THE BETA-PHASEOLIN GENE IS FLANKED BY MATRIX ATTACHMENT REGIONS

The beta-phaseolin promoter yields high levels of spatially and tempor ally regulated expression in transgenic plants. It is shown that eleme nts within 5' and 3' regions flanking the coding sequence bind strongl y and specifically to matrix preparations from tobacco nuclei. Both of these matrix attachment regions (MARs) are A/T-rich and contain topoi somerase II, A-box, T-box and MAR motifs frequently found in MARs in D rosophila and other organisms. The beta-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 bi nding to nuclear matrix preparations and contained consensus MAR motif s. A beta-phaseolin promoter-GUS fusion construct (p1470/phas) that in cluded the MAR elements exhibited higher expression levels and lower p lant-to-plant variability in stably transformed tobacco plants than di d a similar construct lacking MAR sequences. Seeds of plants containin g two intact copies of the insert had higher expression levels than di d 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 sen sitivity associated with the onset of phaseolin transcription in bean and with the consistently high and position-independent expression obs erved in transgenic plants from the beta-phaseolin promoter. The resul ts suggest that the native beta-phaseolin gene is located on a small c hromatin loop that functions as an active and independent domain.