Cloning and characterization of the 5-methylcytosine methyltransferase gene in maize (Zea mays) plants and tissue cultures

The genomic sequence of maize containing the methyltransferase gene called Zmet1 was successfully cloned and sequenced. Seven clones were identified from a genomic library by using a highly conserved region from an Arabidopsis EST homologous to the Met1 methyltransferase gene as a probe. The assembled genomic sequence of four overlapping clones covers both the 5{dollar}spprime{dollar} and 3{dollar}spprime{dollar} flanking regions of the maize methyltransferase gene totaling 7,955 nucleotides. Sequence alignments with the Arabidopsis Met1 cDNA revealed that the open reading frame of Zmet1 encodes a putative protein of 1,525 amino acids, which is interrupted in the genomic sequence by ten introns. Northern analysis confirmed this result by the identification of a single 4.6 kb RNA transcript. The structure of the Zmet1 methylase is similar to the other eukaryotic maintenance methylases; a large N-terminal domain of 1,054 amino acids linked to a smaller C-terminal domain of 471 amino acids by a lysine-rich sequence. Zmet1 is highly expressed in actively dividing cells, namely in seedling tissue and rapidly dividing callus tissue. Restriction analysis suggests that there are at least two Zmet1 loci in the maize genome, one of which maps to the short arm of chromosome 7 in bin 2.; The percent of 5-methylcytosine in maize DNA was shown to be dependent on the type of tissue and the stage in development. Although the amount of repetitive DNA remained stable in the overall G/C to A/T ratio, methylation levels were found to significantly decrease from 15-day-old embryos to one-week-old seedlings. Remethylation occurs between the first and second week of seedling growth. These changes indicate that there are both de novo methylation and demethylation activities in early development. The methylation pattern at four low-copy sequences remained unchanged throughout plant and callus development except for a possible hypermethylation event in the third month of cell culture. However, there was significant demethylation in the repetitive sequences, rDNA and COS 12, throughout an eight-month period in tissue culture. The identification of stages or tissues which are undergoing demethylation and de novo methylation may be important in identifying undiscovered methylase and demethylase enzymes, developmentally regulated genes, as well as interacting proteins that may regulate methylase functions.