A coliphage lambda vector with enhanced biological containment: lambda gtALO.lambda B.

The biological containment of the lambda gt family of cloning vectors has been enhanced by conditionally blocking DNA replication as well as head and tail morphogenesis. The vector, lambda gtALO.lambda B, was constructed by crossing the Oam29, Aama1 and Lam439 mutations into lambda gt.lambda B. The mutation blocking phage DNA replication, Oam29, is suppressed by suII+ or suIII+. The head gene mutation, Aama1, is suppressed by suIII+ but not by suII+ and the tail gene mutation, Lam439, is suppressed by suII+ but not by suIII+. This allows the option of increasing the biological containment by producing heads when a large amount of cloned DNA is being prepared from an individual isolate. A model recombinant, lambda gt Aama1 Lam439 Oam29.KmR' (lambda gtALO.KmR') was constructed and the containment of the vector was evaluated by the series of standardized experiments required for EK2 certification.     

Novel bacteriophage lambda mutation affecting lambda head assembly.

A novel phage lambda mutation, called dc10, which interferes with proper lambda head assembly has been isolated and characterized. Phage lambda carrying this mutation is (i) unable to form plaques at 30 or 37 degrees C but does so at 42 degrees C and (ii) unable to form plaques at 42 degrees C on pN-constitutive hosts. Both properties are due to dc10 since all phage revertants for one phenotype simultaneously lose the other phenotype and vice versa. The dc10 mutation has been mapped in the B gene and has been shown to be dominant over the corresponding wild-type product. At 30 degrees C the dc10 mutation results in the formation of abnormal petit lambda heads made up of pE, pB, pC, and pNu3. Under pN-constitutive conditions, the dc10 mutation results in the formation of abnormal petit lambda heads made of pE, X1, and X2 only. A model to explain the data is presented.     

Sites of contact between lambda operators and lambda repressor.

DNA bearing lambda operator sequences was methylated by dimethyl sulfate (DMS) in the presence or absence of lambda repressor. Under the experimental conditions, DMS methylates only the purine residues. The presence of lambda repressor affects only the methylation of certain G residues in the operators. Repressor blocks the methylation of certain G's and enhances the methylation of other G's. Since the reactive ring-nitrogen of G lies in the major groove of double-stranded DNA, and the reactive ring-nitrogen of A lies in the minor groove, the above results imply that the repressor makes contacts in the major groove of the helix. The repressor effect on G-methylation is sharply confined to the three 17 base pair units within each lambda operator previously proposed as the repressor-binding sites.     

Stability of coliphage lambda DNA replication initiator, the lambda O protein.

The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.     

Restriction alleviation by bacteriophages lambda and lambda reverse.

Deletion analysis indicated that the phage lambda restriction alleviation gene(s) ral resides between the cIII and N genes. The Ral+ phenotype was expressed only when lambda ral+ carried a modification such that it was resistant to restriction by the host specificity system. Under these conditions, Ral function protected superinfecting unmodified phages from restriction by EcoK or EcoB but not from restriction by EcoP1. Ral-protected phage DNA was not concomitantly K and B modified, but rather received only the modification specified by the system of the restricting host. Possible mechanisms for Ral action are discussed. Of the other lambdoid phages tested, the hybrid phage lambda rev had Ral activity, whereas phi 80vir and one lambda-P22 hybrid did not. The restriction alleviation activity of lambda rev called Lar, may be the same as the activity expressed in sbcA- strains of Escherichia coli, but it was functionally separable from exonuclease VIII activity (the product of the recE gene), which is also expressed in sbcA- strains.     

Chromosomal orientation of the lambda light chain locus: V lambda is proximal to C lambda in 22q11.

We have demonstrated that the chromosomal breakpoint at 22q11 of a Burkitt lymphoma cell line (PA682) with an 8;22 translocation interrupts the variable region of the lambda light chain locus. In these cells, all of the C lambda and some V lambda sequences translocate to the 8q+ chromosome whereas some V lambda sequences remain on the 22q-. These results indicate that the lambda light chain locus on the long arm of chromosome 22 is oriented such that V lambda is proximal to C lambda.     

Poisson-Boltzmann analysis of the lambda repressor-operator interaction.

A theoretical study of the ion atmosphere contribution to the binding free energy of the lambda repressor-operator complex is presented. The finite-difference form of the Poisson-Boltzmann equation was solved to calculate the electrostatic interaction energy of the amino-terminal domain of the lambda repressor with a 9 or 45 base pair oligonucleotide. Calculations were performed at various distances between repressor and operator as well as at different salt concentrations to determine ion atmosphere contributions to the total electrostatic interaction. Details in the distribution of charges on DNA and protein atoms had a strong influence on the calculated total interaction energies. In contrast, the calculated salt contributions are relatively insensitive to changes in the details of the charge distribution. The results indicate that the ion atmosphere contribution favors association at all protein-DNA distances studied. The theoretical number of ions released upon repressor-operator binding appears to be in reasonable agreement with experimental data.     

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.     

Recombination of uracil-containing lambda bacteriophages.

