Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants.

Abstract

The 35S promoter of cauliflower mosaic virus (CaMV) is able to confer high-level gene expression in most organs of transgenic plants. A cellular factor from pea and tobacco leaf tissue, which recognizes nucleotides in a tandemly repeated TGACG motif at the -75 region of this promoter, has been detected by DNase I footprinting and gel retardation assays. This factor is named activation sequence factor 1 (ASF-1). A cellular factor binding to the two TGACG motifs can also be detected in tobacco root extracts. Mutations at these motifs inhibit binding of ASF-1 to the 35S promoter in vitro. When examined in transgenic tobacco, these mutations cause a 50% drop in leaf expression of the 35S promoter. In addition, these same mutations attenuate stem and root expression of the 35S promoter about 5-to 10-fold when compared to the level of expression in leaf. In contrast, mutations at two adjacent CCAAT-box-like sequences have no dramatic effect on promoter activity in vivo. A 21-base-pair element containing the two TGACG motifs is sufficient for binding of ASF-1 in vitro when inserted in a green-tissue-speciflic promoter. In vivo, the insertion of an ASF-1 binding site caused high levels of expression in root. Thus, a single factor binding site that is defined by site-specific mutations is shown to be sufficient to alter the expression pattern of promoters in vivo.A lx 2x

The Arabidopsis NPR1/NIM1 Protein Enhances the DNA Binding Activity of a Subgroup of the TGA Family of bZIP Transcription Factors

“…As described in this study, at least one TGA factor (TGA2) is also capable of binding in the EMSA to the SA response element LS7 of the PR-1 gene promoter, which is a gene that is known to be induced during SAR (Ward et al, 1991) and whose expression is abolished in npr1 mutants (Cao et al, 1994;Delaney et al, 1995). Furthermore, TGA factors, such as tobacco TGA1a, act as genuine transcription factors in heterologous and homologous in vitro transcription systems Yamazaki et al, 1990) and can transcriptionally activate as-1-containing promoters in plants (Lam et al, 1989) and yeast (Rüth et al, 1994). Taken together, these results sug- (A) Extracts from wild-type (Wt; lanes 3 and 4) or npr1 mutant (lanes 5 and 6) Arabidopsis plants treated for 1 hr with water (lanes 3 and 5) or with a solution of 0.5 mM SA (lanes 4 and 6) were subjected to an EMSA, using the as-1 element as a probe (lanes 1 to 6).…”

“…To test whether NPR1 behaves similarly to I B ␣ , we investigated the effect of NPR1 on the binding of TGA factors to the as-1 promoter element. This element is one of the best-characterized DNA sequences recognized by the TGA family of transcription factors (Lam et al, 1989). Figure 4 shows results from EMSA experiments performed in the presence or absence of in vitro-translated TGA2, NPR1, or both.…”

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

“…The promoter did not show extensive homology to the promoter of the Ntl03 gene. However, typically a sequence related to an as-1 like element [13], as present in the promoters of genes belonging to the auxin-inducible Ntl03 gene family, was present in the promoter of the AtlO3-1a gene as well (Fig. 2).…”

Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to -6 in transgenic tobacco and banana cells.

“…This region contained a 10 bp sequence, CATGACGTCA, which has strong homology with the 3h end of the 20 bp consensus incorporating the ocselement of the Agrobacterium tumefaciens T-DNA promoters (Bouchez et al, 1989). Internal to this sequence is a TGACG motif (ASF-1) which exists as a tandem repeat in the as-1 element of the CaMV 35S and related promoters (Lam et al, 1989 ;Sanger et al, 1990 ;Verdaguer et al, 1996). The as-1 element is able to confer expression primarily in root tissues, but also interacts synergistically with other cis-elements (Lam et al, 1989) and has been shown to bind AS1 tobacco nuclear factor (Fromm et al, 1989).…”

“…Internal to this sequence is a TGACG motif (ASF-1) which exists as a tandem repeat in the as-1 element of the CaMV 35S and related promoters (Lam et al, 1989 ;Sanger et al, 1990 ;Verdaguer et al, 1996). The as-1 element is able to confer expression primarily in root tissues, but also interacts synergistically with other cis-elements (Lam et al, 1989) and has been shown to bind AS1 tobacco nuclear factor (Fromm et al, 1989). This region also contains a sequence, ACGTCA, with homology to the hexamer motif of plant histone promoters (Mikami et al, 1987).…”