Phage S13 Research Articles

Consequences of Transmutation of 32P or 33P Atoms Incorporated in DNA of Phages S13 or T2: Local Damages and Lethal Effect

Consequences of Transmutation of 32P or 33P Atoms Incorporated in DNA of Phages S13 or T2: Local Damages and Lethal Effect

Consequences of Transmutation of 32P or 33P Atoms Incorporated in DNA of Phages S13 or T2: Local Damages and Lethal Effect

When incorporated into cellular DNA, the transmutations of 32P or 33P provoke a lethal effect as well as single and double strand breaks. In the case of phage S13, characterised by circular single stranded DNA, a single strand break is always a lethal event. Moreover, in the case of phage T2, characterised by a circular double stranded DNA, a double strand break is always a lethal event. However, for 33P a DNA break (single for S13 and double for T2) is never the only lethal event. For 32P the single strand break is indeed the only lethal event for S13, but the double strand break is not the only lethal event for T2.

UV-induced mutagenesis of phage S13 can occur in the absence of the RecA and UmuC proteins of Escherichia coli.

The UV-induced mutagenesis of phage S13 that accompanies Weigle repair is known to require the products of the recA and umuDC genes, as does the UV-induced mutagenesis of the Escherichia coli chromosome. I found that UV-induced mutagenesis of phage S13 occurred in the absence of both the RecA and UmuC functions when the irradiated phage was photoreactivated. Furthermore, UV-induced phage mutations were produced in a recA- umuC- cell even without photoreactivation and in the absence of any other known UV repair mechanism, at a frequency 29% of that found after photoreactivation and 7% of that found after Weigle repair, implying that DNA synthesis can proceed past a dimer at an unexpectedly high frequency even when unaided by the UmuC-RecA SOS repair functions. The unaided DNA synthesis appears capable of producing mutations in the vicinity of a pyrimidine dimer; by aiding synthesis past a dimer, a repair mechanism may disclose a mutation without having any active role in producing it.

Suppression of polar effects of nonsense mutations by ultraviolet irradiation

Nonsense mutations in capsid genes F and G of phages S13 and phi X174 decreased the expression of genes downstream. These polar effects were suppressed by ultraviolet irradiation of the host before infection. Activities of the downstream genes were restored to between 30 and 95% of their normal levels, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral proteins. The effect of ultraviolet irradiation may be to suppress rhodependent termination of transcription. The polarity of one mutation was poorly suppressed by ultraviolet irradiation.

Potential for variability through multiple gene products of bacteriophage phiX174.

The small single-stranded DNA phages phiX174 and S13 produce multiple products of certain phage genes, as observed by electrophoresis on SDS-polyacrylamide slab gels. Two A protein products, two A products and four G products are observed. The multiple gene products may arise from multiple sites for initiation or termination of translation, or by protein modification. Some of the variant products may provide a substitute for heterozygosity without a concomitant increase in the size of the genome.

Gene A protein of bacteriophage S13 is required for singel-stranded DNA synthesis.

The product of gene A of the small icosahedral DNA phage S13 has been found to be needed for single-stranded DNA synthesis in vivo in addition to its previously known role in progeny replicative-form DNA synthesis.

EFFECT OF dna MUTATIONS ON REPLICATION OF S13, φR, AND φKh-1

Host functions involved in multiplication of single-stranded DNA phages S13, φR, and φKh-1 were examined using various DNA replication mutants of E. coli. When the phage-resistant bacteria were to be tested, glycine spheroplasts or Ca2+-treated cells were transfected by the phage DNA. Replication of S13 and φR required host dnaB, dnaC(D), dnaE, dnaG, and dnaZ gene products. On the other hand, growth of φKh-1 was dependent on cellular dnaE, dnaF(nrdA), dnaG, and dnaZ functions, but not on dnaB and dnaC(D) activities. Multiplication of these phages absolutely relied on host rep function. Based on these host factor requirements, relationship among single-stranded DNA phages was discussed. In addition, host range and antigenic property of φKh-1 were compared with those of the related phages.

Bacterial rep- mutations that block development of small DNA bacteriophages late in infection.

