Publications

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 ...	2003	12612607
a highly efficient recombineering-based method for generating conditional knockout mutations.	phage-based escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify dna cloned into plasmids, bacs, or pacs without the need for restriction enzymes or dna ligases. this new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. this method uses homologous recomb ...	2003	12618378
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 ...	2003	12622820
[on parametric stability of gene networks controlling ontogenetic processes].	the problem of evaluating the parametric stability of three models of pro- and eukaryotic gene networks controlling ontogenetic processes has been defined and solved. experimental plans of testing gene networks for parametric stability based on the method of generalized threshold models were developed and realized as a software application. we examined the "sensitivity" of the functioning modes to random variations of the parameters in the three model systems: the system of developmental control ...	2003	12624951
mechanism of inhibition of site-specific recombination by the holliday junction-trapping peptide wkhyny: insights into phage lambda integrase-mediated strand exchange.	holliday junctions are central intermediates in site-specific recombination reactions mediated by tyrosine recombinases. because these intermediates are extremely transient, only artificially assembled holliday junctions have been available for study. we have recently identified hexapeptides that cause the accumulation of natural holliday junctions of bacteriophage lambda integrase (int)-mediated reactions. we now show that one of these peptides acts after the first dna cleavage event to stabili ...	2003	12628247
genotoxicity of water extracts from the river yamuna at mathura, india.	water samples were collected from the river yamuna at mathura, india, and concentrated by using xad resins (amberlite xad-4 and xad-8) and liquid-liquid extraction procedures. the genotoxicities of the extracted water samples were evaluated by the ames salmonella/mammalian microsome test, dna repair of defective mutants, and bacteriophage lambda systems. the results of the salmonella test demonstrated that the xad-concentrated water samples had maximum mutagenicity with the ta98 strain, both wit ...	2003	12635095
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 ...	2003	12654908
[pleiotropic function of phosphoenolpyruvate-dependent phosphotransferase system in bacteria. report 1].	modern data (collected mainly in the 1998-2001 studies) about the transport of carbohydrates in bacteria, about the regulation of utilization of sugars via the glycolytic pathway as well as about the regulation of transformation of pyruvat into the products of secondary metabolism and of tricarboxylic acid cycle are presented in the survey. issues, related with the regulation of synthesis of enzymes involved in the last mentioned process, are discussed in detail. besides, the key pathways pertai ...	2003	12656043
arm sequences contribute to the architecture and catalytic function of a lambda integrase-holliday junction complex.	lambda integrase (int) mediates recombination between attachment sites on lambda phage and e. coli dnas. with the assistance of accessory proteins that induce dna loops, int bridges pairs of distinct arm- and core-type dna binding sites to form synapsed recombination complexes, which then recombine via a holliday junction (hj) intermediate. we show that, in addition to promoting the proper positioning of int protomers, the arm sequences facilitate the catalytic activities of the int tetramer, in ...	2003	12667459
bacteriophage spp1 chu is an alkaline exonuclease in the synexo family of viral two-component recombinases.	many dna viruses concatemerize their genomes as a prerequisite to packaging into capsids. concatemerization arises from either replication or homologous recombination. replication is already the target of many antiviral drugs, and viral recombinases are an attractive target for drug design, particularly for combination therapy with replication inhibitors, due to their important supporting role in viral growth. to dissect the molecular mechanisms of viral recombination, we and others previously i ...	2003	12670970
mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination.	bacteriophage lambda integrase (int) catalyzes site-specific recombination between pairs of attachment (att) sites. the att sites contain weak int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. we have characterized a number of mutant int proteins with substitutions at positions s282 (s282a, s282f, and s282t), s286 (s286a, s286l, and s286t), and r293 (r293e, r293k, and r293q). we investigated the core- and arm-binding p ...	2003	12670991
basic peptides as functional components of non-viral gene transfer vehicles.	improving the performance of non-viral gene-delivery vehicles that consist of synthetic compounds and nucleic acids is a key to successful gene therapy. supplementing synthetic vehicles with various biological functions by using natural or artificial peptides is a promising approach with which to achieve this goal. one of the obstacles hindering this effort is that some of the potentially useful peptides, especially those with many basic amino acid residues, interfere with the formation of the c ...	2003	12678853
phage hk022 nun protein represses translation of phage lambda n (transcription termination/translation repression).	the n-terminal arginine-rich motif of phage hk022 nun protein binds to nut sequences in phage lambda nascent transcripts and induces transcription termination. interactions between the nun c terminus and rna polymerase as well as the dna template are required for termination. we have isolated nun c-terminal point and deletion mutants that are unable to block transcription. the mutants bind nut rna and inhibit translation of the lambda n gene. thus hk022 excludes lambda both by terminating transc ...	2003	12684530
