Comparative Genomic Analysis of Several Deep-sea Bacterial Species Using Homing Endonuclease Digestion and Pulse Field Electrophoresis
Maria SMORAWINSKA*, Kaoru NAKASONE, Chiaki KATO, and Koki HORIKOSHI
The DEEPSTAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka 237, Japan
A new class of sequence-specific endonucleases was recently discovered (reviewed in 1-3). These "homing endonucleases" are classified as enzymes whose catalytic activities result in self-propagation. Homing endonucleases have potential for analysis of large genomes, because their unique recognition sequences are usually composed of 12-40 nucleotides far more than those recognized by type II restriction enzymes. The I-CeuI homing endonuclease is encoded by the fifth intron in the chloroplast large subunit rRNA gene of the green alga Clamydomonas eugametos, introduces a double strand break in a 26 base pair sequence present near the insertion site of the intron in cognate intronless allele (4). The genomes of enteric bacteria like Escherichia coli K12 and Salmonella typhimurium are cut by I-CeuI at seven sites corresponding to all rrn operons (5). No other sites are Cut. Since rRNA-determining genes are very highly conserved, I-CeuI would be expected to cleave the DNA of a wide range of bacteria in the rRNA genes.
We used the I-CeuI homing endonuclease for genome analysis of several deep- sea adapted bacteria ranging from obligatory barophilic to barotolerant strains isolated in our laboratory (6). Preliminary results show that deep-sea isolates are cut by I-CeuI in at least 9 sites, which may correspond to 9 rrn operons.
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