Several related mutants of Escherichia coli C have been isolated that block the growth of the small icosahedral DNA phages phiX174 and S13 late in infection. Phage G6 is also blocked, at a stage not yet known. Growth of the filamentous phage M13, though not blocked, is affected in these strains. These host mutations co-transduce with ilv at high frequency, as do rep- mutations. However, the new mutants, designated groL-, differ from previously studied rep- mutants in that they permit synthesis of progeny replicative-form DNA. The groL- mutants are blocked in synthesis of stable single-stranded DNA of phiX174 and related phages. They are gro+ for P2. Evidence that groL- mutations and rep- mutations are in the same gene is presented. Spontaneous mutants (ogr) of phiX174, S13, and the G phages can grow on groL- strains. The ogr mutations are located in the phage's major capsid gene, F, as determined by complementation tests. There are numerous sites for mutation to ogr. Some mutations in genes A and F interfere with the ogr property when combined with an ogr mutation on the same genome. The ogr mutations are cis acting in a groL- cell; i.e., an ogr mutant gives very poor rescue of a non-ogr mutant. The wild-type form of each G phage appears to be naturally in the ogr mutant state for one or more groL- strains. It is suggested that a complex between F and rep proteins is involved in phage maturation. The A protein appears to interact with this complex.

Recognition properties of the beta subunit of Escherichia coli ribonucleic acid polymerase.

Changes in the phage protein patterns obtained by gel electrophoresis of extracts from phage S13 and phiX174 infection of rifampin-resistant hosts suggest that the beta subunit of ribonucleic acid polymerase of Escherichia coli has a function in the recognition of promoter or terminator sites or both. The altered protein patterns also provide information on the location of some ribonucleic acid polymerase recognition signals in S13 deoxyribonucleic acid. There is a promoter site before gene A, which lies either in gene H or between H and A. There is evidence for a promotor between genes C and D or in gene C. There is either a terminator or a promoter somewhere between the end of gene D and the beginning of gene F.

Reinitiation mutants of gene B of bacteriophage S13 that mimic gene A mutants in blocking replicative form DNA synthesis

Several mutations in gene B of phage S13 appear to shorten the B protein by elimination of an N-terminal fragment, without destroying the B protein function. The shortened B protein resulting from each of these mutations can block the unique DNA-nicking properties of the S13 gene A protein. Because of the block in gene A function, normal gene B protein may have a function in phage DNA synthesis in addition to its known role in catalyzing capsid assembly. From gel electrophoresis the mutant B protein is estimated to be shorter than the normal S13 B protein by 1720 ± 70 daltons and is therefore believed to be an internal reinitiation fragment. The reinitiated fragments are functional and are made in about twice the amount of the normal B protein. The phage mutants which yield the reinitiation fragments are double mutants, each phage containing the same gene B nonsense mutation and each appearing to contain a different compensating gene B mutation. Various data support the assumption that the compensating mutations are frame-shifts, including the fact that suppression does not restore the normal-sized B protein. The reinitiation is assumed to occur at a pre-existing out-of-phase initiator codon, near the nonsense triplet; the correct reading frame would then be restored by each of the several different compensating mutations. The position of the normal S13 B protein in the gel electrophoresis pattern has been located both by elimination and shifting of the B peak, using appropriate amber mutants. The molecular weight of the S13 B protein is about 17,200, and is 2100 daltons less than the B protein of phage φX174; the S13 B protein can nevertheless substitute for the φX 174 B protein. Thus substantial portions of the B protein can be deleted without destroying its function.

The gene H spike protein of bacteriophages ∅X174 and S13: II. Relation to synthesis of the parental replicative form

Several molecules of the ∅X174 gene H protein, a component of the phage spike, become firmly associated with the host cell upon infection and resist protease treatment of the cell. The gene H protein was recovered in association with the phage DNA converted to the duplex replicative form and was bound to several different regions of the DNA. Similar results were obtained with the gene H protein of the related phage S13. Cells infected with a temperature-sensitive mutant of S13 in gene H failed to make parental replicative form at the restrictive temperature, although phage binding and eclipse occurred at normal levels. Since phage penetration is coupled to replication and the gene H protein is recovered tightly bound to the parental replicative form, this protein is presumed to participate in the first stage of DNA synthesis. These and related findings form the basis for a general proposal of a “pilot protein” as a minor component of the virus with multiple functions in adsorption, penetration, and early intracellular stages of viral chromosome expression.

