Ultraviolet reactivation of lambda phage: assay of infectivity of DNA molecules by spheroplast transfection

Prior irradiation of non-lysogenic bacteria by ultraviolet light leads to an increase in the viability of infecting irradiated λ phage (ultraviolet reactivation). Similarly, u.v. irradiation of wild type or uvrD bacteria lysogenic for λcIind^? increased the fraction of closed circular duplex phage DNA molecules formed after infection with u.v.-irradiated λ phage. The closed circular molecules isolated from the irradiated lysogens were shown to be free from u.v. damage by a spheroplast transfection assay. The increase of closed circular molecules is sufficient to explain the ultraviolet reactivation observed by the increase of viability of irradiated phage.In ultraviolet reactivation, damage must be erased on irradiated DNA molecules and the repair is independent of total replication of phage genomes, exchange of sister chromatids or recombination between phage genomes. Protein synthesis is necessary to increase the level of closed circular molecules of irradiated λ phage after irradiation of bacteria.     

Filter-supported preparation of lambda phage DNA

A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.     

Rapid isolation of phage lambda DNA

A modified procedure in two versions (micro, for 10 ml of phage lysate, and macro, for 200-500 ml) is described for preparing lambda phage DNA. The main advantage of the modified method is that it gives a possibility to isolate high-quality DNA from lambda phage lysates in 2-3 hrs. Only standard solutions (TE, NaCl, SDS, MgCl2, EDTA, RNAse A) were used throughout the whole protocol. Incubation with DNAse I and proteinase K was omitted and in microvariant concentration of the phage by PEG 6000 was excluded. Digestion by RNAse A was performed in solution with EDTA and SDS and leads to RNA degradation. The yields of DNA (0.5-2 micrograms per ml of L-broth) are similar to those obtained by other methods. DNA quality is better than in the samples of DNA prepared by other express-methods and practically the same as after CsCl centrifugation. DNA can be used for splitting by restriction enzymes, cloning and gene library construction.     

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.     

Isolation and structure of phage lambda head-mutant DNA

High molecular weight DNA accumulates in bacteria in which λ is multiplying but cannot complete the formation of new phage particles due to a defect in head assembly. Accumulated λ DNA has been isolated from induced mitomycin C-treated lysogens by means of a shift in buoyant density labels from heavy to light and fractionation by density-gradient sedimentation for completely light DNA. Head formation was blocked in these lysogens by amber mutations in genes D or E, which specify the two major head proteins. The purified DNA is at least 80% λ by DNA-DNA hybridization and some preparations are close to 100% λ by this test.     

Forces controlling the rate of DNA ejection from phage lambda

The goal of this work was to investigate how internal and external forces acting on DNA affect the rate of genome ejection from bacteriophage lambda after the ejection is triggered in vitro by a lambda receptor. The rate of ejection was measured with time-resolved static and dynamic light scattering, while varying such parameters as temperature and packaged DNA length, as well as adding DNA-binding proteins to the host solution. We found that temperature has a strong effect on the ejection rate, with an exponential increase of the initial ejection rate as a function of temperature. This can possibly be explained by the temperature-induced conformational changes in the tail pore-forming proteins where the "open" conformation dominates over "closed", at elevated temperatures. The DNA length also had an effect on initial ejection rate, with a nearly linear dependence comparing the three different genomes (37.7, 45.7 and 48.5 kb DNA), with faster ejection rate for longer genomes. Since the initial rate of ejection increases in an almost direct relationship with the length of the genome, the total time needed to eject DNA completely appeared to be nearly constant for all three DNA length phage mutants. The increased initial rate of ejection with increasing DNA length is due to the increased DNA bending and inter-strand repulsion forces for the longer DNA chains. Finally, we also show that addition of non-specific DNA-binding proteins (HU and DNase I) increases the rate of ejection by exerting additional "pulling" forces on the DNA that is being ejected.     

Interactions between phage lambda replication proteins, lambda DNA and minicell membrane

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.     

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.     

Tests of spool models for DNA packaging in phage lambda

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine.In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map.In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with ³²P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found.These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on λ DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982).A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into λ phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method. Sites of AMT crosslinking appear duplex; uncrosslinked regions appear as single-stranded loops. AMT is found to intercalate throughout the λ DNA. Patterns of reacted sites appear different from one DNA molecule to the next, and no consistent pattern can be found. More extensive intercalation occurs with the deletion mutant λb221 than with phage of wild-type DNA length, and free DNA shows much more reaction than the DNA inside either phage type. In order for intercalation to occur, the DNA helix must unwind and become further extended. This experiment shows that regions throughout the entire DNA molecule can unwind and be extended by intercalation, which is not confined to a single DNA segment or to segments in one half of the DNA molecule, as would be expected for the two spool models if only the DNA in contact with the capsid were accessible to the dye.     

Rapid restriction mapping of DNA cloned in lambda phage vectors

A protocol for the rapid restriction mapping of phage λ clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive λ DNA termini by hybridisation with [³²P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the “ladder” of partial digestion products.     

Involvement of DNA replication in phage lambda Red-mediated homologous recombination

Crosses between a non-replicating linear bacteriophage lambda chromosome and a replicating plasmid bearing a short cloned segment of lambda DNA were monitored by extracting DNA from infected cells, and analysing it via restriction endonuclease digestion and Southern blots. Recombinant formation resulting from the action of the Red homologous recombination system, observed directly in this way, was found to be fast, efficient, independent of the bacterial recA function and highly dependent upon replication of the target plasmid. These features of the experimental system faithfully model Red-mediated recombination in a lytically infected cell in which phage DNA replication is occurring. Neither of the previously established mechanisms by which the Red system can operate – strand annealing or strand invasion – accounts well for these findings. A third mechanism, replisome invasion, involving replication directly in the recombination mechanism, is invoked as an alternative.     

The fate of phage lambda DNA in lambda-infected minicells.

The fate of phage lambda DNA in lambda-infected Escherichia coli minicells harboring the plasmid ColE1, and in plasmid-free minicells, were studied. Binding of lambda DNA to the minicell membrane, and formation of the supercoiled covalently-closed circular structure has been demonstrated. Phage infection abolishes plasmid DNA synthesis. Only a very slight, non-replicative lambda DNA synthesis occurs, soon after infection. This synthesis is associated with fragments of lambda DNA arising during, or soon after its penetration.