SEARCH · PubMed Health

Results for “Bacteriophage lambda”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 919 records · Page 51Linked to original sources

PubMed1999

Bacteriophage T4 gp2 interferes with cell viability and with bacteriophage lambda Red recombination.

The T4 head protein, gp2, promotes head-tail joining during phage morphogenesis and is also incorporated into the phage head. It protects the injected DNA from degradation by exonuclease V during the subsequent infection. In this study, we show that recombinant gp2, a very basic protein, rapidly kills the cells in which it is expressed. To further illustrate the protectiveness of gp2 for DNA termini, we compare the effect of gp2 expression on Red-mediated and Int-mediated recombination. Red-mediated recombination is nonspecific and requires the transient formation of double-stranded DNA termini. Int-mediated recombination, on the other hand, is site specific and does not require chromosomal termini. Red-mediated recombination is inhibited to a much greater extent than is Int-mediated recombination. We conclude from the results of these physiological and genetic experiments that T4 gp2 expression, like Mu Gam expression, kills bacteria by binding to double-stranded DNA termini, the most likely mode for its protection of entering phage DNA from exonuclease V.

Bacteriophage T4↗

PubMed1984

An integration-proficient int mutant of bacteriophage lambda.

We have isolated and characterized a novel int mutant of phage lambda. This mutant promotes efficient recombination between the phage and bacterial attachment sites, but, unlike wild type, does not promote efficient recombination of any other pair of attachment sites tested in most conditions. In particular, recombination between two phage or two prophage attachment sites is poor relative to the wild type frequency. We attribute this unusual phenotype to differences in the distribution of int protein binding sites among different attachment sites (Ross and Landy 1982, 1983). We suggest that int protein molecules bound to one of two recombining DNAs interact with empty sites or with bound proteins on the other, and that the mutant protein acts efficiently only if the distribution of protein binding sites within the two attachment sites is that of the attP-attB pair. Similar discrimination among attachment site pairs by wild type int protein may also modulate recombination frequencies.

Bacteriophage lambda↗

PubMed1984

Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda.

Abortive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage lambda PRE promoter by cII gene product. Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cII protein by factors of 4-16 relative to the wild-type affinity. Kinetic analyses of transcription initiation in the presence and absence of cII protein demonstrated that five of the six mutations did not significantly affect the intrinsic interaction of RNA polymerase with PRE. Thus, these mutations differ from other cy mutations, including those in the -35 consensus region, which affect the formation of polymerase-PRE closed complexes or the isomerization of closed complexes to open complexes but do not affect the binding of cII protein. A sixth T-T-G-C mutation, cy3001, may affect intrinsic initiation by RNA polymerase as well as cII binding.

Bacteriophage lambda↗

PubMed1983

Role of the Xis protein of bacteriophage lambda in a specific reactive complex at the attR prophage attachment site.

Phage lambda controls its integration and excision by differential catalysis of the forward and reverse reactions. The lambda Int protein is required for both directions, but Xis for excision only. Previous electron microscopic observations have shown that Int protein forms a stable, condensed protein-DNA complex with the phage (attP) and prophage left (attL) substrate sites, but not with the host (attB) or prophage right (attR) sites. We have found that Int and Xis together produce a stable, condensed complex with attR. The attR complex involves the P region DNA to the left of the crossover point (O site). In contrast, the attP complex includes DNA on both sides of the crossover point (P and P'), and the attL structure involves the P' DNA to the right of O. In the presence of Int and Xis, the attL and attR sites form a paired structure. We conclude that the role of Xis is to provide a distinct reactive structure at attR, allowing attL and attR to pair efficiently.

Bacteriophage lambda↗

PubMed1998

ATP-reactive sites in the bacteriophage lambda packaging protein terminase lie in the N-termini of its subunits, gpA and gpNu1.

