Insertion mutant of bacteriophage f1 sensitive to EcoRI.
The nucleotide sequence A-A-T-T was inserted into the intergenic region of the f1 genome at a site cleaved by Hae III (cleavage sequence (G-G-C-C). The resultant viable phage mutant (R199) contains a single site sensitive to the restriction endonuclease EcoRI (cleavage sequence G-A-A-T-T-C). This phage is sensitive to EcoRI restriction and modification in vivo and in vitro. Its potential for use as a cloning vector has been tested by construction in vitro of an f1/pBR322 chimeric phage. The four bases inserted into wild-type f1 to generate the R199 mutant came from a small restriction fragment obtained by digesting plasmid pBR322 with EcoRI and HindIII. The use of this linker prepared from a biological substrate is an example of a technique for constructing restriction enzyme sites in vitro. It is presented as an alternative to the use of synthetic linkers and should be generally applicable.
D001483:Base Sequence; D003090:Coliphages / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000378:metabolism*; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005814:Genes, Viral; D009154:Mutation; D010957:Plasmids
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004279:DNA, Viral; D004262:DNA Restriction Enzymes