Biochem Biophys Res Commun, 2003 Aug 8, 307(4), 791 - 6 Use of cyclic peptide phage display library for the identification of a CD45RC epitope expressed on murine B cells and their precursors; Czompoly T et al.; The alternative splicing and variable expression of the exons near to the N-terminus of the leukocyte common antigen (L-CA, CD45) result in distinct extracellular isoforms expressed by cells with different functional and developmental properties . Here we report the tissue reactivity pattern and epitope specificity of a novel rat monoclonal antibody (IBL-8) against a restricted epitope of mouse CD45 . We found that this mAb reacts with an epitope displayed by B cells and their precursors (both in newborn spleen and adult bone marrow) . Moreover, peripheral CD8-positive T cells were also recognised at an intermediate intensity, whereas the CD4 T cell subset was weakly reactive . The epitope of this mAb was determined with M13 filamentous phages that display cysteine constrained nonapeptides on their coat proteins . The isolated bacteriophages expressing the putative epitope showed an isoform-specific inhibition of the binding of exon-specific mAbs . Deduced amino acid sequence data of these phages indicate that the epitope recognised by the IBL-8 mAb lies at the 136-144 region of the mouse CD45 molecule within its C exon, with a TAFP consensus sequence at its centre. Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9250 - 5 Epub 2003 Jul 22. Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase; Davydova EK et al.; Coliphage N4 virion RNA polymerase (vRNAP), the most distantly related member of the T7-like family of RNA polymerases, is responsible for transcription of the early genes of the linear double-stranded DNA phage genome . Escherichia coli single-stranded DNA-binding protein (EcoSSB) is required for N4 early transcription in vivo, as well as for in vitro transcription on super-coiled DNA templates containing vRNAP promoters . In contrast to other DNA-dependent RNA polymerases, vRNAP initiates transcription on single-stranded, promoter-containing templates with in vivo specificity; however, the RNA product is not displaced, thus limiting template usage to one round . We show that EcoSSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling . EcoSSB binds to the template and to the nascent transcript and prevents the formation of a transcriptionally inert RNA:DNA hybrid . Using C-terminally truncated EcoSSB mutant proteins, human mitochondrial SSB (Hsmt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hsmt-EcoSSB chimera, we have mapped a determinant of template recycling to the C-terminal amino acids of EcoSSB . T7 RNAP contains an amino-terminal domain responsible for binding the RNA product as it exits from the enzyme . No sequence similarity to this domain exists in vRNAP . Hereby, we propose a unique role for EcoSSB: It functionally substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding. J Mol Biol, 2003 Aug 1, 331(1), 255 - 62 Sequence context strongly modulates association of polar residues in transmembrane helices; Dawson JP et al.; Polar residues are capable of mediating the association of membrane-embedded helices through the formation of side-chain/side-chain inter-helical hydrogen bonds . However, the extent to which native van der Waals packing of the residues surrounding the polar locus can enhance, or interfere with, the interaction of polar residues has not yet been studied . We examined the propensities of four polar residues (aspartic acid, asparagine, glutamic acid, and glutamine) to promote self-association of transmembrane (TM) domains in several biologically derived sequence environments, including (i) . four naturally occurring TM domains that contain a Glu or Gln residue (Tnf5/CD40 ligand, C79a/Ig-alpha, C79b/Ig-beta, and Fut3/alpha-fucosyltransferase); and (ii) . variants of bacteriophage M13 major coat protein TM segment with Asp and Asn at interfacial and non-interfacial positions . Self-association was quantified by the TOXCAT assay, which measures TM helix self-oligomerization in the Escherichia coli inner membrane . While an appropriately placed polar residue was found in several cases to significantly stabilize TM helix-helix interactions through the formation of an interhelical hydrogen bond, in other cases the strongly polar residues did not enhance the association of the two helices . Overall, these results suggest that an innate structural mechanism may operate to control non-specific association of membrane-embedded polar residues. J Mol Biol, 2003 Aug 1, 331(1), 139 - 54 Defining the ATPase center of bacteriophage T4 DNA packaging machine: requirement for a catalytic glutamate residue in the large terminase protein gp17; Goetzinger KR et al.; Double-stranded DNA packaging in icosahedral bacteriophages is driven by an ATPase-coupled packaging machine constituted by the portal