Publications

fragmentation of the crispr-cas type i-b signature protein cas8b.	crispr arrays are transcribed into long precursor rna species, which are further processed into mature crispr rnas (crrnas). cas proteins utilize these crrnas, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. type i crispr-cas systems are defined by the presence of different cascade interference complexes containing large and small subunits that play major roles during target dna selection.	2017	28238733
display of hiv-1 envelope protein on lambda phage scaffold as a vaccine platform.	the generation of a strong antibody response to target antigens is a major goal for vaccine development. here we describe the display of the human immunodeficiency virus (hiv) envelope spike protein (env) on a virus-like scaffold provided by the lambda phage capsid. phage vectors, in general, have advantages over mammalian virus vectors due to their genetic tractability, inexpensive production, suitability for scale-up, as well as their physical stability, making them an attractive vaccine platf ...	2017	28374253
dna topology and the initiation of virus dna packaging.	during progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. for large dsdna viruses, including tailed bacteriophages and herpesviruses, immature viral dna is recognized and translocated into a preformed icosahedral shell, the prohead. recognition involves specific interactions between the viral packaging enzyme, terminase, and viral dna recognition sites. generally, viral dna is recognized by terminase's small subunit (ters). the ...	2016	27144448
why be temperate: lessons from bacteriophage λ.	many pathogens have evolved the ability to induce latent infections of their hosts. the bacteriophage λ is a classical model for exploring the regulation and the evolution of latency. here, i review recent experimental studies on phage λ that identify specific conditions promoting the evolution of lysogenic life cycles. in addition, i present specific adaptations of phage λ that allow this virus to react plastically to variations in the environment and to reactivate its lytic life cycle. all of ...	2016	26946976
ecological speciation of bacteriophage lambda in allopatry and sympatry.	understanding the conditions that allow speciation to occur is difficult because most research has focused on either long-lived organisms or asexual microorganisms. we propagated bacteriophage λ, a virus with rapid generations and frequent recombination, on two escherichia coli host genotypes that expressed either the lamb or ompf receptor. when supplied with either single host (allopatry), phage λ improved its binding to the available receptor while losing its ability to use the alternative. wh ...	2016	27884940
carriage of λ latent virus is costly for its bacterial host due to frequent reactivation in monoxenic mouse intestine.	temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. although these prophages are generally considered to increase their hosts' fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. here, we investigated the impact of prophage carriage in the gastroi ...	2016	26871586
influence of internal dna pressure on stability and infectivity of phage λ.	viruses must remain infectious while in harsh extracellular environments. an important aspect of viral particle stability for double-stranded dna viruses is the energetically unfavorable state of the tightly confined dna chain within the virus capsid creating pressures of tens of atmospheres. here, we study the influence of internal genome pressure on the thermal stability of viral particles. using differential scanning calorimetry to monitor genome loss upon heating, we find that internal press ...	2015	26254570
exploring the balance between dna pressure and capsid stability in herpesviruses and phages.	we have recently shown in both herpesviruses and phages that packaged viral dna creates a pressure of tens of atmospheres pushing against the interior capsid wall. for the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized dna from the capsid. furthermore, using a new calorimetric assay to accurately determine the temperature inducing dna release, we found a direct influence of internal dna pressure on the stability of th ...	2015	26136570
bacteriophage lambda: early pioneer and still relevant.	molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of dna virus assembly and of the molecular nature of lysogeny. the development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their effor ...	2015	25742714
bacteriophage spp1 tail tube protein self-assembles into β-structure-rich tubes.	the majority of known bacteriophages have long tails that serve for bacterial target recognition and viral dna delivery into the host. these structures form a tube from the viral capsid to the bacterial cell. the tube is formed primarily by a helical array of tail tube protein (ttp) subunits. in phages with a contractile tail, the ttp tube is surrounded by a sheath structure. here, we report the first evidence that a phage ttp, gp17.1 of siphophage spp1, self-assembles into long tubes in the abs ...	2015	25525268
recombination promoted by dna viruses: phage λ to herpes simplex virus.	the purpose of this review is to explore recombination strategies in dna viruses. homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. recombination and dna replication are interconnected, with recombination being essential for repairing dna damage and supporting replication of the viral genome. recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understan ...	2014	25002096
enhanced cell immune responses to hepatitis c virus core by novel heterologous dna prime/lambda nanoparticles boost in mice.	hepatitis c virus (hcv) is a worldwide problem which does not have an effective vaccine and more than 170 million people worldwide are chronically infected by hcv. t cell responses are associated with spontaneous clearance of hcv infection. we report here the development of recombinant lambda bacteriophage nanoparticles encoding hcv core antigen. the aim of this study was to investigate the antigen-specific immune responses triggered in mice by different prime-boost combinations of dna and lambd ...	2014	24752903
stochastic holin expression can account for lysis time variation in the bacteriophage λ.	the inherent stochastic nature of biochemical processes can drive differences in gene expression between otherwise identical cells. while cell-to-cell variability in gene expression has received much attention, randomness in timing of events has been less studied. we investigate event timing at the single-cell level in a simple system, the lytic pathway of the bacterial virus phage λ. in individual cells, lysis occurs on average at 65 min, with an s.d. of 3.5 min. interestingly, mutations in the ...	2014	24718449
