Genetic Map of Bacteriophage phiX174

Bacteriophage X174 temperature-sensitive and nonsense mutations in eight cistrons were mapped by using two-, three-, and four-factor genetic crosses. The genetic map is circular with a total length of 24 × 10⁻⁴wt recombinants per progeny phage. The cistron order is D-E-F-G-H-A-B-C. High negative interference is seen, consistent with a small closed circular deoxyribonucleic acid molecule as a genome.     

Bacteriophage phiX174 growth in an Escherichia coli dnaIts mutant, KS810.

A bacteriophage phiX174-sensitive Escherichia coli dnaIts mutant, KS810, was constructed and growth of phiX174 in the cells was investigated. phiX174 and phiX174am3trD could grow normally at 43 degrees C as well as 27 degrees C, therefore we conclude that the growth of bacteriophage phiX174 is not dependent upon the host dnaI gene product.     

Non-Continuous Translation of Coat Protein and Ribonucleic Acid Polymerase Cistrons in MS2 Bacteriophage Ribonucleic Acid

In an MS2 phage ribonucleic acid (RNA)-directed in vitro protein-synthesizing system, the coat protein cistron and the adjacent RNA polymerase cistron are translated non-continuously. The ribosomes which have completed the synthesis of coat protein dissociate from the MS2 RNA and do not read through the intercistronic gap. Translation of the adjacent RNA polymerase cistron requires ribosomes other than those translating the coat protein cistron.     

Bacteriophage phiX174 single-stranded viral DNA synthesis in temperature-sensitive dnaB and dna C mutants of Escherichia coli.

We asked if phiX174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chlorampheicol that had been added at various times after infection to specifically stop this stage of phiX174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of phiX174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of phiX174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of phiX174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, phiX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of phiX174 DNA synthesis are not necessarily mutually exclusive.     

Process of infection with bacteriophage phiX174. XXXVII. RNA metabolism in phiX174-infected cells.

The RNA produced in vivo from bacteriophage phiX174 DNA has been analyzed by polyacrylamide-agarose gel electrophoresis and sedimentation in dimethyl sulfoxide gradients, and the results of Hayashi and Hayashi (1970) have been confirmed and extended. An efficient procedure for recovery of RNA from gels, followed by a hybridization assay, has indicated the presence in infected cells of 18 distinct RNA species with sizes up to and greater than the unit (viral) length. The sizes of phiX mRNA's were similar irrespective of whether material was analyzed on gels or in dimethyl sulfoxide gradients. When virus-induced RNA was detected by a double-label method, seven additional low-molecular weight species were observed on gels and the resolution of dimethyl sulfoxide gradients was enhanced. The present results lend support to aspects of the model of Hayashi and Hayashi (1970) for the generation of these discrete mRNA species; an alternative model is also discussed.     

The mechanism of replication of phiX174 single-stranded DNA. X. Distribution of the gaps in nascent phiX174 replicative form DNA

When the enzyme rhodanese (EC 2. 8. 1. 1) is digested with trypsin under controlled conditions, the parent protein is converted from a polypeptide of molecular weight 32,600 to a polypeptide of molecular weight 28,800. This proteolytic conversion occurs with no loss of rhodanese activity. In fact, preliminary results indicate that the polypeptide produced by proteolysis has higher sulfur transferase activity than the native rhodanese.     

Dimeric circular duplex DNA of bacteriophage phiX174 and recombination

Summary Bacteriophage X174 replicative from DNA (RF DNA) was formed in the presence of chloramphenicol at a concentration of 40 g per ml and isolated at 12 and at 55 min. after infection. The component I RF DNA (double stranded covalently closed and twisted form) was separated and divided into a monomer and multimer (dimer) fraction.The frequency of recombinants found after phage formation in the chloramphenicol treated cells and that found after spheroplast infection with the monomer molecules both increase with the time of RF formation. However, the frequency of recombinant molecules among the dimers remained constant. This finding is explained by the hypothesis that two separate mechanisms act in X174 recombination, one of which is restricted to the formation of dimers.Irradiation with UV of phage prior to infection showed that the frequency of recombinants in monomers increased, as the recombination frequency of phage after (a single) growth (step) did, but that neither the frequency of recombinant molecules in dimers is raised, nor the frequency of dimers. Using a recombination negative host the frequency of recombinant dimer molecules was three to fourfold decreased, whereas the frequency of dimers was only slightly lower (relative to the normal host). These results support the hypothesis mentioned above and moreover lend support to the view that the greater part of the dimers is not formed by recombination events.     

Origin and direction phiX174 double- and single-stranded DNA synthesis

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.     

Genetic Expression in Heterozygous Replicative Form Molecules of phiX174

Heterozygous replicative form molecules of bacteriophage X174 deoxyribonucleic acid (DNA) have been constructed in vitro. These are composed of viral strands extracted from purified preparations of phage bearing ts mutations and complementary strands of either half length or full length synthesized with purified DNA polymerase, in vitro, on DNA from am3 phage. In infections with such heterozygous DNA, involving mutations in each of four different cistrons, phage with the genotype of the complementary strand comprised 1 to 20% of the total phage produced by a spheroplast population. From single-burst analysis of the progeny from DNA heterozygous in one cistron (B), it appears that those phage with the genotype of the complementary strand arise as major components in a small proportion of the infected cells rather than comprising a minor component in most cells. The implications of such a pattern of expression are discussed with respect to mechanisms of phage DNA synthesis.     

Identification of lysis protein E of bacteriophage phiX174.

The product of gene E, the lysis gene of phiX174, has been identified as a distinct band in a sodium dodecyl sulfate-gel electropherogram. The position of the band is consistent with the molecular weight of 10,589 calculated from the nucleotide sequence of the gene. The band is eliminated by a nonsense mutation in gene E. It is estimated that roughly 100 to 300 molecules of E protein are made in an infected cell; this appears to be less than one-tenth the amount of protein made by gene D, in which gene E is wholly contained.