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 ...	2003	12694740
maltodextrin transport through lamb.	the trimeric protein lamb of e. coli k12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane and acts as a non-specific porin for small hydrophilic molecules. lamb serves also as a specific cell surface receptor for phages, including phage lambda. each monomer consists of an eighteen-stranded antiparallel beta-barrel with nine surface loops (l1 to l9). three loops fold into the beta-barrel, with loop l3 constricting the channel abou ...	2003	12700071
genetic screen for monitoring hepatitis c virus ns3 serine protease activity.	we have developed a genetic system to monitor the activity of the hepatitis c virus (hcv) ns3 serine protease. this genetic system is based on the bacteriophage lambda regulatory circuit where the viral repressor ci is specifically cleaved to initiate the switch from lysogeny to lytic infection. an hcv protease-specific target, ns5a-5b, was inserted into the lambda phage ci repressor. the target specificity of the hcv ns5a-5b repressor was evaluated by coexpression of this repressor with a beta- ...	2003	12709356
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 ...	2003	12715152
stability of monomeric cro variants: isoenergetic transformation of a type i' to a type ii' beta-hairpin by single amino acid replacements.	the thermodynamic stabilities of three monomeric variants of the bacteriophage lambda cro repressor that differ only in the sequence of two amino acids at the apex of an engineered beta-hairpin have been determined. the sequences of the turns are evk-xx-evk, where the two central residues are dg, gg, and gt, respectively. standard-state unfolding free energies, determined from circular dichroism measurements as a function of urea concentration, range from 2.4 to 2.7 kcal/mole, while those determ ...	2003	12717034
novel transgenic rat for in vivo genotoxicity assays using 6-thioguanine and spi- selection.	transgenic rodents are valuable models for investigating the genotoxicity of chemicals in vivo. here, we report the establishment of a novel transgenic rat for genotoxicity analysis. in this model, about 10 copies of lambdaeg10 dna carrying the gpt gene of e. coli and the red/gam genes of lambda phage are integrated per haploid genome of sprague-dawley rats at position 4q24-q31. after recovery of lambdaeg10 phage, point mutations in the gpt gene and deletions in the red/gam genes are identified ...	2003	12717780
the escherichia coli fis protein stimulates bacteriophage lambda integrative recombination in vitro.	the escherichia coli nucleoid-associated protein fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. while purified fis protein was shown to stimulate in vitro excision, fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. we demonstrate here that e. coli ...	2003	12730167
interplay between dnaa and seqa proteins during regulation of bacteriophage lambda pr promoter activity.	dnaa and seqa proteins are main regulators (positive and negative, respectively) of the chromosome replication in escherichia coli. nevertheless, both these replication regulators were found recently to be also transcription factors. interestingly, both dnaa and seqa control activity of the bacteriophage lambdap(r) promoter by binding downstream of the transcription start site, which is unusual among prokaryotic systems. here we asked what are functional relationships between these two transcrip ...	2003	12742018
hardware (dna) circuits.	a scheme is presented whereby a new genetic control circuit can be introduced into an organism, permitting the experimenter to turn the expression of a given gene (or set of genes) on or off at will. the proposed scheme involves a positive feedback loop--here, a positive regulator, the cii protein of phage lambda, with its structural gene engineered so as to require cii for its expression. this feedback loop creates the possibility of two stable steady states, with gene cii on or off. genes adde ...	2003	12754939
rna polymerase mutations that impair conversion to a termination-resistant complex by q antiterminator proteins.	bacteriophage lambda q-protein stably binds and modifies rna polymerase (rnap) to a termination-resistant form. we describe amino acid substitutions in rnap that disrupt q-mediated antitermination in vivo and in vitro. the positions of these substitutions in the modeled rnap/dna/rna ternary elongation complex, and their biochemical properties, suggest that they do not define a binding site for q in rnap, but instead act by impairing interactions among core rnap subunits and nucleic acids that ar ...	2003	12756229
immunity profiles of wild-type and recombinant shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens.	the pathogenicity of shiga-like toxin (stx)-producing escherichia coli (stec), notably serotype o157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx(1) and/or stx(2)) that are known to be carried on temperate lambdoid bacteriophages. stx phages were isolated from different stec strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx(1) or stx(2) genes. ...	2003	12761125
parametric stability evaluation in computer experiments on the mathematical model of drosophila control gene subnetwork.	using the method of generalized threshold models, the problem is formulated and solved to evaluate the parametric stability of the model of a gene subnetwork controlling the early ontogenesis of the fruit fly drosophila melanogaster. computer experiments have been performed to test the parametric stability of the model. quantitative evaluations have been obtained for parametric stability of the drosophila gene subnetwork in nuclei along the embryo's anterior-posterior axis. the results of comput ...	2003	12762850