a touch of glue to complete bacteriophage assembly: the tail-to-head joining protein (thjp) family.	bacteriophage spp1 is a nanomachine built to infect the bacterium bacillus subtilis. the phage particle is composed of an icosahedric capsid, which contains the viral dna, and a long non-contractile tail. capsids and tails are produced in infected cells by two distinct morphogenetic pathways. characterization of the suppressor-sensitive mutant spp1sus82 showed that it produces dna-filled capsids and tails but is unable to assemble complete virions. its purified tails have a normal length but lac ...	2014	24443902
λ phage nanobioparticle expressing apoptin efficiently suppress human breast carcinoma tumor growth in vivo.	using phages is a novel field of cancer therapy and phage nanobioparticles (nbps) such as λ phage could be modified to deliver and express genetic cassettes into eukaryotic cells safely in contrast with animal viruses. apoptin, a protein from chicken anemia virus (cav) has the ability to specifically induce apoptosis only in carcinoma cells. we presented a safe method of breast tumor therapy via the apoptin expressing λ nbps. here, we constructed a λ zap-cmv-apoptin recombinant nbp and investiga ...	2013	24278212
recombinant λ bacteriophage displaying nanobody towards third domain of her-2 epitope inhibits proliferation of breast carcinoma skbr-3 cell line.	phage display of many nanobodies via filamentous phage in combination with helper phage has been reported by many scientists. the aim of this study was to produce lambda (λ) bacteriophage displaying high-affinity nanobody against her-2 expressing breast carcinoma cells. bacteriophage λ is a temperate phage with inherent biological safety in mammalian cells. here we report the construction of a recombinant λ phage that efficiently expresses specific nanobody towards third domain of her-2 target o ...	2013	23224340
improved quantitative pcr protocols for adenovirus and cmv with an internal inhibition control system and automated nucleic acid isolation.	with the establishment of routine virus load (dnaemia) screening for human adenovirus (hadv) and cytomegalovirus (cmv) in post-transplant care quality standards for quantitative pcr-assays are increasing. established real-time pcr assays were improved with a fully automated dna-extraction and with a competitive internal control dna packaged into a lambda phage which serves as an extraction and amplification control in each sample. hadv and cmv dna were detected and quantified simultaneously in v ...	2012	22499011
competing pathways control host resistance to virus via trna modification and programmed ribosomal frameshifting.	viral infection depends on a complex interplay between host and viral factors. here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, trna modification, competitive binding, and programmed ribosomal frameshifting (prf). we first demonstrate that the iron-sulfur cluster biosynthesis pathway in escherichia coli exerts a protective effect during lambda phage infection, while a trna thiolation pathway enhances viral infection. we show that trna(lys) uridine ...	2012	22294093
repeatability and contingency in the evolution of a key innovation in phage lambda.	the processes responsible for the evolution of key innovations, whereby lineages acquire qualitatively new functions that expand their ecological opportunities, remain poorly understood. we examined how a virus, bacteriophage λ, evolved to infect its host, escherichia coli, through a novel pathway. natural selection promoted the fixation of mutations in the virus's host-recognition protein, j, that improved fitness on the original receptor, lamb, and set the stage for other mutations that allowe ...	2012	22282803
plug-and-play, infrared, laser-mediated pcr in a microfluidic chip.	microfluidic polymerase chain reaction (pcr) systems have set milestones for small volume (100 nl-5 μl), amplification speed (100-400 s), and on-chip integration of upstream and downstream sample handling including purification and electrophoretic separation functionality. in practice, the microfluidic chips in these systems require either insertion of thermocouples or calibration prior to every amplification. these factors can offset the speed advantages of microfluidic pcr and have likely hind ...	2012	22218821
expression, purification, and characterization of authentic mouse prolactin obtained in escherichia coli periplasmic space.	prolactin (prl) is a pleiotropic hormone produced by lactotroph cells of the anterior pituitary gland and is mainly related to lactation control and reproduction. recombinant mouse prolactin (r-mprl), never obtained in its authentic form, can be very useful for research and tests in animal models, in which human prolactin (hprl) is usually employed in a heterologous mode. synthesis of r-mprl was carried out here via secretion in escherichia coli periplasmic space using a plasmid containing mprl ...	2012	23586827
capstan friction model for dna ejection from bacteriophages.	bacteriophages infect cells by attaching to the outer membrane and injecting their dna into the cell. the phage dna is then transcribed by the cell's transcription machinery. a number of physical mechanisms by which dna can be translocated from the phage capsid into the cell have been identified. a fast ejection driven by the elastic and electrostatic potential energy of the compacted dna within the viral capsid appears to be used by most phages, at least to initiate infection. in recent in vitr ...	2012	23368388
recovery of small dna fragments from serum using compaction precipitation.	while most nucleic acids are intracellular, trace amounts of deoxyribonucleic acid (dna) and ribonucleic acid (rna), including micro rnas, can also be found in peripheral blood. many studies have suggested the potential utility of these circulating nucleic acids in prenatal diagnosis, early cancer detection, and the diagnosis of infectious diseases. however, dna circulating in blood is usually present at very low concentrations (ng/ml), and is in the form of relatively small fragments (<1,000 bp ...	2012	23284792
hsm - a hybrid system based approach for modelling intracellular networks.	the paper proposes a hybrid system based approach for modelling of intracellular networks and introduces a restricted subclass of hybrid systems - hsm - with an objective of still being able to provide sufficient power for the modelling of biological systems, while imposing some restrictions that facilitate analysis of systems described by such models. the use of hybrid system based models has become increasingly popular, likely due to the facts that: 1) they provide sufficiently powerful mathem ...	2012	23266641
graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on λ phage.	as native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. in this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, egfp. to achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the ...	2012	23027705
thermodynamic characterization of viral procapsid expansion into a functional capsid shell.	the assembly of "complex" dna viruses such as the herpesviruses and many tailed bacteriophages includes a dna packaging step where the viral genome is inserted into a preformed procapsid shell. packaging triggers a remarkable capsid expansion transition that results in thinning of the shell and an increase in capsid volume to accept the full-length genome. this transition is considered irreversible; however, here we demonstrate that the phage λ procapsid can be expanded with urea in vitro and th ...	2012	22365932
canine hepacivirus ns3 serine protease can cleave the human adaptor proteins mavs and trif.	canine hepacivirus (chv) was recently identified in domestic dogs and horses. the finding that chv is genetically the virus most closely related to hepatitis c virus (hcv) has raised the question of whether hcv might have evolved as the result of close contact between dogs and/or horses and humans. the aim of this study was to investigate whether the ns3/4a serine protease of chv specifically cleaves human mitochondrial antiviral signaling protein (mavs) and toll-il-1 receptor domain-containing ...	2012	22870331
Logical Modelling of Gene Regulatory Networks with GINsim.	Discrete mathematical formalisms are well adapted to model large biological networks, for which detailed kinetic data are scarce. This chapter introduces the reader to a well-established qualitative (logical) framework for the modelling of regulatory networks. Relying on GINsim, a software implementing this logical formalism, we guide the reader step by step towards the definition and the analysis of a simple model of the lysis-lysogeny decision in the bacteriophage ?.	2012	22144167
challenging packaging limits and infectivity of phage λ	the terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. yet, these motors are hindered at the end of the packaging process by the progressive buildup of a force-resisting packaging associated with already packaged dna. in this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be ...	2011	22108169
gold micro-flowers: one-step fabrication of efficient, highly reproducible surface-enhanced raman spectroscopy platform.	we present a new method enabling simultaneous synthesis and deposition of gold micro-flowers (aumfs) on solid substrates in a one-pot process that uses two reagents, auric acid and hydroxylamine hydrochloride, in aqueous reaction mixture. the aumfs deposited onto the substrate form mechanically stable gold layer of expanded nanostructured surface. the morphology of the aumfs depends on and can be controlled by the composition of the reaction solution as well as by the reaction time. the nanostru ...	2011	22081763
effects of macromolecular crowding on genetic networks.	the intracellular environment is crowded with proteins, dna, and other macromolecules. under physiological conditions, macromolecular crowding can alter both molecular diffusion and the equilibria of bimolecular reactions and therefore is likely to have a significant effect on the function of biochemical networks. we propose a simple way to model the effects of macromolecular crowding on biochemical networks via an appropriate scaling of bimolecular association and dissociation rates. we use thi ...	2011	22208186
a novel gateway®-compatible binary vector allows direct selection of recombinant clones in agrobacterium tumefaciens.	abstract:	2011	22145613
Chemical coupling as a potent strategy for preparation of targeted bacteriophage-derived gene nanocarriers into eukaryotic cells.	The ability to direct efficiently and specifically carriers toward target cells and express the transgene of interest is a critical step in gene therapy trails. The display of targeting molecules on the surface of phage particles might represent a potent solution. In the present study, we evaluated a chemical coupling strategy for displaying human holotransferrin as a targeting molecule on the surface of phage lambda particles for specifically delivering green fluorescent protein (GFP) encoding ...	2011	22002551
Quantification of trace-level DNA by real-time whole genome amplification.	Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole ...	2011	22174862
Following cell-fate in E. coli after infection by phage lambda.	The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant). We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells. Here, we d ...	2011	22025187
A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.	Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. He ...	2011	21858126
fractal dimension of an intrinsically disordered protein: small-angle x-ray scattering and computational study of the bacteriophage λ n protein.	small-angle x-ray scattering (saxs) was used to characterize the bacteriophage λ n protein, a 107 residue intrinsically disordered protein (idp) that functions as a transcriptional antitermination factor. the saxs data were used to estimate both the average radius of gyration and the fractal dimension, a measure of the protein's internal scaling properties, under a variety of solution conditions. in the absence of denaturants, the radius of gyration was 38 ± 3.5 å and the fractal dimension was 1 ...	2011	21936008
energy-independent helicase activity of a viral genome packaging motor.	the assembly of complex double-stranded dna viruses includes a genome packaging step where viral dna is translocated into the confines of a preformed procapsid shell. in most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (dna maturation) and translocation of the duplex into the capsid (dna packaging). bacteriophage λ terminase site-specific ...	2011	22191393
bacteria-based in vivo peptide library screening using biopanning approach.	traditionally, library screening has been performed to identify biologically active agents including small molecules or peptides that inhibit target proteins or molecules with therapeutic interests. due to its chemical nature, library screening is usually performed under in vitro environments using purified proteins and molecules. however, active agents identified from in vitro screenings often fail to exhibit biological activities in cells. to overcome this inherent limitation, we have develope ...	2011	22068505
