Cloning of a cDNA encoding DNA topoisomerase I in Daucus carota and expression analysis in relation to cell proliferation.

DNA topoisomerase I is an enzyme involved in several processes related to DNA metabolism. Despite the physiological importance, the regulation of top1 gene expression has not yet been investigated in plants. In order to monitor the possible correlation between levels of top1 transcripts and the proliferative state of the cell, two partially overlapping cDNAs encoding DNA topoisomerase I from Daucus carota have been isolated from a poly(A)(+)-primed library, using an Arabidopsis thaliana probe, and from a cDNA library spanning the 5' region of the top1 transcript, which was constructed using an antisense specific oligonucleotide. The top1 nucleotide sequence encoded an open reading frame of 2370 bp, predicting a protein of 90 kDa. The deduced amino acid sequence showed a similarity of 51% with A. thaliana, 41% with S. cerevisiae, 40% with S. pombe and 31% with H. sapiens, respectively. Southern blot analysis, performed under moderate stringency conditions, showed the presence of a single-copy gene. Evaluation of the top1 mRNA steady-state level revealed, besides a constitutive expression in vegetative carrot tissues, an induced expression related to cell proliferation.