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PubMed1986

A study of phi X-174 DNA torus and lambda DNA torus tertiary structure and the implications for DNA self-assembly.

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386-48000 bp). With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.

Bacteriophage lambda↗
PubMed1979

Bacteriophage lambda-E. coli K12 vector-host system for gene cloning and expression under lactose promoter control: I. DNA fragment insertion at the lacZ EcoRI restriction site.

Bacteriophage lambda vectors, derived from lambda plac5 were constructed. Their genomes have only one EcoRI restriction site, located near the end of the beta-galactosidase gene. Recombinants, constructed in vitro, having a DNA fragment inserted in the EcoRI site, are lac- and can be easily recognized. Expression of such foreign genes is then under the control of the lac promoter. Mutations Qam73 and Sam7 greatly increase the amount of beta-galactosidase synthesized by the vector bacteriophage. The lambda ZEQS vector has been certified B2 (EK2) by the French control commission "Recombinaisons génétiques in vitro".

Coliphages↗
PubMed2003

A conformational switch controls the DNA cleavage activity of lambda integrase.

The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements. We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond. In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis. A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA. Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site. The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule. The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.

Bacteriophage lambda↗
PubMed1989

Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda.

A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire. The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation. It was possible to generate, in 2 weeks, large numbers of monoclonal Fab fragments against a transition state analog hapten. The methods described may supersede present-day hybridoma technology and facilitate the production of catalytic and other antibodies.

Amino Acid Sequence↗
PubMed1985

Evidence that ribosomal protein S10 itself is a cellular component necessary for transcription antitermination by phage lambda N protein.

Bacteriophage lambda N gene product acts to modify host RNA polymerase allowing the formation of a termination-resistant transcription apparatus. Previous studies have demonstrated that the nusE71 mutation that has altered the ribosomal protein S10 prevents N action in vivo. Using a coupled transcription-translation system, we demonstrate here that purified S10 protein as well as the 30S ribosomal subunit is sufficient to restore N activity in the nusE mutant extract, allowing antitermination of Rho-dependent and Rho-independent terminators. This provides direct biochemical evidence that the S10 protein itself is one of the cellular components necessary for the formation of an antitermination apparatus.

Bacterial Proteins↗
PubMed1993

A comparison of human and bovine Escherichia coli O157:H7 isolates by toxin genotype, plasmid profile, and bacteriophage lambda-restriction fragment length polymorphism profile.

Foods of bovine origin have been linked to human disease outbreaks caused by Escherichia coli O157:H7 and may be linked to the more common sporadic cases as well. In this study, E. coli O157:H7 from the bovine reservoir (22 isolates: 12 from dairy and 10 from beef breed cows) and from human patients (50 isolates from sporadic human infections) were compared using Shiga-like toxin genotypes, plasmid profiles, and DNA restriction fragment length polymorphisms identified with a bacteriophage lambda probe (lambda-RFLP). Twenty-three lambda-RFLP profiles, 4 Shiga-like toxin genotypes, and 8 plasmid profiles were identified among the isolates tested. Together the typing methods distinguished 43 strains, of which 3 were isolated from both humans (5 isolates) and cattle (6 isolates; 5 from dairy herds). These data demonstrate the value of lambda-RFLP as a means of strain identification for E. coli O157:H7.

Animals↗
PubMed1981

Cell lysis by induction of cloned lambda lysis genes.

The lysis gene region of bacteriophage lambda, including genes S, R, and Rz, was cloned into the plasmid pBH20. In the recombinant plasmid, the lysis genes are expressed under the control of the lacOP region. Induction of this "lysis operon" with the lac inducer, IPTG, under conditions where transcription from the lacOP region is not subject to catabolite repression, results in a sharply defined lysis after 35 min. Premature lysis can be accomplished by cyanide, chloramphenicol, or chloroform, exactly as in bacteriophage lambda infected cells. The lysis gene region of an S- mutant was also cloned into pBH20. Induction of the S- lysis operon has no apparent effect on culture growth; however, large quantities of bacteriolytic activity accumulate intracellularly. Neither cyanide nor chloramphenicol causes lysis in the induced S- clones. Thus premature lysis appears to be entirely an S-dependent phenomenon. A model for the control of lysis in bacteriophage lambda infections is presented in which it is the accumulation of the S gene product in competition with a host "anti-S" protein that determines lysis timing.

Bacteriophage lambda↗
PubMed2006

The cytotoxic activity of the bacteriophage lambda-holin protein reduces tumour growth rates in mammary cancer cell xenograft models.