monoclonal antibodies bind a snp-sensitive epitope that is present uniquely in mycobacterium avium subspecies paratuberculosis.	due to a close genetic relatedness, there is no known antibody that detects mycobacterium avium subspecies paratuberculosis (map), which causes johne's disease in cattle and sheep, and does not cross-react with other m. avium subspecies. in the present study, a monoclonal antibody (mab; 17a12) was identified from mice immunized with a cell membrane fraction of map strain k-10. this antibody is 100% specific as it detected a 25-kda protein in all 29 map whole cell lysates, but did not bind to any ...	2011	21845186
the bacteriophage lambda gpnu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst.	procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. the essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded dna viruses. typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. the scaffolding proteins are essential to ...	2011	21821043
Peptide vaccination is superior to genetic vaccination using a recombineered bacteriophage ? subunit vaccine.	Genetic immunization holds promise as a vaccination method, but has so far proven ineffective in large primate and human trials. Herein, we examined the relative merits of genetic immunization and peptide immunization using bacteriophage ?. Bacteriophage ? has proven effective in immune challenge models using both immunization methods, but there has never been a direct comparison of efficacy and of the quality of immune response. In the current study, this vector was produced using a combination ...	2011	22210400
Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis.	Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced ß-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Ex ...	2011	21893587
Protection of mice against Chlamydophila abortus infection with a bacteriophage-mediated DNA vaccine expressing the major outer membrane protein.	A bacteriophage-delivered DNA vaccine against Chlamydophila abortus was constructed by cloning a eukaryotic cassette containing the ompA gene (which expresses the Major Outer Membrane Protein) into a bacteriophage lambda vector. Four groups, each of 20 BALB/c mice were inoculated separately with the phage vaccine, a conventional DNA vaccine based on the same ompA expression cassette, a live attenuated vaccine (strain 1B) or the empty phage vector. The phage and DNA vaccines and empty phage vecto ...	2011	21872342
Deletion of one nucleotide within the homonucleotide tract present in the hsdS gene alters the DNA sequence specificity of type I restriction-modification system NgoAV.	As a result of a frameshift mutation, the hsdS locus of the NgoAV type IC restriction and modification (RM) system comprises two genes, hsdS(NgoAV1) and hsdS(NgoAV2). The specificity subunit, HsdS(NgoAV), the product of the hsdS(NgoAV1) gene, is a naturally truncated form of an archetypal specificity subunit (208 N-terminal amino acids instead of 410). The presence of a homonucleotide tract of seven guanines (poly[G]) at the 3' end of the hsdS(NgoAV1) gene makes the NgoAV system a strong candida ...	2011	21984785
[anaerobic synthesis of succinic acid by escherichia coli strains with activated nad+ reducing pyruvate dehydrogenase complex].	effect of constitutive expression of the aceef-lpda operon genes coding for the enzymes of nad+ reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acids from glucose by recombinant escherichia coli strains was studied. basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids by deletion of the genes acka, pta, poxb, and ldha (sgmo.1) in e. coli strain mg 1655 cells and additional introduction of the bacillus s ...	2011	21950115
The ß-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the a-subunit genes.	ß-Conglycinin, a major seed protein in soybean, is composed of a, a', and ß subunits sharing a high homology among them. Despite its many health benefits, ß-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the a, a', and ß subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, 'QT2', lacks all of the ß-conglycinin subunits, and the deficien ...	2011	22193750
the protein interaction map of bacteriophage lambda.	bacteriophage lambda is a model phage for most other dsdna phages and has been studied for over 60 years. although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. for a complete understanding we need to know all interactions among its proteins. we have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. we set out to find out how many protein-prot ...	2011	21943085
Fitting experimental transcription data with a comprehensive template-dependent modular kinetic model.	In the companion article, we developed a modular scheme for representing the kinetics of transcription elongation by RNA polymerase. As an example of how to use these approaches, in this article we use a comprehensive modular model of this sort to fit experimental transcript elongation results obtained on the canonical tR2 template of phage ? by means of complementary bulk gel electrophoresis and surface plasmon resonance assays. The gel electrophoresis results, obtained in experiments quenched ...	2011	21889454
Active Bax and Bak are functional holins.	The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. Here, we express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the ? bacteriophage (?) holin. As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysin, a muralytic enzyme, escapes the cytoplas ...	2011	22006182
multilevel autoregulation of λ repressor protein ci by dna looping in vitro.	the prophage state of bacteriophage λ is extremely stable and is maintained by a highly regulated level of λ repressor protein, ci, which represses lytic functions. ci regulates its own synthesis in a lysogen by activating and repressing its promoter, p(rm). ci participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening dna. we investigated the roles of each individual site under conditions that permitted dna loop forma ...	2011	21873207
revisiting the nmr structure of the ultrafast downhill folding protein gpw from bacteriophage λ.	gpw is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. previous nmr studies revealed a novel α+β fold for this protein. recent experiments have shown that gpw folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding). these features make gpw a highly desirable target for further experimental and computational folding studies. as a step in that direction, we have re-determined the high-resolution structure of gpw by multidim ...	2011	22087227
