characterization of werner syndrome protein dna helicase activity: directionality, substrate dependence and stimulation by replication protein a.werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. the wild type werner syndrome protein (wrn) has been demonstrated to exhibit dna helicase activity in vitro. here we report further biochemical characterization of the wrn helicase. the enzyme unwinds double-stranded dna, translocating 3'-->5' on the enzyme-bound strand. hydrolysis of datp or atp, and to a lesser extent hydrolysis of dctp or ctp, supports wrn-catalyzed st ...19989611231
cloning and characterization of the dnak heat shock operon of the marine bacterium vibrio harveyi.we cloned the dna region of the vibrio harveyi chromosome containing the heat shock genes dnak and dnaj and sequenced them. these genes are arranged in the chromosome in the order dnak-dnaj, as in other proteobacteria of the alpha and gamma subdivisions. the dnak gene is 1923 nucleotides in length and codes for a protein of 640 amino acid residues, with a predicted molecular mass of 69,076 da and 81.2% similarity to the dnak protein of escherichia coli. the v. harveyi dnaj gene has a coding sequ ...19989747709
l-atp is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity?we demonstrate that l-atp is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-atp, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-atp strongly inhibits (ki 80 microm) the synthesis of rna primers catalysed by dna primase associated with human ...19999895305
characteristics of gene 28 product, the constituent of the central part of bacteriophage t4 baseplate.the phage td gene 28 product has been partially characterized and its biological role has been examined. it was found to be a protein with a molecular size of 24 kda which cosediments with the membrane fraction of the bacterial extracts and could only be washed out by a 0.2% sarcosyl solution. other observations indicate that gp 28 has a majority of hydrophobic residues on its surface and forms a homotrimeric complex in the absence of other phage proteins. the product was finally identified as a ...19989990707
dna polymerase mutagenic bypass and proofreading of endogenous dna lesions.dna polymerases differentiate between correct and incorrect substrates during synthesis on undamaged dna templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision. recent research examining dna polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms. inhibition of dna synthesis results from correct polymerase discrimination against utilization of geometrically incorrect temp ...199910064863
x-ray structure of t4 endonuclease vii: a dna junction resolvase with a novel fold and unusual domain-swapped dimer architecture.phage t4 endonuclease vii (endo vii), the first enzyme shown to resolve holliday junctions, recognizes a broad spectrum of dna substrates ranging from branched dnas to single base mismatches. we have determined the crystal structures of the ca2+-bound wild-type and the inactive n62d mutant enzymes at 2.4 and 2.1 a, respectively. the endo vii monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping. the major dimerization elements are two pairs of antipara ...199910075917
activity losses among t4 lysozyme variants after adsorption to colloidal silica.maintaining a specific molecular conformation is essential for the proper functioning of an enzyme. a substantial loss of catalytic activity can occur from the displacement caused by even a single amino acid substitution. activity may also be lost as an enzyme undergoes a conformational change during adsorption. in this study, we investigated the effect of thermostability on the activities of three t4 lysozyme variants after adsorption to 9 nm colloidal silica particles. less-stable t4 lysozyme ...199810099305
the non-enzymatic microbicidal activity of lysozymes.t4 lysozyme was thought to destroy bacteria by its muramidase activity. however, we demonstrate here that amphipathic helix stretches in the c-terminus of t4 lysozyme mediate its bactericidal and fungistatic activities. in heat-denatured t4 lysozyme, the enzymatic activity is completely abolished but unexpectedly, the antimicrobial functions remain preserved. small synthetic peptides corresponding to amphipathic c-terminal domains of t4 lysozyme show a microbicidal activity. its membrane disturb ...199910338111
inhibition of escherichia coli rna polymerase by bacteriophage t7 gene 2 protein.the 64 amino acid residue product of bacteriophage t7 gene 2 (gp2) binds the escherichia coli rna polymerase and inhibits transcription. we localized the gp2 binding site to within 53 amino acid residues in the functionally dispensable region of the rna polymerase beta' subunit. we investigated the effect of gp2 on transcription at a -10/-35 promoter and at an "extended -10" promoter. our results indicate that binding of gp2 to the sigma70holoenzyme (esigma70) prevents promoter recognition at -1 ...199910369763
lysis and lysis inhibition in bacteriophage t4: rv mutations reside in the holin t gene.upon infecting populations of susceptible host cells, t-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37 degrees c after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells. the timing of lysis depends upon gene t and upon one or more rapid-lysis (r) genes whose inactivation prevents lysis inhibition. t encodes a holin that mediates the mo ...199910400598
comparison of the assembly of the bacteriophage t4 clamp loader complex (gp44/62) expressed in a cis versus trans genomic configuration.proper formation of the bacteriophage t4 dna polymerase holoenzyme requires a wide spectrum of protein-protein and protein-dna interactions among the dna polymerase gp43, the sliding clamp gp45, and gp44/62, the clamp loader complex (clc). the 44 and 62 proteins associate to form a tight complex maintained in a 4:1 ratio. the 44 and 62 genes are adjacent to each other on the t4 genome, are cotranscribed, and are translationally coupled. it has been suggested that translational coupling may play ...199910405357
