Insertion Mutant of Bacteriophage f1 Sensitive to EcoRI

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 76; no. 6; pp. 2699 - 2702
• Main Authors: Boeke, Jef D., Vovis, Gerald F., Zinder, Norton D.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.06.1979; National Acad Sciences
• Subjects: Bacteriophages; Base Sequence; Coliphages - metabolism; DNA; DNA Restriction Enzymes; DNA, Bacterial - metabolism; DNA, Recombinant; DNA, Viral - metabolism; Enzymes; Escherichia coli - metabolism; Gels; Genes, Viral; Genomes; Intergenic DNA; Ligation; Molecules; Mutation; Plasmids; Single stranded DNA
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.76.6.2699

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$\overset \downarrow \to{-}$C-C) The resultant viable phage mutant (R199) contains a single site sensitive to the restriction endonuclease EcoRI (cleavage sequence G$\overset \downarrow \to{-}$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.