reengineering cro protein functional specificity with an evolutionary code.	cro proteins from different lambdoid bacteriophages are extremely variable in their target consensus dna sequences and constitute an excellent model for evolution of transcription factor specificity. we experimentally tested a bioinformatically derived evolutionary code relating switches between pairs of amino acids at three recognition helix sites in cro proteins to switches between pairs of nucleotide bases in the cognate consensus dna half-sites. we generated all eight possible code variants ...	2011	21945527
[verification of a decrease in the rigidity of the phage lambda dna polymeric chain in low ionic strength aqueous solutions by testing the polymer-polymer interlink interactions].	changes in the rigidity of the polymetric chain of phage lambda double-strand dna have been studied by laser correlation spectroscopy. it was shown that, as the ionic strength increases, the effect of the screening of the hydrodynamic interaction of the links of the polymeric chain specific for polymeric coils arises in a dna solution. it is assumed that the screening occurs when the threshold of the overlapping of dna coils is achieved. the overlapping of coils is the result of a previously obs ...	2011	21950067
linear nicking endonuclease-mediated strand-displacement dna amplification.	we describe a method for linear isothermal dna amplification using nicking endonuclease-mediated strand displacement by a dna polymerase. the nicking of one strand of a dna target by the endonuclease produces a primer for the polymerase to initiate synthesis. as the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. the combined continuous repetitive action of nicking by the endonuclease and strand-displacement synt ...	2011	21342654
domain interactions of the transcription-translation coupling factor escherichia coli nusg are intermolecular and transient.	the bacterial transcription factor nusg (n-utilization substance g) is suggested to act as a key coupling factor between transcription and translation [burmann, schweimer, luo, wahl, stitt, gottesman and rösch (2010) science 328, 501-504] and contributes to phage ?-mediated antitermination in escherichia coli that enables read-through of early transcription termination sites. e. coli nusg consists of two structurally and functionally distinct domains that are connected through a flexible linker. ...	2011	21345171
selective inactivation of a human neuronal silencing phosphatase by a small molecule inhibitor.	the unstructured c-terminal domain (ctd) of eukaryotic rna polymerase ii dynamically regulates the process of transcription by recruiting different factors to nascent mrna through its multiple phosphorylation patterns. a newly discovered class of phosphatases, the human small c-terminal domain phosphatases (scp's), specifically dephosphorylates phosphorylated ser(5) (phospho.ser5) of the tandem heptad repeats of the ctd of rna polymerase ii. scp's also function as transcription regulators that e ...	2011	21348431
construction and characterization of a cdna library from wheat infected with fusarium graminearum fg 2.	total rna from wheat spikes infected with f. graminearum fg2 was extracted and the mrna was purified. switching mechanism at 5' end of the rna transcript (smart) technique and cds ill/3' primer were used for first-strand cdna synthesis using reverse transcriptase by rt-pcr. primer extension polymerase chain reaction was used to construct the double-strand cdna that was digested by proteinase k, then by sfi i and fractionated. cdnas longer than 0.5 kb were collected and ligated to ?triplex2 vecto ...	2011	21340003
salt-dependent dna-dna spacings in intact bacteriophage ? reflect relative importance of dna self-repulsion and bending energies.	using solution synchrotron x-ray scattering, we measure the variation of dna-dna d spacings in bacteriophage ? with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5×10(3) base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. from the decrease in d spacings with increasing salt, we deduce the relative contributions of dna self-repulsion and bending to the energetics of packaged phage genomes. we quantify the dna-dna interaction energies within the intact phage by combini ...	2011	21405253
role of dlp12 lysis genes in escherichia coli biofilm formation.	phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. one defective lambdoid phage carried by escherichia coli k-12 is dlp12. among the genes found in dlp12 are essd, ybcs and rzpd/rzod, which are homologues of the lambda phage genes encoding cell-lysis proteins (s, r and rz/rz(1)). the role that these dlp12 lysis genes play in biofilm formation was examined in deletion mutants of e. coli phl628, a curli-o ...	2011	21415116
a type iv modification-dependent restriction enzyme sauusi from staphylococcus aureus subsp. aureus usa300.	a gene encoding a putative dna helicase from staphylococcus aureus usa300 was cloned and expressed in escherichia coli. the protein was purified to over 90% purity by chromatography. the purified enzyme, sauusi, predominantly cleaves modified dna containing 5mc and 5-hydroxymethylcytosine. cleavage of 5mc-modified plasmids indicated that the sites s5mcngs (s?=?c or g) are preferentially digested. the endonuclease activity requires the presence of adenosine triphosphate (atp) or datp whereas the ...	2011	21421560
to lyse or not to lyse: transient-mediated stochastic fate determination in cells infected by bacteriophages.	cell fate determination is usually described as the result of the stochastic dynamics of gene regulatory networks (grns) reaching one of multiple steady-states each of which corresponds to a specific decision. however, the fate of a cell is determined in finite time suggesting the importance of transient dynamics in cellular decision making. here we consider cellular decision making as resulting from first passage processes of regulatory proteins and examine the effect of transient dynamics with ...	2011	21423715
probing the viral metallome: searching for metalloproteins in bacteriophage ?-- the hunt begins.	although the proteome and genome of bacteriophages are well developed, there is little knowledge about metals and their interactions with the phages, even though metals have been observed in stabilizing phage particles. with expanding studies of phage display and its promising applications, metalloprotein investigations in the bacteriophage areas are necessary to understand whether or not metalloproteins are included in the viral coat proteome. since these virus studies are still in their infanc ...	2011	21423961
single virus genomics: a new tool for virus discovery.	whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. however, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. we report here on a new approach called 'single virus genomics', which enabled the isolation and complete genome sequencing of the first single virus particle. a mixed assemblage ...	2011	21436882
