[Enlargement of the host area of bacteriophage lambda for Escherichia coli B].
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Bacteriophage lambda↗
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Site-specific recombination of bacteriophage lambda. The change in topological linking number associated with exchange of DNA strands.
The changes in supercoiling that accompany site-specific recombination have been measured. In each experiment, the substrate was a circle that contained two attachment sites oriented as an inverted repeat; recombination between the sites inverts one segment of the circle with respect to the other. Using conditions developed in the accompanying work, a measurable amount of the recombinant is in the form of unknotted, simple circles. The difference between the topological linking number of this product relative to that of the substrate can be determined directly from the change in mobility during agarose gel electrophoresis. With partially supercoiled substrates, both integrative and excisive recombination are characterized by a unique change in linking number, a relaxation of two topological turns. For excisive recombination, it has been possible to study closed circular substrates that lack supercoils. In this case, changes in linking number of both +2 and -2 are observed. These results are used to evaluate various proposals for synapsis and strand exchange in bacteriophage lambda site-specific recombination.
Attachment Sites, Microbiological↗
Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor.
Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for genome packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (de Beer et al. (2002) Mol. Cell 9, 981-991). We further proposed that gpNu1 and IHF cooperatively bind and bend viral DNA to regulate the assembly of the packaging motor. Here, we characterize cooperative gpNu1 and IHF binding to the cos site in lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay. These studies provide direct experimental support for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA. Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. Thus, specific recognition of viral DNA by the packaging apparatus is mediated by both DNA sequence information and by structural alteration of the duplex. The relevance of these results with respect to the assembly of a viral DNA-packaging motor is discussed.
Bacteriophage lambda↗
Sequence specificity of mutagenesis in the cI gene of bacteriophage lambda.
Studies of DNA base sequence alterations have shown that for every agent the mutagenic process is specific with respect to the types of base changes induced and the location of the changes in the DNA. Analysis of the types of mutations produced by mutagenic agents can provide insight into the mechanism of mutation and can suggest which DNA lesions may be involved in the actual mutagenic event. We have developed a system for the analysis of chemically induced base sequence alterations in the cI repressor gene of bacteriophage lambda using DNA sequencing techniques. To illustrate the utility of this type of analysis, we present the results obtained with ultraviolet light (UV). Irradiation of target DNA with UV alone, or UV followed by photoreactivating light (which removes dimers), produces mostly transitions at pyrimidine-pyrimidine sites. Conversely, irradiation with 313 nm light plus acetophenone (which produces only thymine dimers) produces mostly transversions at low efficiency. This and other evidence suggests that the actual premutagenic UV lesion in E. coli may not be pyrimidine-pyrimidine dimers, but rather pyr(6-4)pyo photoproducts.
Bacteriophage lambda↗
Crystallization and preliminary X-ray analysis of bacteriophage lambda lysozyme in which all tryptophans have been replaced by aza-tryptophans.
After many unsuccessful attempts to crystallize the bacteriophage lambda lysozyme, a mutant where all the tryptophan residues have been replaced by aza-tryptophans has been crystallized by the vapor-diffusion method. The crystals are orthorhombic and belong to space group P2(1)2(1)2(1) with cell dimensions a = 73.01, b = 78.80, c = 82.31 A. Diffraction data were collected using synchrotron radiation sources. Crystals diffract to a resolution of 2.3 A. Data from two different platinum derivatives were also recorded to 2.8 and 2.5 A, respectively.
Journal Article↗
Solid-phase sequence analysis of polypeptides eluted from polyacrylamide gels. An aid to interpretation of DNA sequences exemplified by the Escherichia coli unc operon and bacteriophage lambda.
An approach to sequencing proteins by the solid-phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described. Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels. They are detected with Coomassie blue, eluted, selectively reacted with porous glass derivatives and sequenced in their amino-terminal regions with the aid of a new microsequencer. Alternatively they can be analysed or digested with enzymes and fingerprinted. It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences. Nine genes, four from the unc operon of Escherichia coli encoding the alpha, beta, gamma and epsilon subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.
Adenosine Triphosphatases↗
Bacteriophage lambda and plasmid vectors, allowing fusion of cloned genes in each of the three translational phases.
