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Nucleic Acids Research, 1989, Vol. 17, No. 9 3479-3490
© 1989

CHEMISTRY

Tracking bacterial DNA replication forks in vivo by pulsed field gel electrophoresis

Misao Ohki* and Cassandra L. Smith1

Biology Division, National Cancer Center Research Institute Tsukiji 5-1-1, Chuo-ku, Tokyo 1042, Japan 1Departments of Microbiology and Psychiatry, College of Physicians and Surgeons, Columbia University New York, NY 10032, USA

Received November 1, 1988. Revised March 28, 1989. Accepted March 28, 1989.

The location of chromosomal DNA replication forks was identified in synchronously replicating E. coli cultures by pulse labeling DNA at specific times with 14C-thymidine and following incorporation of radionucleotide into genomic Not I restriction fragments. This technique could be used to characterize chromosomal DNA replication, to characterize mutations which affect this process, to identify the location of DNA replication origins and termini as well as aid in the construction of macrorestriction maps. Here, we further characterize the DNA replication mutations divE and dnaK and preliminarily characterize the genomic organization of E. coli isolate 15.

*Present address: Department of Immunology and Virology, Saitama Cancer Center Research Institute, Ina-machi, Saitamaken 362, Japan.


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