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 469 records · Page 26Linked to original sources

PubMed1989

Overexpression of N antitermination proteins of bacteriophages lambda, 21, and P22: loss of N protein specificity.

The N protein of bacteriophage lambda (N lambda) modifies Escherichia coli RNA polymerase in such a way that it transcribes through termination signals, a process called antitermination. N antitermination normally occurs only if the template contains a specific utilization or nut site upstream of the terminators and only in the presence of host-encoded Nus proteins. The lambda-related phages 21 and P22 produce N analogs, N21 and N22, but these require different nut sites and show a different pattern of functional interaction with one of the Nus factors, NusA, according to whether this protein is of E. coli or Salmonella origin (NusAEc or NusASal). We report the overproduction of N lambda, N21, or N22, each of which was induced by isopropyl-beta-D-thiogalactopyranoside at 37 degrees C from its cloned position downstream from ptac on a high-expression plasmid, each in a host that provided NusAEc or NusASal. Overproduction of each of these N proteins resulted in relaxed specificity for nut, which was shown by the ability to complement N mutants of heterologous phages; NusA specificity was determined by the N type that was present in these complementation tests. We also observed that excess N was able to suppress transcriptional polarity in the particular case of cloned 'trpA, the last gene of the tryptophan operon, although there was no effect on polarity within chromosomal trpE. Such polarity is attributed to the presence of cryptic intragenic terminators that become exposed in the absence of translation. Because there is no known nut site cis to 'trpA, we suggest that the 'trpA segment itself fortuitously contains a nut sequence that is able to function with excess N of any of the types tested and with either NusAEc or NusASal. We also found that excess N of any specificity, or even inactive N with missense mutation, could cause an increase in the level of NusAEc or NusASal, possibly because interaction between N and NusA, but independent of nut, whether functional or not, interferes with the autoregulation of NusA synthesis. These observations highlight the importance of protein concentration for the specificity of interactions both with other proteins and with nucleic acids. They also indicate that the interaction between N and NusA requires nut participation both for specificity and functionality.

Cloning, Molecular↗

PubMed1979

Specificity of the bacteriophage lambda N gene product (pN): nut sequences are necessary and sufficient for antitermination by pN.

We have cloned the nutR site together with the tR1 site of bacteriophage lambda in the E. coli galactose operon to examine whether the lambda promoter sequences PR and PL are involved in the recognition specificity of the lambda N gene product (pN). We first constructed a derivative of plasmid pBR322 in which the expression of the tetracycline genes (tet) is controlled by the gal promoter (Pgal). This new plasmid contains a unique Hind III site between Pgal and tet into which the nutR and tR1 sites were introduced. The order of the relevant genetic markers in this second plasmid is Pgal-nutR-tR1-tet. Cells transformed with this plasmid express tet only if pN is provided and if the plasmid contains an intact gal promoter. Our data suggest that transcription which originates at Pgal is modified by pN at nutR, enabling it to pass through tR1 into tet. We conclude that promoters do not play a specific role in pN recognition and that nut sequences are both necessary and sufficient for pN action.

Bacteriophage lambda↗

PubMed1984

Coupling with packaging explains apparent nonreciprocality of Chi-stimulated recombination of bacteriophage lambda by RecA and RecBC functions.

Chi (chi, 5'-GCTGGTGG) is a recombinator in RecA- and RecBC-mediated recombination in Escherichia coli. In vegetative recombination between two bacteriophage lambda strains, one with and the other without Chi (a+ chi +b- X a- chi 0b+), the chi-containing recombinant (a- chi +b-) is less abundant than the non-chi-containing recombinant (a+ chi 0b+). Previously this was taken was evidence for nonreciprocality of chi-stimulated exchange. This inequality, however, is now seen to result from an event at cos (lambda's packaging origin) that both activates Chi and initiates DNA packaging. An event at rightward cos leads to activation of leftward chi on the same chromosome for an exchange to its left. From the resulting circulating dimer (--cos-a+- chi 0-b+-cos-a-- chi +-b- --), the cos that activated chi is more likely to be used for rightward packaging initiation than is the cos from the other parent. Consistent with this coupling model is "biased packaging" in lambda carrying two cos sites per monomer genome. When their maturation is dependent on dimerization by chi-stimulated exchange, the phage particles result more often from packaging from the cos that activates chi than from packaging from the other cos. Since Chi activation and packaging can be uncoupled, we infer that some early and reversible step in packaging activates chi. A strong candidate for this step is a double-strand break at cos that provides an oriented entry site for a recombinase.