gene 61.3 of bacteriophage t4 is the spackle gene.the bacteriophage t4 e gene encodes lysozyme (e-lysozyme), which releases progeny phage after normal infection of escherichia coli cells. a mutation in the spackle gene suppresses the defect in e-lysozyme (emrich, 1968). the spackle gene was mapped between genes 41 and 61, but its precise location has not previously been determined. in the current study, we constructed an amber mutant of gene 61.3, amst14, by site-directed mutagenesis. the gene 61.3 mutant shares phenotypes with spackle mutants: ...199910417260
detection of tamoxifen-dna adducts on laci genes using dna polymerase stop assay.tamoxifen forms dna adducts in rat liver and causes an increased mutation frequency at the laci genes in the livers of lambda/laci transgenic rats. although an elevated occurrence of endometrial cancer is found in a small proportion of breast cancer patients treated with tamoxifen, there is conflicting evidence on whether or not low levels of dna adducts are formed in humans.199910493022
folding of coliphage t4 short tail fiber in vitro. analysing the role of a bacteriophage-encoded chaperone.the morphogenesis of the escherichia coli bacteriophage t4 depends on the presence of helper proteins which are not components of the mature virion. two bacteriophage-encoded proteins, p57 and p38, are required for the assembly of the bacteriophage t4 tail fibers. in the absence of p57, two polypeptides of the long fiber (p34 and p37) and that of the short tail fiber (p12) fail to trimerize. instead they form water-insoluble aggregates. co-expression of the genes 12 and 57 in vivo caused the for ...199910504409
the structure of bacteriophage t4 gene product 9: the trigger for tail contraction.the t4 bacteriophage consists of a head, filled with double-stranded dna, and a complex contractile tail required for the ejection of the viral genome into the escherichia coli host. the tail has a baseplate to whïch are attached six long and six short tail fibers. these fibers are the sensing devices for recognizing the host. when activated by attachment to cell receptors, the fibers cause a conformational transition in the baseplate and subsequently in the tail sheath, which initiates dna ejec ...199910545330
the stem hairpin loop structure of p2sp1 rna is required for rna-cleaving activity.we studied the hairpin-loop structure of an rna fragment (guuucguacaaac) (r13) with the sequence corresponding to the self-cleavage domain in the precursor of an rna molecule from bacteriophage t4-infected escherichia coli cells (p2sp1 rna). in order to determine the influence of the hairpin-loop structure on these sequence-specific cleavage reactions, we have synthesized oligoribonucleotides containing hairpin-loop, double-helical stem-loop, and single-stranded rna structures. the cleavage was ...199910673038
dinucleotide repeat expansion catalyzed by bacteriophage t4 dna polymerase in vitro.dna replication normally occurs with high fidelity, but certain "slippery" regions of dna with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. we have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. the primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine ca repeats; nonrepeat sequences flank the dinucleotide repeats. dna polymerases are expected to repair ...200010924513
function of tyrosyl-trna synthetase in splicing group i introns: an induced-fit model for binding to the p4-p6 domain based on analysis of mutations at the junction of the p4-p6 stacked helices.we used an escherichia coli genetic assay based on the phage t4 td intron to test the ability of the neurospora crassa mitochondrial tyrosyl-trna synthetase (cyt-18 protein) to suppress mutations that cause structural defects around its binding site in the p4-p6 domain of the group i intron catalytic core. we analyzed all possible combinations of nucleotides at either p4 bp-1 or p6 bp-1, which together form the junction of the p4-p6 stacked helices, and looked for synergistic effects in double m ...200010926509
structure of bacteriophage t4 gene product 11, the interface between the baseplate and short tail fibers.bacteriophage t4, like all other viruses, is required to be stable while being transmitted from host to host, but also is poised to eject efficiently and rapidly its double-stranded dna genome to initiate infection. the latter is coordinated by the recognition of receptors on escherichia coli cells by the long tail fibers and subsequent irreversible attachment by the short tail fibers. these fibers are attached to the baseplate, a multi-subunit assembly at the distal end of the tail. recognition ...200010966799
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
the structure of udp-n-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases.bacterial udp-n-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at c-2 of udp-n-acetylglucosamine (udp-glcnac) and thereby provides bacteria with udp-n-acetylmannosamine (udp-mannac), the activated donor of mannac residues. mannac is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as streptococcus pneumoniae types 19f and 19a. we have determined the x-ray structure (2.5 a) of udp-glcnac 2-epimera ...200011106477
role of the substrate conformation and of the s1 protein in the cleavage efficiency of the t4 endoribonuclease regb.the t4 endoribonuclease regb is involved in the inactivation of the phage early messengers. it cuts specifically in the middle of ggag sequences found in early messenger intergenic regions but not ggag sequences located in coding sequences or in late messengers. in vitro regb activity is very low but is enhanced by a factor up to 100 by the ribosomal protein s1. in the absence of clear sequence motif distinguishing substrate and non-substrate ggag-containing rnas, we postulated the existence of ...200111118457
inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure.we have studied the inactivation of six gram-negative bacteria (escherichia coli, pseudomonas fluorescens, salmonella enterica serovar typhimurium, salmonella enteritidis, shigella sonnei, and shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage t4 lysozyme. none of these compounds had a bactericidal or bacteriostatic effect on ...200111133464
