Asymmetric information transfer during φX174 DNA replication

By annealing the single-stranded DNA of φX174 phage with the complementary strand obtained from the replicative form of this phage, heteroduplex RF was prepared with a genetic marker in the viral strand, which is different from that in the complementary strand. Heteroduplex RF was indistinguishable from native RF in buoyant density in CsCl, in contour length in the electron microscope and in hydroxylamine sensitivity. Heteroduplex RF is infectious to spheroplasts of the appropriate bacterial host. Infected spheroplasts yield a burst of approximately equal quantities of phage of the plus- and minus-strand type and the majority of the spheroplasts produces a single type of phage. This and other evidence indicates that the heteroduplex region is repaired before replication. The ultraviolet sensitivity of heteroduplex RF DNA is intermediate between that of single- and double-stranded φX DNA suggesting that repair of a heteroduplex region involves the degradation of a large single-strand segment

φX 174 replicative form DNA replication, origin and direction

Heteroduplex replicative form of bacteriophage φX174 was prepared by annealing minus strand isolated from wild-type φX RF† with plus strand DNA, obtained from temperature-sensitive and amber mutants of φX in different cistrons. Three different kinds of bursts were produced by spheroplasts infected with heteroduplex RF: bursts with only wild-type phage, bursts with only mutant phage and bursts with wild-type and mutant phage. The frequencies of these bursts for RF with heteroduplex regions in different cistrons were determined in single-burst experiments under permissive conditions. A gradient in the percentage of spheroplasts producing both genotypes, depending on the position of the mutation in the plus strand of heteroduplex RF on the genetic map of φX phage, was observed. The direction of replication of double-stranded φX DNA as well as the origin of replication were deduced from this gradient

On the active site of horse-liver ali esterase II. Amino acid sequence in the DFP-binding site of the enzyme

The structure of a diisopropylphosphoryl-containing peptide obtained by digestion of DFP-inhibited horse-liver ali esterase by pepsin was established as follows: gly-glu-DP·O·ser-ala-gly-gly-(glu, ser). © 1959

Bacteriophage φX174 RF DNA replication in vivo : A study by electron microscopy

We have investigated bacteriophage φX174 RF † DNA replication by electron microscopy. Three different, types of replicative intermediates were observed: rolling circles, partially duplex DNA circles and structures consisting of two DNA circles connected at a single point. Rolling circles with a single-stranded or partially double-stranded DNA tail were both observed. After cleavage of the rolling circles with the restriction endonuclease from Providentia stuartii 164 (PstI) the startpoint of rolling circle replication could be located at 21 map units from the PstI cleavage site in agreement with the previously determined position of the origin of φX RF DNA replication. Partially duplex DNA circles consist of circular viral DNA strands and incomplete complementary DNA strands. After cleavage of these molecules with PstI information about the startpoints of the synthesis of the complementary DNA strand was obtained. The connected DNA circles always contain one completely double-stranded DNA circle whereas the other circle consists of either single-stranded, partially duplex or completely duplex DNA. Part of the duplex-to-duplex DNA circles represent the well-known figure eight or catenated circular dimers. The other connected DNA circles presumably represent replication intermediates which arise by the association of the end of the genome length tail of the rolling circle with the origin-terminus region. This is suggested by the fact that the point of contact between the two DNA circles is located at approximately 21 map units from the Pst1 cleavage site, i.e. at the origin-terminus region of the φX genome. The connected DNA circles may be intermediates in the circularization and cleavage of the genome-length tail of the rolling circles in vivo. A model for φX174 RF DNA replication in vivo summarizing the data obtained by biochemical (Baas et al., 1978) and electron microscopic analysis of replicative intermediates is presented (Fig. 9)

On the active site of horse-liver ali esterase I. Reaction of the enzyme with diisopropylphosphorofluoridate

Horse-liver ali esterase reacts with DFP to form the enzymically inactive DP-enzyme. With isonitrosoacetone diisopropylphosphate is released from the inhibited enzyme; this is accompanied by a recovery of the enzymic activity. In order to investigate the chemical nature of the DFP-binding site of ali esterase the DP-enzyme was digested with pepsin. Essentially one DP-peptide was formed which contained per mole of DP-group the following moles of amino acid residues: alanine (1), glutamic acid or glutamine (2), glycine (3), and serine (2). © 1959

Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA

Replicating Ad5 DNA was isolated from nuclei of infected KB cells and studied by electron microscopy. Branched as well as unbranched linear intermediates were observed containing extended regions of single-stranded DNA. The relationship between the branched and unbranched structures was studied during synchronized synthesis in the first round of replication after release of hydroxyurea inhibition. The branched intermediates represented early and the unbranched intermediates late stages in the replication cycle. Digestion of the branched intermediates with Eco RI endonuclease revealed that replication had started at the molecular right end (the A-T-rich end). This was confirmed by partial denaturation mapping of branched intermediates. These results confirm and extend a tentative model on the mechanism of Ad5 DNA replication

The effect of chloramphenicol on synthesis of ΦX 174-specific proteins and detection of the cistron A protein

Synthesis of ΦX 174-specific proteins in Escherichia coli H 502 was examined on sodium dodecyl sulphate-acrylamide gels by coelectrophoresis of proteins from [3H]leucine-labelled infected cells and [14C]leucine-labelled reference cells, which had been infected with ultraviolet-light irradiated phage. Addition of 35 μg/ml of chloramphenicol during infection reduced the synthesis of ΦX-specific proteins of cistrons A, F, H and D to different degrees. The greatest reduction is found in synthesis of the cistron F product (coat protein), while synthesis of cistron D protein (single-strand DNA synthesis) is relatively resistant to the drug. Mutants in cistron A, which are unable to replicate RF, show a great reduction in the synthesis of all ΦX-specific proteins upon infection in the presence of chloramphenicol which made the identification of the cistron A protein unfeasible under these conditions. In the absence of chloramphenicol the cistron A product was identified as a protein with a molecular weight of 55 000. The cistron A protein is always strongly associated with the cell membrane

Comparison of the G4 and φX174 phage genomes by electron microscopy

The order of G4 Haemophilus influenzae Rd (HindII) DNA fragments was mapped by electron microscopy. G4 HindII DNA fragments were annealed to φX174 viral singles-tranded DNA circles and found to partially hybridize. Eight to eighteen percent of the φX174 DNA circles had a G4 DNA fragment attached, depending upon the fragment and the time of annealing. The G4 HindII restriction enzyme cleavage map was aligned with the φX174 restriction enzyme cleavage map by analyzing under the electron microscope partial heteroduplex molecules consisting of φX174 single-stranded viral DNA circles annealed with φX174 Arthrobacter luteus (AluI) DNA fragments as markers and G4 HindII DNA fragments. These results indicate that the gene order of the bacteriophages G4 and φX174 is the same. Despite the high frequency of φX174/G4 heteroduplex DNA molecules observed under the electron microscope, heterologous marker rescue experiments with G4 DNA fragments and φX174 mutant viral DNA strand circles were not successful, except in the case of a φX174 amber mutation in gene E (am3)