Isolation, characterization and baculovirus-mediated expression of the cDNA encoding cytosine DNA methyltransferase from Pisum sativum.

摘要

A series of overlapping clones complementary to the Arabidopsis cytosine-5 DNA methyltransferase (C-5 MTase) was isolated from pea cDNA libraries. The assembled nucleic acid sequence contained an open reading frame of 4761 bp encoding a protein of 1554 amino acids. Like other eukaryotic C-5 MTases, the inferred protein contains a presumed regulatory N-terminal region linked to a catalytic C-terminal domain, which has eight of the ten conserved motifs found in prokaryotic C-5 MTases. The pea C-5 MTase shows 65% identity at the nucleotide level and 61% identity at the protein level with the Arabidopsis C-5 MTase. The catalytic domain of the pea enzyme shares 78% identity with Arabidopsis and～52% identity with murine and human C-5 MTases, includingthe relative position of the proline-cysteine dipeptides of the catalytic centre. Using the conserved region of the cDNA as a probe, a transcript of 5 kb was identified. Southern blot analysis of pea genomic DNA with the above probe indicated the presence of a single gene. Using poly(A)+ RNA from different developmental stages and different tissues, it was observed that expression is confined mostly to the rapidly dividing tissues of the plant. Expression of this assembled cDNA in a baculovirus system gave a protein of～174 kDa. The expressed protein can be cross-linked, in an AdoMet-dependent manner, to duplex oligonucleotide substrates containing FdC in place of target cytosines in either CG or CAG/CTG sequences. Nucleotide sequence data have been submitted to the EMBL/GenBank/DDBJ databases under the accession number AF034419.