tetracycline-regulated reporter gene expression in the moss Physcomitrella patens

“…Tc added to the recovery medium after transformation reduced expression from pGLtVT to background levels (2 nmol MU min-1 mg-l). As this plasmid carries a hpt hygromycin- pGL2 [20] pTopl0 [28] ptetVP16 [28] pGLtVT This paper, [20,28] resistance gene it was possible to select stable transformants and study the Topl0 system in these. Such transformants subcultured for several weeks alternately on selective (30mg/1 hygromycin) and non-selective media in the presence of Tc, were finally transferred to -Tc media for 3 days prior to MUG assay.…”

“…24 h on -T c . Although the speed of downregulation following addition of Tc is likely to be very rapid on account of the binding avidity of TetR and the speed of penetration into the cells [27,28], the stability of the GUS enzyme prevented our measuring this (Fig. 3e).…”

“…As the affinity of the repressor for Tc is high [27], very low levels of exogenous Tc (1 mg/1) are sufficient for dissociation of the repressor from the promoter in planta. Such low levels of Tc have negligible physiological impact [8,9,28]. The Tc-inducible system requires a strong promoter driving TetR expression because high levels of repressor protein are needed for efficient displacement of the transcriptional initiation complex.…”

“…The Tc-inducible system requires a strong promoter driving TetR expression because high levels of repressor protein are needed for efficient displacement of the transcriptional initiation complex. This potential problem has been circumvented by fusing TetR to the VP16 transcriptional activation domain from Herpes simplex, thereby turning the repressor into an activator, tTA [ 11,28]. As illustrated in Fig.…”

“…The chimeric molecule, tTA, a fusion of the E. coli TetR molecule and the Herpes simplex virus activator VP 16, binds to the Top l0 promoter and initiates transcription. In the presence of Tc, however, tTA binding to the promoter is impeded, abolishing gene expression[28]. (nospA, polyadenylation signal of the nos gene; uidA, #-glucuronidase gene of E. coli; 35S pA, polyadenylation signal of CaMV 35S gene).…”