substitutions in bacteriophage t4 asia and escherichia coli sigma(70) that suppress t4 mota activation mutations.bacteriophage t4 middle-mode transcription requires two phage-encoded proteins, the mota transcription factor and asia coactivator, along with escherichia coli rna polymerase holoenzyme containing the sigma(70) subunit. a mota positive control (pc) mutant, mota-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. separate genetic selections isolated two asia mutants (s22f and q51e) and five sigma(70) mutants (y571c, y571h, d570n, l595p, and s60 ...200111244069
expression and properties of bacteriophage t4 gene product 11.a plasmid vector for expression of bacteriophage t4 gene product 11 (gp11) in e. coli cells has been constructed. gp11 is a baseplate protein that connects short tail fibers providing irreversible adsorption of the virus on a cell. a method based on chromatography on hydroxyapatite has been developed for purification of recombinant gp11. the protein is active in an in vitro complementation assay and transforms defective phage particles lacking gp11 into infective ones. gel filtration data sugges ...200111255120
thermodynamic molecular switch in macromolecular is known that most living systems can live and operate optimally only at a sharply defined temperature, or over a limited temperature range, at best, which implies that many basic biochemical interactions exhibit a well-defined gibbs free energy minimum as a function of temperature. the gibbs free energy change, deltag(o) (t), for biological systems shows a complicated behavior, in which deltag(o)(t) changes from positive to negative, then reaches a negative value of maximum magnitude (favora ...200011325035
replisome-mediated dna replication.the elaborate process of genomic replication requires a large collection of proteins properly assembled at a dna replication fork. several decades of research on the bacterium escherichia coli and its bacteriophages t4 and t7 have defined the roles of many proteins central to dna replication. these three different prokaryotic replication systems use the same fundamental components for synthesis at a moving dna replication fork even though the number and nature of some individual proteins are dif ...200111395406
identification of important amino acid residues that modulate binding of escherichia coli groel to its various cochaperones.genetic experiments have shown that the groel/groes chaperone machine of escherichia coli is absolutely essential, not only for bacterial growth but also for the propagation of many bacteriophages including lambda. the virulent bacteriophages t4 and rb49 are independent of the host groes function, because they encode their own cochaperone proteins, gp31 and coco, respectively. e. coli groel44 mutant bacteria do not form colonies above 42 degrees nor do they propagate bacteriophages lambda, t4, o ...200111404317
bacteriophage t4 multiplication in a glucose-limited escherichia coli escherichia coli k-12 biofilm was grown at a dilution rate of 0.028 h(-1) for 48 h in a glucose-limited chemostat coupled to a modified robbins' device to determine its susceptibility to infection by bacteriophage t4. bacteriophage t4 at a multiplicity of infection (moi) of 10 caused a log reduction in biofilm density (expressed as colony forming units (cfu) per cm2) at 90 min postinfection. after 6 h, a net decrease and equilibrium in viral titer was seen. when biofilms were exposed to t4 ph ...200111547890
solution structure and stability of the anti-sigma factor asia: implications for novel functions.anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors. the bacteriophage t4 anti-sigma factor asia is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of escherichia coli, sigma(70). asia is an all-helical, symmetric dimer in solution. the solution structure of the asia dimer reveals a n ...200211830637
the cell surface protein ag43 facilitates phage infection of escherichia coli in the presence of bile salts and 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
the evolutionary and integrative roles of transthyretin in thyroid hormone larger mammals, thyroid hormone-binding plasma proteins are albumin, transthyretin (ttr) and thyroxine (t4)-binding globulin. they differ characteristically in affinities and release rates for t4 and triiodothyronine (t3). together, they form a 'buffering' system counteracting thyroid hormone permeation from aqueous to lipid phases. evolution led to important differences in the expression pattern of these three proteins in tissues. in adult liver, ttr is only made in eutherians and herbivorou ...200212379491
mutational and functional analysis of a segment of the sigma family bacteriophage t4 late promoter recognition protein gp55.bacteriophage t4 late promoters, which consist of a simple 8-base pair tata box, are recognized by the gene 55 protein (gp55), a small, highly diverged member of the sigma family proteins that replaces sigma(70) during the final phase of the t4 multiplication cycle. a 16-amino acid segment of gp55 that is proposed to be homologous to the sigma(70) region 2.2 has been subjected to alanine scanning and other mutagenesis. the corresponding proteins have been examined in vitro for binding to escheri ...200312496274
purification of histidine-tagged t4 rna ligase from e. we report the construction of a histidine-tagged t4 rna ligase expression plasmid (prht4). the construct, when overexpressed in bl21 (de3) cells, allows the preparation of large quantities of t4 rna ligase in high purity using only a single purification column. the histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. a simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found f ...200212503310
[action of spirulina platensis on bacterial viruses].the impact of the biomass of the blue-green microalga (cyanobacterium) s. platensis on bacteriophage t4 (bacterial virus) has been evaluated. the study revealed that the addition of s. platensis biomass into the agar nutrient medium, followed by sterilization with 2% chloroform and thermal treatment, produced an inhibiting or stimulating effect on the reproduction of the bacteriophage in escherichia coli b cells, depending on the concentration of s. platensis and the multiplicity of phage infect ...200212506621
