Process of Infection with Bacteriophage φX174 XXXIII. Templates for the Synthesis of Single-Stranded Deoxyribonucleic Acid

The origin of the templates for the synthesis of {varphi}X174 progeny single-stranded deoxyribonucleic acid was studied by means of the mutagenic activity associated with the decay of incorporated 3H-labeled 5-cytosine. The results indicate that the single-strand synthesis occurs in an asymmetric semiconservative manner using as template the complementary strands of the pool of replicative from molecules accumulated during the eclipse period. These complementary strands are repeatedly used as templates, and there is no detectable preferential use of complementary strand templates made early in the eclipse versus those made late

Process of infection with bacteriophage phi X 174 XXXVIII. Replication of phi chi 174 replicative form in vivo

The replication of bacteriophage phi X 174 replicative-form DNA has been studied by structural analysis of pulse-labeled replicative-intermediate molecules. Such intermediates were identified by pulse-labeling with [13H]thymidine and separated into four major fractions (A, B, C, and D) in a propidium diiodide-cesium chloride buoyand density gradient. Sedimentation analysis of each of these fractions suggests the following features of phi X replicative-form DNA replication in vivo. (i) At the end of one cycle of replication, one daughter replicative form (RFII) contains a nascent plus (+) strand of the unit viral length, and the other daughter RFII contains small fragments of nascent minus (-) strand. (ii) Asymmetry is also associated with production of the first supercoiled RFI after addition of pulse label in that only the minus strand becomes radioactive. (iii) A supercoiled DNA (RFI') seems to occur in vivo. This DNA is observed at a position of greater density in a propidium diiodide-cesium chloride buoyant density gradient than normal RFI. (iv) A novel DNA component is observed, at a density greater than RFI, which releases, in alkali, a plus strand longer (1.5 to 1.7 times) than the unit viral length. These results are discussed in terms of the possible sequence of events in phi X 174 replicative-form replication in vivo

A cleavage map of bacteriophage Phi-X174 genome

Restriction endonucleases isolated from Hemophilus influenzae, Hemophilus parainfluenzae, and Hemophilus aegyptius were used to cleave phi-X174 replicative form DNA into three sets of specific DNA fragments. The order of these fragments in the phi-X replicative form molecule was determined by (1) analysis of partial digest products, (2) analysis of overlapping sets of fragments produced by two different restrictive enzymes. On the basis of these results, a detailed physical map of the phi-X174 genome has been constructed with respect to the cleavage sites of all three enzymes

Class of {varphi}X174 Mutants Relatively Deficient in Synthesis of Viral RNA

Nonpermissive cells infected with {varphi}X174 gene D amber mutants synthesized some sixfold less viral RNA than permissive cells. The decrease was unaffected by increasing the multiplicity of infection and was a consequence of an overall decrease in all viral RNA species. It is suggested that the gene D product may function in replicative form DNA unwinding to expose the template for transcription

The Process of Infection with Bacteriophage øX174 XIII. Evidence for an Essential Bacterial "Site"

The burst of a starved bacterium infected with several øX174 bacteriophage was usually found to contain genetic traits of only one of the possible parents; less often, two phage multiplied in the same host cell. Unstarved cells, in contrast, supported the growth of at least four parental phage types. The unproductive phage seemed to be able to undergo the intracellular transition from parental single-stranded deoxyribonucleic acid to the double-stranded "replicative form" (RF). These results are taken to mean that some bacterial factor required for a step between RF synthesis and maturation of progeny is limited in starved cells

The process of infection with bacteriophage phiX174, XXI. Replication and fate of the replicative form

Previous publications have described the formation of a double-stranded DNA intermediate(1) (replicative form, RF) during the replication of bacteriophage phiX174, the semiconservative replication at a bacterial "site"(2) of that RF containing the parental DNA strand,(3,4) and the persistence of the parental DNA strand at the site, accompanied by the release of the daughter RF.(5) The nascent RF molecules first appear as RFII,(6,7) and are rapidly converted to RFI. This paper considers the structure of the nascent RF molecules in more detail and inquires into the fate of the daughter RF molecules during the subsequent period of progeny single-strand DNA formation. A succeeding paper(8) will consider the process of formation of the progeny single-strands

Nature of {varphi}X174 Linear DNA from a DNA Ligase-Defective Host

Linear DNAs have been prepared from {varphi}X phage and from {varphi}X RF II (double-stranded circular form of {varphi}X DNA, formed during infection and nicked in one or both strands) molecules derived from infection at the restrictive temperature of Escherichia coli ts7, a host mutant with a temperature-sensitive DNA ligase activity. The linear DNA from these phages can be circularized by annealing with fragments of {varphi}X RF DNA produced by the Haemophilus influenzae restriction nuclease. The circularization experiment indicated that the site of breakage of the linear phage DNAs is not unique nor confined to a particular region of the genome. These linear DNAs were less than 0.1% as infective as circular phage DNA. The linear, positive strand of late RF II DNA, however, is uniquely nicked in the region of the {varphi}X genome corresponding to cistron A. Although a low level of infectivity is associated with the linear DNA derived from late RF II, this infectivity appears to be a result of the association of linear positive and linear negative strands during the infectivity assay

The process of infection with bacteriophage phiX174, XXII. Synthesis of progeny single-stranded DNA

In a previous article,(1) it was demonstrated that of the labeled thymidine incorporated into the replicative form (RF) of phiX-DNA during the period of RF replication (3-12 or 15 min after infection), approximately half was transferred to virus during the subsequent period of single-strand DNA synthesis, while half remained in RF molecules. In this paper experiments are presented which permit a definition of the label remaining in the RF and which clarify the role of the RF in the synthesis of the progeny single-stranded DNA

The process of infection with bacteriophage phiX174, XXX. Replication of double-stranded phiX DNA

Intermediates involved in the replication of double-stranded phiX174 RF DNA have been identified and partially characterized. Analysis of pulse-labeled RF DNA suggests that the synthesis of progeny RF molecules involves, in part, the addition of nucleotides to linear complementary strands on a circular parental strand as template, so as to produce intermediate DNA strands of greater than viral length. Electron microscopy reveals DNA rings with "tails" and "double rings," which could be the intermediate structures. A model is postulated for the replication process