Functional relationship between bacteriophages G4 and phi X174.

Mutants of bacteriophage G4 were isolated and characterized, and their mutations were mapped. They constitute six different genes, namely, A, B, E, F, G, and H. The functional relationship with bacteriophage phi X174 was determined by complementation experiments using amber mutants of phi X and amber mutants of G4. Bacteriophage phi X was able to use the products of G4 genes E, F, G, and H. In bacteriophage G4, however, only the phi X gene H product was functional.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

In vivo methylation of bacteriophage phi X174 DNA.

A mutant (designated mec(-)) has been isolated from Escherichia coli C which has lost DNA-cytosine methylase activity and the ability to protect phage lambda against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an E. coli K-12 mec(-) mutant; thus, the E. coli C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C mec(+) and C mec(-) cells infected with bacteriophage phiX174. phiX. mec(-) RFI is sensitive to in vitro cleavage by R.EcoRII and is cut twice to produce two fragments of almost equal size. In contrast, phiX.mec(+) RFI is relatively resistant to in vitro cleavage by R.EcoRII. R.BstI, which cleaves mec(+)/RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R. EcoRII. These results demonstrate that phiX.mec(+) RFI is methylated in vivo by the host mec(+) enzyme and that this methylation protects the DNA against cleavage by R.EcoRII. This is consistent with the known location of two mec(+)/ RII sequences (viz., [Formula: see text]) on the phiX174 map. Mature singlestranded virion DNA was isolated from phiX174 propagated in C mec(+) or C mec(-) in the presence of l-[methyl-(3)H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that phiX.mec(+) DNA had a 10-fold-higher ratio of [(3)H]5-methylcytosine to [(3)H]cytosine compared to phiX.mec(-). Since phiX.mec(+) contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of phiX174 is mediated by the host mec(+) enzyme only. These results are not consistent with the conclusions of previous reports that phiX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.     

Stability of bacteriophage phi X174-specific mRNA in vivo.

Different-size species of phi X174-specific mRNA's decayed exponentially, with half-lives ranging from 4.5 to 11 min.     

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.     

The mechanism of replication of phi X 174. XVIII. Gene A and A* proteins of phi X 174 bind tightly to phi X 174 replicative form DNA

Evidence is presented that the gene A and A * proteins of bacteriophage phi X 174 form covalent associations with the 5' ends of the DNA molecules when superhelical phi X replicative form DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5' ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative form DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAse digestion revealed that it was composed almost entirely polypeptides with electrophoretic mobilities similar to those of the gene A and A * proteins. We speculate that the gene * protein may be essential for normal progeny single-stranded DNA synthesis in vivo.     

Functional relationship between the J proteins of bacteriophages phi X174 and G4 during phage morphogenesis.

The functions of the small DNA-binding protein, gpJ, of bacteriophages phi X174 and G4 were examined by in vivo cross-complementation and sucrose gradient sedimentation. The morphogenetic roles of the two proteins may differ. The phi X174 J protein may be required for the formation or stabilization of the phi X174 prohead.     

Initiation and termination of the bacteriophage phi X174 rolling circle DNA replication in vivo: packaging of plasmid single-stranded DNA into bacteriophage phi X174 coats.

The bacteriophage phi X174 viral (+) origin when inserted in a plasmid can interact in vivo with the A protein produced by infecting phi X174 phages. A consequence of this interaction is packaging of single-stranded plasmid DNA into preformed phage coats resulting in infective particles (1). This property was used to study morphogenesis and to analyse the signals for initiation and termination of the rolling circle DNA replication in vivo. It is shown that the size of the DNA had a strong effect on the encapsidation by the phage coats and the infectivity of the particle. Termination was analysed by using plasmids with two phi X (+) origins either in the same orientation or in opposite orientation. Both origins were used with equal frequency. Initiation at one origin resulted in very efficient termination (greater than 96%) at the second origin in the case of two origins in the same orientation. When the two (+) origins have opposite orientations, no correct termination was observed. The second origin in the opposite strand effectively inhibits (greater than 98%) the normal DNA synthesis; i.e. the covalently bound A protein present in the replication fork interacts with the (+) origin sequence in the opposite strand.     

Radiological mapping of functional transcription units of bacteriophages phiX174 and S13. The function of proximal and distal promoters.

We have found that the nearest promoter is not always the primary promoter for making translatable message. The technique of ultraviolet mapping was used to determine the location of promoter sites for translated mRNA coded for by bacteriophages φX174 and S13. The method is based on the theory that the “target size” for u.v. inactivation of expression of a gene is proportional to the distance between the promoter and the 3′ end of the gene. This method has revealed an expected and some unexpected locations for the promoters responsible for gene expression. Ultraviolet-survival curves for expression of phage genes were interpreted in the following way. The contiguous genes D, F, G and H are expressed as a unit under the control of a promoter located near gene D. However, gene B (and probably the adjacent genes K and C) are controlled by a promoter distant from gene B, possibly in the region of gene H, rather than from a promoter located just before gene B. Likewise, gene A is controlled by a promoter distant from gene A.     

DNA heteroduplex analysis of the relation between bacteriophages phiX174 and S.13

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.     

Transfer of genome fragments in double infection of E. coli with T5 and phi X174 bacteriophages.

Double infection of Escherichia coli by two DNA phages (phi X174 and T5) resulted in encapsidation into T5 particles of T5 DNA containing linked fragments of phi X174 DNA. The phi X474 sequences in T5 "hybrid" DNA were detected by RNA-DNA hybridization.     

Comparison of the relative mutagenicities of O-alkylthymines site-specifically incorporated into phi X174 DNA

The relative mutagenicities of O-alkylthymine-DNA adducts were analyzed in vivo by site-specific mutagenesis. Purified DNA polymerases were used to incorporate O4-methyl (Me)-, O4-ethyl (Et)-, O4-isopropyl (iPr)-, or O2-Me-dTTP onto the 3' terminus of a synthetic oligonucleotide (15-mer) hybridized to phi X174 am3 DNA. The product oligonucleotides were further extended in the presence of unmodified dNTPs to yield 21-mers containing single O-alkylthymine adducts opposite the adenine residue of the bacteriophage amber codon. Polyacrylamide gel electrophoresis and nearest-neighbor analyses confirmed the identities and nucleotide positions of the adducts. Transfection and replication of the site-specifically alkylated DNAs in ada- Escherichia coli (defective in the alkyltransferase capable of repairing O4-alkylthymine-DNA adducts) yielded mutant progeny phage with reversion frequencies of: O4-Me-dThd (19.5 X 10(-6) ) greater than O4-Et-dThd (7.5 X 10(-6) ) greater than O4-iPr-dThd (3.0 X 10(-6) ) greater than or equal to O2-Me-dThd (1.0 X 10(-6) ) approximately equal to dThd (2.0 X 10(-6) ). None of the adducts produced mutations above background following replication in ada+ E. coli. DNA sequence analyses of 40 independently isolated mutant phage derived from the O4-Me- or O4-Et-dThd-containing DNAs showed that all mutants contained guanine residues opposite the original site of the alkylthymines. These data are consistent with a mechanism of mutagenesis involving the formation of O4-alkyl-T.G base pairs during DNA replication in E. coli and suggest that the formation of A.T----G.C transition mutations is characteristic of mutagenesis by O4-Me- and O4-Et-dThds in vivo.     

Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.