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.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

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.     

Studies on radiation-sensitive mutants of E. coli. II. Breakage and repair of ultraviolet irradiated intracellular DNA of phage lambda

Summary It has been shown that linear DNA molecules of phage are converted to the twisted circular structure (species I) by covalent closure of the both strands at the cohesive ends after infection to the immune bacteria and that the twisted circular molecules are transformed to the circular form (species II) by a single-strand break in one of the strands of their DNA. This system offers a very sensitive method to study on the strand breaks or their repair. For characterization of the defects of ultraviolet sensitive strains, the structural changes of ultraviolet irradiated DNA in these strains were studied.Ultraviolet irradiation to phage greatly reduced the extent of conversion of the molecules to the species I in the uvrD mutant while the irradiation showed little effect on the conversion in the uvrA, B and C mutants. When infected bacteria carrying species I molecules were irradiated, the species I molecules in the uvrD mutant were disrupted while most of the molecules in the uvrA, B and C mutants kept the structure. These results indicate that in the irradiated DNA strand breaks are rarely introduced or, if introduced, repaired rapidly in the uvrA, B and C mutants and they are introduced in the uvrD mutant leading to the degradation of the DNA. These results provide a firm evidence that the defect of the uvrD mutant is different from other Her^- mutants and in the process of repair synthesis.Ultraviolet irradiation to the uvrD mutants promote the formation of the species I molecules from the infected irradiated -DNA.Such effect was not observed with the uvrA mutant. Since the uvrD mutant has UV reactivation capacity and the uvrA mutant has not, the above phenomenon is probably caused by UV reactivation and may provide a more direct method to study the mechanisms of UV reactivation than the plaque assay.Abbreviations used UV Ultraviolet light - UVr Ultraviolet light reactivation This work was aided in part by a research grant GM 08384 from the United States Public Health Service.     

Damaged-site independent mutagenesis of phage lambda produced by inducible error-prone repair

The existence of damaged-site independent mutagenesis is confirmed here by scoring the appearance of clear-plaque (c-) or virulent (vir) forward mutations on intact (non-irradiated) phage lambda grown on UV-irradiated E. coli K12 hosts. The mutation frequency was measured as a function of the incubation time between the occurrence of host DNA lesions and phage infection. The time course of mutagenesis of intact phage followed the induction pattern observed upon UV-reactivation of UV-damaged phage by Defais et al. (1976). Intact phage did not mutate in UV-irradiated hosts carrying the uvm-25 mutation known to prevent the occurrence of UV-reactivation. These findings suggest that damaged-site independent mutagenesis results from inducible error-prone repair. Clear-plaque mutations arising on intact phage were mostly found in phage bursts consisting of clear and turbid plaque formers whereas UV-damaged phage gave rise to mostly clear-plaque formers. Contrarily to damaged-site dependent mutagenesis, damaged-site independent mutagenesis can arise even at late times during the phage replication cycle. Our data indicate that about half of the phage mutations that arise upon UV-reactivation are damaged-site independent mutations. Replication of intact phage DNA in a host during induction of SOS functions provides a sensitive assay for the detection of damaged-site independent mutagenesis.     

Mutagenesis of ultraviolet-irradiated lambda phage by host cell irradiation: induction of Weigle mutagenesis is not an all-or-none process

Summary Ultraviolet mutagenesis of lambda phage to clear plaque formers is the same in the total phage population and in subpopulations of phage which have also mutated to gam ^- or at an amber codon. This is true for phage assayed in host cells in which Weigle mutagenesis has been either partially induced by low levels of ultraviolet irradiation, or fully induced by higher levels. If induction of Weigle mutagenesis were all-or-none, clear plaque formers in phage subpopulations selected for another mutation elsewhere would come mainly from induced cells; then the clear plaque mutation rate would always be that for fully induced host cells. Therefore, induction requires more than one lesion in host cell DNA.Although thymine starvation of cells induces synthesis of recA protein, it does not induce Weigle mutagenesis; in fact starvation inhibits induction of this process on subsequent ultraviolet irradiation of the cells.     

Substrate of a UV-induced repair system providing for W-reactivation of lambda phage]

Escherichia coli uvrA, polA and uvrD cells carrying non-UV-inducible prophage lambdac1857ind- were infected with 3H-thymidine labelled homoimmune phage lambdac1857, and the effect of UV-irradiation of super-infecting phage and lysogenic bacterial cells on the content of intracellular covalently-closed lambda DNA circles (cccDNA) and pyrimidine dimer content in lambda DNA are studied. UV-irradiation of host cells results in two-fold increase of relative content of cccDNA of UV-irradiated phage lambda in uvrD mutant, while there is no such an effect in uvrA and polA mutants. In UV-irradiated or intact uvrA lysogens cccDNA molecules, forming after the infection with UV-irradiated phage lambda, contain pyrimidine dimers, but in uvrD mutant cccDNA in free of dimers. The data indicate that the repair system induced by UV-irradiation of uvrA and polA cells acts exclusively on the DNA defects appearing after (or in the course) of phage genomes replication. UV-inducible repair system in uvrD mutant can operate also on some intermediates of abortive excision repair, possibly on long single straided excision gaps.     

Reactivation and mutagenesis of UV irradiated lambda phage in bacteria treated with platinum (II) compounds

Treatment of wild type Escherichia coli with cis -Pt(NH3)2Cl2 increased the survival and frequency of clear plaques formation of lambda phage damaged by UV radiation. The reactivation process was present in an uvrA mutant and abolished in a lexA host. Trans-Pt(NH3)2Cl2 and [Pt(dien) Cl]Cl (dien = 2HN-CH2-CH2NH-CH2-CH2-NH2) which, inhibited DNA synthesis less than the cis isomer or not at all, respectively, induced only a slight increase in survival of UV irradiated phage while mutagenesis was not affected. A relation exists between the reactivation of UV damaged phage in bacteria treated with these three compounds and their recently reported abilities to inhibit DNA synthesis and induce recA protein.     

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.     

Enlarged model of lambda phage ontogenesis

An enlarged threshold model of the Regulatory System of Development of λ -Phage (RSDP λ-2) is built. It includes 15 synthetic blocks of proteins and mRNAs and four blocks corresponding to the other ontogenetic processes: two-stage replication, integration and excision of phage genome, formation of oligomers of regulatory proteins, regulation of bacteriallysis. By way of computer simulation of the RSDP A-2 model the dynamics of concentrations of all main proteins, respective fractions of mRNAs and DNA are described in the lytic and lysogenic regimes of phage ontogenesis. Results obtained are in good agreement with available experimental data. The dependence of a portion (%) of lysogenic responses on the multiplicity $\text{k}$ of phage infection of bacterial culture, is built. This curve has a maximum point in accordance with the experimental data of P. Kourilsky (1973).     

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.     

Enlarged model of lambda phage ontogenesis

An enlarged threshold model of the Regulatory System of Development of λ-Phage (RSDP λ-2) is built. It includes 15 synthetic blocks of proteins and mRNAs and four blocks corresponding to the other ontogenetic processes: two-stage replication, integration and excision of phage genome, formation of oligomers of regulatory proteins, regulation of bacteriallysis. By way of computer simulation of the RSDP λ-2 model the dynamics of concentrations of all main proteins, respective fractions of mRNAs and DNA are described in the lytic and lysogenic regimes of phage ontogenesis. Results obtained are in good agreement with available experimental data. The dependence of a portion (%) of lysogenic responses on the multiplicity $\text{k}$ of phage infection of bacterial culture, is built. This curve has a maximum point in accordance with the experimental data of P. Kourilsky (1973).