Faculty Bibliography

Simple methods for introducing one or two extra codons of genetic information into the f1 genome in vitro have been devised. The methods use various combinations of enzymes to insert three or six base-pairs into the RF1 DNA of the bacteriophage. Since such insertions do not cause frameshifts in coding regions, a number of these mutants are viable. Several such mutants were mapped and characterized. The methods described and variations of them can be applied to other circular DNA genomes.

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.