Lysogenization by bacteriophage lambda IV inhibition of phage DNA synthesis by the products of genes cII and cIII

In direct measurements of phage λ DNA synthesis, we have detected an inhibition caused by the cII and cIII gene products. This inhibition was more clearly observed when P amber phages were grown in a permissive host, presumably because of the limitation in DNA synthesis due to uncomplete suppression. The inhibition takes place in cells infected at high multiplicity, but not in cells infected at low multiplicity. To explain these findings, we propose a model in which the bacterial population is heterogeneous with respect to its ability to support phage DNA synthesis. An initial limitation caused by host factors would be amplified by the action of the cII and cIII products, at high multiplicity only, and the resulting inhibition would be essential in the « choicetowards lysogeny.     

Induction of phage lambda by transferred irradiated colI DNA

Summary Induction of prophage in a lysogenic recipient cell may be brought about by mating with ultraviolet irradiated donor cells carrying a transmissible plasmid such as colI (indirect induction). Doses of irradiation to the colI donor required to bring about indirect induction of lysogenic recipient cells reduce to an undetectable level the transfer of viable colI factors to non-lysogenic cells. It is shown by autoradiography that this irradiation does not significantly affect the frequency or amount of colI DNA transfer. Nalidixic acid prevents transfer of colI DNA and also prevents indirect induction. It is concluded that indirect induction results from the transfer of a UV-damaged colI factor. Transfer of the radiation-damaged colI factor to non-lysogenic cells leads to inhibition of division of these cells resulting in filament formation, but has little if any effect on cellular DNA replication.     

Mutagenic effects of gamma-rays and incorporated 8-3H-purines on extracellular lambda phage: influence of mutY and mutM host mutations

The lethal and mutagenic effects on phage lambdacI857 of 60Co gamma-rays and of decay of 3H incorporated into phage DNA both as 8-3H-deoxyadenosine and 8-3H-deoxyguanosine (using 8-3H-adenine as a labelled DNA precursor) were studied on four isogenic Escherichia coli strains: AB1157 M(+)Y(+) (wild type, mutM(+) mutY(+)), AB1157 M(-)Y(+) (mutM::kan mutY(+) mutant deficient in the formamidopyrimidine-DNA glycosylase MutM), AB1157 M(+)Y(-) (mutM(+) mutY mutant deficient in the A:G mismatch DNA glycosylase MutY), and AB1157 M(-)Y(-) (mutM::kan mutY double mutant deficient in both DNA glycosylases). The main products of transmutation component of 3H decay in position 8 of purine residues are 8-oxo-7, 8-dihydroadenine (8-oxoA) and 8-oxo-7,8-dihydroguanine (8-oxoG), the latter being responsible for the most part of the mutagenic effect. The lethal effects of both gamma-rays and tritium decay virtually did not depend on the repair phenotypes of the host strains used. Therefore, the MutM and MutY glycosylases are not involved in the repair of lethal DNA damages induced by ionizing radiation or by the transmutation component of 3H decay in purine residues of phage DNA. The efficiencies of mutagenic action of 3H-purines E(m) (frequencies of c-mutations per one 3H decay in phage genome) were 2.4-, 3.8- and 55-fold higher in the M(-)Y(+), M(+)Y(-) and M(-)Y(-) mutants, respectively, in comparison to the wild-type host. The mutagenic efficiencies E(m) for gamma-rays were nearly identical in the M(+)Y(+) and M(-)Y(+) hosts, but were increased 1.8- and 8.3-fold, respectively, in the M(+)Y(-) and M(-)Y(-) mutants. These data suggest that: (1) the MutY and MutM DNA glycosylases are important for prevention of mutations caused not only by spontaneous oxidation of guanine residues, but also by ionizing radiation or by decay of 3H incorporated into purine bases of DNA; (2) the MutY and MutM enzymes functionally cooperate in elimination of mutagenic damages induced by these agents.     

Induction of prophage lambda by gamma-rays, mitomycin C and tif; repressor cleavage studied by immunoprecipitation.

Summary The fate of the repressor on induction of E. coli lysogens has been studied after -irradiation, mitomycin C treatment or by temperature step-up of a tif-1 mutant. The repressor and a fragment of the repressor were selectively precipitated from labelled cell lysates by anti-repressor serum and subjected to gel electrophoresis and autoradiography. Induction of lysogens by all of these treatments leads to cleavage of the repressor. Repressor cleavage closely parallels the inactivation of its DNA binding properties and seems to be the general fate of the repressor on induction, irrespective of the nature of the inducing treatment.The effect of chloramphenicol on the cleavage has been studied in order to assess the requirement for inducible protein synthesis in this process. In contrast to results obtained by mitomycin C treatment, tif mediated cleavage of the repressor does not require the induction of new protein synthesis. Although inactivation of the DNA binding properties of the repressor after induction by -irradiation does not appear to be inhibited by chloramphenicol (West, Powell and Emmerson, 1975), cleavage of the repressor is substantially inhibited by this compound. The possible implications of these findings are discussed in relation to inducible repair functions and the mechanism of repressor inactivation.     

