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PubMed1998

rexB of bacteriophage lambda is an anti-cell death gene.

In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.

Adenosine Triphosphatases↗

PubMed1974

Protein fusion: a novel reaction in bacteriophage lambda head assembly.

Parts of two phage-coded head proteins, pE and pC, become fused during bacteriophage lambda head assembly. pE is the main structural component of lambda heads and pC is a minor head protein that is not found as such in mature heads. The bond joining the two proteins appears to be covalent and is not a disulfide bond. Only a specific subset of the sequences of each protein is found in the fusion products, and these sequences are found in the products in equimolar amounts. Two nearly identical fusion products; X1 and X2, are detected; X2 is slightly smaller than X1 and appears to be a proteolytic cleavage product of X1. The fusion reaction probably takes place on a nascent head structure.

Autoradiography↗

PubMed1975

The synthesis of a DNA duplex corresponding to the icosanucleotide sequence at the 5' end of messenger RNA from the gene N of bacteriophage lambda.

In connection with work on the nucleotide sequence of the promoter for the gene N of bacteriophage lambda as well as a study of the mechanism of transcription, a 20-unit long DNA duplex corresponding to the known sequence at the 5' end of the above gene transcript has been synthesized. For synthesis, the required duplex was divided into the following deoxyribooligonucleotides: a) the dodecanucleotide, d-A-T-C-A-G-C-A-G-G-A-C-G (II); b) the octanucleotide, d-C-A-C-T-G-A-C-C- (IV); c) the hexanucleotide, d-G-C-T-G-A-rU (I); and d) dodecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T (III). All of the four olignucleotides were chemically synthesized and characterized by extensive chromatographic and fingerprinting methods (after labeling the 5' ends with[32P]phosphate group). Longer polynucleotides (an icosa- and an octadecanucleotide) were prepared by polynucleotide ligase-catalyzed joining of segments I and III and by joining segments II and IV. The use of the octadecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, in work on the sequence analysis of the promoter is described in the accompanying paper. The octadecanucleotide and icosanucleotide were hybridized together to give the double-stranded duplex.

Adenosine Triphosphate↗

PubMed1993

The solution structure of the Oct-1 POU-specific domain reveals a striking similarity to the bacteriophage lambda repressor DNA-binding domain.

The POU-specific (POUs) domain, in association with a POU-type homeodomain, forms the bipartite DNA-binding POU domain. The solution structure of the Oct-1 POUs domain has been determined by multidimensional nuclear magnetic resonance spectroscopy and consists of four alpha helices surrounding a conserved hydrophobic core. The POUs domain is structurally similar to the DNA-binding domains of the bacteriophage lambda and 434 repressors and 434 Cro. These domains exhibit superimposable helix-turn-helix (HTH) motifs, except that in the POUs domain, the first helix and the linker to the second helix of the motif are extended. The conserved structural features have been used to propose a plausible model for DNA binding by the POUs domain. A human dwarfism mutation that affects positive control in the related POU domain protein Pit-1 maps to the same region of the HTH motif as do positive control mutations in lambda repressor.

Amino Acid Sequence↗

PubMed1996

Identification of an UP element within the IHF binding site at the PL1-PL2 tandem promoter of bacteriophage lambda.

An UP element defines a supplementary promoter element located upstream of the -35 region that stimulates transcription by interacting with the C-terminal domain of the RNA polymerase alpha subunit (alpha CTD). The alpha CTD also responds to various transcription activators, including the integration host factor protein, IHF, in the stimulation of the bacteriophage lambda PL promoter. PL consists of the tandem PL1-PL2 promoters where PL1 is stimulated and PL2 is repressed by IHF. We identified a functional UP element that binds the alpha subunit of RNA polymerase and is located in the region from -36 to -60 relative to the PL2 start site. PL2 expression requires the presence of the UP element and requires an intact alpha CTD. The UP element is nested within the DNA region protected by IHF against DNase I digestion. We used mutational analysis to identify the IHF recognition sequence which was found to be located downstream of the UP element, overlapping the -35 region of PL2. The possible function of the complex structure of the PL promoter is discussed.

Amino Acid Sequence↗

PubMed2004

Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination.

There are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means. Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome. We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes.

Adenoviridae↗

PubMed1996

Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target.

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lacI, making the cII assay an attractive alternative for mutant phage recovery for any lambda-based mouse mutagenesis assay system. The frequencies of lambda cII- mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C-->A/T transitions, a large fraction of which occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.

Animals↗

PubMed2000

Changes in the 17 bp spacer in the P(R) promoter of bacteriophage lambda affect steps in open complex formation that precede DNA strand separation.

