Molecular cloning and in vitro transcription of Bacillus subtilis plasmid in Escherichia coli View Full Text


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Article Info

DATE

1977-01

AUTHORS

Sueharu Horinouchi, Takeshi Uozumi, Takayuki Hoshino, Akio Ozaki, Sadayo Nakajima, Teruhiko Beppu, Kei Arima

ABSTRACT

A composite plasmid (pAT2010) has been constructed in vitro from RSF2124 and Bacillus subtilis IFO3022 plasmid (pAT1060) by covalent joining of the two DNA molecules by means of Escherichia coli DNA ligase through the cohesive ends generated by restriction endonuclease RI (EcoRI) cleavage. The composite plasmid was selected by transformation of E. coli C600 r−m− with the ligated mixture affer enrichment for composite plasmid by preparative agarose gel electrophoresis, and plating of the transformants on a medium containing ampicillin and colicin El. Treatment of the composite plasmid with EcoRI yielded two fragments corresponding to the linear forms of the parental plasmids.The composite plasmids replicated as biologically functionally units in E. coli, and expressed genetic information carried by RSF2124. In the presence of chloramphenicol, the composite plasmids continued to replicate and the copy number gradually increased. Such nature of replication in the presence of chloramphenicol is characteristic to RSF2124 derived from colicin El factor, and so it is suggested that the replicator of RSF2124 is functional in the composite plasmid.The composite plasmid was found to synthesize mRNA of B. subtilis plasmid in cell-free extracts of E. coli, by hybridization of the mRNA to the original plasmid DNA of pAT1060. More... »

PAGES

175-182

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Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/bf00267395

DOI

http://dx.doi.org/10.1007/bf00267395

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1024458373

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/414072


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