ATP-reactive sites in terminase and its subunits have been successfully identified using three different affinity analogs of ATP (2-and 8-azidoATP and FITC) GpA, the larger subunit of terminase, was shown to have a higher affinity for these analogs than gpNu1, the smaller subunit. The suitability of these reagents as affinity analogs of ATP was demonstrated by ATP protection experiments and in vitro assays done with the modified proteins. These analogs were thus shown to modify the ATP-reactive sites. The results obtained from these experiments also indicate the importance of subunit-subunit interactions in the holoenzyme. Terminase, gpA, and gpNu1 were modified with these analogs and the ATP-reactive sites were identified by isolating the modified peptide by reverse-phase chromatography. The sequence analysis of the modified peptides indicates a region including amino acids 18-35 in the N-terminus of gpNu1 and a region including amino acids 59-85 in the N-terminus of gpA as being the ATP-reactive sites.

Adenosine Triphosphatases↗

PubMed1985

A CII-responsive promoter within the Q gene of bacteriophage lambda.

A site within the phage lambda Q gene shares homology with the CII-activated promoters, pE and pI, and is oriented in the direction opposite to that of Q gene transcription. A DNA fragment containing this site can serve as a template for CII-activated transcription in vitro. To ask if this presumptive CII control site functions as a CII-activated promoter in vivo, a restriction fragment containing this promoter has been cloned on a plasmid so that synthesis of beta-galactosidase will be under its control. When CII protein is supplied in trans from a compatible plasmid, this promoter, designated PaQ, is activated to produce beta-galactosidase. A promoter positioned within the Q gene which can be activated by CII protein to initiate transcription in an anti-sense direction should result in an interference with Q gene expression, enhancing CII regulation of late functions, and adding to the list of known CII controls on the lysogenic response.

Bacteriophage lambda↗

PubMed1982

The receptor of bacteriophage lambda: evidence for a biosynthesis dependent on lipid synthesis.

Inhibition of lipid synthesis in cerulenin-treated cells or in a mutant strain defective in sn-glycerol-3-phosphate acyltransferase after glycerol deprivation, results in a marked decrease of insertion of lamB protein into the outer membrane. No lambda receptor was found in any other cell compartment or in the medium under these conditions. The LamB protein synthesis was inhibited by about 70% in the absence of lipid synthesis. The residual 30% protein produced during inhibition of fatty-acid or phospholipid synthesis, was probably incorporated into the outer membrane since no further incorporation was observed after resumption of these syntheses. Besides OmpF and OmpC protein [Bocquet-Pagès, C., Lazdunski, C., and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105-111], at least four other proteins of the outer membrane are also subject to alteration of levels in the absence of lipid synthesis. Under these conditions the uptake of maltose, like the uptake of 5'AMP [Bocquet-Pagès, C., Lazdunski, C., and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105-111], was inhibited as much as 60%. These results are discussed with regard to the biosynthesis and assembly of the outer membrane proteins.

Bacterial Outer Membrane Proteins↗

PubMed1989

DNA looping induced by bacteriophage lambda O protein: implications for formation of higher order structures at the lambda origin of replication.

A plasmid has been constructed, pOri2, which contains two lambda replication origin sequences separated by 1068 bp; both lambda sequences having the same orientation. When lambda initiation protein O is reacted with linearized pOri2 and examined by electron microscopy it is found to contain a looped area in which two parts of the plasmid are bound together by the O protein complex. Length measurements show that the O protein binds at the expected positions of the lambda origin sequences and that the looped area represents the DNA segment between the two O protein binding domains. Similar looping occurs in reactions with supercoiled pOri2 or if an amino-terminal fragment of O protein is used. When looped molecules are reacted with psoralen, crosslinked by irradiation with uv light, and then denatured, it is found that the looped area is more thermostable than the rest of the molecule. This indicates that the DNA within the looped segment is torsionally constrained while that outside the loop is free to rotate and suggests that simultaneous binding of O to two origins fixes the linkage number of the intervening DNA. The double origin binding ability of O may be diagnostic of the details of the reaction of O with a single origin sequence. A model is presented that rests on the assumption that O can produce microscopic looping between O protein binding sites within a single ori sequence.