Tau plots and temperature-shift experiments were used to determine which step in the formation of transcriptionally-competent open complexes is affected by changing the length of the 17 bp spacer separating the -10 and -35 consensus regions of the P(R) promoter of bacteriophage lambda. Abortive initiation assays at 37 degrees C indicate that the primary effect of insertion of a base-pair, thereby increasing spacer length to 18 bp, is a decrease in k(f), the rate constant for conversion from closed (RP(c)) to open (RP(o)) complexes, by approximately a factor of 4. The mutation did not significantly affect K(B), the equilibrium constant for formation of closed complexes, and decreased K(B)k(f) by a factor of 3. Deletion of a bp to create a 16 bp spacer had a much greater effect, decreasing the measured value of k(f) by a factor of about 25 to 30, and K(B)k(f) by a factor of 7 to 8. When the values of the parameters for the deletion mutant were corrected for incomplete occupancy of RP(o) at equilibrium, the effects of the deletion were even greater. In particular, the corrected value of K(B)k(f) was about 15 times lower than the corresponding value for two promoters with wild-type spacing. Based on temperature shift experiments, the changes in spacer length did not affect the equilibrium at 20 degrees C between RP(i), a stable intermediate in which DNA strands are not separated, and RP(o). Although differential sensitivity of single-stranded bases to KMnO(4) indicated that in about 20% of the open complexes at 20 degrees C the DNA strands are not fully separated (RP(o1)), the distribution between these complexes and RP(o2) (DNA strands fully separated) was also not affected significantly by changes in spacer length. Thus, changes in spacer length primarily affect k(2), the rate constant for conversion of RP(c) to RP(i), which corresponds to a nucleation of DNA strand-separation. Application of published data and/or algorithms for determining effects of nucleotide sequence on twist angle or rise at individual bp steps does not provide a simple explanation of the difference in promoter strength between P(R) derivatives with 16 bp spacing and those with 18 bp spacing.

Algorithms↗

PubMed1990

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library.

We have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and kappa light-chain variable and constant region domains, were inserted into modified bacteriophage lambda expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. We estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.

Amino Acid Sequence↗

PubMed1988

OR3 operator of bacteriophage lambda in a 23 base-pair DNA fragment: sequence-specific 1H NMR assignments for the non-labile protons and comparison with the isolated 17 base-pair operator.

Sequence-specific 1H NMR assignments are presented for a non-selfcomplementary 23-base-pair DNA duplex of molecular weight 15,000 daltons, containing the OR3 repressor binding site of bacteriophage lambda as the central core. The NMR techniques used were mainly phase-sensitive two-dimensional NOE and 2Q spectroscopy, the latter to overcome overlap problems within the spectral region of the deoxyribose spin-systems. Direct sequential NOE connectivities are observed between adenine 2 H and deoxyribose 1' protons. We propose the use of these connectivities as a check of the assignments of C1' and A2 protons, which have independently been derived via other assignment pathways.

Bacteriophage lambda↗

PubMed2000

Dissection of bacteriophage lambda site-specific recombination using synthetic peptide combinatorial libraries.

A wide variety of tools have been used to dissect biochemical pathways, inhibitors being chief among them. Combinatorial approaches have made the search for inhibitors much more efficient. We have applied such an approach to identify hexapeptides which inhibit different steps in a site-specific recombination reaction mediated by the bacteriophage lambda integrase protein. Integrase's mechanism is still incompletely understood, in large part because several pathway intermediates remain hard to isolate. Integrase-catalyzed recombination is very efficient, but if blocked, it is highly reversible to substrates; this combination makes some intermediates exceedingly transient. We have used synthetic peptide combinatorial libraries to screen for hexapeptides that affect the recombination pathway at different stages, and have identified two families of peptides: one probably blocks DNA cleavage, the other may stabilize the Holliday junction intermediates. These peptides do not resemble parts of integrase or any of the other helper functions in the pathway. The deconvolution of hexapeptide libraries based both on inhibition of an enzymatic reaction as well as on accumulation of reaction intermediates is a novel approach to finding useful tools for dissecting a biochemical pathway.

Alanine↗

PubMed1976

Construction of plasmids carrying the cI gene of bacteriophage lambda.

By techniques of recombination in vitro, we have constructed a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon. Strains carrying this plasmid overproduce lambda repressor. This functional cI gene was reconstituted by joining DNA fragments bearing different parts of that gene. Flush end fusion techniques, involving no sequence overlap, were necessary for the construction; in certain cases, the abutting of the DNA molecules bearing ends generated by different restriction endonucleases creates a sequence at the junction which is recognized by one of the restriction endonucleases.

Coliphages↗

PubMed1972

Plasmid-controlled variation in the content of methylated bases in bacteriophage lambda deoxyribonucleic acid.

The N(6)-methyladenine (MeAde) and 5-methylcytosine (MeC) contents in deoxyribonucleic acid (DNA) of bacteriophage lambda has been analyzed as a function of host specificity. The following facts have emerged: (i) lambda grown on strains harboring the P1 prophage contain ca. 70 more MeAde residues/DNA molecule than lambda grown either in the P1-sensitive parent, or in a P1 immune-defective lysogen which does not confer P1 modification; (ii) lambda grown on strains harboring the N-3 drug-resistance factor contain ca. 60 more MeC residues/DNA molecule than lambda grown on the parental strain lacking the factor; (iii) lambda grown in Escherichia coli B strains is devoid of MeC, whereas lambda grown in a B (N-3) host contains a high level of MeC; (iv) the MeAde content in lambda DNA is not affected by the N-3 factor. These results suggest that P1 controls an adenine-specific DNA methylase, and that the N-3 plasmid controls a cytosine-specific DNA methylase. The N-3 factor has been observed previously to direct cytosine-specific methylation of phage P22 DNA and E. coli B DNA in vivo; in vitro studies presented here demonstrate this activity.

