In the present study, the isolation and characterization of two distinct cDNAs that code for carrot DNA (cytosine-5)-methyltransferase (DNA-METase) are reported. The screening of a cDNA library with a carrot genomic DNA fragment, previously obtained by PCR using degenerate primers, has led to the isolation of clones that belong to two distinct classes of genes (Met1 and Met2) which differ in sequence and size. Met1-5 and Met2-21 derived amino acid sequences are more than 85% identical for most of the polypeptide and completely diverge at the N-terminus. The larger size of the Met2-21 cDNA is due to the presence of nearly perfect fivefold repeat of a 171bp sequence present only once in the Met1-5 cDNA. Northern and in situ hybridization analyses with young carrot plants and somatic embryos indicate that both genes are maximally expressed in proliferating cells (suspension cells, meristems and leaf primordial), but differ quantitatively and spatially in their mode of expression. Polyclonal antibodies were raised in rabbit using fusion proteins corresponding to the regulatory and catalytic regions of the most highly expressed gene (Met1-5). In nuclear carrot extracts, both antibodies were found to recognize a band of about 200 kDa along with some additional bands of lower size. These results provide the first direct demonstration that DNA-METases of a higher eukaryote are encoded by a gene family.
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