Robert C. Warner

Figure-8 configuration of dimers of S13 and φX174 replicative form DNA☆

Abstract Evidence from electron microscopy of the replicative form of S13 and φX174 DNA shows the presence of a “figure-8” configuration. This species consists of two monomer length and one dimer length circular strands in covalently closed circular form and containing a fused junction that divides the molecule into two equal circular segments. Its existence is supported by the demonstration that it is converted by digestion with the restriction endonuclease of Hemophilus influenzae strain Rd to α- and X-shaped forms that retain the fused junction, and by examination by electron microscopy in the presence of ethidium bromide, which eliminates tangling and accidental overlays of parts of the DNA molecules. Kgure-8s are present to the extent of about 5% of the dimers present in replicative form DNA. They are proposed to be intermediates in genetic recombination in S13 and φX174.

Physical studies on the size and structure of the covalently closed circular chloroplast DNA from higher plants.

The size and structure of the covalently closed circular chloroplast DNAs (ctDNA) from pea, lettuce, and spinach plants, have been studied by analytical ultracentrifugation. The values of so20,w,Na+ of the native and denatured forms of the open and closed circular DNAs from these plants have been determined. The absolute molecular weight of purified closed circular pea ctDNA monomers has been determined by buoyant equilibrium sedimentation to be 89.1 (S.D. +/- 0.7)-10(6). The value of the so20,w,Na+ of open circular pea ctDNA and its molecular weight, in conjunction with corresponding values for other sizes of circular DNA, has been used to derive an empirical relationship between so20,w,Na+ and molecular weight for open circular DNAs. Using this relationship, the molecular weights of lettuce and spinach ctDNAs have been determined to be 98.2 (S.D. +/- 1.5)-10(6) and 97.2 (S.D. +/- 1.5)-10(6), respectively. At pH values 12.7 and 13, closed circular lettuce and pea ctDNAs have been found to exist as mixtures of reversibly and irreversibly denatured closed circular DNAs.

The molecular size of the herpes simplex virus type 1 genome.

Abstract The Facial (type 1) strain of Herpes Simplex virus (HSV) can be isolated free of contaminating host cell DNA from HeLa cells. The molecular weight (MW) of HSV-DNA isolated from purified virions has been measured using sedimentation analysis, equilibrium band-width measurements, electron microscopic contour length measurements relative to φX-174 RF DNA, and comparative reassociation kinetics using T4-DNA and pea chloroplast DNA as standards. There is excellent agreement between values determined by all these methods. The best average value from all methods for the MW of HSV-DNA is 82 ± 5 × 10 6 . There is no evidence that the high G + C content of HSV-DNA yields any systematic error in molecular weight determinations using any of the methods described in this work.

Agarose gel electrophoresis of circular DNA of replicative form of bacteriophage G4

Abstract Agarose gel electrophoresis can be used to fractionate the replicative form of DNA (RF-DNA) of bacteriophage G4 according to both the conformation and the molecular weight of the component molecules. We have examined the effect of gel concentration, current density, and temperature on the degree of separation of the different species. Optimum results are obtained by manipulation of the gel concentration and control of temperature and current density. Purification of figure-8 DNA has been accomplished in a two-stage procedure which separates figure-8s from catenated dimers. The viral and complementary strands of G4 RE-DNA can also be fractionated. Several methods for the recovery of DNA from agarose gels have been tested and their efficiency evaluated.

Pseudolysogeny of Azotobacter phages

Abstract The establishment of a pseudolysogenic state accompanied by a phenotypic conversion in Azotobacter vinelandii strain O by phages A14, A21, A31, and A41 has been identified. Host cells can be recovered from the pseudolysogens by cultivation in phage-specific antiserum. Pseudolysogens continually give rise at a low rate to phage as a result of the occasional initiation of a lytic burst. As a result of the establishment of the pseudolysogenic state the host cells lose their polysaccharide coat, become flagellated and motile and acquire a yellow pigmented appearance. The four phages, although they differ serologically and molecularly, give rise to converted states that are indistinguishable except by the identification of the phage that is produced by it. On repeated subculturing each of the pseudolysogens will give rise to a stable or permanently converted cell that has the phenotype of the pseudolysogen, but from which it is no longer possible to obtain either host cells or phage.

Pseudolysogenic conversion of Azotobacter vinelandii by phage A21 and the formation of a stably converted form.

Abstract Phenotypically converted pseudolysogens are formed when Azotobacter vinelandii strain O is infected with phage A21. The segregation of phage-sensitive host cells that occurs when the pseudolysogen is grown in phage-specific antiserum was studied in two isolates of pseudolysogens that had different rates of segregation. The segregation appears to be based on the random partitioning at cell division of a number of carrier particles such that a daughter cell that does not receive one reverts to the host phenotype. Values for the copy number for two isolates were determined by analysis of the kinetics of segregation. The copy number of 5 for the more rapidly segregating isolate is in agreement with a determination by hybridization that there are about four copies of phage-specific DNA per cell. In a process independent of segregation, pseudolysogens can become stably converted to a species designated here as PC cells. These cells retain the converted phenotype of the pseudolysogen but they can no longer produce phage or segregate host cells. They contain less than one genome equivalent of phage DNA. Some observations have been made on the conditions of formation of PC cells and on their loss of phage-forming ability, but the basis of the underlying process has not been clarified.