arm site independence of coliphage hk022 integrase in human cells.	the integrase encoded by the lambdoid phage hk022 (int-hk022) resembles its coliphage ? counterpart (int-?) in the roles of the cognate dna arm binding sites and in controlling the direction of the reaction. we show here that within mammalian cells, int-hk022 does not exhibit such a control. rather, int-hk022 recombined between all ten possible pairwise att site combinations, including attb × attb that was more effective than the conventional integrative attp × attb reaction. we further show tha ...	2011	21442327
recombination phenotypes of escherichia coli grea mutants.	the elongation factor grea binds to rna polymerase and modulates transcriptional pausing. some recent research suggests that the primary role of grea may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with rna polymerase, and which might otherwise collapse. replication fork collapse is known to generate dsdna breaks, which can be recombinogenic. it follows that grea malfunction could have consequences affecting homologous recombinati ...	2011	21453489
identification of antigenic proteins in trichomonas vaginalis.	trichomoniasis is a sexually transmitted disease due to infection with trichomonas vaginalis, and it can cause serious consequences for women's health. to study the virulence factors of this pathogen, t. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of t. vaginalis. the t. vaginalis expression library was constructed by cloning the cdna derived from mrna of t. vaginalis into a phage ? uni-zap xr vector, and then used for immunoscreeni ...	2011	21461274
determinants of gas-phase disassembly behavior in homodimeric protein complexes with related yet divergent structures.	the overall structure of a protein-protein complex reflects an intricate arrangement of noncovalent interactions. whereas intramolecular interactions confer secondary and tertiary structure to individual subunits, intermolecular interactions lead to quaternary structure-the ordered aggregation of separate polypeptide chains into multisubunit assemblies. the specific ensemble of noncovalent contacts dictates the stability of subunit folds, enforces protein-protein binding specificity, and determi ...	2011	21486017
lambda-display: a powerful tool for antigen discovery.	since its introduction in 1985, phage display technology has been successfully used in projects aimed at deciphering biological processes and isolating molecules of practical value in several applications. bacteriophage lambda, representing a classical molecular cloning and expression system has also been exploited for generating large combinatorial libraries of small peptides and protein domains exposed on its capsid. more recently, lambda display has been consistently and successfully employed ...	2011	21490557
functional characterization of an alkaline exonuclease and single strand annealing protein from the sxt genetic element of vibrio cholerae.	abstract: background: sxt is an integrating conjugative element (ice) originally isolated from vibrio cholerae, the bacterial pathogen that causes cholera. it houses multiple antibiotic and heavy metal resistance genes on its ca. 100kb circular double stranded dna (dsdna) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. here, we characterize the activities of an alkaline exonuclease (s066, sxt-exo) and single ...	2011	21501469
analysis of the fluctuations of a single-tethered, quantum-dot labeled dna molecule in shear flow.	a novel technique for analyzing the conformational fluctuations of a single, surface-tethered dna molecule by fluorescence microscopy is reported. attaching a nanometer-sized fluorescent quantum dot to the free end of a λ-phage dna molecule allows us to study the fluctuations of a native dna molecule without the mechanical properties being altered by fluorescent dye staining. we report on the investigation of single-tethered dna in both the unperturbed and the shear flow induced stretched state. ...	2011	21508487
secondary structure of double-stranded dna under stretching: elucidation of the stretched form.	almost two decades ago, measurements of force versus extension on isolated double-stranded dna molecules revealed a force plateau. this unusual stretching phenomenon in dna suggests that the long molecules may be extended from the usual b form into a new conformation. different models have been proposed to describe the nature of dna in its stretched form, s-dna. using atomic force microscopy combined with a molecular combing method, we identified the structure of λ-phage dna for different stretc ...	2011	21517521
nmr structure of the bordetella bronchiseptica protein np_888769.1 establishes a new phage-related protein family pf13554.	the solution structure of the hypothetical phage-related protein np_888769.1 from the gram-negative bacterium bordetella bronchoseptica contains a well-structured core comprising a five-stranded, antiparallel β-sheet packed on one side against two α-helices and a short β-hairpin with three flexibly disordered loops extending from the central β-sheet. a homology search with the software dali identified two protein data bank deposits with z-scores > 8, where both of these proteins have less than 8 ...	2011	21520320
decision making in living cells: lessons from a simple system.	the life cycle of bacteriophage lambda serves as a simplified paradigm for cell-fate decisions. the ongoing quantitative, high-resolution experimental investigation of this life cycle has produced some important insights in recent years. these insights have to do with the way cells choose among alternative fates, how they maintain long-term memory of their gene-expression state, and how they switch from one stable state to another. the recent studies have highlighted the role of spatiotemporal e ...	2011	21545284
the lysis-lysogeny decision of bacteriophage 933w: a 933w repressor-mediated long distance loop has no role in regulating 933w prm activity.	our data show that unlike bacteriophage λ, repressor bound at o(l) of bacteriophage 933w has no role regulating 933w repressor occupancy of 933w o(r)3 or the transcriptional activity of 933w p(rm). this finding suggests that a cooperative long-range loop between repressor tetramers bound at o(r) and o(l) does not form in bacteriophage 933w. nonetheless, 933w forms lysogens and 933w prophage display a threshold response to uv induction similar to related lambdoid phages. hence, the long range loo ...	2011	21551291
dynamic solid phase dna extraction and pcr amplification in polyester-toner based microchip.	a variety of substrates have been used for fabrication of microchips for dna extraction, pcr amplification, and dna fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (pet) microfluidic devices with channel depths on the order of tens of micrometers. here, we describe a novel a ...	2011	21557576
