Publications

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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 ...201122191393
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 ...201121415116
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 ...201121826554
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 ...201121602355
fine tuning of the e. coli nusb:nuse complex affinity to boxa rna is required for processive antitermination.phage lambda propagation in escherichia coli host cells requires transcription antitermination on the lambda chromosome mediated by lambdan protein and four host nus factors, nusa, b, e (ribosomal s10) and g. interaction of e. coli nusb:nuse heterodimer with the single stranded boxa motif of lambdanutl or lambdanutr rna is crucial for this reaction. similarly, binding of nusb:nuse to a boxa motif is essential to suppress transcription termination in the ribosomal rna (rrn) operons. we used fluor ...201019854945
tuning a genetic switch: experimental evolution and natural variation of prophage induction.genetic switches allow organisms to modulate their phenotype in response to environmental changes. understanding the evolutionary processes by which switches are tuned is central to understanding how phenotypic variation is realized. prophage induction by phage lambda is the classic example of a genetic switch and allows lambda to move between two different modes of transmission: as a lysogen it reproduces vertically as a component of the host genome; as a free phage it reproduces horizontally b ...201019891623
dna heats up: energetics of genome ejection from phage revealed by isothermal titration calorimetry.most bacteriophages are known to inject their double-stranded dna into bacteria upon receptor binding in an essentially spontaneous way. this downhill thermodynamic process from the intact virion to the empty viral capsid plus released dna is made possible by the energy stored during active packaging of the genome into the capsid. only indirect measurements of this energy have been available until now, using either single-molecule or osmotic suppression techniques. in this work, we describe for ...201019969001
escherichia coli mw005: lambda red-mediated recombineering and copy-number induction of oriv-equipped constructs in a single host.escherichia coli strain el350 contains chromosomally integrated phage lambda red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. bac and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the dna is difficult to isolate in high yield and purity. to overcome this limitation vectors, e.g. pcc1fos, have been constructed that contain the additional replica ...201020350301
a forward-genetic screen and dynamic analysis of lambda phage host-dependencies reveals an extensive interaction network and a new anti-viral strategy.latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. this screen identified 57 escherichia coli (e. coli) genes--over half of which have not been previously associated with infection--that when knocked out inhibited lambda phage's ability to replicate. our results demonstrate a highly integrated network betwee ...201020628568
reca-independent single-stranded dna oligonucleotide-mediated mutagenesis.the expression of beta, the single-stranded annealing protein (ssap) of bacteriophage lambda in escherichia coli promotes high levels of oligonucleotide (oligo)-mediated mutagenesis and offers a quick way to create single or multiple base pair insertions, deletions, or substitutions in the bacterial chromosome. high rates of mutagenesis can be obtained by the use of mismatch repair (mmr)-resistant mismatches or mmr-deficient hosts, which allow for the isolation of unselected mutations. it has re ...201020711416
sequence-specific recognition of dna by the c-terminal domain of nucleoid-associated protein h-ns.the molecular determinants necessary and sufficient for recognition of its specific dna target are contained in the c-terminal domain (h-nsctd) of nucleoid-associated protein h-ns. h-nsctd protects from dnasei cleavage a few short dna segments of the h-ns-sensitive hns promoter whose sequences closely match the recently identified h-ns consensus motif (tcg(t/a)t(a/t)aatt) and, alone or fused to the protein oligomerization domain of phage lambda ci repressor, inhibits transcription from the hns p ...200919740756
cleavage of bacteriophage lambda ci repressor involves the reca c-terminal domain.the sos response to dna damage in escherichia coli involves at least 43 genes, all under the control of the lexa repressor. activation of these genes occurs when the lexa repressor cleaves itself, a reaction catalyzed by an active, extended reca filament formed on dna. it has been shown that the lexa repressor binds within the deep groove of this nucleoprotein filament, and presumably, cleavage occurs in this groove. bacteriophages, such as lambda, have repressors (ci) that are structural homolo ...200919013467
production of double-stranded rna for interference with tmv infection utilizing a bacterial prokaryotic expression system.in many species, the introduction of double-stranded rna (dsrna) induces potent and specific gene silencing, a phenomenon called rna interference (rnai). rnai is the process of sequence-specific, posttranscriptional gene silencing (ptgs) in animals and plants, mediated by dsrna homologous to the silenced genes. in plants, ptgs is part of a defense mechanism against virus infection, and dsrna is the pivotal factor that induces gene silencing. here, we report an efficient method that can produce d ...200919330324
high adsorption rate is detrimental to bacteriophage fitness in a biofilm-like environment.bacterial biofilm is ubiquitous in nature. however, it is not clear how this crowded habitat would impact the evolution of bacteriophage (phage) life history traits. in this study, we constructed isogenic lambda phage strains that only differed in their adsorption rates, because of the presence/absence of extra side tail fibers or improved tail fiber j, and maker states. the high cell density and viscosity of the biofilm environment was approximated by the standard double-layer agar plate. the p ...200919804637
characterization and validation of a bioluminescent bioreporter for the direct detection of escherichia coli.a two-component bacteriophage-based bioluminescent reporter system was developed for the detection of escherichia coli in environmental samples. the bioreporter system consists of a luxi integrated lambda bacteriophage and a lux-based bioluminescent reporter cell that responds to the infection event through acyl-homoserine lactone (ahl) mediated quorum sensing and bioluminescent signal stimulation. this work addresses the ability of the bioreporter system to detect and quantify the target pathog ...200818593593
importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class a penicillin-binding protein 1b of escherichia coli.the peptidoglycan glycosyltransferase (gt) module of class a penicillin-binding proteins (pbps) and monofunctional gts catalyze glycan chain elongation of the bacterial cell wall. these enzymes belong to the gt51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. in this work, we have systematically modified all the conserved amino acid residues of the gt module of escherichia coli class a pbp1b by site-directed mutagenesis and deter ...200818701463
a biotin interference assay highlights two different asymmetric interaction profiles for lambda integrase arm-type binding sites in integrative versus excisive recombination.the site-specific recombinase integrase encoded by bacteriophage lambda promotes integration and excision of the viral chromosome into and out of its escherichia coli host chromosome through a holliday junction recombination intermediate. this intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the holliday junction dna and via its amino-terminal domains to distal "arm-type" sites. the two classes of integrase binding sit ...200818319248
development of a bacterial cloning vector for expression of scorpion toxins for biotechnological studies.scorpion venoms contain toxic peptides that recognize k(+) channels of excitable and non-excitable cells. these toxins comprise three structurally distinct groups designated alpha-ktx, beta-ktx, and gamma-ktx. it is highly desirable to develop systems for the expression of these toxins for further physiological and structural studies. in this work, an expression vector (ptev3) was constructed by inserting protein d (major capsid of phage lambda) and tev protease recognition site into plasmid pet ...200817904381
identification and functional analysis of the rz/rz1-like accessory lysis genes in the membrane-containing bacteriophage prd1.bacteriophage prd1 is a tailless membrane-containing double-stranded (ds) dna virus infecting a variety of gram-negative bacteria. in order to affect cell lysis, like most dsdna phages, prd1 uses the holin-endolysin system. in this study, we identified two accessory lysis genes, xxxvi and xxxvii, coding for proteins p36 and p37, respectively. using genetic complementation assays, we show that protein pair p36/p37 is a functional and interchangeable analogue of the rz/rz1 of bacteriophage lambda. ...200818366440
removal of deoxyinosine from the escherichia coli chromosome as studied by oligonucleotide transformation.deoxyinosine (di) is produced in dna by the hydrolytic or nitrosative deamination of deoxyadenosine. it is excised in a repair pathway that is initiated by endonuclease v, the product of the nfi gene. the repair was studied in vivo using high-efficiency oligonucleotide transformation mediated by the beta protein of bacteriophage lambda in a mismatch repair-deficient host. escherichia coli was transformed with oligonucleotides containing a selectable a-g base substitution mutation. when the mutag ...200817981100
analysis of some phenotypic traits of feces-borne temperate lambdoid bacteriophages from different immunity groups: a high incidence of cor+, fhua-dependent phages.a group of previously isolated heterogeneous mep lambdoid phages (43) from 19 different immunity groups for phage infection was further characterized to gain insight into some phenotypic traits and to assess their relationship with phage lambda. interestingly, the fhua host receptor was required by the majority of mep phages (37 out of 43; approximately 85%). the cor gene, which has been reported to be involved in fhua-dependent exclusion of lambdoid phages, was also found in most of the fhua-de ...200818516490
fis targets assembly of the xis nucleoprotein filament to promote excisive recombination by phage lambda.the phage-encoded xis protein is the major determinant controlling the direction of recombination in phage lambda. xis is a winged-helix dna binding protein that cooperatively binds to the attr recombination site to generate a curved microfilament, which promotes assembly of the excisive intasome but inhibits formation of an integrative intasome. we find that lambda synthesizes surprisingly high levels of xis immediately upon prophage induction when excision rates are maximal. however, because o ...200717275024
structure of the cooperative xis-dna complex reveals a micronucleoprotein filament that regulates phage lambda intasome assembly.the dna architectural protein xis regulates the construction of higher-order nucleoprotein intasomes that integrate and excise the genome of phage lambda from the escherichia coli chromosome. xis modulates the directionality of site-specific recombination by stimulating phage excision 10(6)-fold, while simultaneously inhibiting phage reintegration. control is exerted by cooperatively assembling onto a approximately 35-bp dna regulatory element, which it distorts to preferentially stabilize an ex ...200717287355
switch from theta to sigma replication of bacteriophage lambda dna: factors involved in the process and a model for its regulation.bacteriophage lambda genome is one of the classical model replicons in studies on the regulation of dna replication. moreover, since genes coding for shiga toxins are located in genomes of lambdoid phages, understanding of mechanisms controlling lambda dna replication may be of bio-medical importance. during lytic development of bacteriophage lambda, its genome is replicated according to the theta (circle-to-circle) mode early after infection, and then it is switched to the sigma (rolling circle ...200717377819
multicopy plasmid modification with phage lambda red recombineering.recombineering, in vivo genetic engineering using the bacteriophage lambda red generalized recombination system, was used to create various modifications of a multicopy plasmid derived from pbr322. all genetic modifications possible on the escherichia coli chromosome and on bacterial artificial chromosomes (bacs) are also possible on multicopy plasmids and are obtained with similar frequencies to their chromosomal counterparts, including creation of point mutations (5-10% unselected frequency), ...200717434584
on kinetics of phage adsorption.adsorption of lambda-phage on sensitive bacteria escherichia coli is a classical problem but not all issues have been resolved. one of the outstanding problems is the rate of adsorption, which in some cases appears to exceed the theoretical limit imposed by the law of random diffusion. we revisit this problem by conducting experiments along with new theoretical analyses. our measurements show that upon incubating lambda-phage with bacteria ymel, the population of unbound phage in a salt buffer d ...200717434950