Bacteriophage lambda↗

PubMed1982

A chain of interlinked genes in the ninR region of bacteriophage lambda.

The 3612-bp DNA sequence of the phage lambda P-Q (ninR) region contains a series of nine open reading frames in a distinctly overlapping pattern: ATGA sequence modules occur at the boundaries of consecutive genes and are able to serve both as terminator (TGA) and (re)initiator (ATG) codons for most of the adjacent frames. Together with genes O, P, and Q, the newly detected ren and ninA through ninH constitute a series of twelve closely linked genes in the pR operon. Based upon the available evidence for several of the nin proteins, and on plasmid expression data, we conclude that at least the larger nin genes, and probably all of the newly detected open reading frames code for proteins. The nin5 deletion of 2803 bp is a frame-to-frame fusion of ren and ninH, and covers the t R2 termination signal located near its left boundary, immediately behind the ren gene. The possible significance of the observed chain of closely interlinked genes for the regulation of Q expression is discussed.

Bacteriophage lambda↗

PubMed1983

Knotting of DNA caused by a genetic rearrangement. Evidence for a nucleosome-like structure in site-specific recombination of bacteriophage lambda.

Intramolecular recombination between two attachment sites on a circular substrate can invert one segment of the circle with respect to the other. We have studied the topological form of the products of such site-specific inversion as a function of two parameters of the substrate circle: the degree of supercoiling and the distance between the recombining sites. For both integrative and excisive recombination, supercoiled substrates produced knotted recombinants; the complexity of the knots reflects the distance separating the sites. This confirms and extends earlier observations and supports the hypothesis that random interwrapping of segments of the double-helical substrate persists during recombination. For integrative recombination, we find that even at conditions that should limit random interwrapping, absence of supercoiling and very short separation between attachment sites, only about one-half of the recombinant products are simple circles and the rest are knotted. Under the same conditions, excisive recombination yields only simple circular inverted recombinants. We propose that the excess knotting that characterizes integrative recombination reflects the requirement for wrapping of one attachment site, presumably attP, into a nucleosome-like structure. This hypothesis accounts for both the frequency of knots and the observation that the extra knots are trefoils rather than more complex forms.

Attachment Sites, Microbiological↗

PubMed1988

Effects of DNA heterologies on bacteriophage lambda packaging.

We have examined the impact of DNA heterologies on the packaging of lambda DNA in vitro. Heterology-containing DNA molecules were constructed by denaturing and reannealing a mixture of DNA from cI+ phage and DNA front phage carrying small insertion or deletion mutations in the cI gene. We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch. In contrast, with a heterology of 26-bp heterozygous plaque formers are rare. In principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsulate the DNA or a failure to inject the packaged DNA on infection. Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage. Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus The size of the connector portal could be the basis for this limit.

Bacteriophage lambda↗

PubMed2004

Nonspecific binding of the OR repressors CI and Cro of bacteriophage lambda.

We estimate the Gibbs free energy for nonspecific binding (DeltaGNSB) to the Escherichia coli DNA for two regulatory proteins of the lambda phage, CI and Cro. By means of a statistical-mechanical approach, we calculate the cI and cro activities associated with the operator OR of an introduced lambda phage genome (prophage). In this statistical model we apply in vitro-measured binding free energies to fit in vivo experimental data for cI and cro activities, respectively, where DeltaGNSB is introduced as a free (fitting) parameter. Without nonspecific binding included in the model, the quality of the description is fairly poor, whereas data are nicely correlating with our model with nonspecific binding included over the entire data range. The obtained values of DeltaGNSB are -4.1+/-0.9 kcal/mol, for CI, and -4.2+/-0.8 kcal/mol, for Cro. In particular, in a lysogen (approximately 250 CI monomers per cell) we conclude that 86% of the total CI in the cell is nonspecifically bound, leaving on average around 10 CI dimers freely available in the E. coli cytoplasma. These findings corroborate the view that due to low free cellular particle numbers a dynamical analysis of genetic regulation at OR and comparable systems should include a stochastic component. In addition, we perform a stability analysis of the OR system in the presence of nonspecific binding.