Adenine↗

PubMed1978

The bacteriophage lambda int gene product. A filter assay for genetic recombination, purification of int, and specific binding to DNA.

Bacteriophage lambda int gene is required for the integration of viral DNA into the chromosome of Escherichia coli. We have extensively purified the product of the int gene (Int) from a lysogen of E. coli that constitutively expresses this gene. Int was assayed by its ability to promote integrative recombination of supertwisted substrate DNA in vitro using a new method based on filter trapping of a recombinant product DNA. In order to catalyze integrative recombination, Int must be supplemented by other factors that can be extracted from bacterial host cells. By itself, purified Int does not demonstrate detectable endonuclease, exonuclease, or nicking-closing activities. However, Int does make stable complexes with double-stranded lambda-DNA containing an attachment site, the region at which recombination takes place. No stable complexes are observed between Int and lambda-DNA without an attachment site or between Int and DNA containing the bacterial site of integration. Int, therefore, appears to be a specificity element that relies on additional factor(s) to provide or activate the catalytic functions required for recombination.

Chromosomes, Bacterial↗

PubMedDate not supplied

Determination of bacteriophage lambda tail length by a protein ruler.

How the size and shape of living structures are determined by genetic information is one of the fundamental problems in biology. Here I describe a study in which the size of a biological supramolecular structure was changed in a predictable way by in vitro genetics, with the size both before and after manipulation being exactly determined. I have studied the tail of bacteriophage lambda, whose length is determined by the length of the 'ruler protein', the product of gene H. The length of the tail can be decreased or increased by deleting the middle part of gene H or by forming a small duplication there, and the length of the tail is proportional to the size of the protein. These results can be regarded as a special case of protein engineering, namely supramolecular protein engineering.

Bacteriophage lambda↗

PubMed1994

Inactivation and repair of bacteriophage lambda by furfural.

Furfural is a dietary mutagen and is present in various frequently consumed food products. In earlier work we have shown that it induces single strand breaks in duplex DNA which occur preferentially in AT base pairs. Experiments on the conservation of EcoRI cleavage site in mutant plasmids further established that repair of furfural damaged plasmid DNA occurs on propagation in the host cells. In this paper it is shown that the strand scissions induced by furfural in DNA account for its biological activity as assayed by inactivation of bacteriophage lambda. Using various repair defective mutants of E. coli we have shown that DNA repair predominantly involves the polA pathway.

Bacteriophage lambda↗

PubMed1987

Sequence of amino acids in lamB responsible for spontaneous ejection of bacteriophage lambda DNA.

The DNA sequence of the promoter-distal half of lamB from Shigella sonnei 3070 has been determined and compared with the known sequence for the Escherichia coli K12 gene. The only predicted amino acid changes in this region of LamB, the receptor protein for bacteriophage lambda, lie between positions 381 and 390, where seven of the ten amino acids are altered. Evidence is presented that indicates that this region is responsible for the ability of the S. sonnei receptor, but not the E. coli receptor, to trigger spontaneous ejection of DNA from the bacteriophage in vitro. DNA injection in vivo must be more complex and involve also the host Pel protein and the lambda tail proteins gpJ, gpH, and gpV.

Amino Acid Sequence↗

PubMed1993

Kinetic characterization of the ATPase activity of the DNA packaging enzyme from bacteriophage lambda.

Terminases are enzymes common to all of the complex double-stranded DNA viruses and are required for viral assembly. These enzymes function to excise a single viral genome from a concatemeric DNA precursor and package it into a preformed protective protein shell or capsid. ATP hydrolysis by these enzymes has been described and appears to be critical to the packaging process. We have previously characterized the endonuclease activity of purified terminase from bacteriophage lambda [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065], and we describe here a kinetic characterization of the ATPase activity of the enzyme. lambda Terminase possesses a DNA-stimulated ATPase activity and hydrolyzes ATP to ADP and Pi. This activity requires divalent metal and is supported by all of the group IIa metals examined, as well as Mn2+. The reaction is also stimulated by NaCl, GTP, and dGTP. Of note is that neither of the guanosine nucleotides is hydrolyzed by the enzyme, while dATP is hydrolyzed at a rate comparable to that of ATP. Kinetic analysis of the ATPase activity revealed two apparent binding sites for ATP hydrolysis. The high-affinity site (Km = 5 microM) and low-affinity site (Km approximately 1.3 mM) hydrolyze ATP with kcat = 3 and 16 min-1, respectively. While the high-affinity site is unaffected by the presence of DNA, ATP hydrolysis at the low-affinity site is stimulated by DNA, which results from both a decrease in the Km and a concomitant increase in the kcat of the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