modelling the stability of stx lysogens.shiga-toxin-converting bacteriophages (stx phages) are temperate phages of escherichia coli, and can cause severe human disease. the spread of shiga toxins by stx phages is directly linked to lysogen stability because toxins are only synthesized and released once the lytic cycle is initiated. lysogens of stx phages are known to be less stable than those of the related lambda phage; this is often described in terms of a 'hair-trigger' molecular switch from lysogeny to lysis. we have developed a m ...200717604057
probing the antiprotease activity of lambdaciii, an inhibitor of the escherichia coli metalloprotease hflb (ftsh).the ciii protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous escherichia coli metalloprotease hflb (ftsh). this inhibition results in the stabilization of transcription factor lambdacii, thereby helping the phage to lysogenize the host bacterium. lambdaciii, a small (54-residue) protein of unknown structure, also protects sigma(32), another specific substrate of hflb. in order to understand the details of the inhibitory mechanism of ciii, we cloned and expres ...200717890311
chromosomal model for analysis of a long ctg/cag tract stability in wild-type escherichia coli and its nucleotide excision repair mutants.many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy, and friedreich's ataxia, are associated with expansions of the triplet repeat sequences (trs) (cgg/ccg, ctg/cag, and gaa/ttc) within or near specific genes. mechanisms that mediate mutations of trs include dna replication, repair, and gene conversion and (or) recombination. the involvement of the repair systems in trs instability was investigated in escherichia coli on plasmid models, and the results s ...200717898841
comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure.the antibacterial working range of six lysozymes was tested under ambient and high pressure, on a panel of five gram-positive (enterococcus faecalis, bacillus subtilis, listeria innocua, staphylococcus aureus and micrococcus lysodeikticus) and five gram-negative bacteria (yersinia enterocolitica, shigella flexneri, escherichia coli o157:h7, pseudomonas aeruginosa and salmonella typhimurium). the lysozymes included two that are commercially available (hen egg white lysozyme or hewl, and mutanolys ...200616487612
a novel method for evaluating free radical scavenging abilities of antioxidants using ultraviolet induction of bacteriophage lambda.a novel biological method used to evaluate free radical scavenging abilities of antioxidants using ultraviolet (uv) induction of bacteriophage lambda in lysogenic escherichia coli kappa12 (lambda+) has been developed. this method is based on the induction of bacteriophage lambda from lysogenic cycle to lytic cycle by ultraviolet irradiation, and formation of free radicals during the course of induction. in the experiments, 10(8)cells/ml and 30s (39j/m2) were determined as the cell density of the ...200616574238
display of aggregation-prone ligand binding domain of human ppar gamma on surface of bacteriophage lambda.to display the aggregation-prone ligand binding domain (lbd) of the human peroxisome proliferator-activated receptor gamma (ppargamma) on the surface of bacteriophages to establish an easy screening assay for the identification of ppargamma ligands.200616364215
rapid allelic exchange in enterohemorrhagic escherichia coli (ehec) and other e. coli using lambda red recombination.this unit describes an allelic exchange system for enterohemorrhagic e. coli (ehec), and similar pathogenic species of bacteria. the phage lambda red recombination system is expressed from a plasmid, inducing a hyper-recombinogenic state where electroporated pcr-generated substrates recombine with the bacterial chromosome at high efficiency. the technique can be used to substitute a drug marker for the gene of interest, or used to generate a clean in-frame deletion of the target gene. single gen ...200618770591
gene-specific random mutagenesis of escherichia coli in vivo: isolation of temperature-sensitive mutations in the acyl carrier protein of fatty acid synthesis.acyl carrier proteins (acps) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. moreover, recent data indicate that the acyl carrier protein of escherichia coli has a large protein interaction network that extends beyond lipid synthesis. despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (acpp) that encodes acp have been isolated. we report the isolation of three such mutants by a new appro ...200616352845
cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts.we have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and micrococcus lysodeikticus with chloroform/tris-hcl buffer (chloroform/buffer). the lysozymes included two that are commercially available (hen egg white lysozyme or hewl, and mutanolysin from streptomyces globisporus or m1l), and four that were chromatographically purified (bacteriophage lambda lysozyme or lal, bacteriophage t4 lysozyme or ...200616684100
overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.during the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. bacterial systems remain most attractive due to low cost, high productivity, and rapid use. however, the rational choice of the adequate promoter system and host for a specific protein of interest remains difficult. this review gives an overview of the most commonly used systems: as hosts, bacillus brevis, bacillus megaterium, bacillus subtilis, caulobacter crescentus, other str ...200616791589
[bacteriophage lambda dna replication--new discoveries made using an old experimental model].bacteriophage lamda is a model in molecular biology studies since over fifty years. nevertheless, studies of recent years (similarly to previous time periods) resulted in many new experimental results which not only expanded our knowledge on molecular mechanisms of functions of this virus, but also shed new light on general rules of the transduction and transfer of genetic information. in this review, we present recent achievements of studies on mechanisms of regulation of bacteriophage lamda dn ...200616869300
stimulation of the lambda pr promoter by escherichia coli seqa protein requires downstream gatc sequences and involves late stages of transcription initiation.escherichia coli seqa protein is a major negative regulator of chromosomal dna replication acting by sequestration, and thus inactivation, of newly formed oric regions. however, other activities of this protein have been discovered recently, one of which is regulation of transcription. seqa has been demonstrated to be a specific transcription factor acting at bacteriophage lambda promoters p(i), p(aq) and p(r). while seqa-mediated stimulation of p(i) and p(aq) occurs by facilitating functions of ...200617005979
