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Engineering New Restriction Endonucleases
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Restriction endonucleases are of great importance to molecular DNA cloning, DNA diagnostics and genengineering. There is an ever increasing demand for a larger repertoire of recognition specificitiesendonuclease field is at point where the engineering of cleavage specificities is approachable. BamHfor engineering new specificities. It has been cloned, overexpressed and crystallized. BamHI bindingdeficient (cat-) variants have been isolated by using an in vivo transcriptional interference selectthat only cats variants can lead to spectinomycin resistance (spR). By inserting the BamHI recognitiinto an antisense promoter, transcription can be regulated. Binding of the cats BamHI to this operatthereby relieving the transcriptional interference resulting in spR. The 1.95 Angstroms resolution tstructure of BamHI has led to a determination of four amino acid residues within a seven amino acidrecognition. Cassette mutagenesis which varies all four DNA recognizing residues of a cats BamHI varrelatively few mutants (-160,000). Among these mutants, those which bind to anti-sense promoter operthan GGATCC can easily be selected. After selection of altered binding variants, cleavage will be recar mutation.
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