CARROT DNA-METHYLTRANSFERASE IS ENCODED BY 2 CLASSES OF GENES WITH DIFFERING PATTERNS OF EXPRESSION

In the present study, the isolation and characterization of two distin ct cDNAs that code for carrot DNA (cytosine-5)-methyltransferase (DNA- METase) are reported. The screening of a cDNA library with a carrot ge nomic DNA fragment, previously obtained by PCR using degenerate primer s, has led to the isolation of clones that belong to two distinct clas ses 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 p erfect fivefold repeat of a 171bp sequence present only once in the Me t1-5 cDNA. Northern and in situ hybridization analyses with young carr ot plants and somatic embryos indicate that both genes are maximally e xpressed 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 p roteins corresponding to the regulatory and catalytic regions of the m ost highly expressed gene (Met1-5). In nuclear carrot extracts, both a ntibodies were found to recognize a band of about 200 kDa along with s ome additional bands of lower size. These results provide the first di rect demonstration that DNA-METases of a higher eukaryote are encoded by a gene family.