seasonal change and fate of coliphages infected to escherichia coli o157:h7 in a wastewater treatment plant.seasonal change of virulent phage infected to two e. coli o157:h7 strains (o:157-phage) in the influent of a domestic wastewater treatment plant in the central part of japan and fate of o:157-phage in the plant were monitored almost monthly from march 2001 to february 2002. coliphage infected to nonpathogenic e. coli o157:h7 atcc43888 (43888-phage) was detected for 1 year. on the other hand, phage infected to pathogenic e. coli o157:h7 edl933 (edl-phage) was detected intermittently. concentratio ...200312553989
interaction of t4 asia with its target sites in the rna polymerase sigma70 subunit leads to distinct and opposite effects on transcription.bacteriophage t4 asia is a homodimeric protein that orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of escherichia coli rna polymerase holoenzyme (esigma(70)) and preventing promoter complex formation on most e.coli and early t4 promoters. in addition, esigma(70)asia, but not esigma(70), is a substrate of transcription activation by t4-encoded dna-binding protein mota, a co-activator of transcription from middle ...200312581632
engineering of bacteriophage t4 tail sheath protein.gene product 18 (gp18, 659 amino acids) forms bacteriophage t4 contractile tail sheath. recombinant protein assembles into different length polysheaths during expression in the cell, which complicates the preparation of protein crystals for its spatial structure determination. to design soluble monomeric gp18 mutants unable to form polysheaths and useful for crystallization, we have used bal31 nuclease for generation deletions inside gene 18 encoding the ile507-gly530 region. small deletions in ...200212600265
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
purification of the bacteriophage t4 type ii dna topoisomerase. 199912844873
theoretical studies on solvation contribution to the thermodynamic stability of mutants of lysozyme t4.atomic solvation parameters (asps) are widely used to estimate the solvation contribution to the thermodynamic stability of proteins as well as the free energy of association for protein-ligand complexes. in view of discrepancies in the results of free energies of solvation of folding for various proteins obtained using different atomic solvation parameter sets, systematic studies have been carried out for the calculation of accessible surface area and the changes in free energy of solvation of ...200312874374
study on interaction between t4 phage and escherichia coli b by microcalorimetric method.the process that t4 phages multiply in host cells of escherichia coli b was determined using lkb-2277 bioactivity monitor by means of stopped-flow method, and the growth was measured turbidometrically at the same time at 37 degrees c. by analyzing thermo-curves, quantitative parameters could be obtained to characterize the interactions of host cells and phages. the parameters such as k(a), p(max), g etc. change regularly with the decrease of multiplicity of infection (moi) value. infection-lysis ...200312951222
the delayed origin of mutants induced by exposure of extracellular phage t4 to ethyl methane sulfonate. 196113708170
the physical characterization of dna molecules released from t2 and t4 bacteriophage. 196113776441
inhibition of deoxyribonucleic acid-directed ribonucleic acid polymerase in escherichia coli after infection with bacteriophage t4. 196414166762
the course of infection with abnormal bacteriophage t4 containing non-glucosylated dna on escherichia coli strains. 196414202283
glucosylation of deoxyribonucleic acid. iii. alpha- and beta-glucosyl transferases from t4-infected escherichia coli. 196214452558
the kinetics of parental deoxyribonucleic acid replication, deoxyguanylate kinase formation and 32p-inactivation of the parental virus in escherichia coli infected with bacteriophage t4. 196114459099
the nature of the "deletion" mutants in the rii region of phage t4. 196114480261
rna metabolism in escherichia coli infected with bacteriophage t4. inhibition of host ribosomal and soluble rna synthesis by phage and effect of chloromycetin. 196214480263
blocking the t4 lysis inhibition phenotype.nonlysogenic escherichia coli k cells exhibit a delay in lysis when infected by t4rii phage termed lysis inhibition (lin). e. coli k cells expressing lambda rexb from either a prophage defective for rexa, or a multicopy plasmid supported t4rii infection, but prevented the establishment of lin. in addition, e. coli null mutations in either the periplasmic "tail-specific protease" tsp, or the 10sa rna ssra, completely blocked the establishment of lin following t4 infections. the expression of rexb ...200314637004
the structure of escherichia coli rusa endonuclease reveals a new holliday junction dna binding fold.holliday junction resolution performed by a variety of structure-specific endonucleases is a key step in dna recombination and repair. it is believed that all resolvases carry out their reaction chemistries in a similar fashion, utilizing a divalent cation to facilitate the hydrolysis of the phosphodiester backbone of the dna, but their architecture varies. to date, with the exception of bacteriophage t4 endonuclease vii, each of the known resolvase enzyme structures has been categorized into on ...200314656440
separation and recovery of dna fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide).we have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for dna electrophoresis. the aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees c, whereas it reverted into a solution upon cooling. linear double-stranded dna molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the dna length. th ...200314656528
the neurospora crassa cyt-18 protein c-terminal rna-binding domain helps stabilize interdomain tertiary interactions in group i introns.the neurospora crassa mitochondrial tyrosyl-trna synthetase (cyt-18 protein) promotes the splicing of group i introns by stabilizing the catalytically active rna structure. to accomplish this, cyt-18 recognizes conserved structural features of group i intron rnas using regions of the n-terminal nucleotide-binding fold, intermediate alpha-helical, and c-terminal rna-binding domains that also function in binding trna(tyr). curiously, whereas the splicing of the n. crassa mitochondrial large subuni ...200415037773