BACKGROUND: The potential use of gene therapy for cancer treatment is being intensively studied. One approach utilises the expression of genes encoding cytotoxic proteins. Such proteins can affect cellular viability, for example by inhibiting the translation machinery or disturbing membrane integrity. The bacteriophage Lambda (lambda)-holin protein is known to form a lesion in the cytoplasmic membrane of E. coli, triggering bacterial cell lysis and thereby enabling the release of new bacteriophage particles. The aim of this study was to evaluate whether the lambda-holin protein has a cytotoxic impact on eukaryotic cells and whether it holds potential as a new therapeutic protein for cancer gene therapy. METHODS: To explore this possibility, stably transfected human cell lines were established that harbour a tetracycline (Tet)-inducible system for controlled expression of the lambda-holin gene. The effect of the lambda-holin protein on eukaryotic cells was studied in vitro by applying several viability assays. We also investigated the effect of lambda-holin gene expression in vivo using a human breast cancer cell tumour xenograft as well as a syngeneic mammary adenocarcinoma mouse model. RESULTS: The lambda-holin-encoding gene was inducibly expressed in eukaryotic cells in vitro. Expression led to a substantial reduction of cell viability of more than 98%. In mouse models, lambda-holin-expressing tumour cell xenografts revealed significantly reduced growth rates in comparison to xenografts not expressing the lambda-holin gene. CONCLUSIONS: The lambda-holin protein is cytotoxic for eukaryotic cells in vitro and inhibits tumour growth in vivo suggesting potential therapeutic use in cancer gene therapy.

Animals↗
PubMed1978

Adjacent insertion sequences IS2 and IS5 in bacteriophage Mu mutants and an IS5 in a lambda darg bacteriophage.

Using electron microscopic heteroduplex analysis, we have demonstrated that an insertion found in a Mu prophage and in some infectious. Mu deletion-substitution mutants derived from it consists of bacterial insertion sequence IS2 linked directly to IS5. Other infectious Mu mutants derived from the same lysogen have only IS5 or a portion of IS2. In addition, we have found that an independent insertion in a transducing phage, lambda 13 dargB2, is IS5. The ends of IS5 are short, inverted duplications of each other. These observations support the notion that the DNA insertion previously designated IS5 on the basis of a single example in lambda KH100 is a bona fide bacterial insertion sequence.

Coliphages↗
PubMed1989

A simple purification procedure for lambda gt bacteriophage DNA with hybridization size screening for isolation of longest length cDNA clones.

An improved procedure for isolating lambda DNA and screening lambda gt10 or lambda gt11 libraries is described. Recombinant lambda gt11 bacteriophage particles (150,000) were amplified on three agarose plates (50,000 per plate) with Escherichia coli Y1090 as plating bacteria. After confluent lysis, recombinant bacteriophage was extracted with SM buffer. Bacterial debris was removed by centrifugation. A small aliquot of amplified lambda gt11 bacteriophage was kept to rescreen the bacteriophage, should a large or full-length clone be found to be present, after analysis of the size of the cDNA inserts. The major portion of the bacteriophage particles was purified by treatment with equilibrated DEAE-cellulose, pH 7.5. Purified phage particles were precipitated with polyethylene glycol from the DEAE supernatant and extracted with phenol, phenol-chloroform, and chloroform. Such lambda gt11 DNA was readily digested with EcoRI. Liberated insert cDNA was separated on 1.2% agarose gels, transferred onto a nylon membrane, and hybridized with an alkaline phosphatase cDNA probe in an iterative procedure that allows isolation of the largest cDNA clones present in the library. We have used this procedure to isolate a full-length alkaline phosphatase cDNA. The method is quick, reliable, and less costly than conventional procedures for the isolation of full-length cDNAs.

Bacteriophage lambda↗
PubMed2004

Complex spatial distribution and dynamics of an abundant Escherichia coli outer membrane protein, LamB.

Advanced techniques for observing protein localization in live bacteria show that the distributions are dynamic. For technical reasons, most such techniques have not been applied to outer membrane proteins in Gram-negative bacteria. We have developed two novel live-cell imaging techniques to observe the surface distribution of LamB, an abundant integral outer membrane protein in Escherichia coli responsible for maltose uptake and for attachment of bacteriophage lambda. Using fluorescently labelled bacteriophage lambda tails, we quantitatively described the spatial distribution and dynamic movement of LamB in the outer membrane. LamB accumulated in spiral patterns. The distribution depended on cell length and changed rapidly. The majority of the protein diffused along spirals extending across the cell body. Tracking single particles, we found that there are two populations of LamB--one shows very restricted diffusion and the other shows greater mobility. The presence of two populations recalls the partitioning of eukaryotic membrane proteins between 'mobile' and 'immobile' populations. In this study, we have demonstrated that LamB moves along the bacterial surface and that these movements are restricted by an underlying dynamic spiral pattern.