amidase domains from bacterial and phage autolysins define a family of gamma-d,l-glutamate-specific amidohydrolases.	several phage-encoded peptidoglycan hydrolases have been found to share a conserved amidase domain with a variety of bacterial autolysins (n-acetylmuramoyl-l-alanine amidases), bacterial and eukaryotic glutathionylspermidine amidases, gamma-d-glutamyl-l-diamino acid endopeptidase and nlp/p60 family proteins. all these proteins contain conserved cysteine and histidine residues and hydrolyze gamma-glutamyl-containing substrates. these cysteine residues have been shown to be essential for activity ...	2003	12765833
dynamics and dna substrate recognition by the catalytic domain of lambda integrase.	bacteriophage lambda integrase (lambda-int) is the prototypical member of a large family of enzymes that catalyze site-specific dna recombination via the formation of a holliday junction intermediate. dna strand cleavage by lambda-int is mediated by nucleophilic attack on the scissile phosphate by a conserved tyrosine residue, forming an intermediate with the enzyme covalently attached to the 3'-end of the cleaved strand via a phosphotyrosine linkage. the crystal structure of the catalytic domai ...	2003	12767827
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 ...	2003	12768624
recombineering with overlapping single-stranded dna oligonucleotides: testing a recombination intermediate.	a phage lambda-based recombination system, red, can be used for high-efficiency mutagenesis, repair, and engineering of chromosomal or episomal dna in vivo in escherichia coli. when long linear double-stranded dna with short flanking homologies to their targets are used for the recombination, the lambda exo, beta, and gam proteins are required. the current model is: (i) gam inhibits the host recbcd activity, thereby protecting the dna substrate for recombination; (ii) exo degrades from each dna ...	2003	12771385
cloning and characterization of the groe heat-shock operon of the marine bacterium vibrio harveyi.	the dna region of the vibrio harveyi chromosome containing the heat-shock genes groes and groel was cloned, and the genes were sequenced. these genes are arranged in the chromosome in the order groes-groel. northern hybridization experiments with rna from v. harveyi and a dna probe carrying both groes and groel genes showed a single, heat-inducible transcript of approximately 2200 nt, indicating that these genes form an operon. primer extension analysis revealed a strong, heat-inducible transcri ...	2003	12777488
ests from the basidiomycete schizophyllum commune grown on nitrogen-replete and nitrogen-limited media.	lambda phage cdna libraries were constructed using mrnas from the basidiomycete schizophyllum commune grown on media with high or low nitrogen concentrations. a total of 440 clones were sequenced, representing 373 distinct transcripts. of these, 166 showed significant similarity to annotated genes in genbank. those that could be tentatively identified using blast searches were classified by function using the gene ontology (go) database. genes with products involved in cell-cycle processes were ...	2003	12781677
dynamics of large semiflexible chains probed by fluorescence correlation spectroscopy.	fluorescence correlation spectroscopy was used to probe the dynamics of lambda-phage dna in aqueous solution labeled with the randomly intercalating dye toto. the linear macromolecules (i). carry more than one chromophore and (ii). are larger than the waist of the focal volume. the correlation function decays significantly faster than expected for a stiff globule of corresponding size but is in good agreement with the dynamic model of semiflexible chains including hydrodynamic interactions. as t ...	2003	12786596
role of e.coli transcription-repair coupling factor mfd in nun-mediated transcription termination.	phage hk022 nun protein excludes phage lambda by binding nascent lambda-nut rna and inducing termination and transcript release. in contrast, in a purified in vitro system, nun arrests transcription on lambdadna templates without dissociation of the transcription elongation complex (tec). our evidence indicates that transcription-repair coupling factor (mfd) frees nun-arrested rna polymerase. the activity of nun is enhanced in an mfd-null mutant, consistent with prolonged association of nun with ...	2003	12787667
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 ...	2003	12795410
analysis of insertion into secondary attachment sites by phage lambda and by int mutants with altered recombination specificity.	when phage lambda lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. here, we characterize the secondary sites that are preferred by wild-type lambda and by lambda int mutants with altered insertion specificity. the sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. the imperfect inverted repeats of th ...	2003	12798688
nano-scale imaging of chromosomes and dna by scanning near-field optical/atomic force microscopy.	nano-scale structures of the yoyo-1-stained barley chromosomes and lambda-phage dna were investigated by scanning near-field optical/atomic force microscopy (snom/afm). this technique enabled precise analysis of fluorescence structural images in relation to the morphology of the biomaterials. the results suggested that the fluorescence intensity does not always correspond to topographic height of the chromosomes, but roughly reflects the local amount and/or density of dna. various sizes of the b ...	2003	12801660
the herpes simplex virus type 1 alkaline nuclease and single-stranded dna binding protein mediate strand exchange in vitro.	the replication of herpes simplex virus type 1 (hsv-1) dna is associated with a high degree of homologous recombination. while cellular enzymes may take part in mediating this recombination, we present evidence for an hsv-1-encoded recombinase activity. hsv-1 alkaline nuclease, encoded by the ul12 gene, is a 5'-->3' exonuclease that shares homology with redalpha, commonly known as lambda exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. the hsv-1 single-s ...	2003	12805441
