Abstract
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.
Highlights
From the $Department of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco, California 94143 and the §Departmentof Microbiology and Immunology, Duke University Medical Center and the Duke University Program in Genetics, Durham, NorthCarolina 27710
I / S Vector System Requirements-As depicted in Fig. 1,the successful development of an I/S vector system for T4 bacteriophage demands that a number of requirements be satisfied
4) The integration of the plasmid must not increase the length of the phage so much that it prevents the packaging of a complete, intact chromosome into thephage head
Summary
Same as pBSCO but with Bal31-generatedfrd deletions (see Fig. 4) Same as pBSPLO+/- but with the E. coli rrnB T1 transcriptiotnerminator Same as pBSPLO+/-ter+/- but containing the 381-bp frd fragment of pBSfs381. 1.1-kb3 ’ is the 1.1-kbT4 EcoRI fragment from pKK025 that contains the gene 34 tertiary origin of replication. 250-bp 3 ’ is the 250-bp T4 HindIII-EcoRI fragment from pKKO61 that contains the gene 34 tertiary origin of replication. Substitution of the vector-borne T4 target sequence for its chromosomal counterpart involved first selecting for integrant phage on lawns of E. coli BE. The integrants were propagated under nonselective conditions (host CR63), which permitted the vector sequence as well as one copy of the duplicated target sequence to segregate out of the chromosome. For this purpose, an overnight culture of CR63 (supD) was diluted 1:200 and grown for 105 min.
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