CLONING OF UPSTREAM SEQUENCES RESPONSIBLE FOR CELL-CYCLE REGULATION OF THE NICOTIANA-SYLVESTRIS CYCB1-1 GENE

To understand the mechanisms involved in the regulation of the mitotic cyclin B Nicta; CycB1;1 expression, we have cloned the Nicotiana sylv estris cyclin gene, Nicsy; CycB1;1, whose coding sequence is homologou s to that of Nicta;CycB1;1 cDNA. The structure and the function of its 5'-flanking region, 1149 bp upstream of the first start codon, was an alysed. By producing stably transformed cells of a synchronized cultur e with the Nicsy;CycB1;1 promoter/beta-glucuronidase (gus) reporter ge ne fusion, we demonstrate that the 1149 bp promoter fragment mediates a gus transcriptional oscillation, indistinguishable from that of endo genous Nicsy;CycB1;1 cyclin B transcripts. Transient GUS activity in B Y-2 protoplasts reveals that promoter activity is considerably reduced by shortening the 5'-flanking region to 538 or 320 bp. Furthermore, t he 320 bp fragment no longer mediates the observed transcriptional reg ulation of the 1149 bp Nicsy;CycB1;1 promoter in BY-2 protoplasts isol ated from synchronized cells.