Isolation and functional analysis of the 5'-flanking region of carrot top1b gene coding for the b isoform of DNA topoisomerase I

We have isolated and functionally characterized the promoter region of the top1beta gene encoding carrot (Daucus carota) DNA topoisomerase Ibeta. The major transcription start site was mapped by primer extension analysis 164 nt upstream the ATG translation start codon. Sequence analysis of the 5'-upstream region of the gene revealed the presence of a canonical TATA-like box at position -35 bp and several cis-acting sequences, including a (CT)n element in the leader region of the gene, a myb-related motif and the Dof element NtBBF-1, which correlate with the inducible expression pattern of this gene. Functional reporter analysis of the top1beta 5'-flanking region was performed in both carrot and Arabidopsis thaliana transfected protoplasts. The region at -719 to +161 was sufficient to confer high expression level in both species. The transient expression assay in protoplasts induced to stop dividing confirmed that the promoter, whose activity is low in quiescent cells, is activated when protoplasts are induced to proliferate by exogenous application of growth factors.