Isolation and Characterization of an Unknown Restriction Endonuclease

Currently, there are approx 3000 restriction endonucleases known, recognizing 235 different sequences (1 ). Although primarily found in bacteria, they also exist in archaea, viruses, and eukaryotes. An estimated 25% of bacteria examined contain at least one restriction endonuclease (2 ), and therefore the probability of encountering new ones is relatively high. Indeed, many new enzymes have been “discovered” in contaminated bacterial cultures. The presence of three restriction activities in a single organism is not unusual. Neisseria strains appear to be particularly rich in restriction endonucleases and their corresponding methyltransferases. As many as seven different endonucleases from a single strain have been idenitified through cloning (3 ). The first enzyme discovered which recognizes a unique sequence, although it may not be commercially available or commonly known, is designated the prototype. Although few new prototypes have been discovered recently, two potential four-base palindromes and seven potential six-base palindromes are not cleaved by any known restriction endonucleases. Most databases are arranged alphabetically by prototype, with isoschizomers listed under the prototype heading. A database of all known endonucleases, maintained by Dr. Richard J. Roberts, is available at http://www.neb.com/rebase . A number of formats are available, and references are provided. Detailed information on restriction endonuclease biology, classification, structure, specificity, and catalytic mechanism is provided elsewhere in this book (see Chapters 19 , 27 , and 29 ).