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Nucleic Acids Research, 1989, Vol. 17, No. 23 9749-9759
© 1989

MOLECULAR BIOLOGY

The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Echerichia coli DNA topoisomerase I deletion mutant

Kazuo Shishido, Satoshi Ishii and Naoki Komiyama*

Department of Life Science, Faculty of Science, Tokyo institute of Technology Nagatsuta, Midori-ku, Yokohama 227, Japan

Received August 8, 1989. Revised November 9, 1989. Accepted November 9, 1989.

Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987). These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA. Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting. When the entire tet region is present in a native orientation, the level of knotting is highest. Inactivating the tet promoter is manifested by a middle level of knotting. For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining. Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting. Deleting the ampicillin resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains. A possible mechanism of regulation of plasmid knotting is discussed.

*Present adresses: Meiji institute of Health Science, 540 Naruda, Odawara-shi, Kanagawa 250 Japan


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