Vspl methylase belongs to m6A-gamma class of adenine methylases.

Bacterial DNA-methyltransferases are responsible for site-specific modification of DNA. They transfer the methyl-group from Sadenosylmethionine (SAM) to DNA with formation of N6-methyladenosine (m6A), 5-methylcytosine (m5C) or N4-methylcytosine (m4Q (1). Lately nucleotide structures of more than one hundred genes of different methyltransferases have been determined (2). Analysis of deduced amino acid sequences has revealed two conservative motifs of bacterial methylases (2). m6A-Methylases are characterized by two patterns of amino acids D/NPPY and P-G-G and their mutual positions in the gene define these enzymes as alpha, beta or gamma class of m6A-methyltransferase (2,3). We have successfully isolated a genomic clone Vibrio species strain 343 which encodes methyltransferase and prevents plasmid and bacterial DNA degradation by restriction endonuclease Vspl. Plasmid pMVS16 containing the methylase gene was isolated from a genomic library constructed by ligating a partial Sau3AI digest of Vibrio species strain 343 genomic DNA into the SalGI site of plasmid pMTL22 (4). Before ligation, the ends of DNA fragments were partially restored by Klenow fragment in the presence of dGTP + dATP and dTTP + dCTP, respectively. The DNA from plasmids library has been cleaved completely by Vspl and this digest has been used for retransformation of E.coli RR1 strain. Plasmid and genomic DNA isolated from E.coli RR1 harboring plasmid pMVS16 was resistant to cleavage by Vspl. The plasmid pMVS7 was constructed by ligation of Pstl-PstI fragment of pMVS16 containing methylase gene into the PstI site of pMLT22. The sequence analysis of this gene has been made. There are three open reading frames starting from nucleotides #503, #557, #812 and ending at #1780, which encode 47.5, 45.5 and 36.1 kd proteins respectively. The Shine-Dalgarno signal presents in the last two open reading frames. Gel-filtration of native methylase Vspl showed a molecular weight of 42.7 kd. Thus, the open reading frame of the methylase gene is #557-1780 and it encodes a 408 amino acid protein. The comparison of deduced amino acid structure of Vsplmethylase and others has been done according to Klimasauscas et al. [2] and new entries have been added: Dpnl (5), Ecal (6), EcoR124/3 (7), Fokl (8), Hindi (9) and Rsrl (10). All the data are presented in Figure 1. According to mutual positions of two conservative domains this enzyme belongs to m6A-gamma class. M.VspI has a NPPW-motif instead of an NPPY one, but replacement of aromatic amino acid tyrosine by another aromatic amino acid tryptophan might be insignificant. REFERENCES