Bacterial Proteins↗

PubMed1998

Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda.

We describe the construction and characterization of a hepatitis C virus (HCV) cDNA expression library displayed as a fusion to the carboxy terminus of the capsid protein D of bacteriophage lambda. cDNA inserts were obtained by tagged random-priming of the HCV genome and cloned into a lambda vector from which chimeric phage bearing both wild-type D protein and D fusion products on the capsid surface were produced. The resulting library was affinity-selected with anti-HCV human monoclonal antibodies recognizing linear or conformational epitopes, and human sera from HCV-infected patients. Selection was monitored by immuno-screening experiments, ELISA, and sequence analysis of positive clones. The performance of this library was compared with two additional HCV cDNA display libraries generated as N-terminal fusions to the III and VIII capsid proteins of filamentous phage M13. The results obtained demonstrate the great potential of the lambda display system for constructing complex cDNA libraries for natural ligand discovery.

Antibodies, Monoclonal↗

PubMed1991

Structure of the bacteriophage lambda cohesive end site. Genetic analysis of the site (cosN) at which nicks are introduced by terminase.

A collection of mutations affecting the site (cosN) at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces nicks to generate mature lambda chromosomes has been studied. A good correlation was found for mutational effects on burst size, accumulation of unused proheads, packaging of DNA into heads and cos cutting by terminase in vitro, indicating that defective cosN cleavage by terminase is the molecular explanation for the phenotypic effects of the mutations. Although the base-pairs of cosN display partial twofold rotational symmetry, cosN was found to be asymmetric functionally. Certain mutations to the left side of the center of rotational symmetry have more pronounced phenotypic effects than rotationally symmetric mutations to the right. The cosN11G mutation has no phenotypic effects when present as a single mutation, but does affect DNA packaging and cosN cutting in the presence of the symmetrically disposed cosN2C mutation. Mutations that decrease cosN cleavage result in the accumulation of unexpanded proheads, indicating that prohead expansion depends on cosN cutting.

Bacteriophage lambda↗

PubMed1982

Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda.

Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein. Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site. Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed. Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells. The different applications of these plasmids for expression of cloned genes are discussed.

Bacteriophage lambda↗

PubMed1981

Multiply branched replicative intermediates in E. coli and bacteriophage lambda.

Multiple branched DNA fragments present in a fast sedimenting complex comprising a minute fraction of the E. coli genome have been isolated. Similar structures were also observed among bacteriophage lambda DNA replicative intermediates after infection of synchronized E. coli cells. These structures were found to be associated with the amino acid and thymidine starvation steps required for synchronization and originate either by initiation from secondary sites or by snap-back of daughter strands containing substantial single stranded regions in the vicinity of the growing point.

Bacteriophage lambda↗

PubMed1996

Synthesis of the bacteriophage lambda P protein in amino acid-starved Escherichia coli cells.

It was demonstrated previously that in isoleucine-starved Escherichia coli relA mutants harboring a plasmid derived from bacteriophage lambda the lambda O protein is not synthesized. However, a protein which coprecipited with the lambda O during immunoprecipitation with anti-lambda O serum was synthesized during the relaxed response. Here we found that this protein is the lambda P gene product. Despite significant inhibition of transcription from the pR promoter (which produces mRNA for the lambda P protein synthesis) during the stringent response, the lambda P protein was efficiently synthesized in relA- as well as relA+ strains starved for isoleucine, threonine and histidine, whereas the synthesis was negligible during starvation for arginine and leucine. The synthesis of the lambda P protein in amino acid-starved cells is sensitive to rifampicin. Thus we presume that this phenomenon is not caused by eventual increased stability of the lambda P mRNA but rather is an effect of preferential translation of this mRNA and incorporation of limited amount of amino acids arising in the starved cells as a result of intracellular proteolysis. One of possible explanations of the mechanism of this phenomenon may suggest that the same signals can be recognized in both prokaryotic and eukaryotic cells during initiation of translation at non-AUG codons.