identification of essential residues within lit, a cell death peptidase of escherichia coli k-12.bacteriophage exclusion is a suicide response to viral infection. in strains of escherichia coli k-12 infected with t4 phage this process is mediated by the host-encoded lit peptidase. lit is activated by a unique sequence in the major head protein of the t4 phage (the gol sequence) which then cleaves site-specifically the host translation factor ef-tu, ultimately leading to cell death. lit has very low sequence identity with other peptidases, with only a putative metallopeptidase motif, h(160)e ...200415196039
the pcna-rfc families of dna clamps and clamp loaders.the proliferating cell nuclear antigen pcna functions at multiple levels in directing dna metabolic pathways. unbound to dna, pcna promotes localization of replication factors with a consensus pcna-binding domain to replication factories. when bound to dna, pcna organizes various proteins involved in dna replication, dna repair, dna modification, and chromatin modeling. its modification by ubiquitin directs the cellular response to dna damage. the ring-like pcna homotrimer encircles double-stran ...200415210332
the site-specific incorporation of p-iodo-l-phenylalanine into proteins for structure determination.a recently developed method makes it possible to genetically encode unnatural amino acids with diverse physical, chemical or biological properties in escherichia coli and yeast. we now show that this technology can be used to efficiently and site-specifically incorporate p-iodo-l-phenylalanine (iodophe) into proteins in response to an amber tag codon. the selective introduction of the anomalously scattering iodine atom into proteins should facilitate single-wavelength anomalous dispersion experi ...200415378068
the role of an activating peptide in protease-mediated suicide of escherichia coli k12.activation of latent proteinases ensures that the timing of proteolysis is regulated precisely, a process that generally involves proteolytic excision of a pro-region or a tightly bound inhibitor. here we define the activation mechanism for lit, a dormant suicide proteinase in escherichia coli k-12. previous work has shown that gol, a short sequence within the major capsid protein gp23, activates lit during the latter stages of t4 phage infection. this results in cell death and exclusion of the ...200515501819
divergence of the mrna targets for the ssb proteins of bacteriophages t4 and rb69.the single-strand binding (ssb) protein of phage t4 (t4 gp32, product of gene 32) is a mrna-specific autogenous translational repressor, in addition to being a sequence-independent ssdna-binding protein that participates in phage dna replication, repair and recombination. it is not clear how this physiologically essential protein distinguishes between specific rna and nonspecific nucleic acid targets. here, we present phylogenetic evidence suggesting that ssdna and specific rna bind the same gp3 ...200415507125
isolation of escherichia coli bacteriophages from the stool of pediatric diarrhea patients in bangladesh.a 3-week coliphage survey was conducted in stool samples from 140 bangladeshi children hospitalized with severe diarrhea. on the escherichia coli indicator strain k803, all but one phage isolate had 170-kb genomes and the morphology of t4 phage. in spot tests, the individual t4-like phages infected up to 27 out of 40 diarrhea-associated e. coli, representing 22 o serotypes and various virulence factors; only five of them were not infected by any of these new phages. a combination of diagnostic p ...200415576777
gpwac of the t4-type bacteriophages: structure, function, and evolution of a segmented coiled-coil protein that controls viral infectivity.the wac gene product (gpwac) or fibritin of bacteriophage t4 forms the six fibers that radiate from the phage neck. during phage morphogenesis these whiskers bind the long tail fibers (ltfs) and facilitate their attachment to the phage baseplate. after the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the ltfs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. a comparative analysis of the sequences of 5 wac ...200515659683
whole plasmid mutagenic pcr for directed protein evolution.protein function can be engineered through iterated cycles of random mutagenesis and screening (directed evolution). optimization of protein expression is essential for the development of sensitive and precise high throughput assays. here we optimize the performance of a plasmid-borne escherichia coli lacz gene in two rounds of directed evolution. first, its promoter was "randomized" by whole plasmid polymerase chain reaction (pcr) and intra-molecular self-ligation. a genetically stable constitu ...200515857786
an altered-specificity dna-binding mutant of escherichia coli sigma70 facilitates the analysis of sigma70 function in vivo.the sigma subunit of bacterial rna polymerase is strictly required for promoter recognition. the primary (housekeeping) sigma factor of escherichia coli, sigma(70), is responsible for most of the gene expression in exponentially growing cells. the fact that sigma(70) is an essential protein has complicated efforts to genetically dissect the functions of sigma(70). to facilitate the analysis of sigma(70) function in vivo, we isolated an altered-specificity dna-binding mutant of sigma(70), sigma(7 ...200515882415
mechanisms associated with acanthamoeba castellanii (t4) phagocytosis.using fluorescein isothiocyanate (fitc)-labelled escherichia coli, phagocytosis in acanthamoeba is studied. this assay is based on the quenching effect of trypan blue on fitc-labelled e. coli. only intracellular e. coli retain their fluorescence, which are easily discriminated from non-fluorescent adherent bacteria. acanthamoeba uptake of e. coli is significantly reduced in the presence of genistein, a protein tyrosine kinase inhibitor. in contrast, sodium orthovanadate (protein tyrosine phospha ...200515940518