We have constructed vectors from bacteriophage lambda and from plasmid pBR322 having a single EcoRI restriction site which is immediately downstream from the lac UV5 promotor. Each vector allows the fusion of a cloned gene to the lac Z gene in a different phase relative to the translation initiation codon of the lac Z gene. These vectors were constructed through modification of the initial EcoRI restriction site by S1 endonuclease treatment and then addition of octadeoxyribonucleotides (EcoRI linkers), which shifted the restriction site by 2 or 4 nucleotides. Used in combination these vectors should allow translation of a cloned gene in any one of the three coding phases. The bacteriophages vectors are certified as B2 (EK2) safety level vectors by the French "recombinaison génétique in vitro" committee (D.G.R.S.T.).
Base Sequence↗
Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter.
By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.
Bacteriophage lambda↗
A mutation affecting the DNA content of bacteriophage lambda and its lysogenizing properties.
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Bacteriophage lambda↗
[Spontaneous induction of the development of bacteriophage lambda during genetic recombination in Escherichia coli K12].
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Bacteriophage lambda↗
Effects on protein structure and function of replacing tryptophan with 5-hydroxytryptophan: single-tryptophan mutants of the N-terminal domain of the bacteriophage lambda repressor.
Conformational energy computations have been carried out on the N-acetyl-N'-methylamide of 5-hydroxytryptophan (5OH-Trp) using ECEPP/3. As observed with tryptophan (Trp), the most preferred conformation about the C alpha-C beta bond of the side chain is g+ or t. This preference is reduced to only the t conformational state when 5-hydroxyTrp is in the middle of a right-handed poly(L-alanine) alpha-helix. A similar result has been obtained with Trp [Piela et al. (1987), Biopolymers 1987, 1273-1286]. These results suggest that replacement of Trp by its analog 5-hydroxyTrp may be tolerated in an alpha-helix. To test this hypothesis, we have replaced Trp by 5OH-Trp in the fifth helices of two functionally active mutants of the N-terminal domain of the bacteriophage lambda repressor. Computations on the packing of these helices have shown that no significant structural changes results from the replacement of Trp by 5OH-Trp. The DNA-binding activity of these mutants, as assessed indirectly through geometrical parameters, is also unaltered.
5-Hydroxytryptophan↗
Temperature-inducible gene expression in Bacillus subtilis mediated by the cI857-encoded repressor of bacteriophage lambda.
An efficient system to control the expression of cloned genes in Bacillus subtilis was established by introducing the Escherichia coli bacteriophage lambda cI857 repressor-pR promoter system into this host. A staphylokinase reporter gene (sak42D), which was fused to the lambda pR promoter was constitutively expressed in B. subtilis even when the cI857 gene was present on the same plasmid. S1 nuclease mapping of the transcription start point confirmed that the pR promoter was active in B. subtilis. Constitutive expression under pR-control in B. subtilis was, therefore, likely to result from a lack of repressor formation caused by the inefficiency of cI857 expression signals in the Gram+ host. This lack of repressor synthesis was overcome by fusing the cI857 gene to sak42D transcription and translation signals which have previously been shown to function efficiently in B. subtilis. Plasmids carrying the cI857 gene together with an alpha-amylase-encoding gene (amy) under pR-control mediated temperature-inducible amy expression at 37 degrees C and 42 degrees C. The high repression factor (greater than or equal to 1400) was comparable to the OR efficiencies reported in E. coli.
Amylases↗
Synthesis of bacteriophage lambda DNA in vitro: requirement for O and P gene products.
We have developed a cell-free system to study bacteriophage lambda DNA replication. Maximal DNA synthesis in vitro requires the four deoxynucleoside triphosphates, ATP, and exogenous lambda DNA. DNA synthesis requires the products of the phage O and P genes but is not inhibited by lambda repressor. The kinetics of synthesis is linear for 10-15 min; however, the product of synthesis amounts to only 0.5-1% of the added template DNA. As judged by isopycnic analysis, extensive regions of the template are copied. Sedimentation analysis indicates that all of the product consists of short (11S) DNA chains. Fractions partially purified from lambdaO(+)P(+)-infected cell extracts will complement extracts prepared from lambdaO(-) or lambdaP(-)-infected cells.
Adenosine Triphosphate↗
Rec-mediated recombinational hot spot activity in bacteriophage lambda. I. Hot spot activity associated with spi-deletions and bio substitutions.