rna-mediated destabilization of the sigma(70) region 4/beta flap interaction facilitates engagement of rna polymerase by the q antiterminator.the bacterial rna polymerase (rnap) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. during early elongation, the stability of interactions between sigma(70) (the primary sigma factor in escherichia coli) and core decreases due to an ordered displacement of segments of sigma(70) from core triggered by growth of the nascent rna. here we demonstrate that the nascent rna-mediated de ...200617081994
role of c-terminal residues in oligomerization and stability of lambda cii: implications for lysis-lysogeny decision of the phage.a crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein cii, which has several interesting properties. it promotes lysogeny through activation of three phage promoters p(e), p(i) and p(aq), recognizing a direct repeat sequence ttgcn6ttgc at each. the three-dimensional structure of cii, a homo-tetramer of 97 residue subunits, is unknown. it is an unstable protein in vivo, being rapidly degraded by the host protease hflb (ftsh). this instability is e ...200515571724
the structure of the excisionase (xis) protein from conjugative transposon tn916 provides insights into the regulation of heterobivalent tyrosine recombinases.heterobivalent tyrosine recombinases play a prominent role in numerous bacteriophage and transposon recombination systems. their enzymatic activities are frequently regulated at a structural level by excisionase factors, which alter the ability of the recombinase to assemble into higher-order recombinogenic nucleoprotein structures. the tn916 conjugative transposon spreads antibiotic resistance in pathogenic bacteria and is mobilized by a heterobivalent recombinase (tn916int), whose activity is ...200515733914
the e. coli nusa carboxy-terminal domains are structurally similar and show specific rnap- and lambdan interaction.the carboxy-terminal domain of the transcription factor escherichia coli nusa, nusactd, interacts with the protein n of bacteriophage lambda, lambdan, and the carboxyl terminus of the e. coli rna polymerase alpha subunit, alphactd. we solved the solution structure of the unbound nusactd with high-resolution nuclear magnetic resonance (nmr). additionally, we investigated the binding sites of lambdan and alphactd on nusactd using nmr titrations. the solution structure of nusactd shows two structur ...200515987884
two-stage continuous operation of recombinant escherichia coli using the bacteriophage lambda q- vector.a two-stage continuous culture of escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. a phage lambda vector with a q(-) mutation was used to enhance the expression of the lambda system. the optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned ...200516096763
[red/et recombination and its biomedical applications].red/et recombination, a powerful homologous recombination system based on the red operon of lambda phage or rece/ rect from rac phage, provides an innovative approach for dna engineering. deletion, insertion and mutation can be quickly and precisely performed on the target gene mediated by red/et recombination with pcr derived dna fragments or oligonucleotides. this technical platform has extensive applications in biomedical field including bacterial artificial chromosome modification, gene knoc ...200516108384
functional analysis of the lysis genes of staphylococcus aureus phage p68 in escherichia coli.double-stranded dna phages of both gram-positive and gram-negative bacteria typically use a holin-endolysin system to achieve lysis of their host. in this study, the lysis genes of staphylococcus aureus phage p68 were characterized. p68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of s. aureus. another gene, hol15, was identified embedded in the -1 reading frame at the 3' end of lys16. the deduced hol15 protein ha ...200516000723
assignments of 1h and 15n resonances of the bacteriophage lambda capsid stabilizing protein gpd. 200414739644
a novel cell-free protein synthesis system.an efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. using the new energy source, 3-phosphoglycerate (3-pga), protein synthesis continues beyond 2 h. in contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (pep) under identical reaction conditions. this improvement results in the production of twice the amount of protein obtained with pep as an energy source. we have ...200415163516
the recognition and modification sites for the bacterial type i restriction systems kpnai, styseai, styseni and stysgi.using an in vivo plasmid transformation method, we have determined the dna sequences recognized by the kpnai, styseai, styseni and stysgi r-m systems from klebsiella oxytoca strain m5a1, salmonella eastbourne, salmonella enteritidis and salmonella gelsenkirchen, respectively. these type i restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their dna recognition sequ ...200415199175
translation repression by an rna polymerase elongation complex.bacteriophage lambda n and bacterial nus proteins together with a unique site nut in the leader of the early viral n gene transcript bind rna polymerase (rnap) and form a highly processive antitermination complex; n bound at nut also represses n translation. in this study, we investigate whether n and nut cause n translation repression as part of the antitermination complex by testing conditions that inhibit the formation of the n-modified transcription complex for their effect on n-mediated tra ...200415255895
nonspecific binding of the or repressors ci and cro of bacteriophage lambda.we estimate the gibbs free energy for nonspecific binding (deltagnsb) to the escherichia coli dna for two regulatory proteins of the lambda phage, ci and cro. by means of a statistical-mechanical approach, we calculate the ci and cro activities associated with the operator or of an introduced lambda phage genome (prophage). in this statistical model we apply in vitro-measured binding free energies to fit in vivo experimental data for ci and cro activities, respectively, where deltagnsb is introd ...200415488529
electrokinetic bioprocessor for concentrating cells and molecules.bioprocessors for concentrating bioparticles, such as cells and molecules, are commonly needed in bioanalysis systems. in this microfluidic processor, a global flow field generated by ac electroosmosis transports the embedded particles to the regions near the electrode surface. the processor then utilizes electrophoretic and dielectrophoretic forces, which are effective in short range, to trap the target cells and molecules on the electrode surface. by optimizing the operating parameters, we hav ...200415571340