adenylate kinase of escherichia coli, a component of the phage t4 dntp synthetase complex.adenylate kinase, which catalyzes the reversible atp-dependent phosphorylation of amp to adp and damp to dadp, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. lu and inouye (lu, q., and inouye, m. (1996) proc. natl. acad. sci. u. s. a. 93, 5720-5725) showed that an escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. bacteriophage t4 can reproduce in an es ...200515941717
in vitro synthesis of enzymes of the tryptophan operon of escherichia coli. evidence for positive control of transcription.a protein fraction, called at (= anti termination) factor, has been isolated from extracts of e. coli and partially purified. the at factor stimulates the synthesis in vitro of anthranilate synthetase, an enzyme encoded by two genes of the tryptophan (trp) operon, but has no effect on the synthesis of t7 rna polymerase and other t7- and t4 coded proteins. the at factor stimulates the synthesis of trp mrna; it has no effect on the translation of trp mrna. we conclude that in vitro transcription o ...197516094972
the mono-adp-ribosyltransferases alt and modb of bacteriophage t4: target proteins identified.infection of escherichia coli by bacteriophage t4 leads to the expression of three phage mono-adp-ribosyltransferases (namely, alt, moda, and modb), each of which modifies a distinct group of host proteins. to improve understanding of these interactions and their consequences for the t4 replication cycle, we used high-resolution two-dimensional gel electrophoresis and mass-spectrometry to identify some of the putative target proteins adp-ribosylated in vitro by alt (total approximately 27) and m ...200516112649
crystal structure of a dna polymerase sliding clamp from a gram-positive bacterium.sliding dna clamps are processivity factors that are required for efficient dna replication. dna polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle dna and thereby link the polymerase enzyme to the dna substrate. although the structures of sliding clamps from gram-negative bacteria (e. coli), eukaryotes, archaea, and t4-like bacteriophages are well-known, the structure of a sliding clamp from gram-positive bacteria has not been reported prev ...200616403212
the bacteriophage t4 inhibitor and coactivator asia inhibits escherichia coli rna polymerase more rapidly in the absence of sigma70 region 1.1: evidence that region 1.1 stabilizes the interaction between sigma70 and core.the n-terminal region (region 1.1) of sigma70, the primary sigma subunit of escherichia coli rna polymerase, is a negatively charged domain that affects the dna binding properties of sigma70 regions 2 and 4. region 1.1 prevents the interaction of free sigma70 with dna and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. the bacteriophage t4 asia protein is an inhibitor of sigma70-dependent transcription from promoters that require an interaction betwee ...200616452409
[t4-type bacteriophages: ubiquitous components of the "dark matter" of the biosphere]. 200616457745
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
both regions 4.1 and 4.2 of e. coli sigma(70) are together required for binding to bacteriophage t4 asia in vivo.the t4 asia is an anti-sigma factor encoded by one of the early genes of bacteriophage t4. it has been shown that asia inhibits transcription from promoters containing -10 and -35 consensus sequence by binding to sigma(70) of e. coli. binding of asia to sigma(70) in vivo, in e. coli, leads to inhibition of transcription of essential genes resulting in killing of the organism. by using various in vitro methods, the region of sigma(70) binding to asia have been mapped to domain 4.2. additionally, ...200616545925
the role of an upstream promoter interaction in initiation of bacterial transcription.the bacterial rna polymerase (rnap) recognizes promoters through sequence-specific contacts of its promoter-specificity components (sigma) with two dna sequence motifs. contacts with the upstream ('-35') promoter motif are made by sigma domain 4 attached to the flap domain of the rnap beta subunit. bacteriophage t4 late promoters consist solely of an extended downstream ('-10') motif specifically recognized by the t4 gene 55 protein (gp55). low level basal transcription is sustained by gp55-rnap ...200616601684
direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage t4.the genome of bacteriophage t4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. the t4dnl (t4 dna ligase) and two rna ligases [t4rnl1 (t4 rna ligase 1) and t4rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. to unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity ...200616671895
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
nonsense mutants in the rii a cistron of bacteriophage t4.after in vitro treatment of bacteriophage t4 with hydroxylamine (ha), 54 nonsense mutants in the rii a cistron were isolated. these mutants were characterized by growth on suppressor strains of escherichia coli, and the mutational sites were mapped in the rii a cistron. twenty-five (9 sites) were amber (uag), 20 (6 sites) were opal (uga), and 9 (6 sites) were ochre (uaa). mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably inv ...196916789112
[construction of recombinant gst-rcas1 fusion gene and its expression in e. coli].to construct the recombinant plasmid of rcas1, to express and purify its fusion protein gst-rcas1, and to investigate its biological function.200616924700
biochemical analysis of the substrate specificity and sequence preference of endonuclease iv from bacteriophage t4, a dc-specific endonuclease implicated in restriction of dc-substituted t4 dna synthesis.endonuclease iv encoded by denb of bacteriophage t4 is implicated in restriction of deoxycytidine (dc)-containing dna in the host escherichia coli. the enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in e.coli cells, and was purified to homogeneity. the purified enzyme showed high activity with single-stranded (ss) dna and denatured dc-substituted t4 genomic double-stranded (ds) dna but exhibited no activity with dsd ...200616971463
