'Stop-codon-specific' restriction endonucleases: their use in mapping and gene manipulation.

Abstract Certain restriction endonucleases recognise target sequences that contain the stop triplet TAG and are commonly either 4 or 6 bp in length. Interestingly, these restriction targets do not occur at the frequency expected on the basis of base composition and size. For example, the tetranucleotide Mae I recognition sequence (CTAG) occurs considerably less commonly (5–8-fold) in the genome of Escherichia coli (and many other eubacteria) than expected from mononucleotide frequencies. This surprising rarity is particularly evident in protein-encoding genes and is largely dictated by codon usage. Thus, amber (TAG) nonsense mutations frequently give rise to novel Mae I (CTAG) sites which are unique within a translated region. Such amber/ Mae I sites, whether arising spontaneously or created in vitro by site-directed mutagenesis, act as a useful physical marker for the presence of the nonsense mutation and are a convenient startpoint for a range of diverse procedures. These features provide a useful supplement to protein engineering methods which use nonsense suppression to mediate amino acid replacements.