Amino Acid Sequence↗

PubMed1979

The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic.

Nucleotide sequence has been determined for the restriction fragments and cloned DNA from the pL-N-tL1 region of bacteriophage lambda. A unique reading frame for the N gene is defined by the absence of natural nonsense codons and by the presence of seven nonsense codons generated by mutations in N. This reading frame is initiated at two alternative ATG codons, the second of which is probably the in vivo translation start. Reading is stopped at a single TAG codon. The protein coded is therefore 133 or, more probably, 107 amino acids long, rich in lysine, arginine and proline.

Bacteriophage lambda↗

PubMed1986

The production of generalized transducing phage by bacteriophage lambda.

Generalized transduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage lambda to assess some of the factors that affect that process. The results of those experiments indicate: the production of generalized transducing particles by bacteriophage lambda is inhibited by the phage lambda exonuclease (Exo). Also inhibited by lambda Exo is the production of lambda docR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of lambda docL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by lambda Exo; lambda-generalized transducing particles are not detected in induced lysis-defective (S-) lambda lysogens until about 60-90 min after prophage induction. Since wild-type lambda would normally lyse cells by 60 min, the production of lambda-generalized transducing particles depends on the phage being lysis-defective; if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10-20-fold range. In contrast, if transducing lysates are prepared by the induction of a lambda lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers; if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell. Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA. These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by lambda, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather recognize some feature of the DNA tht is sensitive to lambda exonuclease, such as a nick or a double-stranded cut.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage lambda↗

PubMed1982

High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter.

Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.

Antibody Specificity↗

PubMed1994

Synthesis and assembly of the F0 proton channel from F0 genes cloned into bacteriophage lambda and integrated into the Escherichia coli chromosome.

The promoter region and the first four genes of the Escherichia coli proton-translocating ATPase (unc) operon, uncIBEF, were cloned into bacteriophage lambda, enabling this region to be recombined into an unc-deleted E. coli chromosome at the lambda att site. The resultant E. coli strain, carrying single-copy F0 genes, was tested for synthesis and assembly of functional F0 proton channels. Membranes isolated from this strain contained all three F0 subunits and were capable of binding purified F1 and reconstituting F1F0-dependent energy coupling activities. The presence of these F0 sectors did not affect cell growth or membrane proton permeability assayed by fluorescence quenching. When compared with wild type membranes, membranes from the single-copy F0 strain contained less a and b subunits. When the single-copy lambda F0 strain was transformed with an F1 plasmid, the cells became phenotypically and biochemically Unc+, with membrane-bound ATPase and ATP synthase activities that were 50-60% of wild type. The results demonstrate that F0 produced from single-copy genes in the absence of F1 is membrane-bound and functional (i.e. reconstitutable) but not freely permeable to protons. The presence of F1 genes and/or subunits during F0 synthesis and assembly both increases the relative amounts of membrane-bound a and b subunits and produces an F0 sector more like that found in wild type cells than is produced from the single-copy F0 genes alone.

Adenosine Triphosphate↗

PubMed2000

Affinity selection of DNA-binding proteins displayed on bacteriophage lambda.

Two transcription factors, human ATF1, its DNA-binding domain (ATF1BD), and the DNA-binding domain (GAL4BD) of the yeast GAL4 protein, were displayed on the surface of bacteriophage lambda vectors and efficiently selected by DNA fragments immobilized in microtiter wells. The DNA-binding proteins are fused to the carboxy terminus of the tail protein gpV and head protein gpD of the vectors, lambdafoo and lambdafooDc, respectively. After a single round of affinity selection, the fusion phages were successfully enriched 60- to 4,000-fold over the vector phages. Further, the GAL4BD fusion phages were enriched 5- and 15-fold by affinity selection using specific DNA as probes over nonspecific DNA when expressed on lambdafooDc and lambdafoo, respectively. The ATF1BD fusion phages were also sequence-specifically enriched greater than 4-fold when displayed on lambdafoo. These results suggest that the lambdafoo display system is useful for in vitro studying of protein-DNA interactions and may be applied to screening of DNA-binding protein from complex cDNA libraries through DNA-binding affinity.