Mutagenic effect of o-methylhydroxylamine on the prophage and extracellular phage lambda

Induction of c-mutations in extracellular bacteriophage and prophage lambda cI857 ind-treated with 1 M O-methylhydroxylamine (OMHA) at 32 degrees and pH 5.6 has been studied. The frequency of c-mutations increases proportionally to the time of treatment of extracellular phage and does not depend on cellular recA+ or polA+ functions and on induction of SOS-repair system caused by UV-irradiation of host cells. Prophage is inactivated and mutagenized approximately 10-fold faster than extracellular phage immediately after treatment of lysogenic cells during prophage induction. Thus, prophage survival does not depend on repair functions of the host cells, and the frequency of c-mutations in recA and, especially, in polA lysogens is significantly lower, than in the wild-type cells.Delayed thermoinduction (90 min) of prophage causes significant enhancement of survival and decreases the frequency of c-mutations in all strains studied. Preliminary treatment of non-lysogens with OMHA does not increase the frequency of c-mutations in undamaged phage or in phage treated with OMHA in vitro.     

Isolation of lambda phage DNA by hydroxylapatite chromatography

A simple and rapid (1 day) method for preparation of lambda phage DNA was proposed. The method included two main steps: (a) growth and lysis of bacteria containing lambda phage and (b) purification of lambda phage DNA by hydroxylapatite chromatography. The phage DNA prepared by this method was intact and free of RNA, proteins, and bacterial DNA.     

Lethal and mutagenic action of incorporated 5-3H-cytosine on extracellular phage lambda

A study was made of the lethal and mutagenic effects on extracellular phage gamma of 5-3H-cytosine incorporated into DNA. The efficiencies of inactivation by incorporated 3H were equal for 5-3H-cytosine and [3H-methyl]-thymidine, but the yield of c-mutations for the former was 14 times higher. The lethal and mutagenic effects of incorporated 5-3H-cytosine did not depend on ung mutation of host cells which caused a deficiency in uracil-DNA-glycosylase. The mutagenic effect was not enhanced when SOS-repair system was induced by UV-radiation. The mutagenic effect of 5-3H-cytosine was associated with the modified mispairing bases but not with uracil residues.     

Protection of irradiated lambda phage by methylated proflavine

Summary The lethal action of U.V. on bacteriophage is diminished by pretreatment with methylated proflavine.The effective distance over which each dye molecule inhibits dimer formation is calculated to be 5 to 7.5 base pairs.     

Analysis of a temperature sensitive mutation in gene cII of bacteriophage lambda

Summary The mutation cIIts612 was found to map outside the immunity region of phage imm21 hybrid. As expected of a cII mutation, cIIts612 is unable to stimulate either cI repressor or Int synthesis during the establishment of lysogeny. These results indicate that part of the cII gene of is homologous to that of imm21 phage.     

Induction of mutation in phage T4 by extracellular treatment with methylene blue and visible light

Summary Photodynamic inactivation with methylene blue and light (MB+Li) of maximally sensitized phages T4 and T4rII nearly followed single-hit kinetics. Not so mutation induction, which in one phage (T4rII N₁₇) tested showed multi-hit kinetics. This difference and the fact that T4rII phages with a GC base pair at the site of the rII-mutation showed no enhanced backmutation when compared to rII phages with an AT base pair or to sign mutants suggests that there is no guanine specificity for MB+Li-induced mutation in phage T4.     

Induction of lambda prophage by furazolidone

A dose-dependent prophage induction by furazolidone exhibited a gradual rise to a maximum, corresponding to an exposure dose of 1.2 microgram/ml X h and a gradual fall thereafter. A 2-3-fold higher level of induction was achieved when the lysogens were treated with furazolidone in the presence of a metabolizing mixture. A maximum of about 70% efficiency of induction was achieved. Kinetics of prophage induction by any concentration of furazolidone exhibited a common pattern, viz., an initial rise for 15-20 min, then a plateau extending up to about 60 min and a faster rise thereafter. Higher concentrations of the drug (10 micrograms/ml) exhibited a toxic effect. Chloramphenicol at a concentration of 20 micrograms/ml inhibited the furazolidone-induced prophage induction, the plaque-forming units gradually decreasing from several minutes after the chloramphenicol treatment. The burst size of the lysogens was not significantly affected by treatment with 2 micrograms/ml of furazolidone up to a period of about 10 min, but thereafter, decreased faster with the duration of furazolidone treatment. The "latent period' of induction decreased linearly with the duration of furazolidone treatment.     

