Scientific papers // The enzymes genes cloning and study
Cloning and characterization of a new site specific methyl- directed DNA endonuclease EcoBLI recognizing 5’- G(5mC)NGC-3’/3’-CGN(5mC)G-5’
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D.A.Gonchar, V.A.Chernukhin, M.A.Abdurashitov, E.V.Kileva, V.S.Dedkov, N.A.Mikhnenkova, E.N.Lomakovskaya, S.G.Udalyeva, S.Kh. Degtyarev
BioTechnology : An Indian Journal, Volume 12,Issue 4, Pages 175-181
A gene coding BisI, site specific 5mC-directed DNA endonuclease recognizing DNA sequence 5’-G(5mC)NGC- 3’/3’-CGN(5mC)G-5’, was recently identified in the sequenced genome of the strain-producer Bacillus subtilis T30. In this work we have undertaken a search of bisI gene homologues among the sequenced genomes of enterobacteria. DNA analysis has revealed a small group of highly homologous ORFs with unknown function including one ORF in DNA of well-known strain E.coli. This ORF WP 001276099.1 from E.coli BL21 (DE3) was amplified and cloned. An obtained recombinant strain E.coli pEcoBLI produces MD-endonuclease named EcoBLI. The new enzyme has the same substrate specificity as BisI MD-endonuclease. Thus, ORF WP 001276099.1 from E.coli BL21 (DE3) encodes site-specific 5mC-directed DNA-endonuclease EcoBLI recognizing and cleaving DNA sequence as indicated by arrows 5’-G(5mC)^NGC-3’/3’-CGN^(5mC)G-5’.
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Cloning and Study of New DNA Methyltransferase M.FatI Modifying Cytosine in a Recognition Site CATG
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Dedkov V.S., Gonchar D.A., Abdurashitov M.A., Udalyeva S.G., Urumceva L.A., Chernukhin V.A., Mutylo G.V., Degtyarev S.Kh
Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol 6 (6), 2015 P. 1341-1348
A fragment of Flavobacterium aquatile NL3 DNA carrying the gene of DNA methyltransferase M.FatI was cloned in pUC19 plasmid. DNA was sequenced and M.FatI gene was analyzed. A recombinant strain Esherichia coli was grown up and the enzyme was purified. M.FatI specificity was determined by a blocking of some restriction endonucleases and computer modeling. It’s well known that M.NlaIII produces 5’-C(m6A)TG- 3’, whereas FatI MTase modifies the cytosine residue with formation 5’-(m5C)ATG-3’. The sensitivity of restriction endonucleases to FatI-methylation has been studied. Keywords: gene cloning, enzyme isolation, bacterial DNA methyltransferase, enzyme specificity, restriction endonuclease, methylation sensitivity
Multiplicity of Site-specific DNA Methyltransferases of the BstF5l Restriction-Modification System
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Translated from MOLECULAR BIOLOGY (Russia) Vol. 34 No. 3, 443-447, 2000
A fragment located downstream of the genes for DNA methyltransferases of Bacillus stearothermophilus F5 (M.BstF5I-1 and M.BstF5I-2) was sequenced. The fragment contains a gene for another methylase. M.BstF5I-3. structurally and functionally similar to the N-terminal domain of M.FokI. Thus, in contrast to other restriction-modification systems, the BstF5I system includes three methylases, two being homologous to the individual M.FokI domains.
The Second Methyltransferase of the BstF5I Restriction–Modification System Is Homologous to the C-Terminal Domains of FokI and StsI Methylases
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Translated from Molekulyarnaya Biologiya, Vol. 34, No. 1, 2000, pp. 87–94
The bstF5IM-2 gene for the second DNA methyltransferase of Bacillus stearothermophilus F5 (M.BstF5I-2) was cloned and sequenced. On the chromosome, the gene is located downstream of the gene for M.BstF5I-1 and is oriented similarly. The protein encoded has conserved regions specific for adenine methylases of subclass D12. M.BstF5I-2 is homologous to the C-terminal domains of M.FokI and M.StsI and recognizes the same sites. M.BstF5I-1 modifies the DNA upper strand and M.BstF5I-2 modifies the other strand in the recognition site.