The β‐phaseolin gene is flanked by matrix attachment regions

Abstract

The β‐phaseolin promoter yields high levels of spatially and temporally regulated expression in transgenic plants. It is shown that elements within 5′ and 3′ regions flanking the coding sequence bind strongly and specifically to matrix preparations from tobacco nuclei. Both of these matrix attachment regions (MARs) are A/T‐rich and contain topoisomerase II, Abox, T‐box and MAR motifs frequently found in MARs in Drosophila and other organisms. The β‐phaseolin 5′ MAR is located on a 1047 bp fragment with a core element of 673 bp (positions −1096 to −424), that includes a 55 bp region (−682 to −628) previously shown to function as an enhancer. A 1205 bp fragment (positions +1879 to +3083) 3′ of the phaseolin coding region also showed strong and specific binding to nuclear matrix preparations and contained consensus MAR motifs. A β‐phaseolin promoter‐GUS fusion construct (p1470/phas) that included the MAR elements exhibited higher expression levels and lower plant‐to‐plant variability in stably transformed tobacco plants than did a similar construct lacking MAR sequences. Seeds of plants containing two intact copies of the insert had higher expression levels than did those of plants containing a single copy. These findings, which are the first to report a plant gene being flanked both 5′ and 3′ by MARs, are in accordance with previous observations of increased DNase I sensitivity associated with the onset of phaseolin transcription in bean and with the consistently high and position‐independent expression observed in transgenic plants from the β‐phaseolin promoter. The results suggest that the native β‐phaseolin gene is located on a small chromatin loop that functions as an active and independent domain.