Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli |
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Authors: | Arvydas Janulaitis Petras Povilionis Kestutis Sasnauskas |
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Institution: | Laboratory of Genetic Engineering, Institute of Applied Enzymology, 232028 Vilnius Lithuanian SSR, U.S.S.R. |
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Abstract: | The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity. |
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Keywords: | Recombinant DNA pBR322 expression in foreign host bp base pairs EtBr ethidium bromide kb kilobase pairs r-m restriction modification SDS sodium dodecyl sulfate SSC 0 15 M NaCl+0 015 M Na 3-citrate pH 7 8 U units |
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