Cloning, sequencing, overproduction, and purification of M. CviBI (GANTC) methyltransferase from Chlorella virus NC-1A [corrected].
We have cloned and sequenced the cvibIM gene from Chlorella virus NC-1A by selecting for the modification phenotype. The modification gene was cloned on a 7-kb BamHI fragment inserted into the BamHI site of the pUC13 plasmid. The cvibIM gene was localized at the 3' end of this fragment. Sequencing of this region revealed a large open reading frame that codes for methyltransferase (MTase; symbol M.) (predicting 260 amino acids). M.CviBI (GANTC) aa sequence is homologous to M.Dam(GATC), M.DpnII(GATC), and M.T4 (GATC), and not so to M.HinfI(GANTC), M.HhaII (GANTC), and M.DpnA(GATC). We also describe the use of the polymerase chain reaction technique to alter transcriptional and translational signals surrounding this gene so as to achieve overexpression in Escherichia coli. This construct yields M.CviBI at 2-3% of the total cellular protein. The MTase was purified by phosphocellulose, DEAE, and gel filtration chromatography. Its size by SDS-PAGE is approx. 28 kDa, in good agreement with that predicted from the nucleotide sequence.
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Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
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Chandrasegaran
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002708:Chlorella; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004267:DNA Viruses / Q000201:enzymology* / Q000235:genetics; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017386:Sequence Homology, Amino Acid; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic
D004279:DNA, Viral; C077993:DNA modification methylase CviBI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)