restoration of gene function by homologous recombination: from pcr to gene expression in one step.we have developed a simple method for single-step cloning of any pcr product into a plasmid. a novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. in this method a dna fragment is amplified by pcr with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdapr or rrna1 promoter regions. the resulting pcr product is then electroporated into an escherichia coli ...200415574912
bacteriophage hk022 nun protein: a specific transcription termination factor that excludes bacteriophage lambda. 200314712713
role of the cgta gene function in dna replication of extrachromosomal elements in escherichia coli.the cgta gene codes for a common gtp-binding protein whose homologues were found in all prokaryotic and eukaryotic organisms investigated so far. although cgta is an essential gene in most bacterial species, its precise functions in the regulation of cellular processes are largely unknown. in escherichia coli, dysfunction or overexpression of the cgta gene causes problems in various chromosomal functions, like synchronization of dna replication initiation and partitioning of daughter chromosomes ...200312826057
osmotic pressure inhibition of dna ejection from phage.bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. we demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. in the particular case of bacteriophage lambda, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by ...200312881484
pathogenic leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.proteins with bacterial immunoglobulin-like (big) domains, such as the yersinia pseudotuberculosis invasin and escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. here, we report the identification and characterization of a new family of big domain proteins, referred to as lig (leptospiral ig-like) proteins, in pathogenic leptospira. screening of l. interrogans and l. kirschneri expression libraries with sera from leptospirosis patien ...200312890019
the pair of arginine codons aga agg close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating peptidyl-trnaarg4.to analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives. the lambda int gene has a high frequency of rare ata, aga and agg codons; two of them (aga agg) located at positions 3 and 4 of the int open reading frame (orf). escherichia coli pth (rap) cells, which are defective in peptidyl-trna hydrolase (pth) activity, are more susceptible to the inhibitory effe ...200312890027
the mobile element is1207 of brevibacterium lactofermentum atcc21086: isolation and use in the construction of tn5531, a versatile transposon for insertional mutagenesis of corynebacterium glutamicum.is1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an escherichia coli phage lambda ci gene integrated in the corynebacterium brevibacterium lactofermentum atcc21086 genome. we examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two is1207 sequences. one of these, the tn5531 transposon, transposed efficiently in corynebacterium glutamicum. a replicative and a non-repl ...200312948647
on the origin of unusual mutations recovered from the laci transgenic assay.amongst approximately 25,000 mutants recovered from tissues of the laci mouse and rat transgenic mutation assay, we identified seven mutants that carry changes that are unlike the majority of mutations that are normally recovered in these systems. the recovered mutants feature replacements and insertions of sequences that originate in the animal's genome, in the bacteriophage lambda construct that harbors the laci gene, and in the genome of the e. coli plating host. these mutants demonstrate tha ...200312694740
seqa-mediated stimulation of a promoter activity by facilitating functions of a transcription activator.it was demonstrated recently that the seqa protein, a main negative regulator of escherichia coli chromosome replication initiation, is also a specific transcription factor. seqa specifically activates the bacteriophage lambda pr promoter while revealing no significant effect on the activity of another lambda promoter, pl. here, we demonstrate that lysogenization by bacteriophage lambda is impaired in e. coli seqa mutants. genetic analysis demonstrated that cii-mediated activation of the phage p ...200312622820
the optimal eukaryotic signal for translation initiation from non-aug codons, present upstream of bacteriophage lambda p cistron, is inactive in escherichia coli.expression of the replication genes of bacteriophage lambda, o and p, is believed to be translationally coupled. however, it was previously noted that, under conditions of amino acid starvation, when o is not synthesized, p continues to be expressed at a relatively high level. the results presented in this report, contrary to the previously presented hypothesis, suggest that an agacuggau sequence (an optimal context for translation initiation from non-aug codons in eukaryotes, and present upstre ...200312813564
constitutive versus thermoinducible expression of heterologous proteins in escherichia coli based on strong pr,pl promoters from phage lambda.constitutive and thermoinducible expression plasmids based on strong p(r),p(l) promoters from phage lambda were compared for production of tnf-alpha and its analogs under various conditions. much higher accumulation of tnf was obtained in a constitutive system, so the wider applicability of such systems was studied. in constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not r ...200312768624
stationary phase-like properties of the bacteriophage lambda rex exclusion phenotype.the rex genes of bacteriophage lambda were found to protect lysogenic escherichia colik host cells against killing by phage t4 rii, when compared in parallel to isogenic rex(-) lysogens and nonlysogens. this protective effect was abrogated upon mutation of the host stationary-phase sigma factor rpos. rex(+) lysogens infected by t4 rii contracted, formed aggregates and shed flagella, thus resembling cells entering stationary phase. these phenotypes were accentuated in nonlysogenic cells carrying ...200312715152
the mechanism of regulation of bacteriophage lambda pr promoter activity by escherichia coli dnaa protein.apart from its function as an initiator of dna replication, the escherichia coli dnaa protein is also a specific transcription factor. it activates and represses a number of promoters. however, mechanisms of transcription stimulation by dnaa remained unknown. bacteriophage lambda pr promoter is one of the promoters activated by dnaa. it was reported previously that dnaa binds downstream of the pr promoter and perhaps interacts with the rna polymerase beta subunit. here we demonstrate that dnaa p ...200312654908
