In vivo detection of regulatory factor binding sites of Arabidopsis thaliana Adh.

In vivo footprinting experiments have been used to analyze the binding of trans-acting regulatory factors in the 5' flanking region upstream of the alcohol dehydrogenase (Adh) gene from Arabidopsis thaliana. Protein-DNA interactions were detected by dimethyl sulfate footprinting and genomic sequencing, using an A. thaliana cell suspension culture that constitutively expressed the Adh gene. Several distinct footprinting domains have been characterized, and the potential effects of the corresponding trans-acting factors have been inferred from a comparison with data from the maize alcohol dehydrogenase-1 (Adh1) gene. One binding site is similar in sequence to one of the anaerobic response elements (ARE) of the maize gene, which has also been shown to bind to a trans-acting factor. Several of the remaining binding sites apparently represent a class of elements sharing the sequence 5'-GTGG-3' within their footprint.Comparisons with maize Adh1 in vivo protein interactions reveal that the elements of Adh promoter structure are highly conserved, but the relative and absolute positions of the elements are variable.

Architecture of a plant promoter: S1 nuclease hypersensitive features of maize Adh1.

S1 nuclease was used to probe the architectural characteristics of the maize alcohol dehydrogenase-1 gene promoter. Three sites were identified as hypersensitive to S1 digestion in supercoiled, but not linear plasmids containing the Adh1 gene. The sites mapped to areas located 65, 330 and 800 base pairs 5' to the start of transcription. In each case, the strand specific nicking pattern was determined with nucleotide level precision. The -65 site was found to be a homopurine/homopyrimidine tract. The -330 site mapped to the boundaries of a region of high Z-DNA potential and the -800 site mapped to a non-descript sequence. The possible biological significance of these sites is discussed.