W-mutagenesis in the bisulfite-treated lambda phage

Survival of phage lambda cI857 inactivated by bisulfite (pH 5.6, 37 degrees C) is higher (the dose modification factor approx. 1.2) and frequency of bisulfite-induced c-mutations 2-4-fold lower on the lawn of the wild-type strain ung+, as compared to ung-1 mutant deficient in uracil-DNA glycosylase. Irradiation of host cells by a moderate UV dose inducing SOS repair system enhances the frequency of bisulfite-induced c-mutations 2-3-fold in the wild-type (ung+) host, but not in the ung-1 mutant. It is suggested that W-mutagenesis in bisulfite-treated lambda phage in the ung+ cells is due to SOS repair of apyrimidinic sites which are produced during excision of uracil residues, the products of cytosine deamination.     

The production of generalized transducing phage by bacteriophage lambda

Generalized tranduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage λ to assess some of the factors that affect that process. The results of those experiments indicate: (1) the production of generalized transducing particles by bacteriophage λ is inhibited by the phage λ exonuclease (Exo). Also inhibited by λ Exo is the production of λdocR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of λdocL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by λ Exo; (2) λ-generalized transducing particles are not detected in induced lysis-defective (S⁻) λ lysogens until about 60–90 min after prophage induction. Since wild-type λ would normally lyse cells by 60 min, the production of λ-generalized transducing particles depends on the phage being lysis-defective; (3) if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10–20-fold range. In contrast, if transducing lysates are prepared by the induction of a λ lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers ; (4) if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; (5) measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell. Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA.

These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by λ, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather     

Rapid construction of adenoviral vectors by lambda phage genetics

Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.     

Characterization of dnaA gene carried by lambda transducing phage

Summary Specialized transducing phages dnaA were obtained by inducing lysogens in which tna was integrated at the tnaA region of the Escherichia coli chromosome; the tnA region is located in the vicinity of the dnaA gene. The dnaA ^- deletion derivatives of dnaA were isolated from the lysate of dnaA grown on bacteria carrying a transposon Tn3.The structures of various transducing phages thus obtained were determined by heteroduplex DNA mapping. From these results, the transducing fragment of 13.8-kb-long was divided into nine domains. Upon infection of UV-irradiated cells with the phage, production of polypeptides of 49 kD and 42 kD was specifically associated with infections by the dnaA and recF transducing phages. Polypeptides of 49 kD and 42 kD appeared to be coded for by dnaA and recF genes, respectively. The dnaA gene was assigned to the region of 2.8-kb-long which extends by 2.4 kb in the counterclockwise direction on the E. coli genetic map and 0.4 kb in the opposite direction, as measured from the nearest HindIII site close to the tnaA gene. The recF gene was also discovered to lie very close to dnaA in the order of tnaA-dnaA-recF.Merogenotes heterozygous for the dnaA gene were constructed by introducing F100-12 carrying dnaA into the recipients with different mutations at or near dnaA. For combinations, F(dnaA ⁺)/dnaA46 and F(dna ⁺)/dna-83, dnaA ⁺ was trans-dominant, whereas the dnaA ⁺ was recessive for F(dnaA ⁺)/dna-5. For F(dnaA ⁺)/dna-167, the result of the transdominance test was affected by the growth media employed; dnaA ⁺ was dominant on a -broth plate, and dna-167 was dominant on an M9-minimal plate. Thus, transdominance of dnaA ⁺ in heterozygotes is affected by difference in mutations and growth media.     

Analysis of cosmids using linearization by phage lambda terminase

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage λ terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested' with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage λ (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.     

Dimeric intermediates of recombination in phage lambda

Biparental lambda phage DNA dimers formed by the Rec recombination system of E. coli were isolated in the absence of DNA replication and phage maturation. The RecA but not the RecB gene is required for dimer formation. Dimers are primarily circular but can also be branched circular or linear. In circular dimers the crossover points are distributed uniformly along the chromosome, even in the presence of the RecB-dependent Chi recombinational hotspots. Thus in the absence of DNA synthesis and maturation, the Rec system can act reciprocally both in the presence and absence of the RecB gene; this lack of RecB participation accounts for the observed lack of Chi activity.