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 ...	2003	12813073
weak yet distinct mutagenicity of acrylamide in mammalian cells.	despite concern raised with the announcement that common heating processes such as frying introduce acrylamide, a known rodent carcinogen, into food, the mutagenicity of acrylamide in mammalian dna is controversial.	2003	12813172
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 ...	2003	12813564
the rna-protein complex: direct probing of the interfacial recognition dynamics and its correlation with biological functions.	the n protein from bacteriophage lambda is a key regulator of transcription antitermination. it specifically recognizes a nascent mrna stem loop termed boxb, enabling rna polymerase to read through downstream terminators processively. the stacking interaction between trp-18 of wt n protein and a7 of boxb rna is crucial for efficient antitermination. here, we report on the direct probing of the dynamics for this interfacial binding and the correlation of the dynamics with biological functions. sp ...	2003	12815093
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 ...	2003	12826057
development of a novel method of lytic phage delivery by use of a bacteriophage p22 site-specific recombination system.	bacteriophage therapy represents a potential alternative to the use of antibiotics to control proliferation of pathogenic bacteria. as an alternative to the strategy where a limited number of doses of large numbers of lytic bacteriophages are administered, a novel method delivery system was developed so that phages are continually released into the culture. specifically, a non-pathogenic escherichia coli strain was constructed that was lysogenic for a lytic mutant of bacteriophage lambda. this l ...	2003	12829296
identification of the lambda integrase surface that interacts with xis reveals a residue that is also critical for int dimer formation.	lambda integrase (int) is a heterobivalent dna-binding protein that together with the accessory dna-bending proteins ihf, fis, and xis, forms the higher-order protein-dna complexes that execute integrative and excisive recombination at specific loci on the chromosomes of phage lambda and its escherichia coli host. the large carboxyl-terminal domain of int is responsible for binding to core-type dna sites and catalysis of dna cleavage and ligation reactions. the small amino-terminal domain (resid ...	2003	12832614
crystallization and preliminary x-ray crystallographic analysis of the excisionase-dna complex from bacteriophage lambda.	bacteriophage lambda uses an elegantly regulated and highly directional site-specific dna-recombination reaction to integrate and excise its genome. a critical regulator of this process is the phage-encoded excisionase (xis) protein, which dramatically stimulates excision by orchestrating the assembly of a higher order nucleoprotein structure that excises the prophage. the xis protein stabilizes this recombination intermediate by substantially altering the trajectory of viral dna and by cooperat ...	2003	12832771
genotoxic hazards of long-term application of wastewater on agricultural soil.	in the city of aligarh (india), wastewater coming from both industrial and domestic sources and without any treatment is used to irrigate the agricultural crops. this practice has been polluting the soil, and the pollutants could possibly reach the food chain. for the above reason, soil irrigated with wastewater was sampled and monitored for the presence of genotoxic agents using three biological assays namely ames salmonella/mammalian microsome test, survival of sos defective e. coli k-12 mutan ...	2003	12834763
genetic analysis of bacteriophage lambdan-dependent antitermination suggests a possible role for the rna polymerase alpha subunit in facilitating specific functions of nusa and nuse.	a role for the escherichia coli rna polymerase alpha subunit in transcription antitermination dependent on bacteriophage lambda n protein has been previously inferred from the isolation of rpoa mutants that alter the efficiency of this process. this report describes studies on the efficiency of n-dependent transcription antitermination in a strain containing the rpoa341 mutation, which interferes with this process. the effect of mutations in genes coding for different nus factors and/or plasmids ...	2003	12845423
genome of xanthomonas oryzae bacteriophage xp10: an odd t-odd phage.	xp10 is a lytic bacteriophage of the phytopathogenic bacterium xanthomonas oryzae. though morphologically xp10 belongs to the syphoviridae family, it encodes its own single-subunit rna polymerase characteristic of t7-like phages of the podoviridae family. here, we report the determination and analysis of the 44,373 bp sequence of the xp10 genome. the genome is a linear, double-stranded dna molecule with 3' cohesive overhangs and no terminal repeats or redundancies. half of the xp10 genome contai ...	2003	12850143
interactions between rickettsiae and dermacentor variabilis ticks: analysis of gene expression.	tick-borne spotted fever group rickettsiae are maintained in nature primarily through transstadial and transovarial transmission in their vector. in the tick, dermacentor variabilis, the infection of, and persistence within, the ovaries are critical steps in the transmission cycle of rickettsiae from one generation to the next. although ixodid ticks can experimentally maintain several species of rickettsiae, often only a single species is maintained transovarially. the molecular mechanisms under ...	2003	12860691
