Sequence, internal homology and high-level expression of the gene for a DNA-(cytosineN4)-methyltransferase, M-PvuII

Abstract

The base sequence of the pvuIIM gene has been determined. This gene codes for a DNA-(cytosine N4)-methyltransferase, M.cntdot.Pvu II. The base sequence contains a single large open reading frame that predicts a 38.3kDa polypeptide, consistent with experimental data. The pvuIIM gene contains some sequences common to DNA methyltransferases in general, but includes none of the sequences specifically conserved among DNA-(cytosine 5)-methyltransferases. The pvuIIM sequence also reveals an internal homology at the amino acid level, each half of which spans over 100 amino acids and is itself homologous to the sequences of some DNA-(adenine N6)-methyltransferases. A derivative of the pvuIIM plasmid was constructed to allow high-level production of M.cntdot.Pvu II. Specifically, the composite Ptac promoter was inserted 5'' to pvuIIM, intervening DNA was deleted, and the resulting construct was used to transform an mcrB lacl-q strain of Escherichia coli. When this transformant was induced with isopropyl-B-D-galactopyranoside (IPTG), growth rapidly ceased and M.cntdot.Pvu II accumulated to the point of comprising over 10% of the total soluble protein.