Cloning and characterization of the O-methyltransferase I gene (dmtA) fromAspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis

O-Methyltransferase I catalyzes both the conversion of demethylsterigmatocy stin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocys tin to dihydrosterigmatocystin during aflatoxin biosynthesis, In this study , both genomic cloning and cDNA cloning of the gene encoding O-methyltransf erase I were accomplished by using PCR strategies, such as conventional PCR based on the N-terminal amino acid sequence of the purified enzyme, 5' and 3' rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by using Aspergillus parasiticus NIAH-26, A comparison of the genomic sequences with the cDNA of the dmtA re gion revealed that the coding region is interrupted by three short introns, The cDNA of the dmtA gene is 1,373 bp long and encodes a 386-amino-acid pr otein with a deduced molecular weight of 43,023, which is consistent with t he molecular weight of the protein determined by sodium dodecyl sulfate-pol yacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the Aspergillus nidulans StcP protein, whereas the N-terminal half of dmtA exhibits 73.0% identity with the 5' flanking region of the stcP gene, suggesting that translation o f the stcP gene may start at a site upstream from methionine that is differ ent from the site that has been suggested previously, Also, an examination of the 5' and 3' flanking regions of the dmtA gene in which TAIL-PCR was us ed demonstrated that the dmtA gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the omtA and ord-2 genes, Northern blotting revealed that expression of the dmtA gene is influenced by both medium composition and culture temperature and that the pattern cor relates with the patterns observed for other genes in the aflatoxin gene cl uster. Furthermore, Southern blotting and PCR analyses of the dmtA gt:ne sh owed that a dmtA homolog is present in Aspergillus oryzae SYS-2.