Bacteriophage lambda↗

PubMed1982

Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination.

Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.

Bacteriophage lambda↗

PubMed1985

High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda.

Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using lambda pL promoter expression vectors. In a first step we constructed endRI::lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing EcoRI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the overproduced EcoRI endonuclease.

Animals↗

PubMed1993

Interference by PR-bound RNA polymerase with PRM function in vitro. Modulation by the bacteriophage lambda cI protein.

Activation of the weak PRM promoter by cI protein is an essential process in the establishment of lysogeny. Much evidence has accumulated that cI protein binds cooperatively to the operators OR1 and OR2 and that protein at the OR2 site contacts RNA polymerase to facilitate open complex formation at the PRM promoter. We had shown previously in vitro that RNA polymerase situated at the nearby PR promoter could interfere with open complex formation at PRM and that an additional mechanism of PRM activation in vitro involved cI-mediated RNA polymerase exclusion from PR. Here we further characterize this second indirect mode of activation. We demonstrate the addition of cI and inactivation of the PR promoter activate open complex formation at PRM similarly over the temperature range from 37 to 20 degrees C in which the extent of activation decreases from 8- to 2-fold. We also show that the binding of cI protein to OR1 is sufficient to effect an increase in the rate of synthesis of abortive RNA products at PRM. This result is difficult to explain based on direct cI-RNA polymerase contacts alone but is readily interpreted in terms of our previously proposed model involving the exclusion of an interfering RNA polymerase from binding at PR.

Bacteriophage lambda↗

PubMed1980

The phage promoter responsible for the expression of the inserted beta-galactosidase gene in bacteriophage lambda plac5.

The lac transducing phage, lambda plac5, carries a segment of the E. coli lac operon on the left side of the b2 region of the lambda phage. In the absence of additional cyclic AMP, beta-galactosidase can only be expressed from the phage promoter, and the expression of the inserted lac promoter is suppressed. This phage promoter responsible for beta-galactosidase synthesis is shown to be under the control of the cI and N gene products; however, the repressive action of the cro gene product at high multiplicity of infection is not observed although some turn off at very late time is detected. To pin down this phage promoter, results described in this communication and those described elsewhere can rule out the promoter PI, PR, P'R, and the promoter PL also looks rather unlikely. No firm identification of this phage promoter has been made, but the promoter(s) in the b2 region (the b2 promoter) is proposed. The phage promoter responsible for beta-galacrosidase synthesis is shown to be a weak promoter, requires the Q gene product or one (or more) of the late gene products for activation, and the time of expression is very late.

Bacteriophage lambda↗

PubMed2001

Cell toxicity caused by products of the p(L) operon of bacteriophage lambda.

Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.

Bacterial Proteins↗

PubMed1984

DNA interactions during bacteriophage lambda site-specific recombination.

Extensive research on site-specific recombination has provided many details, particularly with respect to the protein-DNA interactions. However, very little is known about the molecular mechanism of recombination during synapsis and strand exchange. Presumably, these steps of recombination involve various forms of DNA-DNA, DNA-protein, and protein-protein interactions. One stage at which DNA-DNA interactions may be occurring is at the level of synapsis where the recombining DNAs are juxtaposed. In this paper we have presented evidence that homology-dependent DNA interactions do occur within the overlap region before strand exchange. This interaction is presumably at the synaptic stage of recombination. Furthermore, we have demonstrated that the homology-dependent interactions require that only one strand of attB have homology to attP. Another stage in recombination at which DNA-DNA interactions could occur is during strand exchange where complementary strands from the recombining parents are paired and resealed. We have also presented evidence that homology-dependent DNA interactions occur during strand exchange prior to the resealing of the strands and that disruption of this interaction results in nonreciprocal recombination. Taken together, these results suggest that DNA-DNA interactions during reciprocal site-specific recombination occur during at least two stages in the reaction.

Bacteriophage lambda↗