Densities of transducing lambda bacteriophages.
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Bacteriophage lambda↗
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Identification of wild-type or mutant alleles of bacterial genes cloned on a bacteriophage lambda vector: isolation of uvrC(am) and other mutants.
We have identified lambda transducing bacteriophages carrying deoxyribonucleic acid repair or recombination genes of Escherichia coli K-12 by their ability to infect and express their bacterial genes in mutant cells in an agar overlay. This technique has been used to recognize transducing phages carrying uvrC+, ssb+, and other genes and to isolate phages carrying mutant alleles unable to complement ssb or uvrC cells. Several uvrC mutations were obtained which were suppressor sensitive.
Bacteriophage lambda↗
A genetic analysis of primary products of bacteriophage lambda recombination.
Primary products of bacteriophage lambda recombination that display heterozygosity as a consequence of the presence of regions of heteroduplex DNA are rare in standard lambda crosses. Phage manifesting heterozygosity at a given allele are evident when recombinants, emerging from a cross, are selected for an exchange in a neighboring interval. We show that the abundance of such heterozygotes can be increased 10- to 20-fold by selection on an E. coli indicator that is defective in methyl-directed mismatch repair (mutL). Thus, the activity of the methyl-directed mismatch repair system is, at least in part, responsible for the low frequency of detectably heterozygous phage emerging from a standard cross. In a mutL indicator, many primary products of recombination are replicated without the intervention of mismatch repair.--The products of a six-factor phage cross have been plated on a mutL indicator allowing visual detection of those phage products heterozygous for one of the allelic pairs, cI. By genetic analysis, we show that the heteroduplex regions of these primary products of recombination are on the average about 4 kb in length and can include as much as half of the lambda genome.
Bacteriophage lambda↗
Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5.
Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied. Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology. Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments. The data obtained led to the conclusion of site-specific (homologous) character of abnormal excision upon formation of lambda transducing bacteriophages. Possible mechanisms of the excision are discussed.
Bacteriophage lambda↗
Initiation of DNA synthesis on single-stranded DNA templates in vitro promoted by the bacteriophage lambda O and P replication proteins.
The bacteriophage lambda O and P protein replication initiators, in conjunction with six purified Escherichia coli replication proteins, replicate the single-stranded chromosomes of phages M13 and phi X174 to a duplex form. Several discrete steps are involved in this DNA synthesis reaction. In an ATP-dependent step that precedes priming, the lambda O and P proteins interact with the Escherichia coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto DNA coated with single-stranded DNA binding protein. This creates a stable prepriming intermediate, isolable by gel filtration, that is rapidly primed and replicated upon the addition of primase and DNA polymerase III holoenzyme. Each of the eight proteins required for this nonspecific single strand replication reaction also have physiological roles in the replication of the bacteriophage lambda chromosome in vivo. We propose a scheme for the lambda O and P protein-dependent initiation of DNA synthesis that may be relevant to strand initiation events occurring during lambda DNA replication.
Bacteriophage lambda↗
On the internal structure of bacteriophage lambda.
The structure of bacteriophage lambda has been studied by electron microscopy of negatively stained particles. The phage particles will eject their DNA if they are heated or dialyzed against a chelating agent. The ghost particles, so formed, have a channel running down their tails. Since the channel is not visible in normal particles, the channel may be filled with part of the DNA molecule. Up to 30% of the ghosts contain round objects about half the internal diameter of the head. The round objects, called "cores," have the same buoyant density as the coat protein. The core may be a protein spool about which the phage DNA is wound.
Bacteriophages↗
Bacteriophage lambda: the untold story.
The study of bacteriophage lambda has provided key insights into fundamental biological processes. This review recalls some highlights in the history of lambda research, and relates how simple (but elegant) experiments yielded major scientific breakthroughs. What we know about recombination, gene regulation, and protein folding, for example, derives in large part from bacteriophage lambda genetics. Lambda not only represents a model system of scientific logic in a technology-driven age, but continues to reveal new principles of molecular biology.
Bacteriolysis↗
Genetic structure of the replication origin of bacteriophage lambda.
A fragment of bacteriophage lambda DNA produced by the restriction endonuclease Eco RI and extending from the immunity region to a point inside gene O is found to have a fully functional origin of replication. Seven ori- mutations of lambda cluster in a small region just to the left of the Eco RI cleavage site which defines the right end of this fragment. These mutations lie within gene O.
Chromosome Mapping↗
[C1 and cro repressors of lambda phages. Isolation of strains of bacteriophage lambda imm434 superproducing repressor cro].
