Carrot cells contain two top1 genes having the coding capacity for two distinct DNA topoisomerases I

Five DNA topoisomerase I cDNA clones were isolated from a carrot (Daucus ca rota) cDNA library and two classes of nucleotide sequences were found. One component of the first class, pTop9, perfectly matches the open reading fra me of pTop28, a truncated top1 cDNA previously described, and extended it b y 594 nucleotides (top1 alpha). A member of the second class, pTop11, conta ins an open reading frame 2727 bp long (top1 beta) with a coding capacity f or a second putative DNA topoisomerase I of 101 kDa. Both pTop9 and pTop11 clones are full length cDNAs. The two deduced amino acid sequences share a relevant similarity (89%) only at the C-terminal domain, whereas the simila rity is reduced to 32% in the N-terminal region. Southern blot analysis and PCR amplification of genomic DNAs from carrot pure lines suggested the pre sence of two distinct loci. Northern blot analysis revealed the presence of two distinct transcripts of 3.0 and 3.2 kb in both cycling and starved cel l populations. Three fusion peptides corresponding to the N-terminal domain of the or and beta forms and from the common C-terminal domain of carrot t opoisomerases I were overexpressed in E. coli cells and used to raise antib odies in rabbit. Immunolocalization seems to suggest the presence of two to poisomerases I in carrot nuclei.