identification and mutational analysis of bacteriophage prd1 holin protein p35.holin proteins are phage-induced integral membrane proteins which regulate the access of lytic enzymes to host cell peptidoglycan at the time of release of progeny viruses by host cell lysis. we describe the identification of the membrane-containing phage prd1 holin gene (gene xxxv). the prd1 holin protein (p35, 12.8 kda) acts similarly to its functional counterpart from phage lambda (gene s), and the defect in prd1 gene xxxv can be corrected by the presence of gene s of lambda. several nonsense ...200312813073
a spring-loaded state of nusg in its functional cycle is suggested by x-ray crystallography and supported by site-directed mutants.transcription factor nusg is present in all prokaryotes, and orthologous proteins have also been identified in yeast and humans. nusg contains a 27-residue kow motif, found in ribosomal protein l24 where it interacts with rrna. nusg in escherichia coli (ecnusg) is an essential protein and functions as a regulator of rho-dependent transcription termination, phage lambda n and rrna transcription antitermination, and phage hk022 nun termination. relative to ecnusg, aquifex aeolicus nusg (aanusg) an ...200312600194
growth-rate dependent rna polyadenylation in escherichia coli.rna polyadenylation occurs not only in eukaryotes but also in bacteria. in prokaryotes, polyadenylated rna molecules are usually degraded more efficiently than non-modified transcripts. here we demonstrate that two transcripts, which were shown previously to be substrates for poly(a) polymerase i (pap i), escherichia coli lpp messenger rna and bacteriophage lambda oop rna, are polyadenylated more efficiently in slowly growing bacteria than in rapidly growing bacteria. intracellular levels of pap ...200312612607
bacteriophage lambda repressor allelic modulation of the rex exclusion phenotype.the sensitivity of delta red-gam delta ren mutants of bacteriophage lambda to rex exclusion by lambda rexa+ rexb+ lysogens is modulated by the prophage ci repressor allele. we show the following: (i) lambda spi156 delta nin5 forms plaques on a ci+-rexa+-rexb+ lysogen with 10(5)-fold higher efficiency than on ci[ts]-rexa+-rexb+ derivatives. (ii) the ci[ts]857 allele augmentation of rex exclusion is recessive to ci+. (iii) the ci857-mediated increase in rex exclusion activity involves the particip ...200312795410
alterations of the portal protein, gpb, of bacteriophage lambda suppress mutations in cosq, the site required for termination of dna packaging.the cosq site of bacteriophage lambda is required for dna packaging termination. previous studies have shown that cosq mutations can be suppressed in three ways: by a local suppressor within cosq, an increase in the length of the lambda chromosome, and missense mutations affecting the prohead's portal protein, gpb. in the present work, revertants of a set of lethal cosq mutants were screened for suppressors. seven new cosq suppressors affected gene b, which encodes the portal protein of the proh ...200212019220
protein-protein and protein-dna interactions of sigma70 region 4 involved in transcription activation by lambdaci.the ci protein of bacteriophage lambda (lambdaci) activates transcription from promoter p(rm) through an acidic patch on the surface of its dna-binding domain. genetic evidence suggests that this acidic patch stimulates transcription from p(rm) through contact with the c-terminal domain (region 4) of the sigma(70) subunit of escherichia coli rna polymerase. here, we identify two basic residues in region 4 of sigma(70) that are critical for lambdaci-mediated activation of transcription from p(rm) ...200212421556
scanning mutagenesis identifies amino acid residues essential for the in vivo activity of the escherichia coli dnaj (hsp40) j-domain.the dnaj (hsp40) cochaperone regulates the dnak (hsp70) chaperone by accelerating atp hydrolysis in a cycle closely linked to substrate binding and release. the j-domain, the signature motif of the hsp40 family, orchestrates interaction with the dnak atpase domain. we studied the j-domain by creating 42 mutant e. coli dnaj variants and examining their phenotypes in various separate in vivo assays, namely, bacterial growth at low and high temperatures, motility, and propagation of bacteriophage l ...200212454054
generation and pcr screening of bacteriophage lambda sublibraries enriched for rare clones (the "sublibrary method"). 200212494671
requirement for nusg for transcription antitermination in vivo by the lambda n protein.transcription antitermination by the bacteriophage lambda n protein is stimulated in vitro by the escherichia coli nusg protein. earlier work suggested that nusg was not required for n activity in vivo. here we present evidence that nusg also stimulates n-mediated transcription antitermination in intact cells.200212029062
the cell surface protein ag43 facilitates phage infection of escherichia coli in the presence of bile salts and carbohydrates.it was found that infection of escherichia coli by bacteriophage lambda is inhibited in the presence of certain bile salts and carbohydrates when cells are in the "off" state for production of the phase-variable cell surface protein antigen 43 (ag43). the inhibition of phage growth was found to be due to a significant impairment in the process of phage adsorption. expression of the gene encoding ag43 (agn43) from a plasmid or inactivation of the oxyr gene (encoding an activator of genes importan ...200211988528
arm-site binding by lambda -integrase: solution structure and functional characterization of its amino-terminal domain.the integrase protein (int) from bacteriophage lambda catalyzes the insertion and excision of the viral genome into and out of escherichia coli. it is a member of the lambda-int family of site-specific recombinases that catalyze a diverse array of dna rearrangements in archaebacteria, eubacteria, and yeast and belongs to the subset of this family that possesses two autonomous dna-binding domains. the heterobivalent properties of int can be decomposed into a carboxyl-terminal domain that executes ...200211904406
rapid construction of adenoviral vectors by lambda phage genetics.continued improvements of adenoviral vectors require the investigation of novel genome configurations. since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid dna, it is possible to separate genome construction from virus production. in this way failure to generate a virus is not associated with an inability to generate the desired genome. we have developed a novel lambda-based system that allows rapid modification of the viral gen ...200211907206