Controlled incorporation of uracil into the deoxyribonucleic acid (DNA) of lambda bacteriophages was achieved by growth on dut ung thy mutants of Escherichia coli. The frequency of substitution of uracil for thymine, estimated by alkaline sucrose sedimentation of phage DNA treated in vitro with uracil DNA glycosylase, ranged from 0.17 to 1.9%. The corresponding ratio between the plating efficiencies on wild-type (Ung+) and glycosylase-deficient (Ung-) bacteria ranged from 0.70 to 0.05. If a single-hit dependence of plating efficiency on uracil content is assumed, the probability that any given uracil residue is lethal is approximately 1% (about one-fifth the probability for a pyrimidine dimer). The effect of uracil on recombination was studied in experiments with lambda tandem duplication phages (ethylenediaminetetraacetic acid [EDTA] sensitive), which are converted to single-copy phages (EDTA resistant) by general recombination. For repressed infections (of homoimmune lysogens), recombination was measured by a two-stage assay (DNA extraction, transfection of spheroplasts, and EDTA treatment). The frequencies observed for uracil-containing phages (2 to 4%) were 5 to 10 times higher than control values. However, comparisons with ultraviolet irradiated phages indicated that uracil residues promoted recombination less than 1/100 as efficiently as ultraviolet-induced lesions. Recombination of uracil-containing phages during repressed infections was negligible in recA and partially reduced in recB bacteria. Recombination was very low in ung cells, suggesting that excision repair was responsible for the stimulation. Interestingly, uracil-stimulated recombination was elevated about twofold in xth bacteria.     

Effect of experimental magnetic storm on the production of lambda phage.

1. Sharp fluctuation of the intensity of the vertical component of the MF amounting to +/- 0.1 Oe changing the sign over each 3 min causes variability of both lysogenic and indicator strains of E. coli. This testifies to an extremely low threshold of their magnetic susceptibility and to biological importance of fluctuations of natural parameters of the geomagnetic field as an ecological factor of the environment. 2. A change in the intensity of the vertical component of the MF, not any higher than +/-0.1 Oe, inhibits phage production in the lysogenic system of E. coli K = 12 lambda and is also reflected in the morphological peculiarities of negative phage colonies as well as in the phage-susceptibility of the E. coli indicator strain.     

Effect of sodium ion on the high-resolution melting of lambda DNA.

A series of high-resolution melting curves were obtained by the continuous direct-derivative method [Blake, R. D. & Lefoley, S. G. (1978) Biochim. Biophys. Acta 518 , 233–246] on lambda DNA (cI₈₅₇S₇ strain) under varying conditions of [Na⁺]. Examination of the denaturation patterns at close intervals of [Na⁺] indicates that frequent changes in mechanism occur below 0.04M Na⁺, while almost none occurs above 0.1M Na⁺. Changes at low [Na⁺] generally occur in an abrupt fashion, in most cases within a 3 mM change in [Na⁺], and in at least one case within 0.6 mM, indicating the balance between alternative mechanisms is frequently quite delicate. These changes involve segments of between 900 and 1500 or more base pairs in length and are therefore not insignificant. Changes at low [Na⁺] reflect a perturbation of the energetic balance between competing mechanisms by weakly screened long-range electrostatic forces. Some perturbation probably also arises from variations in the linear charge density of the double helix induced by the proximity of premelted loop segments; however, this contribution cannot be evaluated without a detailed denaturation map. At high [Na⁺] the mechanism of melting is more conserved, permitting the dependence of subtrasitional melting temperature t_m⁽ⁱ⁾ on [Na⁺] to be examined for almost all 34 ± 2 subtransitions. The G + C composition of segments responsible for each subtransition was determined by a quantitative spectral method. Analysis according to the Manning-Record expression [Manning, G. (1972) Biopolymers 11 , 937–949; Record, M. T., Jr., Anderson, C. F. & Lohman, T. M. (1978) Q. Rev. Biophysics 11 , 103–178] relating ΔH_m and dt_m⁽ⁱ⁾/d log[Na⁺] to the fraction of Na⁺ released during melting, appears to indicate almost 40% more Na⁺ is bound to the single-stranded G and/or C residues than to A and T residues. This is consistent with a much shorter mean axial spacing and higher charge density in the former, particularly single-stranded G residues, which have an extraordinary tendency to stack.     

Specialized transduction with lambda plac5: dependence on recA and on configuration of lac and att lambda.

The construction of lambda plac5 transducing phages carrying various lacZ alleles is described. Genetically disabled (N- N- P-) lambda plac transducing the phages were used to study the dependence of specialized transduction on host RecA function and on the location of the lacZ gene in the recipient strain. In the absence of site-specific recombination at att lambda, transduction was completely dependent on host RecA function. Regardless of the configuration of att lambda, lambda plac transducing phages recombined at a 20- to 50-fold higher frequency with F42 lac than with a lac gene located in the cellular chromosome. Deletion mutants of lacZ in the recipient strain were used to show that the probability of lac recombination resulting from lambda plac infection is apparently proportional to the amount of homology between the parental lacZ genes.     

Purified bacteriophage lambda O protein binds to four repeating sequences at the lambda replication origin.

The bacteriophage lambda O protein is needed for initiation of lambda DNA replication. Several lines of evidence suggest that initiation requires that this protein interacts with a specific sequence called ori (for origin) in lambda DNA. We have purified this protein to near homogeneity and studied the protection against nuclease cleavage of the origin DNA sequences. Our data demonstrate that the O protein binds within an interval of about 95 base pairs (bp), which contains four tandemly arranged 19bp repeating sequences, ATCCCTCAAAACGA (G)GG GAT(A). At a low concentration of O protein, the inner two repeats are primarily covered, while binding to the outer two repeats requires a high concentration of O protein. From the molecular size of O protein (32,000 daltons), and the internal symmetry in each 19bp repeat, we inferred that the O protein may bind in dimeric form, and that the 95bp region may be filled only when four such dimers have bound. This interaction is discussed in connection with the "activation" of the ori by O protein leading to initiation of DNA synthesis.