G.Nigel Godson

A simple method of preparing large amounts of ΦX174 RF I supercoiled DNA

Abstract A reproducible NaCl-sodium dodecyl sulfate precipitation method is described which can easily be used on large quantities of ΦX174 infected cells to produce mg amounts of ΦX174 RF I DNA, essentially uncontaminated with host cell DNA.

Evolution of φX174. Isolation of four new φX-like phages and comparison with φX174

Abstract Many new single-stranded circular DNA-containing icosahedral phages have been isolated using a sucrose density gradient to fractionate the phages present in raw sewage into size classes. All the phages sedimenting at 120S were φX-like; from these, four different classes (called the G-phages) have been characterized. They are icosahedral particles with almost the same density and size as φX174, and they contain single-stranded circular DNA of almost the same size as the φX174 genome. Two of the phages, G4 and G14, are serologically unrelated to φX174 and are restricted by the R-factor restriction system mediated by RI endonuclease. The four new phages all code for the same number of proteins (8 or 9) as φX174, and every protein coded by the G phages has a recognizable φX174 counterpart of approximately the same molecular weight. DNA base homologies between these phages were studied by constructing heteroduplex DNA molecules with φX174 DNA and analyzing the base sequence mismatch with the electron microscope. The two phages (G4 and G14) that appeared by biological criteria and by differences in their proteins to be the most distantly related to φX174 gave heteroduplex DNA molecules with a highly characteristic and ordered pattern of single-stranded DNA loops (5 and 4, respectively), which represent specific regions of the viral DNA that have at least 24% base sequence mismatch compared with φX174 DNA. The two phages (G6 and G13) that by the same criteria appear to be very closely related to φX174, gave a highly random pattern of single-stranded DNA loops throughout the heteroduplex molecule, suggesting that the average base sequence mismatch of almost 24% is distributed throughout the whole molecule. The evolutionary significance of these findings is discussed.

Action of the single-stranded DNA specific nuclease S1 on double-stranded DNA

Abstract The single-stranded DNA specific nuclease S1 (Ando, T. (1966) Biochim. Biophys. Acta 114, 158–168) puts single-stranded breaks into double-stranded ΦX174 and T7 DNAs. The enzyme first converts the supercoiled ΦX174 RF I ★ into the relaxed form RF II, and then puts an increasing number of nicks into both of the DNA strands of the duplex. These breaks can only be observed upon denaturation of the molecule. This double-stranded DNA nicking activity of S1 nuclease is low compared with the single-stranded DNA digestion activity, and during the time it takes to completely digest ΦX174 single-stranded DNA to acid soluble nucleotides (i.e. approx. 5000 phosphodiester bond cleavages), only two to three nicks are put into the double-stranded ΦX174 DNA molecule (i.e. four to six phosphodiester bond cleavages). As the enzyme preparation used gave a single band on a sodium dodecyl sulphate polyacrylamide gel, it is tentatively concluded that the S1 nuclease not only digests single-stranded DNA to nucleotides, but also puts single-strand breaks in double-stranded DNA.

Mutants of Escherichia coli with temperature-sensitive lesions in membrane phospholipid synthesis: Genetic analysis of glycerol-3-phosphate acyltransferase mutants

SummaryEscherichia coli mutants possessing temperature-sensitive lesions in glycerol-3-phosphate acyltransferase, the enzyme catalysing the first step in phospholipid biosynthesis, have been characterized genetically. By recombinational and complementation tests, the mutants have been found to map in a single locus, called plsA, which is cotransducibile with the purE locus and lies between the purE and proC loci at minute 13 on the E. coli genetic map.

Characterization and synthesis of φX174 proteins in ultraviolet-irradiated and unirradiated cells

Abstract φX174 protein synthesis in ultraviolet-irradiated Escherichia coli C hcr − cells and. unirradiated cells was examined on sodium dodecyl sulfate-acrylamide gels. Eight viral proteins were identified and six of these assigned to cistrons. Viral protein synthesis in ultraviolet-irradiated cells is not normal. There is a large accumulation of coat protein and relatively little synthesis of cistron V (single-strand DNA synthesis) protein. The relative amount of the other φX gene products synthesized is also different from that observed during a normal infection of an unirradiated cell.

