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Osmium tetroxide (OsO) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO will also identify other torsional perturbations of DNA. In this study, OsO was used to footprint an AT-rich region (between −780 and −500) of the maize Adh1 promoter. Hyperreactive sites were identified both in vitro and in vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei. [ABSTRACT FROM AUTHOR]

The alcohol dehydrogenase ( Adh) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced by hypoxic stress only in roots of mature plants. Deletions and G-box mutations of the Adh promoter were assayed in Arabidopsis protoplasts by PEG-mediated transient expression. Sequence domains necessary for full gene activity are confined to the 384 bp immediately 5′ to the transcription start site, and deletion to −177 results in >90% reduction in promoter activity. Site-specific mutations of G-box bases result in >60% reduction in activity and disrupt G-box factor binding in vitro. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]