The gene H spike protein of bacteriophages ∅X174 and S13: I. Functions in phage-receptor recognition and in transfection

The gene H protein of bacteriophage ∅X174 is a coat component located in the spikes at the vertices of the polyhedron. In extracts of ∅X174-infected cells, the gene H protein binds to exogenous single-stranded phage DNA enhancing its infectivity in transfection of spheroplasts. This stimulation was not observed with extracts deficient in gene H protein. Transfection by the gene H protein-DNA complex was specifically inhibited by antibody to gene H protein. Lipopolysaccharide derived from ∅X174-sensitive cells blocked transfection by the DNA complex with gene H protein but not by the DNA alone, suggesting that the gene H protein functions as the phage adsorption protein by recognizing the phage lipopolysaccharide receptor. Gene H protein from ∅X174 stimulated the transfection by single-stranded DNA from the closely related phage S13; this stimulation was blocked by antibody to gene H protein and by the ∅X174 lipopolysaccharide receptor. These actions in phage-receptor recognition and in transfection suggest that the gene H protein has multiple functions and behaves as a “pilot protein” in guiding the phage and its DNA through several stages of the infection cycle.

Membrane binding of phage S13 messenger RNA

About 65–70% of the S13-specific messenger RNA synthesized during a short pulse of [ 3H]uridine is found in association with host membrane components, which are defined as materials that form complexes with Sarkosyl-magnesium crystals and band (M-band) in a sucrose density gradient. Approximately 0.18 of this membrane-bound messenger can be released from the membrane by DNase treatment, and this small fraction may be from nascent mRNA chains of various molecular sizes. The membrane-bound phage-specific mRNA is found in polysomes. It is concluded that the cellular membrane contains a complex of structural sites for transcription and translation as well as for replication.

Two proteins of gene A of psiX174.

GENE A of the single strand DNA phages ϕX174 and S13 is the only phage gene required for the replication of the double stranded replicative form (RF) after the complementary strand of DNA is synthesized upon the infecting single strand DNA1–3. It is a cis acting gene, double infection with gene A mutants and mutants in the other phage genes fail to rescue mutant A phage4, suggesting that gene A products can only function upon the genome from which they are synthesized. Gene A is necessary for the detection of a cis related viral strand specific discontinuity in the parental RF5. The gene A region of the ϕX174 genome has been suggested to contain the origin of DNA replication6 and genetic evidence has been presented to support the model that gene A produces two proteins, A and A′ (molecular weight 35,000 and 60–65,000, respectively)7.

Difference between functional and structural integrity of messenger RNA.

Messenger RNA molecules that are structurally stable, as measured by their ability to hybridize to DNA, may nevertheless be considerably less stable in retaining their ability to function in protein synthesis. The structure of the majority of the mRNA of phage S13 decays with a half-life of 10.6 +/- 0.5 min. In contrast, much of the function of the mRNA that is involved in synthesis of a capsid protein (product of the F gene) decays rapidly with a half-life of 1.4 +/- 0.8 min; a residual amount of function decays with a half-life of 14.0 +/- 4.0 min. The measurements were made in the presence of rifampicin, which was used to prevent the formation of new mRNA. A proposed model for the functional decay is based on the polycistronic nature of the mRNA. Degradation of the mRNA would proceed in two steps: the first step would be a fast attack at a region near the 5'-terminus of each molecule that would eliminate the function of the proximal message; the second step would be a slow attack on the remaining messenger molecule precipitating a subsequent rapid degradation of the physical structure.

Mechanism of transcription of bacteriophage S13. II. Inhibition of phage-specific transcription by nalidixic acid.