excretion of human beta-endorphin into culture medium by using outer membrane protein f as a fusion partner in recombinant escherichia coli.escherichia coli bl21 strains were found to excrete a large amount of outer membrane protein f (ompf) into culture medium during high-cell-density cultivation. from this interesting phenomenon, a novel and efficient ompf fusion system was developed for the excretion of recombinant proteins by e. coli. the ompf gene of e. coli bl21(de3) was first knocked out by using the red operon of bacteriophage lambda to construct e. coli mbel-bl101. for the excretion of human beta-endorphin as a model protei ...200212324347
increased bar minigene mrna stability during cell growth inhibition.bacteriophage lambda is unable to grow vegetatively on escherichia coli mutants defective in peptidyl-trna hydrolase (pth) activity. mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to pth-defective cells. two of these wild-type bar regions, bari+ and barii+, contain minigenes with similar aug-aua-stop codon sequ ...200111136457
genetic footprinting in bacteria.in vivo genetic footprinting was developed in the yeast saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. we have developed in vivo genetic footprinting for escherichia coli, a model bacterium and pathogen. we further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of e. coli under a variety of growth conditions. the definitive features of this system incl ...200111160101
bacteriophage lambda: alive and well and still doing its thing.the lambda (lambda) family of bacteriophages continues to provide significant insights into the understanding of basic biological processes, as well as useful technological innovations. areas in which recent advances have occurred include transcription elongation, repressor interactions, genomics and post-transcriptional regulation. the homologous lambda recombination functions have been exploited as an efficient in vivo recombinant engineering system for functional genomic studies. the virulenc ...200111282477
specific and non-specific interactions of integration host factor with dna: thermodynamic evidence for disruption of multiple ihf surface salt-bridges coupled to dna binding.site-specific dna binding of architectural protein integration host factor (ihf) is involved in formation of functional multiprotein-dna assemblies in escherichia coli, while non-specific binding of ihf and other histone-like proteins serves to structure the nucleoid. here, we report an isothermal titration calorimetry study of the thermodynamics of binding ihf to a 34 bp fragment composed entirely of the specific h' site from lambda-phage dna. at low to moderate [k(+)] (60-100 mm), strong compe ...200111428896
purification and crystallization of cii: an unstable transcription activator from phage lambda.the cii protein of the temperate bacteriophage lambda is a transcriptional activator involved in the lysis-lysogeny switch of the phage. it is an unstable protein of 97 amino acids and is known to exist as a tetramer in the native state. the cii gene has been cloned and expressed in escherichia coli using a t7 promoter based over-expression system. the recombinant cii protein has been purified to homogeneity by ammonium sulfate fractionation followed by two steps of ion-exchange chromatography. ...200111689008
a new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody.signal transduction from cell surface receptors to the nucleus is regulated in most part by protein phosphorylation. for the purpose of identification of kinases which play an important role at a particular phosphorylation step in a series of signal transduction pathways, we have developed a new expression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide. we have applied this method for screening kinases which phosphorylate stat3 at seri ...200111150545
what makes the bacteriophage lambda red system useful for genetic engineering: molecular mechanism and biological function.recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. the system, called red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsdna end; beta protein, which binds to ssdna and promotes strand annealing; and gamma protein, which binds to the bacterial recbcd enzyme and inhibits its activities. these proteins induce a 'hyper-rec' state in escherichia col ...200111445160
single-strand interruptions in replicating chromosomes cause double-strand breaks.replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template dna and collapse, generating double-strand ends. to model replication fork collapse in vivo, i constructed phage lambda chromosomes carrying the nicking site of m13 bacteriophage and infected with these substrates escherichia coli cells, producing m13 nicking enzyme. i detected double-strand breaks at the nicking sites in lambda dna purified from these cells. the double-strand break ...200111459959
cell toxicity caused by products of the p(l) operon of bacteriophage lambda.induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(l) operon. we genetically modified the lambda prophage to determine which lambda p(l) operon functions were involved in cell killing. viability assays and flow cytometry were used to monitor cell death and filamentation. the kil gene was shown to cause cell death and filamentation as described previously. another killing ac ...200111470529
expression using vectors with phage lambda regulatory sequences.in the expression system described here, plasmids (pskf) utilize regulatory signals--such as the powerful promoter pl--from the bacteriophage lambda. transcription from pl can be fully repressed and plasmids containing it are thus stabilized by the lambda repressor, ci. the repressor is supplied by an e. coli host which contains a integrated copy of a portion of the lambda genome. this so-called defective lysogen supplies the lambda regulatory proteins ci and n but does not provide the lytic com ...200118265129
mechanism for a transcriptional activator that works at the isomerization step.transcriptional activators in prokaryotes have been shown to stimulate different steps in the initiation process including the initial binding of rna polymerase (rnap) to the promoter and a postbinding step known as the isomerization step. evidence suggests that activators that affect initial binding can work by a cooperative binding mechanism by making energetically favorable contacts with rnap, but the mechanism by which activators affect the isomerization step is unclear. a well-studied examp ...200011087868
replication of orij-based plasmid dna during the stringent and relaxed responses of escherichia coli.the orij-based plasmids contain the origin of dna replication from the cryptic rac prophage, present in the chromosomes of most escherichia coli k-12 strains. the organization of the orij replication region resembles that of the bacteriophage lambda, although sequence similarity is small. here we investigated the regulation of replication of the orij-based plasmid in e. coli rela(+) and rela(-) hosts during amino acid starvation and limitation, i.e., during the stringent and relaxed responses. w ...200010964622