Activating Transcription Factor 1↗

PubMed1988

Activation of recF-dependent recombination in Escherichia coli by bacteriophage lambda- and P22-encoded functions.

Escherichia coli strains bearing wild-type and mutant alleles of various recombination genes, as well as plasmids that express recombination-related genes of bacteriophages lambda and P22, were tested for their proficiency as recipients in Hfr-mediated conjugation. It was found that the homologous recombination systems of both phages could promote recombination in a recB recC mutant host. In addition, the Abc function of P22, but not the Gam function of lambda, was found to inhibit recombination in a wild-type host; however, both Abc and Gam inhibited recombination in a recF mutant host. These observations are interpreted as indicating that the recombination systems of both phages, as well as the RecBCD-modulating functions Abc and Gam, all activate the RecF recombination pathway of E. coli.

Alleles↗

PubMed1984

A portable DNA sequence carrying the cohesive site (cos) of bacteriophage lambda and the mob (mobilization) region of the broad-host-range plasmid RK2: a module for the construction of new cosmids.

A polylinker DNA sequence carrying the cos site of bacteriophage lambda and the mob (oriT) region of the IncP group plasmid RK2 was constructed. This composite polylinker has EcoRI sites at both termini and also unique sites for ClaI, HindIII, PstI and XbaI. The cos-mob region is portable with the use of EcoRI or a combination of EcoRI with ClaI, HindIII or XbaI. Another cos-mob cassette was also constructed from which the cos-mob region can be lifted with HindIII, ClaI or either of these enzymes in combination with others. These cos-mob cassettes can be used in constructing new cosmids that can be mobilized into a variety of Gram-negative bacteria. Using one of these cassettes we have constructed a small IncW group cosmid (11.1 kb) that was mobilizable into Escherichia coli, Rhizobium spp. and Alcaligenes eutrophus at high frequency.

Alcaligenes↗

PubMed1980

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.

Bacteriophage lambda↗

PubMed1970

Control of transcription of the repressor gene in bacteriophage lambda.

The rate of transcription of the structural gene for repressor (cI gene) in bacteriophage lambda is controlled by the amount of active repressor in the cell. When the (reversibly) thermolabile repressor in a bacterium lysogenic for lambdacI(857) is inactivated by heat, the rate of repressor gene transcription immediately falls. If the repressor is renatured, synthesis of repressor messenger is immediately restored to a high rate. Control by the repressor of the rate of transcription of its own structural gene does not depend upon the expression of any other known gene because the control can be observed in an N(-)x(-)lysogen.Measurement of lambda repressor gene transcription was made possible by a two-step hybridization technique utilizing prehybridization to lambdadv plasmid DNA. Application of this technique also showed that the product of the N gene stimulates transcription of the r strand in the region of the cII, O, and P genes.

Coliphages↗

PubMed1976

On the nature of cis-acting regulatory proteins and genetic organization in bacteriophage: the example of gene Q of bacteriophage lambda.

We note the existence of a "partially cis-acting" regulatory protein of bacteriophage lambda: the product of the phage Q gene. We suggest that there may be a complete spectrum from "all cis" to "all trans" for such regulatory proteins. This behavior might arise because a DNA-binding protein either acts at a nearby (cis) site soon after synthesis or becomes "lost" for its trans activity on another genome through nonspecific interactions with DNA. Our proposed explanation provides one evolutionary basis for the linkage of genes for regulatory proteins and the sites at which such proteins act; it also suggests a possible rationale for the "metabolic instability" of certain regulatory proteins.

Coliphages↗