identification of a novel temperature sensitive promoter in cho cells.	abstract: background: the chinese hamster ovary (cho) expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. here we report about the successful identification of an endogenous highly active gene promoter obtained from cho cells which shows conditionally inducible gene expression at reduced temperature. resul ...	2011	21569433
noise-aided computation within a synthetic gene network through morphable and robust logic gates.	an important goal for synthetic biology is to build robust and tunable genetic regulatory networks that are capable of performing assigned operations, usually in the presence of noise. in this work, a synthetic gene network derived from the bacteriophage λ underpins a reconfigurable logic gate wherein we exploit noise and nonlinearity through the application of the logical stochastic resonance paradigm. this biological logic gate can emulate or "morph" the and and or operations through varying i ...	2011	21599203
compromised factor-dependent transcription termination in a nusa mutant of escherichia coli : spectrum of termination efficiencies generated by perturbations of rho, nusg, nusa, and the h-ns family of proteins.	the proteins nusa and nusg, which are essential for viability of wild-type escherichia coli, participate in various post-initiation steps of transcription including elongation, antitermination, and termination. nusg is required along with the essential rho protein for factor-dependent transcription termination (also referred to as polarity), but the role of nusa is less clear with conflicting reports that it both promotes and inhibits the process. in this study, we have found that a recessive mi ...	2011	21602355
a method to generate recombinant salmonella typhi ty21a strains expressing multiple heterologous genes using an improved recombineering strategy.	live attenuated salmonella enterica serovar typhi ty21a (ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. to facilitate the use of ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. the phage λ red homologous recombination system has previously been used in various gram-negative bac ...	2011	21611798
a facile method for the assessment of dna damage induced by uv-activated nanomaterials.	fluorescent microscopy observation of gene-size dna (t4 phage dna or λ phage dna) was used to assess dna damage induced by uv irradiation in the presence of nanomaterials, such as qds (quantum dots: cdse/zns semiconductor nanoparticles), the water-soluble fullerene derivative c(60)(oh)(n) (n = 6-12) and titanium oxide nanoparticles of 25 nm in diameter. the magnitude of dna damage could be simply evaluated based on the degree of shortening of the stretched dna image. this method showed that dna ...	2011	21614347
molecular responses of e. coli caused by heat stress and recombinant protein production during temperature induction.	in a recent review, we discussed the extensively used temperature-inducible expression system, based on the pl and/or pr phage lambda promoters that are finely regulated by the thermo-labile ci857 repressor. in this system, an increase in temperature induces the heterologous protein production and activates the heat shock response, as well as the stringent and sos responses. the same responses are activated just by the overproduction of recombinant protein. all such responses result in a metab ...	2011	21636998
cng site-specific and methyl-sensitive endonuclease wen1 from wheat seedlings.	endonuclease wen1 with apparent molecular mass about 27 kda isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. this is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. the enzyme hydrolyzes deoxyribooligonucleotides of different composition on cng (n is g, a, c, or t) sites by splitting the phosphodiester bond between c and n nucleotide res ...	2011	21639845
genome engineering using targeted oligonucleotide libraries and functional selection.	the ? phage red proteins greatly enhance homologous recombination in escherichia coli. red-mediated recombination or "recombineering" can be used to construct targeted gene deletions as well as to introduce point mutations into the genome. here, we describe our method for scanning mutagenesis using recombineered oligonucleotide libraries. this approach entails randomization of specific codons within a target gene, followed by functional selection to isolate mutants. oligonucleotide library mutag ...	2011	21815087
targeted chromosomal gene knockout using pcr fragments.	the development of recombineering technology has converged to a point that virtually any type of genetic modification can be made in the escherichia coli chromosome. the most straightforward -modification is a chromosomal gene knockout, which is done by electroporation of a pcr fragment that contains a selectable drug marker flanked by 50 bp of target dna. the phage λ red recombination system expressed in vivo from a plasmid promotes deletion of the gene of interest at high efficiency. the combi ...	2011	21815084
factors influencing lysis time stochasticity in bacteriophage lambda.	abstract: background: despite identical genotypes and seemingly uniform environments, stochastic gene expression and other dynamic intracellular processes can produce considerable phenotypic diversity within clonal microbes. one trait that provides a good model to explore the molecular basis of stochastic variation is the timing of host lysis by bacteriophage (phage). results: individual lysis events of thermally-inducible lambda lysogens were observed using a temperature-controlled perfusion ch ...	2011	21810267
specific sorting of single bacterial cells with micro-facs and tyramide signal amplification-fish.	when attempting to probe the genetic makeup of diverse bacterial communities that elude cell culturing, researchers face two primary challenges: isolation of rare bacteria from microbial samples and removal of contaminating cell-free dna. we report a compact, low-cost, and high-performance micro-fabricated fluorescence-activated cell sorting (µfacs) technology in combination with a tyramide signal amplification-fluorescence in situ hybridization (tsa-fish) to address these two challenges. the ts ...	2011	21809842
the drosophila gap gene network is composed of two parallel toggle switches.	drosophila "gap" genes provide the first response to maternal gradients in the early fly embryo. gap genes are expressed in a series of broad bands across the embryo during first hours of development. the gene network controlling the gap gene expression patterns includes inputs from maternal gradients and mutual repression between the gap genes themselves. in this study we propose a modular design for the gap gene network, involving two relatively independent network domains. the core of each ne ...	2011	21747931