A collection of recombinant plasmids directing the increased synthesis of the cro-repressor of lambda imm434 bacteriophage under control of the lacZ promoter of Escherichia coli has been constructed. About 87 000 molecules of cro-repressor monomer per cell, e. g. 0,7% of total cell protein are synthesized by the overproducers. Strain bearing one of such plasmids--pBS71-1. Several recombinant plasmids in which the cro-gene is placed under the control of early genes promoter of lambda phage (pl, pr, prm) have been also prepared. Plasmid pIL206, constructed on the basis of pIL203 vector, was used as a source of lambda-promoters.
Bacteriophage lambda↗
An additional function for bacteriophage lambda rex: the rexB product prevents degradation of the lambda O protein.
The rex operon of bacteriophage lambda excludes the development of several unrelated bacteriophages. Here we present an additional lambda rexB function: it prevents degradation of the short-lived protein lambda O known to be involved in lambda DNA replication. We have shown that it is the product of rexB that is responsible for the stabilization of lambda O: when a nonsense mutation is present in rexB, lambda O protein is labile; suppression of the mutation by the corresponding nonsense suppressor causes partial restabilization of lambda O. lambda rexB also stabilizes lambda O in trans. We discuss our results in relation to the function of rexB in lambda DNA replication and its role in the protein degradation pathways of bacteriophage lambda.
Bacteriophage lambda↗
Role of homology in site-specific recombination of bacteriophage lambda: evidence against joining of cohesive ends.
Bacteriophage lambda integration and excision take place at specific loci called attachment sites. Earlier work has shown that efficient recombination requires the identical sequence to be present in both attachment sites throughout the seven-base-pair region between the points of strand exchange. A plausible model for the role of homology postulates that Int, the site-specific recombinase, makes double-strand breaks at attachment sites such that each broken end has a short single-strand protrusion. Recombination would then depend upon the capacity of these protrusions to form Watson-Crick helices--i.e., to anneal--a process that might require perfect complementarity between the cohesive ends. To test this model, we have studied Int-promoted crosses in which one attachment site is a heteroduplex. Specifically, we constructed sites in which the seven-base-pair region between the points of strand exchange contains one or more noncomplementary pairs. The double-strand break and annealing mechanism predicts that crosses with these heteroduplex sites should yield one completed recombinant and one broken site. We find that such nonreciprocal recombination is uncommon and that the typical outcome of crosses involving a heteroduplex site is a reciprocal recombinant in which both products are resealed. Moreover, the occasional appearance of nonreciprocal products can be explained by our finding that Int can cleave heteroduplex attachment sites after recombination is completed. Taken together, our data strongly indicate that bacteriophage lambda recombination does not proceed by the homology-dependent annealing of cohesive ends; acceptable alternatives for the role of homology are discussed.
Bacteriophage lambda↗
[Bacteriophage lambda:lux: design and expression of bioluminescence in E. coli cells].
The bacteriophages lambda:lux and lambda:luxAB have been constructed by ligation of phage arms generated by BamHI or SalGI restriction endonucleases digestion of EMBL4 to BamHI digested plasmid pF1 lux+ or to SalGI digested plasmid pF2 lambda:luxA+B+. Cells of Escherichia coli prototrophic strain Cs were infected with lambda:lux or lambda:luxAB and intensity of bioluminiscence of the samples registered at different time intervals determined. The signal of bioluminiscence was first detected 15 min after infection and its level increased exponentially thereafter demonstrating replication of the lambda:lux bacteriophages. We have used the recombinant lambda:luxAB bacteriophage to detect the enteric indicator bacteria without enrichment in 15 min, provided that they are present at levels higher than 10(4).
Bacteriophage lambda↗
Conditionally lethal nusAts mutation of Escherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda.
Termination of transcription at bacteriophage lambda terminators as well as at the Escherichia coli trp a attenuator was examined in the conditionally lethal mutant (nusAts11) defective in the NusA protein of E. coli. Experiments using terminator-assay lambda vectors revealed that the efficiency of termination at both rho-dependent (lambda tL1) and rho-independent (lambda tL2 and trp a) terminators decreases in the mutant. The mutation does not block lambda phage growth at either permissive or nonpermissive temperatures, nor does it affect the lambda Q protein antitermination activity at the t6s terminator. These results indicate that NusA is required for transcription termination, and that lambda N and Q-mediated antitermination may not require the NusA protein function in the nusAts11 mutant.
Bacteriophage lambda↗
New map of bacteriophage lambda DNA.
A map of bacteriophage lambda was constructed, including accurate positions for all 41 cut sites made by 12 different restriction enzymes. Over 100 fragments from single, multiple, and partial enzyme digestions were measured versus standards that were calibrated with respect to DNA molecules of known sequence. The data were subjected to least-squares analysis to assign map coordinates. In no case did a fragment size predicted from the map differ from the measurement of the fragment by more than +/- 5%. This low error rate was consistent in all size ranges of fragments. The total length of lambda was calculated as 49,133 nucleotide pairs. This probably is accurate to within 500 base pairs.
