Mechanism of replication of bacteriophage φX174 XXII. Site-specific mutagenesis of the [formula omitted] gene reveals that A ∗ protein is not essential for φX174 DNA replication

Abstract

The A and A ∗ proteins of phage φX174 are encoded in the same reading frame in the viral genome; the smaller A ∗ protein is the result of a translational start signal within the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the φX174 A ∗ gene, previously cloned into pCQV2 under λ repressor control, into a TAG stop codon. The altered A ∗ gene was then inserted back into φX replicative form DNA to produce an amber mutant, φXamA ∗. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su + cells infected with φXamA ∗, φX gene A protein, altered at one amino acid, was synthesized at normal levels; A ∗ protein was not detectable. These observations indicate that the A ∗ protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of φX174 DNA or the packaging of the viral strand under the conditions tested.