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.     

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.     

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.     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Heterologous transfection with bacteriophage phiX174 DNA. Unusual heterogeneous products.

Hydroxylamine-resistant infectious materials (HARIM) synthesized in natural non-host and progeny phage low productive bacterial spheroplasts upon transfection with bacteriophage phiX174 DNA were found to be unusually heterogeneous in their forms. Using Pseudomonas aeruginosa as a source of HARIM, it was shown that they have the following unusual features. (1) Almost all of the HARIM are denser than normal single-stranded (SS)- and double-stranded replicative form (RF)-DNAs of phiX174 found usually in the phage-infected host cells. (2) A great part of these heavy HARIM (approximately 84%) contain a variable length of single-stranded RNA associated with their infectious elements. (3) For most of the HARIM (approximately 80% of total molecules as the infectious elements of the heavy HARIM), the infectious elements are phiX-RFI-DNA. The wide-spread system for phiX-HARIM synthesis was shown to be present in many gram-negative bacterial cells.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Subcellular localization of lethal lysis proteins of bacteriophages lambda and phiX174.

The gene products of the lethal lysis genes S and E of the bacteriophages lambda and phiX174, respectively, were shown to be associated primarily with inner membrane material by isopycnic sucrose gradient centrifugation of lysates of infected cells. A small amount of each polypeptide appeared to be in the outer membrane fraction.     

Heterologous transfection with bacteriophage phiX174 DNA: and improved system.

A highly efficient and much more reproducible system for the heterologous transfection of several kinds of Gram-negative bacterial spheroplasts with bacteriophage phiX174 DNA was established. By mild washing of the speroplasts, the efficiency of transfection of all non-host heterologous bacterial species tested increased one or more orders of magnitude in producing the progeny phages and/or the infectious intermediates. Using the improved heterologous transfection systems, it has become clearer that a strong suppression system operates on the processes of phiX174 progeny phage production and not on those of phiX174 dougle-stranded replicative form DNA synthesis in the heterologous bacterial cells. Similar stimulatory effects of this washing procedure were observed in the homologous transfection. With this improved assay system, even less than 100 molecules of phage phiX174 DNA can be detected and the number of molecules can be determined with accuracy.     

Excision repair of uracil during replication of phiX174 DNA in vitro.

Each of the stages in the replication of ØX174 DNA $\text{in vitro}$, e. g., conversion of circular single stranded parental DNA to the duplex replicative form (SS → RF), replication of the closed circular duplex form (RF → RF), and synthesis of circular single stranded progeny DNA (RF →SS), may be affected by a reduced level of dUTPase. Thus, in enzyme preparations from mutant strains defective in dUTPase ($\text{dut}$^?), the complementary strand synthesized in the SS → RF reaction is abnormally short (7–8S vs. 14S), and the extent of RF replication is decreased 10-fold. Preferential removal of dUTPase during fractionation of enzyme preparations from wild type ($\text{dut}$⁺) cells may produce comparable effects. In particular, the single stranded circular DNA synthesized in the RF → SS reaction by a set of highly purified enzymes is rapidly degraded upon incubation with the less pure enzymes required for its conversion to RF. All of these effects are plausibly accounted for by the incorporation into DNA of uracil from dUTP, possibly present as a contaminant in one or more components of the reaction, followed by excision of the uracil and phosphodiester bond cleavage at the resulting apyrimidinic site.