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 regulati on of top1 gene expression has not yet been investigated in plants. In order to monitor the possible correlation between levels of top1 tran scripts and the proliferative state of the cell, two partially overlap ping cDNAs encoding DNA topoisomerase I from Daucus carota have been i solated from a poly(A)(+)-primed library, using an Arabidopsis thalian a probe, and from a cDNA library spanning the 5' region of the top1 tr anscript, which was constructed using an antisense specific oligonucle otide. The top1 nucleotide sequence encoded an open reading frame of 2 370 bp, predicting a protein of 90 kDa. The deduced amino acid sequenc e showed a similarity of 51% with A. thaliana, 41% with S. cerevisiae, 40% with S. pombe and 31% with H. sapiens, respectively. Southern blo t analysis, performed under moderate stringency conditions, showed the presence of a single-copy gene. Evaluation of the top1 mRNA steady-st ate level revealed, besides a constitutive expression in vegetative ca rrot tissues, an induced expression related to cell proliferation.