double-strand break repair in bacteriophage t4: recombination effects of 3'-5' exonuclease mutations.the role of 3'-5' exonucleases in double-strand break (dsb)-promoted recombination was studied in crosses of bacteriophage t4, in which dsbs were induced site specifically within the riib gene by segc endonuclease in the dna of only one of the parents. frequency of rii+ recombinants was measured in two-factor crosses of the type i x ets1, where ets1 designates an insertion in the riib gene carrying the cleavage site for segc and i's are riib or riia point mutations located at various distances ( ...200617028319
structural and functional studies of regb, a new member of a family of sequence-specific ribonucleases involved in mrna inactivation on the ribosome.the regb endoribonuclease participates in the bacteriophage t4 life cycle by favoring early messenger rna breakdown. regb specifically cleaves ggag sequences found in intergenic regions, mainly in translation initiation sites. its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 s subunit or by ribosomal protein s1. regb has no significant sequence homology to any known protein. here we used nmr to solve the structure of regb and map its interactions with two rna subst ...200717046813
effective inhibition of lytic development of bacteriophages lambda, p1 and t4 by starvation of their host, escherichia coli.bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. they are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection ...200717324284
assaying rna chaperone activity in vivo in bacteria using a ribozyme folding, we report an assay to evaluate the intracellular rna chaperone activity of a protein of interest in vivo in bacterial cells. the method is based on self-splicing of the group i intron, which is located in the thymidylate synthase (td) gene of phage t4. a previously described td mutant (tdsh1) has significantly impaired splicing due to formation of splicing-incompetent alternative structures. in this procedure, overexpression of rna chaperones in the presence of the td mutant sh1 is used to ...200617406411
detecting ultraviolet damage in single dna molecules by atomic force microscopy.we report detection and quantification of ultraviolet (uv) damage in dna at a single molecule level by atomic force microscopy (afm). by combining the supercoiled plasmid relaxation assay with afm imaging, we find that high doses of medium wave ultraviolet (uvb) and short wave ultraviolet (uvc) light not only produce cyclobutane pyrimidine dimers (cpds) as reported but also cause significant dna degradation. specifically, 12.5 kj/m(2) of uvc and 165 kj/m(2) of uvb directly relax 95% and 78% of p ...200717483180
genome analysis of phage js98 defines a fourth major subgroup of t4-like phages in escherichia coli.numerous t4-like escherichia coli phages were isolated from human stool and environmental wastewater samples in bangladesh and switzerland. the sequences of the major head gene (g23) revealed that these coliphages could be placed into four subgroups, represented by the phages t4, rb69, rb49, and js98. thus, js98 defines a new major subgroup of e. coli t4-like phages. we conducted an analysis of the 169-kb js98 genome sequence. overall, 198 of the 266 js98 open reading frames (orfs) shared amino ...200717693496
the ser176 of t4 endonuclease iv is crucial for the restricted and polarized dc-specific cleavage of single-stranded dna implicated in restriction of dc-containing dna in host escherichia coli.endonuclease (endo) iv encoded by denb of bacteriophage t4 is an enzyme that cleaves single-stranded (ss) dna in a dc-specific manner. also the growth of dc-substituted t4 phage and host escherichia coli cells is inhibited by denb expression presumably because of the inhibitory effect on replication of dc-containing dna. recently, we have demonstrated that an efficient cleavage by endo iv occurs exclusively at the 5'-proximal dc (dc1) within a hexameric or an extended sequence consisting of dc r ...200717913749
structure of escherichia coli exonuclease i in complex with thymidine 5' escherichia coli, exonuclease i (exoi) is a monomeric processive 3'-5' exonuclease that degrades single-stranded dna. the enzyme has been implicated as primarily being involved in repairing frameshift mutations. the structure of the enzyme has previously been determined in a hexagonal space group at 2.4 a resolution. here, the structure of exoi in complex with a nucleotide product, thymidine 5'-monophosphate, is described in an orthorhombic space group at 1.5 a resolution. this new high-resol ...200818219121
single-stranded dna-binding protein recruits dna polymerase v to primer termini on reca-coated dna.translesion dna synthesis (tls) by dna polymerase v (polv) in escherichia coli involves accessory proteins, including reca and single-stranded dna-binding protein (ssb). to elucidate the role of ssb in tls we used an in vitro exonuclease protection assay and found that ssb increases the accessibility of 3' primer termini located at abasic sites in reca-coated gapped dna. the mutant ssb-113 protein, which is defective in protein-protein interactions, but not in dna binding, was as effective as wi ...200818223256
detection of escherichia coli with fluorescent labeled phages that have a broad host range to e. coli in sewage water.escherichia coli is used as an indicator microorganism in public health. the conventional way to detect e. coli requires several days to produce a result, because it requires incubation of cells. therefore a rapid and sensitive detection method is needed. t4e-/gfp phage, characterized by suppression of lysozyme and fusion of gfp (green fluorescent protein) to its soc (small outer capsid) protein, was constructed, and it was shown to be able to detect e. coli k12 sensitively within several hours. ...200818225914
the concerted action of lactoferrin and bacteriophages in the clearance of bacteria in sublethally infected mice.both lactoferrin (lf) and bacteriophages are potent antibacterial agents. lf is contained in the secretory fluids of mammals and bacteriophages are specific bacterial viruses.200818268472