Origin and direction of φX174 double- and single-stranded DNA synthesis

Abstract The origin and direction of both φX174 double-stranded and single-stranded DNA synthesis has been determined by pulsing replicating viral DNA molecules with [3H]thymidine for periods of less than one round of DNA synthesis and examining distribution of activity in the Haemophilus influenzae restriction endonuclease (Hin) DNA fragments of these molecules. In early RFI and RFII DNA intermediates in double-stranded DNA replication, gradients of label were observed which started in the R3 fragment (cistron A) and increased towards the R4 fragment (cistron H). The origin of synthesis is near the R4/R3 junction of the R3 fragment. Thus, φX174 double-stranded DNA synthesis proceeds clockwise around the genetic map (5′ → 3′), in one direction only and starting in the region of cistron A, a conclusion consistent with the genetic experiments of Baas & Jansz (1972). Similar experiments with the gapped late RFII DNA molecules that have just completed a round of single-stranded viral DNA synthesis demonstrated that φX174 single-stranded DNA synthesis also has a single origin of replication in the region of cistron A, and that the synthesis moves in the 5′ → 3′ direction, around the genetic map. The gap in both the early and the late RFII DNA molecules also appears to be in the R3 fragment containing cistron A.

Susceptibility of the ∅X-like phages G4 and G14 to R · EcoRI endonuclease

Abstract Unlike ΦX174 and S13, the RF I and RF II DNA of the new ΦX-like phages G4 and G14 (Godson, 1974) are cut by the R1 R-factor restriction enzyme (Eco RI) into unit length RF III linear DNA molecules. The cut is specific and is located in the Haemophilus influenzae restriction enzyme fragments G4 HinB (900 nucleotides) and G14 HinA (1200 nucleotides) of G4 and G14, respectively. Revised sizes of the ΦX174 Hind fragments are given.

Evolution of ∅X174: II. A cleavage map of the G4 phage genome and comparison with the cleavage map of ∅X174

Abstract A physical cleavage map of the ∅X-like phage G4 has been constructed for the DNA fragments obtained using the Haemophilus influenzae (Hind) the Haemophilus aegyptius (Hae) and the Haemophilus parainfluenzae (HpaII) restriction enzymes and the position of the G4 Eco R I restriction site has been located. A comparison of the G4 DNA restriction map with that of ∅X174 shows that while there are no similarities in the pattern of restriction fragments observed on acrylamide gels, the two maps can be superimposed to give a ‘best fit’ with two areas of the genomes showing a considerable similarity in pattern of restriction cuts. These two areas correspond to two of the known ∅X174 promoter regions and to the origin of ∅X174 DNA replication. The areas of the G4 and ∅X174 genomes that show the greatest dissimilarities in restriction cuts correspond to the region of ∅X174 DNA that codes for the virion structural proteins.

DNA heteroduplex analysis of the relation between bacteriophages φX174 and S.13

Abstract When observed in the electron microscope using the isodenaturing methods of Davis & Hyman (1971), only one small segment (4.7 ± 1.9%) of the DNA of phage φX174 is highly homologous with phage S.13 DNA; the rest is partially homologous with an over-all average 36% base mismatch. The two phage DNA molecules appear to be identical in length and have no regions of complete base non-homology. The phage-coded proteins were compared by electrophoresis on slab polyacrylamide gels and only one of the S.13 coded proteins migrated identically with its φX174 counterpart. The other eight S.13 coded proteins varied in size from their φX174 counterparts by +4.6% to −6.0% (± ten amino acid residues). The relevance of these data to the complementation and recombination between these two phages is discussed.

Identification of a X174 coded protein involved in the shut-off of host dna replication

Summary When Escherichia coli is infected with X174, the host cell DNA replication ceases 10 – 12 minutes after infection. This shut-off is prevented by the addition of 30μg/ml chloramphenicol indicating a requirement for post-infection protein synthesis. Some amber mutants in X174 cistron A, which codes for two proteins, do not shut off the host DNA replication. Amber mutants in all other X174 genes shut off host replication. Mutants near the amino-terminal end of cistron A, which still produce the small 35, 000 molecular weight cistron A polypeptide, shut off the host DNA synthesis whereas mutants near the carboxy-terminal end, which do not produce the small A polypeptide do not shut off the host.