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Nucleic Acids Research, 1992, Vol. 20, No. 13 3357-3360
© 1992

MOLECULAR BIOLOGY

Site-specific dissection of E.coli chromosome by {lambda} terminase

Hirokazu Kotani*, Akira Kawamura, Asako Takahashi, Masako Nakatsuji, Nobutsugu Hiraoka, Kazuo Nakajima and Mituru Takanami1

Bioproducts Development Center Takara Shuzo Co. Ltd, Seta 3-4-1, Ohtsu, Shiga 520-21, Japan 1Institute for Chemical Research, Kyoto University Uji, Kyoto 611, Japan

* To whom correspondence should be addressed at: Ishida cho 910-51, Moriyama, Shiga 524, Japan

Received April 7, 1992. Revised June 9, 1992. Accepted June 9, 1992.

We have succeeded the targeted cleavage of chromosomes by {lambda} terminase that introduces double-strand cleavages in DNA recognizing the {lambda} cos sequence. When chromosomal DNAs of various Escherichia coli K-12 strains were subjected to terminase digestion, all were found to contain two common cleavage sites. Therefore, DNAs from {lambda} lysogens in which {lambda} DNA was inserted at different chromosomal sites were specifically cleaved at one more additional site. The two sites, termed ecos1 and ecos2, were mapped at approximately 35.1' and 12.7' of E.coli genetic map. The ecos1 and ecos2 sites were included in qin and qsr' regions, respectively. Therefore, the cleavage sites were associated with cryptic prophages. Sequences at the ecos1 and ecos2 sites showed 98% homology to the {lambda} cos sequence, indicating high fidelity of sequence recognition by the terminase. Since the strategy for integration of a DNA segment into chromosomal DNA through homologous recombination has been established, the dissection method that uses X terminase should be applicable for gene mapping as well as construction of macro-physical maps of larger genomes.


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