electrostatic interactions in a peptide--rna complex.	parallel experimental measurements and theoretical calculations have been used to investigate the energetics of electrostatic interactions in the complex formed between a 22 residue, alpha-helical peptide from the n protein of phage lambda and its cognate 19 nucleotide box b rna hairpin. salt-dependent free energies were measured for both peptide folding from coil to helix and peptide binding to rna, and from these the salt-dependence of binding pre-folded, helical peptide to rna was determined ...	2003	12875837
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 ...	2003	12881484
a conformational switch controls the dna cleavage activity of lambda integrase.	the bacteriophage lambda integrase protein (lambda int) belongs to a family of tyrosine recombinases that catalyze dna rearrangements. we have determined a crystal structure of lambda int complexed with a cleaved dna substrate through a covalent phosphotyrosine bond. in comparison to an earlier unliganded structure, we observe a drastic conformational change in dna-bound lambda int that brings tyr342 into the active site for cleavage of the dna in cis. a flexible linker connects the central and ...	2003	12887904
a simple two-step, 'hit and fix' method to generate subtle mutations in bacs using short denatured pcr fragments.	the bacteriophage lambda recombination system has proven to be a valuable tool for engineering bacterial artificial chromosomes (bac). due to its high efficiency, subtle alterations in the bacs can be generated using oligonucleotides as targeting vectors. since no selection marker is used, recombinant clones are identified utilizing a selective pcr screening method. however, occasionally the selective pcr screening is not feasible. we describe here a two-step 'hit and fix' method that can be rel ...	2003	12888532
combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method.	recombinogenic engineering or recombineering is a powerful new method to engineer dna without the need for restriction enzymes or ligases. we report here a general method for using recombineering to combine overlapping bacterial artificial chromosomes (bacs) to build larger, unified bacs. in order to test the feasibility of using recombineering to combine two large dna fragments (>20 kb), we constructed a unified bac containing the full-length tyrosinase-related protein-1 (tyrp-1) gene from two ...	2003	12888533
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 ...	2003	12890019
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 ...	2003	12890027
establishment and maintenance of hsv latent infection is mediated through correct splicing of the lat primary transcript.	to study the effect of the splicing of hsv-1 latency-associated transcript (lat) on viral latency, we constructed two mutant viruses (fhlambda+ and fhlambda-) in which the 168-bp hpai-hpai fragment within the 2-kb lat intron was replaced by a 447-bp bacteriophage lambda sequence. the lambda dna was inserted in opposite orientations in fhlambda+ and fhlambda-. the mutation in fhlambda+ disrupted the splicing of lat primary transcript and altered both lat exon and intron, whereas the mutation in f ...	2003	12890636
[construction of gene library of arthrobacter bt801 and isolation & expression of hydantoinase gene].	hydantoinase can be widely used in enzymic production of various amino acids. in order to obtain the hydantoinase genes in arthrobacter bt801, its chromatosomal dna is isolated and partialy digested with sau3a i to collect fragments of about 30kb. then, this fragment is inserted into the hpa i and pst i site of cosmid pkc505. the genomic library was thus constructed by packing in vitro with lambda phage package protein and transfecting e. coli dh5alpha. a positive transformant was selected from ...	2003	15969007
bacteriophage hk022 nun protein: a specific transcription termination factor that excludes bacteriophage lambda.		2003	14712713
genetic and biochemical strategies to elucidate the architecture and targets of a processive transcription antiterminator from bacteriophage lambda.		2003	14712720
enhanced levels of lambda red-mediated recombinants in mismatch repair mutants.	homologous recombination can be used to generate recombinants on episomes or directly on the escherichia coli chromosome with pcr products or synthetic single-stranded dna (ssdna) oligonucleotides (oligos). such recombination is possible because bacteriophage lambda-encoded functions, called red, efficiently recombine linear dna with homologies as short as 20-70 bases. this technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homolog ...	2003	14673109
enhancement of the mutagenicity of benzo(a)pyrene diol epoxide by a nonmutagenic dose of ultraviolet a radiation.	we investigated the effects of single and combined exposures to two ubiquitous environmental carcinogens, polycyclic aromatic hydrocarbons and uva radiation, in big blue mouse embryonic fibroblasts. we quantified the cytotoxicity, dna adduct formation, and induction of mutations in the cii transgene in cells treated with a single agent or combinations of agents in both direct and reverse order. mapping of dna adducts by terminal transferase-dependent pcr showed the preferential formation of bulk ...	2003	14695185
isolation and characterization of serine protease gene(s) from perkinsus marinus.	this study reports the first serine protease gene(s) isolated from perkinsus marinus. using universal primers, a 518 bp subtilisin-like serine protease gene fragment was amplified from p. marinus genomic dna and used as a probe to screen a lambda-phage p. marinus genomic library; 2 different lambda-phage clones hybridized to the digoxigenin(dig)-labeled subtilisin-like gene fragment. following subcloning and sequencing of the larger dna fragment, a 1254 bp open reading frame was identified and l ...	2003	14735929
