A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone-inducible transcription from a synthetic promoter (P Top10 ) consisting of seven tet operators upstream of a TATA-box. Tetracycline interferes with induction by negatively regulating the DNA-binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV-driven P Top10 reach from undetectable levels of the reporter enzyme b-glucuronidase in the absence of dexamethasone to induced levels reaching 15±20% of the Cauli¯ower Mosaic Virus 35S promoter (P CaMV35S ). By modifying the sequence of P Top10 , we generated a new target promoter (P Tax ) that is stably expressed over several generations and that can be activated to levels comparable to P CaMV35S , while yielding only slightly elevated background activities.

A series of deletion mutants of a soybean heat shock (hs) gene promoter was generated and linked to the chloramphenicol acetyl transferase (CAT) coding sequence. These chimaeric promoter/reporter gene constructs were introduced into tobacco and thermoregulated expression of CAT activity was examined in leaf extracts. Three different types of gene fusions were tested using two different BIN19 vector constructions: (1) translational fusion between the N-terminus of the protein coding sequence of the heat shock gene Gmhsp17.3-B and CAT; (2) transcriptional fusions between the 5' nontranslated RNA regions of Gmhsp17.3-B and CAT; and (3) promoter fusions joining the hs promoter upstream sequences to the TATA box sequence of the delta CaMV 35S-CATter vector. Alternatively, multiple copies of a synthetic deoxyoligonucleotide with the soybean hs consensus element (HSE2) were used. Heat inducible CAT activities were detected except in plants containing a transcriptional fusion devoid of all but 18 nucleotides at the 5' terminus of the hs gene transcript. CAT activity was detectable in these plants only during the recovery at 25 degrees C after a hs (40 degrees C). Overlapping HSE-like promoter sequences seem to be necessary for the induction of heat inducible transcription of linked genes; synthetic HSE2 sequences have the capacity to reconstitute a hs promoter in combination with a TATA box sequence. Effective translation during hs seems to require sequences in the 5' non-translated leader of the hs protein mRNA; these sequences can be functionally replaced by the 5' leader sequence of the delta CaMV 35S promoter.

A 395 bp fragment located downstream from the soybean heat shock gene Gmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybean in vitro. Chimaeric genes containing the SARL fragment either at one side (5' or 3') or at both sides of a heat shock promoter-regulated beta-glucuronidase reporter gene were constructed. A five- to nine-fold increase of heat-inducible beta-glucuronidase activity was observed in transgenic tobacco plants containing constructs with SARL fragments either at both sides or with at least one SARL copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.

TGA1a and PG13 constitute a family of tobacco basic leucine zipper (bZIP) proteins that bind to activating sequence-1 (as-1), which is one of the multiple regulatory cis elements of the cauliflower mosaic virus (CaMV) 35S promoter. After truncation of the CaMV 35S promoter down to position -90 (CaMV 35S [-90] promoter), transcription stringently depends on the presence of as-1, which is recognized by nuclear DNA binding proteins called ASF-1. The role of the TGA1a/PG13 bZIP family in the formation of ASF-1 and in transcriptional activation of the CaMV 35S (-90) promoter has not yet been demonstrated in vivo. We constructed transgenic tobacco plants expressing a mutant of potato PG13, which lacks its wild-type DNA binding domain. This mutant acts as a trans-dominant inhibitor of ASF-1 formation and of expression from the CaMV 35S (-90) promoter, showing that PG13 can specifically interact with proteins necessary for these processes. Although we did not observe any other obvious phenotypic changes, these transgenic plants are a potentially valuable tool in identifying whether TGA1a and PG13 are involved in controlling promoters encoded in the plant genome.

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