The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome.

A bacteriophage T4 insertion/substitution vector system has been developed as a means of introducing in vitro generated mutations into the T4 chromosome. The insertion/substitution vector is a 2638-base pair plasmid containing the pBR322 origin of replication and ampicillin resistance determinant, a T4 gene 23 promoter/synthetic supF tRNA gene fusion, and a polylinker with eight unique restriction enzyme recognition sites. A T4 chromosomal "target" DNA sequence is cloned into this vector and mutated by standard recombinant DNA techniques. Escherichia coli cells containing this plasmid are then infected with T4 bacteriophage that carry amber mutations in two essential genes. The plasmid integrates into the T4 chromosome by recombination between the plasmid-borne T4 target sequence and its homologous chromosomal counterpart. The resulting phage, termed "integrants," are selectable by the supF-mediated suppression of their two amber mutations. Thus, although the integrants comprise 1-3% or less of the total phage progeny, growth on a nonsuppressing host permits their direct selection. The pure integrant phage can be either analyzed directly for a possible mutant phenotype or transferred to nonselective growth conditions. In the latter case, plasmid-free phage segregants rapidly accumulate due to homologous recombination between the duplicated target sequences surrounding the supF sequence in each integrant chromosome. A major fraction of these segregants will retain the in vitro generated mutation within their otherwise unchanged chromosomes and are isolated as stable mutant bacteriophage. The insertion/substitution vector system thereby allows any in vitro mutated gene to be readily substituted for its wild-type counterpart in the bacteriophage T4 genome.

Duke Scholars

Author Kenneth N. Kreuzer Biochemistry