the processing of high-molecular-weight xylanase (xyne, 110 kda) from aeromonas caviae me-1 to 60-kda xylanase (xyne60) in escherichia coli and purification and characterization of xyne60.	a xylanase gene (xyne) encoding xyne (110 kda) was cloned from a lambda phage genomic library of aeromonas caviae me-1 which is a multiple-xylanase-producing bacterium. upon nucleotide sequence analysis, we found that xyne comprises 2823 by and encodes a protein of 941 amino acid residues (104,153 da), which was similar to endo-beta-1,4-xylanases which are categorized to glycosyl hydrolase family 10. an escherichia coli transformant that harbored pxed30 carrying xyne produced 110-, 84-, 72-, and ...	2003	16233373
an efficient method of constructing homologous recom binant baculovirus with pcr-amplified fragments.	this paper describes a rapid method of constructing homologous recombinant baculovirus ine. coli with pcr-amplified fragments. by using this method, the traditional steps of constructing transfer vector are omitted. the method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. taking hasnpv as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (cm ( r )) to replace ...	2003	21072615
recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes.	an efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in escherichia coli is described. for this purpose, homologous recombination of linear double-stranded targeting dna was mediated by the bacteriophage lambda recombination functions using very short regions of homology. initial manipulation of the incfii target plasmids r1 and r1drd19 indicated that the linear targeting dna should be devoid of all extraneous homologies to the t ...	2002	19709285
in vitro selection of n peptide-binding rna on a quartz-crystal microbalance.	we report here an in vitro selection of a hairpin loop-rna bound to n peptide from bacteriophage lambda on a 27 mhz quartz-crystal microbalance (qcm) to study an interaction between an rna structure and an rna-binding peptide. the n peptide was immobilized on a qcm electrode and specific rnas binding to the peptide were selected from a gnrna pentaloop-randomized rna library with in situ monitoring of selection processes using a qcm. after the 5th round selection, the consensus sequences includin ...	2002	12903178
inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors.	bacteriophage lambda protein phosphatase (lambdapp) with mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (epr) spectroscopy. orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdapp, albeit with inhibition constants (k(i)) that range over 5 orders of magnitude. in addition, small organic anions were tested as ...	2002	11790129
phage genomics: small is beautiful.	the age of genomics dawned only gradually for bacteriophages. it was 1977 when the genome of phage phi x174 was published and 1983 when the "large" genome of phage lambda hit the streets. more recently, the pace has quickened, so that we now have over 100 complete phage genomes and can expect thousands in a very few years. these sequences have been marvelously informative for the biology of the individual phages, but with the advent of high volume sequencing technology, the real excitement for p ...	2002	11792317
detection and evaluation of non-recombinants in cdna libraries by multiple cloning region pcr.	bacteriophages that are routinely used in cdna libraries do not require any biological selection for forming plaques. thus parental non-recombinant phages are always found in variable proportions together with recombinant ones in all cdna libraries. the presence of non-recombinants in significant proportions dilutes the abundance of rare cdna species and makes library screening difficult. if the exact proportion of non-recombinants in a library were known, then one would screen proportionately m ...	2002	11808704
a calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis.	mutations in the tuberous sclerosis 2 (tsc2) gene product have been genetically linked to the pathology of both tuberous sclerosis (tsc) and the gender-specific lung disease, lymphangioleiomyomatosis (lam). both diseases are classified as disorders of cellular migration, proliferation, and differentiation. earlier studies from our laboratory (1) linked tsc2 with steroid/nuclear receptor signaling. studies presented here provide evidence for calmodulin (cam) signaling in the propagation of this t ...	2002	11811958
site-specific photo-cross-linking between lambda integrase and its dna recombination target.	the site-specific recombinase (int) of bacteriophage lambda is a heterobivalent dna-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target dna (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to dna binding at the "core-type" sequences where dna cleavage and ligation are executed. we construct ...	2002	11827961
isolation and characterization of replication-competent molecular dna clones of hiv type 1 crf01_ae with different coreceptor usages.	we have isolated replication-competent molecular clones of hiv-1 circulating recombinant form crf01_ae with different coreceptor usages. after lambda phage cloning of unintegrated circular proviral dnas derived from a crf01_ae strain (hiv-1nh1), isolated in japan, the infectious molecular clone, designated p93jp-nh1, was reconstituted. 93jp-nh1 showed an x4 and r5 phenotype in np2 cell-based coreceptor utilization assays and exerted robust replication in human t cell lines, including mt2, m8166, ...	2002	11839144
the lambda switch: ci closes the gap in autoregulation.	the bacteriophage lambda genetic switch is still yielding surprises. a recent study reveals that a long-range interaction involving proteins bound 2.4 kilobases away from one another on the phage genome mediates negative autoregulation, solving a long-standing puzzle concerning the regulation of lysogeny.	2002	11839286
cloning and sequence analysis of the complementary dna for feline preproparathyroid hormone.	to clone and sequence the cdna for feline preproparathyroid hormone (prepropth) and to compare that sequence with other known parathyroid hormone (pth) sequences.	2002	11843117