In order to survey the distribution along the bacteriophage lambda chromosome of Rec-mediated recombination events, crosses are performed using conditions which block essentially all DNA synthesis. One parent is density-labeled and carries a genetic marker in the left terminal lambda gene (A), while the other parent is unlabeled and carries a genetic marker in the right terminal lambda gene (R). Both parents are deleted for the lambda recombination genes int and red, together with other recombination-associated genes, by virtue of either (1) a pure deletion or (2) a bio insertion-deletion. The distribution in a cesium density gradient of the resulting A+R+ recombinant phage reflects the chromosomal distribution of the recombination events which gave rise to those phage. Crosses employing either of two different pure deletion phage strains exhibit recombinational hot spot activity located near the right end of the lambda chromosome, between the cI and R genes. This hot spot activity persists when unlimited DNA synthesis is allowed. Crosses employing bio1-substituted phage strains exhibit recombinational hot spot activity located to the right of the middle of the chromosome and to the left of the cI gene. Crosses employing either bio1 or bio69-substituted phage strains indicate that the bio-associated hot spot activity occurs in the presence of DNA synthesis, but is dependent on a functional host recB gene.
Centrifugation, Density Gradient↗
Initiation of infection by deoxyribonucleic acid from . . . bacteriophage lambda.
Kinetic studies conducted on the early stages of infection of Escherichia coli K-12 by deoxyribonucleic acid (DNA) isolated from bacteriophage lambda indicate a rapid adsorption of the phage DNA to receptor sites at the bacterial surface prior to deoxyribonuclease-insensitive incorporation. A direct relationship found between the number of DNA molecules adsorbed per bacterium and the multiplicity of helper phage infection indicates a requirement for helper function during the attachment process. An apparent lack of attachment specificity with regard to the source of the DNA preparation, to the size of the inhibiting fragment, to the base ratio of the inhibiting DNA molecule, and to "cohesive" ends suggests a nonspecific interaction between the infectious DNA and the sites of helper phage attachment.
Bacillus subtilis↗
Roles of bacteriophage lambda gene products O and P during early and late phases of infection cycle.
Ring-to-ring (early) replication of bacteriophage lambda DNA was blocked after heat inactivation of the P protein. Rolling circle (late) replication continued for several rounds at the rate reached when the temperature shift was carried out. The same differential effect was observed after inhibition of RNA or protein synthesis during the two different phases of replication. In contrast, inactivation of the O protein resulted in a fast stop of lambda DNA synthesis at early and late times after infection. The results were consistent with the following interpretations. (i) The lambda P gene product plays a role in the initiation of the ring-to-ring replication. (ii) Ring-to-ring replication continues parallel to rolling circle replication, possibly diminishing with time after infection. (iii) The O function is stable in and necessary for the structural integrity of an elongation complex. It is unstable in free form and probably released from such a replication complex after each round of replication at the ring-to-ring stage.
Chloramphenicol↗
Bacteriophage lambda and plasmid pUR288 transgenic fish models for detecting in vivo mutations.
We adapted transgenic rodent mutation assays based on fish carrying bacteriophage lambda and plasmid pUR288 vectors to address the needs for improved methods to assess health risks from exposure to environmental mutagens and also to establish new animal models to study in vivo mutagenesis. The approach entails separating the vectors from fish genomic DNA and then shuttling them into specialized strains of E. coli bacteria to analyze spontaneous and induced mutations in either lacI and cII or lacZ mutational targets. Fish exhibited low frequencies of spontaneous mutants comparable to the sensitivity of transgenic rodent models. Mutations detected after treating fish with chemical mutagens showed concentration-dependent, tissue-specific, and time-dependent relationships. Spontaneous and induced mutational spectra also were consistent with the specificity of known mutagens, further supporting the utility of transgenic fish for studies of in vivo mutagenesis.
Journal Article↗
Kinetics of bacteriophage lambda deoxyribonucleic acid infection of Escherichia coli.
Barnhart, Benjamin J. (Los Alamos Scientific Laboratory, University of California, Los Alamos, N.M.). Kinetics of bacteriophage lambda deoxyribonucleic acid infection of Escherichia coli. J. Bacteriol. 90:1617-1623. 1965.-The kinetics of Escherichia coli K-12 infection by phage lambda deoxyribonucleic acid (DNA) were determined. An initial lag of 55 to 80 sec was found to be the time required for infecting DNA to become deoxyribonuclease-insensitive at 33 C. When cell-DNA interactions were stopped by washing away unbound DNA, the already bound DNA continued to infect the cell at rates described by linear kinetics with no apparent lag. Whereas the lag period was relatively insensitive to DNA and cell concentrations, both the lag and the subsequent linear portions of the rate curves were temperature-sensitive. Cell and DNA dose-response curves prescribed hyperbolic functions. Similarities between lambda DNA infection of E. coli and bacterial transformation systems are discussed.
Coliphages↗