Bacteriophage lambda↗
Inactivation of bacteriophage lambda and lambda DNA by nitrogen mustard.
Bacteriophage lambda and lambda DNA were treated with alkylating agents. The survival of phage was assayed by infectivity and that of DNA by infectivity of phage particles assembled from the DNA in vitro. Phage lambda were more sensitive to nitrogen mustard (C1(CH2)2NMe(CH2)2C1; HN2) than was lambda DNA. The inactivation of lambda DNA was biphasic; the second component of the inactivation was sensitive to mutations allelic for recA, polA and uvrB. This behaviour was not shown by pBR322 plasmid DNA treated with HN2 nor by lambda DNA treated with monofunctional alkylating agents (or HN2 if the second alkylation reaction was stopped by addition of a mercaptan). From Arrhenius plots, the activation energy for the reactions with DNA and interact phage were found to be different. The activation energy for the inactivation of intact phage was the same as that (measured independently) for the predominant reaction (or class of reactions) in which HN2 cross-links DNA to protein in lambda particles. From these data we conclude that the inactivation of lambda by HN2 is due, primarily, to DNA-protein cross-linking. The implications for the mode of action of DNA-reactive bifunctional anti-viral and cytotoxic compounds are discussed.
Antiviral Agents↗
Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product.
The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.
Bacteriophage lambda↗
Host virus interactions in the initiation of bacteriophage lambda DNA replication. Recruitment of Escherichia coli DnaB helicase by lambda P replication protein.
The bacteriophage lambda P protein promoters replication of the phage chromosome by recruiting a key component of the cellular replication machinery to the viral origin. Specifically, P protein delivers one or more molecules of Escherichia coli DnaB helicase to a nucleoprotein structure formed by the lambda O initiator at the lambda replication origin. Using purified proteins, we have examined the features of the pivotal host virus interaction between P and DnaB. These two proteins interact in vitro to form a P.DnaB protein complex that can be resolved by sedimentation or by chromatography on DEAE-cellulose from the individual free proteins. The sedimentation coefficient of the P.DnaB complex, 13 S, suggests a size larger than that of free DnaB hexamer (Mr = 313,600). The P.DnaB complex isolated by glycerol gradient sedimentation contains approximately three protomers of P/DnaB hexamer, consistent with a molecular weight of 393,000. The isolated P.DnaB complex functions in vitro in the initiation of lambda DNA replication. Interaction of P with DnaB strongly suppressed both the intrinsic DNA-dependent ATPase activity of DnaB, as well as the capacity of DnaB to assist E. coli primase in the general priming reaction. Formation of a P.DnaB protein complex also blocked DnaB from functioning in the initiation of E. coli DNA replication in vitro. The physical and functional properties of lambda P protein suggest that it is a viral analogue of the E. coli DnaC replication protein. Like P, DnaC also binds to DnaB (Wickner, S., and Hurwitz, J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925), but unlike P, DnaC stimulates DnaB-mediated general priming. When viral P and bacterial DnaC replication proteins were placed in direct competition with one another for binding to DnaB, the viral protein was clearly predominant. For example, a 5-fold molar excess of DnaC protein only partially reversed the inhibitory effect of P on general priming. Furthermore, when a preformed DnaC.DnaB protein complex was incubated briefly with P protein, it was readily converted into a P.DnaB protein complex and the bulk of the bound DnaC was released as free protein. It is likely that the capacity of the lambda P protein to outcompete the analogous host protein for binding to the bacterial DnaB helicase is the critical molecular event enabling infecting phage to recruit cellular replication proteins required for initiation of DNA synthesis at the viral origin.
Bacterial Proteins↗
Efficient display of scFv antibodies on bacteriophage lambda.
In the present work we demonstrate the efficient display of functional scFv antibodies on the bacteriophage lambda capsid. A single-chain (scFv) anti-CEA antibody gene was cloned in two different vectors to obtain fusion of the scFv antibody to the N- or C-terminus of the bacteriophage lambda capsid protein D (gpD). Lambda bacteriophage assembly occurs in the reducing environment of the cytoplasm; despite this the lambda-displayed anti-CEA antibody fragments retain the capacity to recognize the antigen, indicating correct single-chain antibody folding. Efficient production of functional scFv exposed on lambda capsid with viable antigen binding specificity allowed us to study and compare the capacity of display, the stability of recombinant antibody expression and the assembly efficiency of bacteriophage particles decorated with recombinant antibody fused to the amino- or carboxy-terminus of lambda D protein.
Bacteriophage lambda↗