transcription termination: primary intermediates and secondary adducts.	in living organisms, stable elongation complexes of rna polymerase dissociate at specific template positions in a process of transcription termination. it has been suggested that the dissociation is not the immediate cause of termination but is preceded by catalytic inactivation of the elongation complex. in vitro reducing ionic strength can be used to stabilize very unstable and catalytically inactive complex at the point of termination; the previous biochemical characterization of this complex ...	2002	11856750
the large subunit of bacteriophage lambda's terminase plays a role in dna translocation and packaging termination.	the dna packaging enzyme of bacteriophage lambda, terminase, is a heteromultimer composed of a small subunit, gpnu1, and a large subunit, gpa, products of the nu1 and a genes, respectively. the role of terminase in the initial stages of packaging involving the site-specific binding and cutting of the dna has been well characterized. while it is believed that terminase plays an active role in later post-cleavage stages of packaging, such as the translocation of dna into the head shell, this has n ...	2002	11866517
bacteriophage lambda as a vehicle for peptide and protein display.	bacteriophage lambda has emerged as an alternative vehicle for the surface display of peptides and proteins to the commonly used filamentous phage. there are a number of unique features that make lambda an attractive display vehicle including the ability to display multimeric proteins, no requirement for secretion of the displayed fusion protein and the means to vary the valency of the displayed fusion protein. with its increasing use for cdna encoded display, the lambda display systems will be ...	2002	11883504
complete genomic sequence of sfv, a serotype-converting temperate bacteriophage of shigella flexneri.	bacteriophage sfv is a temperate serotype-converting phage of shigella flexneri. sfv encodes the factors involved in type v o-antigen modification, and the serotype conversion and integration-excision modules of the phage have been isolated and characterized. we now report on the complete sequence of the sfv genome (37,074 bp). a total of 53 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was used to construct a functional map. the gene ...	2002	11889106
elovl1 and p55cdc genes are localized in a tail-to-tail array and are co-expressed in proliferating cells.	elovl1 is a ubiquitously expressed gene, the product of which belongs to a highly conserved family of microsomal enzymes, which are involved in the formation of very long chain fatty acids and sphingolipids in yeast to man. to elucidate the structure and regulation of the elovl gene we have isolated a lambda phage genomic dna clone containing the entire mouse gene and found that elovl1 consists of eight exons that are dispersed over 5.4 kb of genomic sequence. interestingly, sequencing of the la ...	2002	11891222
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 ...	2002	11904406
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 ...	2002	11907206
xis protein binding to the left arm stimulates excision of conjugative transposon tn916.	tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((tn916)int) and excisionase ((tn916)xis) proteins. in this study, we have investigated the role of the (tn916)xis protein in stimulating excisive recombination. the functional relevance of (tn916)xis binding sites on the arms of the transposon has been assessed in vivo using ...	2002	11914339
virus-assisted loading of polymer nanocontainer.	we present a dna-containing polymeric nanocontainer using the self-assembled superstructure of amphiphilic block copolymers in aqueous solutions. to demonstrate that dna translocation is possible across a completely synthetic block copolymer membrane, we have used a phage transfection strategy as a dna-transfer model system. for this purpose the bacterial channel forming protein lamb was reconstituted in aba-triblock copolymer vesicles. the outer membrane protein lamb is a specific transporter f ...	2002	11917114
bacteriophage mu genome sequence: analysis and comparison with mu-like prophages in haemophilus, neisseria and deinococcus.	we report the complete 36,717 bp genome sequence of bacteriophage mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete mu-like prophage genomes found in the genomes of a diverse group of bacteria. the comparative studies confirm that members of the mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups ...	2002	11922669
gene products encoded in the ninr region of phage lambda participate in red-mediated recombination.	the ninr region of phage lambda contains two recombination genes, orf (ninb) and rap (ning), that were previously shown to have roles when the recf and recbcd recombination pathways of e. coli, respectively, operate on phage lambda.	2002	11952832
efficient method to generate homologous recombinant baculovirus genomes in e. coli.	here we describe a convenient method to generate homologous recombinant baculoviral genomes in e. coli. the recombination takes place with the aid of recombination enzymes provided by the phage lambda red system between a bacmid (a baculoviral genome that can replicate in bacteria) and a linear fragment. proof of concept was provided when the cathepsin gene (v-cath) of the helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (hasnpv) was replaced by the chloramphenicol resistance gene ( ...	2002	11962600
regulation of noise in the expression of a single gene.	stochastic mechanisms are ubiquitous in biological systems. biochemical reactions that involve small numbers of molecules are intrinsically noisy, being dominated by large concentration fluctuations. this intrinsic noise has been implicated in the random lysis/lysogeny decision of bacteriophage-lambda, in the loss of synchrony of circadian clocks and in the decrease of precision of cell signals. we sought to quantitatively investigate the extent to which the occurrence of molecular fluctuations ...	2002	11967532