Bacterial Outer Membrane Proteins↗
PubMed2000

A bacteriophage lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease.

Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.

Amino Acid Sequence↗
PubMedDate not supplied

Preparation of a detailed restriction map of the avian leukosis virus MAV-2(O).

Unintegrated MAV-2(O) DNA was isolated from infected chicken embryo fibroblasts and inserted into the lambda bacteriophage vector lambda gtWES lambda B. Three x 10(6) bacteriophage plaques were screened, yielding a total of seven clones, six of which contained DNA representing the complete MAV-2(O) genome. Viral DNA was isolated from four of the clones and was used to transfect chicken embryo fibroblasts. All four clones produced virus as monitored by reverse transcriptase assay. When the four cloned viruses were inoculated into 10-day-old embryos, all hatched chickens developed osteopetrosis. One clone, lambda 9, induced osteopetrosis at a rate of onset and severity identical to that induced by the MAV-2(O) parental stock. This clone was selected for further study. To facilitate restriction mapping, the viral DNA from lambda 9 was subcloned into plasmid vector pUC 12 to construct a plasmid called p9. Cleavage of p9 DNA with single and multiple restriction endonucleases and hybridization with gene-specific probes identified the restriction fragments obtained. A comprehensive restriction map of cloned MAV-2(O) was generated and is compared with published maps and sequences of other avian retroviruses.

Animals↗
PubMed1968

Disruption of T-even bacteriophages by dimethyl sulfoxide.

Dimethyl sulfoxide (DMSO) disrupted T-even bacteriophages as well as lambda bacteriophage. The component substructures of T2L, T4B01, or T6, in particular heads, were readily isolated after treatment with 67% DMSO (v/v). In contrast, concentrations of DMSO above 50% not only separated heads from tails of bacteriophage lambda but led to degradation of the lambda heads. Examination of the isolated free heads of T-even bacteriophage indicated that a distinct neck substructure was attached to one apex of the head. On some free tails a similar neck substructure was also found at the proximal end of the sheath. The dimensions of this neck substructure were found to be about 130 by 180 A; by virtue of its size and morphological attachment to the free heads, it was concluded that this was a distinct substructure and not an extension of the tail tube.

Coliphages↗
PubMed1987

Mismatch repair and recombination in E. coli.

The involvement of the E. coli methyl-directed and very short patch (vsp) mismatch repair systems in bacteriophage lambda recombination has been studied. Genetic crosses and heteroduplex transfection experiments were performed using lambda phages with sequenced mutations in the cl gene. The results indicate that methyl-directed repair does operate during bacteriophage lambda recombination but generally does not contribute to the formation of recombinants involving close markers. Vsp repair apparently acts during bacteriophage lambda recombination to produce recombinants involving close markers because its action does not involve extensive excision tracts. Marker-specific hyperrecombination and the apparent clustering of genetic exchanges in bacteriophage lambda recombination can be accounted for by the action of the vsp repair system.

Bacteriophage lambda↗
PubMed1987

Enzymology of the pre-priming steps in lambda dv DNA replication in vitro.

We have examined some of the early pre-priming steps of bacteriophage lambda dv DNA replication in vitro. Previous experiments have shown that bacteriophage lambda replication requires host RNA polymerase-dependent RNA synthesis near or at the origin of replication (ori lambda) to initiate DNA synthesis. Using a crude Fraction II enzymatic system we have shown that during RNA polymerase action, at least the bacteriophage lambda O and lambda P replication proteins as well as the host dnaB protein must be present to initiate ori lambda-specific DNA replication. The presence of three other host initiation proteins, dnaG primase, dnaJ, and dnaK, is not required during RNA polymerase action. Because of the apparent absence of a requirement for the dnaJ and dnaK pre-priming proteins during the transcriptional activation step, we propose that the early events of lambda dv DNA replication, prior to action by the dnaG primase, can be divided into two recognizable steps: an early step which requires at least RNA polymerase, lambda O, lambda P, and dnaB, and a subsequent step which requires the action of at least the dnaJ and dnaK proteins.

Bacterial Proteins↗