Journal of Experimental Botany, Vol. 51, No. 353, pp. 1979-1990,
December 2000
© 2000 Oxford University Press
Original Papers |
Carrot cells contain two top1 genes having the coding capacity for two distinct DNA topoisomerases I1
2 Department of Genetics and Microbiology ‘A. Buzzati-Traverso’, University of Pavia, Via Abbiategrasso 207, 27100 Pavia, Italy
3 Department of Biology, University of Ferrara, Corso Porta Mare 2, I-44100 Ferrara, Italy
4 Istituto di Mutagenesi e Differenziamento, C N R, via Svezia 2/105, 6100 Pisa, Italy
Five DNA topoisomerase I cDNA clones were isolated from a carrot (Daucus carota) cDNA library and two classes of nucleotide sequences were found. One component of the first class, pTop9, perfectly matches the open reading frame of pTop28, a truncated top1 cDNA previously described, and extended it by 594 nucleotides (top1
). A member of the second class, pTop11, contains an open reading frame 2727 bp long (top1ß) with a coding capacity for 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 similarity is reduced to 32% in the N-terminal region. Southern blot analysis and PCR amplification of genomic DNAs from carrot pure lines suggested the presence 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 cell populations. Three fusion peptides corresponding to the N-terminal domain of the and ß forms and from the common C-terminal domain of carrot topoisomerases I were overexpressed in E. coli cells and used to raise antibodies in rabbit. Immunolocalization seems to suggest the presence of two topoisomerases I in carrot nuclei.
Key words: cDNA cloning, cell proliferation, Daucus carota, heterologous expression, topoisomerase I.
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