Isolation and preliminary characterization of amber mutants of bacteriophage phi W-14 defective in DNA synthesis

Abstract

Of 42 amber mutants of bacteriophage 4W-14, 6 were defective in DNA synthesis. Three of the mutants synthesized DNA in the nonpermissive host, but were defective in post-replicational modification of the DNA. The DNA synthesized by two of these mutants, am36 and am42, contained more thymine and less a-putrescinylthymine than did wild-type DNA; that synthesized by the third mutant, am37, contained the normal amount of thymine, no a-putrescinylthymine, and hydroxymethyluracil. The properties of these mutants suggested that the presence of the normal amount of a-putrescinylthymine in 4W-14 DNA was essential for the production of viable progeny. Three of the mutants, am6, am35, and am45, failed to synthesize DNA in the nonpermissive host. These mutants were analogous to the DNA off mutants of T4. Nonpermissive cells infected with DNA off mutants accumulated dATP, dGTP, dCTP, and hydroxymethyl dUTP, but not dTTP or a-putrescinyldeoxythymidine triphosphate, confirming that both thymine and a-putrescinylthymidine in 4W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level. The synthesis of XW-14 DNA is unusual because (i) thymine is formed from hydroxymethyluracil at the polynucleotide level, (ii) the hypermodification forming a-putrescinylthymine is essential, and (iii) thymine and a-putrescinylthymine must be made in the correct proportions. Complementation tests showed that the mutants defined three genes involved in DNA polymerization and two genes involved in post-replicational modification. Bacteriophage 4W-14 (7) is unusual because

Condensation of bacteriophage .vphi.W14 DNA of varying charge densities by trivalent counterions

“…The function of the putThy in the genome of bacteriophage 0W14 has been the subject of a series of recent studies (Warren, 1980;Miller et al, 1982;Gerhard & Warren, 1982;Scraba et al, 1983; R. A. J. Warren, personal communication).…”

“…Amber 42 is a conditional lethal mutant of 0W14 that under nonpermissive conditions synthesizes a genome with only half the putThy residues of wild type (Miller et al, 1982(Miller et al, , 1983. It packages a piece of DNA that has an average charge density intermediate between wild-type and acetylated 0W14 DNA and is ~10% shorter than a complete genome (Scraba et al, 1983).…”

Polypeptide synthesis directed by bacteriophage phi W-14 and by mutants defective in DNA synthesis

“…Polypeptide synthesis in DNA modification-defective mutants. The DNA synthesized by nonpermissive cells infected with am37 contains 5-[(hydroxymethyl)-O-pyrophosphoryl]uracil (hmPPura) instead of putThy (10); that synthesized by cells infected with am42 or am36 contains only half the wild-type level of putThy and a correspondingly greater amount of thymine (11). The patterns of polypeptide synthesis in cells infected with these mutants were indistinguishable from those in cells infected with wild-type phage (Fig.…”

The Sequence of Two Bacteriophages with Hypermodified Bases Reveals Novel Phage-Host Interactions

“…This was investigated further using conditional lethal (amber) mutants. Several ΦW-14 mutants were affected in phage DNA synthesis [ 32 ]. For example, mutant am37 is defective in PutThy synthesis and abnormally accumulates 5-[(hydroxymethyl)- O -pyrophosphoryl]uracil (HmPPUra) in the newly-synthesized phage DNA [ 33 ].…”