automaton models of computational genetic regulatory networks with combinatorial gene-protein interactions.	in this paper, we present an approach for modeling computational genetic regulatory networks with multi-threshold protein concentration and combinatorial gene-protein interactions. we first present a gene automata model that describes the activities of a single gene, and discuss the construction of a network automaton model that describes the complete behavior of a gene network. to model the gene-protein interaction in the given gene network, we define the basic interaction, in the form of an au ...	2011	21723368
developing an extended genomic engineering approach based on recombineering to knock-in heterologous genes to escherichia coli genome.	most existing genomic engineering protocols for manipulation of escherichia coli are primarily focused on chromosomal gene knockout. in this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage ++ (++ red) and flippase-flippase recognition targets (flp-frt) recombinations. for demonstration purposes, dna operons containing heterologous genes (i.e., pac encoding e. coli penicillin acylase and palb2 encoding pseud ...	2011	21826554
single-virus tracking reveals a spatial receptor-dependent search mechanism.	viral infection begins with the binding of a virus to a specific target on the surface of the host cell, followed by viral genome delivery into the host and a continuation of the infection process. before binding occurs, the virus must first find its receptor by a process whose details are largely unknown. we applied high-resolution fluorescence microscopy and single-particle tracking to elucidate the target-finding process in bacteriophage ++ as it infects an escherichia coli cell. by monitorin ...	2011	21689520
piconewton-millisecond force steps reveal the transition kinetics and mechanism of the double-stranded dna elongation.	we study the kinetics of the overstretching transition in λ-phage double-stranded (ds) dna from the basic conformation (b state) to the 1.7-times longer and partially unwound conformation (s state), using the dual-laser optical tweezers under force-clamp conditions at 25°c. the unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.5-2 pn, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the b-s ...	2011	21843477
reproducible gene targeting in recalcitrant escherichia coli isolates.	abstract:	2011	21696605
a precise excision of the complete epstein-barr virus genome in a plasmid based on a bacterial artificial chromosome.	the maxi-ebv is a bacterial artificial chromosome (bac)-based plasmid that contains the complete epstein-barr virus (ebv) genome of 172 kb. this plasmid also carries an additional cassette of 11.5 kb in size for the expression of a mini f factor, selection markers and gfp. in the intracellular study of ebv infection based on the maxi-ebv system, a parallel control that only contains this 11.5 kb vector is desirable but unavailable. in order to construct the vector in this approach, a clean delet ...	2011	21723326
the lysis cassette of bacteriophage ¤òkmv encodes a signal-arrest-release endolysin and a pinholin.	the lysis cassette of pseudomonas aeruginosa phage ¤òkmv encodes a holin, endolysin, rz and rz1 in the canonical order. it has a tight organization with a high degree of overlapping genes and is highly conserved (between 96 and 100% identity at the protein level) among several other members of the "phikmv-like viruses." the endolysin kmv45 exhibits characteristics as expected for a signal-arrest-release (sar) endolysin, whereas the holin kmv44 is a typical pinholin. kmv45 is initially secreted a ...	2011	21687532
bacteriophage crosstalk: coordination of prophage induction by trans-acting antirepressors.	many species of bacteria harbor multiple prophages in their genomes. prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. the paradigm of prophage induction, as set by the phage lambda model, sees the process initiated by the reca-stimulated self-proteolysis of the phage repressor. here we sh ...	2011	21731505
knockout and pullout recombineering for naturally transformable burkholderia thailandensis and burkholderia pseudomallei.	phage ++-red proteins are powerful tools for pulling and knocking out chromosomal fragments but have been limited to the +¦-proteobacteria. procedures are described here to easily knock out (ko) and pull out (po) chromosomal dna fragments from naturally transformable burkholderia thailandensis and burkholderia pseudomallei. this system takes advantage of published compliant counterselectable and selectable markers (sacb, phes, gat and the arabinose-utilization operon) and ++-red mutant proteins. ...	2011	21738123
biomolecular and structural analyses of cauliflower-like dnas by ultraviolet, circular dichroism, and fluorescence spectroscopies in comparison with natural dna.	cauliflower-like dnas are stem-loop dnas that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dntps) by loop-mediated isothermal amplification (lamp). cauliflower-like dnas have ladder-shape behaviors on gel electrophoresis, and increasing the time of lamp leads to multiplying the repetitions, stem-loops, and electrophoretic bands. cauliflower-like dnas were fabricated via lamp using two loop primers, two bumper primers, dntps, a ++-phage dna template, ...	2011	21738438
chemical mapping of dna and counter-ion content inside phage by energy-filtered tem.	double-stranded dna in many bacterial viruses (phage) is strongly confined, which results in internal genome pressures of tens of atmospheres. this pressure is strongly dependent on local ion concentration and distribution within the viral capsid. here, we have used electron energy loss spectroscopy (eels), energy-filtered tem (eftem) and x-ray energy dispersive spectroscopy to provide such chemical information from the capsid and the phage tail through which dna is injected into the cell. to ac ...	2011	23449697
site-specific binding of short peptides with dna modulated eukaryotic endonuclease activity.	short peptides (2-4 amino acid residues) inhibit or stimulate hydrolysis of λ phage dna by eukaryotic endonucleases wen1 and wen2 depending on dna methylation status. peptide modulation of endonucleases activity most likely appears as a result of their binding to dna. peptides discriminate (recognize) not only certain dna sequences, but also their methylation status. apart from intact dna, the test peptides bind to single-stranded dna structures (oligonucleotides) containing ng- and cg-sites met ...	2011	22442805