identification of acanthamoeba genotype t4 and paravahlkampfia sp. from two clinical this study, two free-living amoebae strains, acanthamoeba genotype t4 and paravahlkampfia sp., which were isolated from keratitis cases are presented. while the acanthamoeba strain was isolated as a single agent, the paravahlkampfia strain was found together with herpes simplex virus. neither of the patients were contact lens wearers, but they did have a history of minor corneal trauma. amoebae were detected on non-nutrient agar covered with escherichia coli. based on pcr-amplified 18s rrna-g ...200818287307
rna base damage and repair.elaborate repair pathways counteract the deleterious effects of dna damage by mechanisms that are understood in reasonable detail. in contrast, repair of damaged rna has not been widely explored. this may be because aberrant rnas are generally assumed to be degraded rather than repaired. the reason for this view is well founded, since conserved surveillance mechanisms that degrade abnormal rnas are thoroughly documented. numerous proteins and protein-rna complexes are involved in the metabolism ...200718289040
atp-induced shrinkage of dna with mukb protein and the mukbef complex of escherichia coli.fluorescence microscopic observation of individual t4 dna molecules revealed that the mukbef complex (bacterial condensin) and its subunit, the mukb (a member of the smc [structural maintenance of chromosomes] superfamily) homodimer, of escherichia coli markedly shrunk large dna molecules in the presence of hydrolyzable atp. in contrast, in the presence of adp or atp-gammas, the conformation of dna was almost not changed. this suggests that the atpase activity of subunit mukb is essential for sh ...200818326568
[comparative study of the resistance of bacteriophaget4, phix174d, ms2 and f2 to gamma radiation].to screen the suitable bacteriophage as virus indicator in irradiation sterilization.200818361821
a basic/hydrophobic cleft of the t4 activator mota interacts with the c-terminus of e.coli sigma70 to activate middle gene transcription.transcriptional activation often employs a direct interaction between an activator and rna polymerase. for activation of its middle genes, bacteriophage t4 appropriates escherichia coli rna polymerase through the action of two phage-encoded proteins, mota and asia. alone, asia inhibits transcription from a large class of host promoters by structurally remodelling region 4 of sigma(70), the primary specificity subunit of e. coli rna polymerase. mota interacts both with sigma(70) region 4 and with ...200818485078
chaperonin complex with a newly folded protein encapsulated in the folding chamber.a subset of essential cellular proteins requires the assistance of chaperonins (in escherichia coli, groel and groes), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. this process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a groel ring. then, binding of atp and co-chaperonin groes to that ring ejects the non-native protein from its binding sites, th ...200919122642
in vitro and in vivo production and purification of circular rna aptamer.rna aptamers are potential candidates for rna therapeutics. they must be clinically modified for medical applications because they are vulnerable to indigenous ribonucleases. since circular rna molecules without any chemical modification are much more stable than linear ones in a cell extract, we report the production of a circular form of streptavidin rna aptamer both in vitro and in vivo. circularization was accomplished by self-splicing permuted intron-exon sequences derived from t4 bacteriop ...200919138712
mechanism of thermal renaturation and hybridization of nucleic acids: kramers' process and universality in watson-crick base pairing.renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. we present here a theoretical investigation of the mechanism of these reactions in vitro under thermal conditions (dilute solutions of single-stranded chains, in the presence of molar concentrations of monovalent salts and at elevated temperatures). the mechanism follows a kramers' process, whereby the complementary chains overcome a potential barrier through brownian motion. the barrier ...200919673131
chemical studies on host-virus interactions : i. the effect of bacteriophage adsorption on the multiplication of its host, escherichia coli b with an appendix giving some data on the composition of the bacteriophage, t2.the addition of active or irradiated t2 bacteriophage and t4 bacteriophage to e. coli b stops bacterial multiplication. the respiratory rate and respiratory quotient of the inhibited bacteria remained at the values observed just before infection. a respiratory rate decrease which occasionally appears can be roughly correlated with change of turbidity of the suspension. an intracellular inhibitor of multiplication appears to be liberated into lysates. a similar substance has been separated from n ...194619871584
using the rate of respiration to monitor events in the infection of escherichia coli cultures by bacteriophage t4.the growing interest in applications of bacteriophages creates a need for improvements in the production processes. continuous monitoring of the phage production is an essential aspect of any control strategy and, at present, there is no completely satisfactory option. the approach presented here uses ir-spectrometry to continuously measure the rate of respiration (co(2) released) of escherichia coli infected by phage t4 at various multiplicities of infection (moi). within the trends in these da ...201020039436
comparison of the molecular influences of no-induced lesions in dna strands on the reactivity of polynucleotide kinases, dna ligases and dna polymerases.nitric oxide (no) causes dna damage, generating xanthine (xan, x) and oxanine (oxa, o) from guanine (gua, g) and hypoxanthine (hyp, h) from adenine (ade, a) by nitrosative oxidation. although these no-induced lesions have been thought to cause mutagenic problems in cellular systems, the influence of these lesions on enzymatic functions has not yet been compared systematically. in this study, we investigated the effect of no-induced lesions on the activities of dna-binding/recognizing enzymes suc ...201020097903
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