M phase-specific activation of the Nicotiana sylvestris cyclin B1 promoter involves multiple regulatory elements

Summary B-type cyclins are cell cycle regulatory proteins specifically expressed in G2-M phases. To understand the mechanisms that regulate their expression, it is important to identify the required promoter element(s). By using synchronized BY-2 cell cultures stably transformed with chimeric genes composed of sequential Nicsy;CycB1; 1 promoter deletions fused to the β-glucuronidase reporter gene (gus), we show that at least five distinct promoter regions are required for maximal M-phase expression. Furthermore, two distinct promoter regions contain sufficient element(s) to induce M-phase-specific expression. In one of these regions, a 23 bp promoter element is able to activate reporter gene expression in cells induced to divide in an orientation-independent manner but without an M-phase specific expression. Therefore, this 23 bp element, which contains a 5 bp element identical to the MYB binding core, is a good upstream activating sequence (UAS) candidate. Moreover, electrophoretic mobility shift assays show that this putative UAS specifically binds protein complexes that appear to differ whether cells are cycling or not. The constitutive transcriptional activation mediated by this UAS would suggest that the Nicsy;CycB1; 1 gene promoter is regulated through an activation/repression mechanism.