endonuclease and helicase activities of bacteriophage lambda terminase: changing nearby residue 515 restores activity to the gpa k497d mutant enzyme.terminase, the dna packaging enzyme of bacteriophage lambda, is a heteromultimer of gpnu1 and gpa subunits. in an earlier investigation, a lethal mutation changing gpa residue 497 from lysine to aspartic acid (k497d) was found to cause a mild change in the high-affinity atpase that resides in gpa and a severe defect in the endonuclease activity of terminase. the k497d terminase efficiently sponsored packaging of mature lambda dna into proheads. in the present work, k497d terminase was found to h ...200011062051
escherichia coli dna polymerase iv mutator activity: genetic requirements and mutational specificity.the dinb gene of escherichia coli is known to be involved in the untargeted mutagenesis of lambda phage. recently, we have demonstrated that this damage-inducible and sos-controlled gene encodes a novel dna polymerase, dna pol iv, which is able to dramatically increase the untargeted mutagenesis of f' plasmid. at the amino acid level, dna pol iv shares sequence homologies with e. coli umuc (dna pol v), rev1p, and rad30p (dna polymerase eta) of saccharomyces cerevisiae and human rad30a (xpv) prot ...200010913093
genetic system for reversible integration of dna constructs and lacz gene fusions into the escherichia coli chromosome.a plasmid system for site-specific integration into and excision and recovery of gene constructs and lacz gene fusions from the escherichia coli chromosome was developed. plasmid suicide vectors utilizing the origin of replication of r6k plasmids and containing the attp sequence of bacteriophage lambda, multiple cloning site, and antibiotic resistance markers facilitate reversible integration into the e. coli chromosome by site-specific recombination. additional vectors permit construction of la ...200010610816
proteolysis of bacteriophage lambda cii by escherichia coli ftsh (hflb).ftsh (hflb) is a conserved, highly specific, atp-dependent protease for which a number of substrates are known. the enzyme participates in the phage lambda lysis-lysogeny decision by degrading the lambda cii transcriptional activator and by its response to inhibition by the lambda ciii gene product. in order to gain further insight into the mechanism of the enzymatic activity of ftsh (hflb), we identified the peptides generated following proteolysis of the phage lambda cii protein. it was found ...200010809689
the crystal structure of nusb from mycobacterium tuberculosis.both prokaryotes and eukaryotes regulate transcription through mechanisms that suppress termination signals. an antitermination mechanism was first characterized in bacteriophage lambda. bacteria have analogous machinery that regulates ribosomal rna transcription and employs host factors, called the n-utilizing (where n stands for the phage lambda n protein) substances (nus), nusa, nusb, nuse and nusg. here we report the crystal structure of nusb from mycobacterium tuberculosis, the bacterium th ...200010881194
1,3-butadiene: cancer, mutations, and adducts. part ii: roles of two metabolites of 1,3-butadiene in mediating its in vivo genotoxicity.1,3-butadiene (bd) is carcinogenic in mice and rats, with mice being more susceptible than rats to its carcinogenic effects. 1,3-butadiene is mutagenic in the bone marrow and spleen cells of b6c3f1 laci transgenic mice. the goal of this research was to assess the roles of two bd metabolites, 1,2-epoxy-3-butene (bdo) and 1,2,3,4-diepoxybutane (bdo2), in the mutagenicity and mutational spectrum of the parent compound bd by determining the mutagenicity and mutational spectra of bdo and bdo2 in huma ...200010925839
detection of beta-amyloid peptide aggregation using dna electrophoresis.dna could readily associate with the aggregated forms of the beta-amyloid peptides beta(1-40) and beta(25-35), giving rise to a shift in the electrophoretic mobility of dna. as a result, dna was retained at the top of a 1% agarose gel. in contrast, the electrophoretic mobility of dna was little influenced by the monomeric forms of beta(1-40) and beta(25-30). dna from different sources such as lambda phage, escherichia coli plasmid, and human gene showed similar results. however, the electrophore ...200010964426
survey and summary: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories.holliday junction resolvases (hjrs) are key enzymes of dna recombination. a detailed computer analysis of the structural and evolutionary relationships of hjrs and related nucleases suggests that the hjr function has evolved independently from at least four distinct structural folds, namely rnase h, endonuclease, endonuclease vii-colicin e and rusa. the endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (vsr) and type ii restrict ...200010982859
addiction modules and programmed cell death and antideath in bacterial cultures.in bacteria, programmed cell death is mediated through "addiction modules" consisting of two genes. the product of the second gene is a stable toxin, whereas the product of the first is a labile antitoxin. here we extensively review what is known about those modules that are borne by one of a number of escherichia coli extrachromosomal elements and are responsible for the postsegregational killing effect. we focus on a recently discovered chromosomally borne regulatable addiction module in e. co ...199910547685
functional importance of regions in escherichia coli elongation factor nusa that interact with rna polymerase, the bacteriophage lambda n protein and rna.the association of the essential escherichia coli protein nusa with rna polymerase increases pausing and the efficiency of termination at intrinsic terminators. nusa is also part of the phage lambda n protein-modified antitermination complex that functions to prevent transcriptional termination. we have investigated the structure of nusa using various deletion fragments of nusa in a variety of in vitro assays. sequence and structural alignments have suggested that nusa has both s1 and kh homolog ...199910564494
regulation of copy number and stability of phage lambda derived ptc lambda 1 plasmid in the light of the dimer/multimer catastrophe hypothesis.the dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned. until now, this hypothesis has been investigated using multicopy cole1 plasmids. however, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the sam ...199910427732
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