Abstract Nalidixic acid has been thought to be a specific inhibitor of DNA synthesis with no effect on RNA and protein synthesis. We find, however, that, in the presence of nalidixic acid, messenger RNA synthesized by phage S13 during a short pulse with [ 3 H]uridine is reduced to 20% of its normal level. The inhibition of DNA synthesis and the resultant reduction in the number of DNA templates available for transcription is not the cause of the inhibition of messenger synthesis, because phage mutants already blocked in DNA replication show the same degree of sensitivity to the drug as the wild type. Nalidixic acid does not enhance degradation of the phage mRNA. By determining the sizes of phage-specific mRNA, we infer that nalidixic acid does not prematurely terminate nascent mRNA chains nor is it specific for certain promoter sites. A spontaneous mutant of Escherichia coli C resistant to 100 μg of the drug per ml shows normal DNA synthesis and phage transcription suggesting that a common step affects both processes. To explain the inhibitory action of nalidixic acid on phage transcription it is proposed that a metabolic product of the drug may behave like an intercalating agent.

A promoter site and polarity gradients in phage S13

Acrylamide gel analysis shows that the polar effect of nonsense mutations in gene F of phage S13 extends to adjacent genes G and H, but not to genes A and B, which follow H in the direction of translation. Therefore, a promoter site must be located before gene A, but not before genes G or H. Degree of polarity was compared with genetic map position for genes F and G. Gene F generates a polarity gradient which is strongly dependent on distance to the end of the gene; in gene G distance to the end of the gene appears to have no effect on degree of polarity.

Discussion de la communication de J. C. Dreyfus

In vitro mutation of phage S13 (and ΦX174), which contain single-stranded DNA, was studied with hyclroxylamine, with ethyl methanesulfonate, and with nitrous acid. Both forward and reverse mutations could be studied selectively. It is argued that the reverse mutations studied are genuine reversions to the original genetic types. From the responses to the three mutagens, the following conclusions were obtained.(1)Hydroxylamine only mutates cytosine to thymine, and is the most specific of the three mutagens in its mutagenic action.(2)Ethyl methanesulfonate and nitrous acid mutate all four bases, but to varying degrees. The mutation of guanine to adenine by nitrous acid was established by extensive tests.(3)For each mutation studied, the altered base could be determined from the response to the three mutagens.(4)Mutation tests showed that only one of the two complementary DNA strands exists in the free phage population.

Identification of the acrylamide gel protein peak for gene C of phages S13 and ∅X174

The functional stabilities of the messages for seven of the nine genes of bacteriophage S13 were determined by measuring the amount of gene-specific translation at different times after the addition of rifampicin to block further initiation of transcription. Although the decay of mRNA function for each gene product tested was exponential, the functional half-lives of the mRNA were not the same for all phage genes, but fell into at least five groups: 6·2 minutes for genes B and C; 4·4 minutes for D;3·8 minutes for F, G and H; 2·5 minutes for A* and 0·65 minutes for A. The overall mass-decay of bulk phage mRNA as determined by hybridization is much slower, with a half-life of 10·6 minutes. The inactivating event for A messages frequently occurs before the RNA polymerase has reached the 3′-end of the gene.The capsid genes F, G and H are contiguous and exhibit polarity, suggesting that the expression of these three genes is regulated in part by a common mRNA functional-decay rate. For the contiguous genes B and C, the similarity of their inactivation rates suggests that they too might show polarity. Amber mutants in B indeed cause at least an 80% reduction in the amount of C protein synthesis. The two gene clusters, B-C and F-G-H, allow recognition of a new type of mRNA unit, characterized by the similar functional stabilities of the component genes. The existence of units of functional stability suggests possible mechanisms for mRNA degradation and the regulation of protein synthesis.

Roles of parental and progeny DNA in 2 mechanisms of phage S13 recombination.

THERE are at least two mechanisms for genetic recombination of phage S13–primary and secondary. They are distinguished by the amount of phage recombination observed in recombination-deficient (recA) bacterial hosts. An S13 cross performed in either of the recA hosts, Escherichia coli JC15531 or AB24632, yields a recombination frequency that is greatly reduced from that found in a rec+ host3, 4; this suggests that the recA gene is required for the primary mechanism of S13 recombination. But even in a recA host the phage undergoes a small residual amount of recombination3 which has been attributed to a minor, secondary mechanism. Apparently the secondary mechanism functions in a recA cell and is only revealed when the primary mechanism is eliminated.