isolation and mapping of self-assembling protein domains encoded by the saccharomyces cerevisiae genome using lambda repressor fusions.	understanding how proteins are able to form stable complexes is of fundamental interest from the perspective of protein structure and function. here we show that lambda repressor fusions can be used to identify and characterize homotypic interaction domains encoded by the genome of saccharomyces cerevisiae, using a selection for polypeptides that can drive the assembly of the dna binding domain of bacteriophage lambda repressor. three high complexity libraries were constructed by cloning random ...	2002	11967834
recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy.	ige-mediated reactions to fish allergens represent one of the most frequent causes of food allergy. we have constructed an expression cdna library from carp (cyprinus carpio) muscle in phage lambda gt11 and used serum ige from a fish allergic patient to isolate 33 cdna clones that coded for two parvalbumin isoforms (cyp c 1.01 and cyp c 1.02) with comparable ige binding capacities. both isoforms represented calcium-binding proteins that belonged to the beta-lineage of parvalbumins. the cyp c 1.0 ...	2002	11971005
excluded volume effects in gene stretching.	we investigate the effects excluded volume on the stretching of a single dna in solution. we find that for small force f, the extension h is not linear in f but proportion to f(gamma), with gamma = (1 - nu)/nu, where nu is the well-known universal correlation length exponent. a freely joint chain model with the segment length chosen to reproduce the small extension behavior gives excellent fit to the experimental data of lambda-phage dna over the whole experimental range. we show that excluded v ...	2002	11979515
application of phage display peptide library to autoimmune diabetes: identification of ia-2/ica512bdc dominant autoantigenic epitopes.	autoantigenic epitope mapping represents a critical issue in autoimmune diseases. the islet tyrosine phosphatase-like protein ia-2/ica512bdc is a major autoantigen in type 1 diabetes (iddm), but the epitopes responsible for autoantibody binding have been only partially defined. the aim of our study was to identify ica512bdc epitopes, and in particular mini-epitopes, utilizing a novel strategy for autoimmune diseases. the study was performed in three sequential steps: (1) construction of a lambda ...	2002	11981830
mapping the c terminal epitope of the alzheimer's disease specific antibody mn423.	the mapping of monoclonal antibody epitopes is now predominantly carried out using molecular diversity techniques, phage display in particular. however, until very recently, phage display methods have been inappropriate for the analysis of epitopes that require a free carboxy terminus. here we describe the use of two different techniques to analyze the known c terminal epitope specificity of mn423, a monoclonal antibody specifically staining truncated tau in alzheimer's brain. using a lambda pha ...	2002	11983234
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 ...	2002	11988528
m-dna is stabilised in g*c tracts or by incorporation of 5-fluorouracil.	m-dna is a complex between the divalent metal ions zn2+, ni2+ and co2+ and duplex dna which forms at a ph of approximately 8.5. the stability and formation of m-dna was monitored with an ethidium fluorescence assay in order to assess the relationship between ph, metal ion concentration, dna concentration and the base composition. the dismutation of calf thymus dna exhibits hysteresis with the formation of m-dna occurring at a higher ph than the reconversion of m-dna back to b-dna. hysteresis is ...	2002	12000844
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 ...	2002	12019220
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.	2002	12029062
identification of the principal serological immunodeterminants of african swine fever virus by screening a virus cdna library with antibody.	protective immunity to african swine fever virus (asfv) may involve a combination of both serological and cellular mechanisms. this work is focused on the identification of the possible relevant serological immunodeterminants of immunity. thus, 14 serological immunodeterminants of asfv have been characterized by exhaustive screening of a representative lambda phage cdna expression library of the tissue culture-adapted ba71v strain of asfv. the library was constructed using rna extracted from ver ...	2002	12029148
stability puzzles in phage lambda.	in the absence of reca-mediated cleavage of the repressor, the lambda prophage is exceptionally stable. we develop a stochastic model that predicts the stability of such epigenetic states from affinities of the molecular components. we find that the stability, in particular, depends on the maximum possible ci protein production, and on the number of translated cro proteins per transcribed mrna. we apply the model to the behavior of recently published mutants of o(r) and find, in particular, that ...	2002	12059600
structures of two major histocompatibility complex class i genes of the rainbow trout (oncorhynchus mykiss).	here we describe two rainbow trout major histocompatibility complex (mhc) class i genes characterized from lambda phage genomic clones prepared from a single fish. clone gc71 contains all exons except a leader peptide-encoding exon. an open reading frame is maintained, and thus the gene mhconmy-u71 could be expressed in this individual. the class i gene found on clone gc41 lacks exons encoding the leader peptide and cytoplasmic domain. this gene, mhconmy-u41p, is a pseudogene due to a deletion i ...	2002	12073148