Dedkov V, Gonchar D, Abdurashitov M, Udalyeva S, Urumceva L, Chernukhin V, Shiryaeva E, Degtyarev S

Language: English
References: 13
Page: 3211-3216
PDF size: 915.43 Kb.

ABSTRACT

A fragment of Arthrobacter luteus B DNA carrying the gene of new DNA methyltransferase M.AluBI was cloned and expressed in Escherichia coli. The recombinant plasmid pM.AluBI-16 contains the M.AluBI gene (1515 bp in length), corresponding to a protein of 504 amino acid residues. The amino acid sequence analysis showed that M.AluBI could be an adenine-(N6)-DNA methyltransferase. A recombinant strain was grown up and the enzyme was purified by a consecutive chromatography on P-11 Phosphocellulose, Heparin-Sepharose, Sephacryl S-200 and Hydroxyapatite. M.AluBI specificity was determined by the original method based on blocking of restriction endonucleases cleavage of overlapped sites and on computer modeling. It was first shown that AluBI MTase modifies the adenine residue with formation of 5´-(m6A)GCT-3´ as opposed to its prototype, M.AluI, producing 5´-AG(m5C)T-3´. A comparative sensitivity analysis of different, well known restriction endonucleases to the methylation by M.AluBI and M.AluI was done using λ and T7 phage DNA. The newly acquired data on methylation sensitivity cold be useful for conducting experiments on DNA digestion with restriction endonucleases, and especially with the particular cleavage sensitivity pattern generated with the M.AluBI methyltransferase enzyme.

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