Azotobacter phages: properties of phages A12, A21, A31, A41 and their constituent DNAs.

Abstract The properties of several Azotobacter vinelandii phages and of the DNA isolated from each have been investigated. Phages A12, A21, and A41 have short noncontractile tails. A12 and A21 have virion weights of 80 × 10 6 daltons and contain 58% DNA. A31 has a long noncontractile tail, a virion weight of 175 × 10 6 daltons and contains 26% DNA. A31 and A41 are unstable in salt solutions of moderate ionic strength. All phages contain linear, double-stranded DNA that sediments as a single component in neutral and alkaline solutions. The molecular weights of A12, A21, and A31 DNAs obtained by equilibrium density gradient sedimentation are (46 ± 7) × 10 6 , (42 ± 5) × 10 6 and (47 ± 5) × 10 6 , respectively. Electron microscope length measurements using the replicative form of ∅X174 DNA as an internal contour length standard yielded molecular weights of (44.3 ± 2) × 10 6 , (45.6 ± 1.2) × 10 6 and (46.5 ± 2.6) × 10 6 , respectively. The buoyant densities of A12 and A21 DNA in CsCl and Cs 2 SO 4 indicate a G + C content of 58% consistent with the T m . For A31 DNA the buoyant densities suggest a G + C content of 52% whereas the T m corresponds to 69% G + C. A12 and A21 DNAs show strand separation in an alkaline CsCl gradient (Δ ρ ⋍0.006 g ml −1 ).

Coinfection of E. coli with phages G4 and φX174: Origin of dimeric replicative form species☆

Abstract The origin of the dimeric species of replicative form (RF) DNA has been investigated by coinfecting E. coli with phages φX174 and G4. The RF DNA from the coinfection was examined by electron microscopy before and after treatment with the EcoRl restriction endonuclease which makes one double-strand scission per monomer length in G4-RF and none in φX174-RF. Recombinant dimer molecules were identified by the unique products resulting from digestion. Circular, catenated, and figure-8 dimers were indicated to be formed both by recombination and replication. Analysis of the tail lengths of a forms derived from homozygous and heterozygous figure-8s demonstrates that branch migration is restricted by the nonhomologous regions of the G4 and φX174 genomes. No bias was detected in figure-8s with respect to the polarity of the strand that is of dimer length.

Mechanism of Genetic Recombination

Genetic studies of the circular single-stranded DNA phages were initiated in the expectation that the smallness of these phages reflected a simplicity conducive to understanding the recombination process on the molecular level. Early extensive efforts just to find genetic recombination failed; the most serious work (Zahler 1958) showed no recombinants at the level of 10 −3 in several crosses of plaque-type mutants of S13. This inability to find genetic recombination was rationalized for several years by inferences from radiosensitivity studies (Tessman et al. 1957) which suggested that ϕ X174 and S13 behaved radiologically like RNA viruses, which are notably single-stranded in their nucleic acid and do not undergo genetic recombination. In the end, however, it turned out that recombination of S13, ϕ X, and G4 was easy to observe even without special selective techniques (Tessman and Tessman 1959). The original success was due to the stimulatory action of UV light, observed first for phage λ (Jacob and Wollman 1955); with UV light the recombination frequencies observed were of the order of 10 −3 , just within the range needed at that time for easy observation. However, UV stimulation is not essential for observing recombinants, and the mechanism of stimulation remains a major puzzle in molecular genetics. The early recombination experiments revealed a useful feature of this phage system: the probability of a mating event (the interaction of two genotypes leading to recombination) is around 0.01. This means that the recombinants emerging from a single cell are, with rare exceptions, the products of a single mating...

Novel dimeric configurations from bacteriophage G4 replicative form DNA

The oligomeric fraction of the replicative form of phage G4 was prepared by sedimentation on three successive CsCl velocity gradients followed by resolution on CsCl-propidium diiodide equilibrium gradients and subfractions through the equilibrium gradients were examined by electron microscopy. The most frequent dimer species were the circular dimer, the singly linked catenane and the figure 8; these occurred in a ratio of 10:3:1. The high enrichment for dimers and other oligomers made possible the observation and the determination of the frequency of occurrence of a number of minor species, some of them of novel configuration. These are (a) dimers similar to figure 8s except containing long, apparently four-stranded junctions common to the two halves (theta forms); (2) dimers similar to those in (1) except that the long junctions separate the two halves (dumbbell forms); (3) multiply catenated dimers with apparent right-handed intertwines; and (4) dimers containing a knot. Theta forms cleaved by EcoRI were shown to be stable under conditions in which EcoRI-treated figure 8s were resolved by branch migration.