protein and two non-structural packaging/terminase proteins assembled at the unique portal vertex of the empty viral capsid . Recent studies show that the N-terminal ATPase site of bacteriophage T4 large terminase protein gp17 is critically required for DNA packaging . It is likely that this is the DNA translocating ATPase that powers directional translocation of DNA into the viral capsid . Defining this ATPase center is therefore fundamentally important to understand the mechanism of ATP-driven DNA translocation in viruses . Using combinatorial mutagenesis and biochemical approaches, we have defined the catalytic carboxylate residue that is required for ATP hydrolysis . Although the original catalytic carboxylate hypothesis suggested the presence of a catalytic glutamate between the Walker A (SRQLGKT(161-167)) and Walker B (MIYID(251-255)) motifs, none of the four candidate glutamic acid residues, E198, E208, E220 and E227, is required for function . However, the E256 residue that is immediately adjacent to the putative Walker B aspartic acid residue (D255) exhibited a phenotypic pattern that is consistent with the catalytic carboxylate function . None of the amino acid substitutions, including the highly conservative D and Q, was tolerated . Biochemical analyses showed that the purified E256V, D, and Q mutant gp17s exhibited a complete loss of gp16-stimulated ATPase activity and in vitro DNA packaging activity, whereas their ATP binding and DNA cleavage functions remained intact . The data suggest that the E256 mutants are trapped in an ATP-bound conformation and are unable to catalyze the ATP hydrolysis-transduction cycle that powers DNA translocation . Thus, this study for the first time identified and characterized a catalytic glutamate residue that is involved in the energy transduction mechanism of a viral DNA packaging machine. Infect Immun, 2003 Aug, 71(8), 4554 - 62 Shiga toxin 2-converting bacteriophages associated with clonal variability in Escherichia coli O157:H7 strains of human origin isolated from a single outbreak; Muniesa M et al.; Shiga toxin 2 (Stx2)-converting bacteriophages induced from 49 strains of Escherichia coli O157:H7 isolated during a recent outbreak of enterocolitis in Spain were examined in an attempt to identify the variability due to the stx(2)-converting phages . The bacterial isolates were divided into low-, medium-, and high-phage-production groups on the basis of the number of phages released after mitomycin C induction . Low- and medium-phage-production isolates harbored two kinds of phages but released only one of them, whereas high-phage-production isolates harbored only one of the two phages . One of the phages, phi SC370, which was detected only in the isolates with two phages, showed similarities with phage 933W . The second phage, phi LC159, differed from phi SC370 in morphology and DNA structure . When both phages were present in the same bacterial chromosome, as occurred in most of the isolates, only phi SC370 was detected in the supernatants of the induced cultures . If phi LC159 was released, its presence was masked by phi SC370 . When phi SC370 was absent, large amounts of phi LC159 were released, suggesting that there was some regulation of phage expression between the two phages . To our knowledge, this is the first description of clonal variability due to phage loss . The higher level of phage production was reflected in the larger amounts of Stx2 toxin produced by the cultures . Some relationship between phage production and the severity of symptoms was observed, and consequently these observations suggest that the virulence of the isolates studied could be related to the variability of the induced stx(2)-converting phages. Poult Sci, 2003 Jul, 82(7), 1108 - 12 Evaluation of aerosol spray and intramuscular injection of bacteriophage to treat an Escherichia coli respiratory infection; Huff WE et al.; Two studies were conducted to determine the efficacy of either aerosol or i.m . injection of bacteriophage to treat an Escherichia coli respiratory infection in broiler chickens . An additional two studies were conducted to enumerate the bacteriophage in the blood of birds at 1, 2, 3, 4, 5, 6, 24, and 48 h after being sprayed or injected i.m . with bacteriophage . Five birds were bled at each period . In study 1, there were 10 treatments with three replicate pens of 10 birds . The treatments consisted of an untreated control, heat-killed bacteriophage spray, active bacteriophage spray, E . coli challenge at 7 d of age, and E . coli challenge followed by spraying the birds with heat-killed bacteriophage or active bacteriophage at 2, 24, or 48 h after challenge . In study 2 there were 11 treatments with three replicate pens of 10 birds per pen . The treatments were untreated controls, birds injected i.m . in the thigh with heat-killed or active bacteriophage, E . coli challenge at 7 d of age, PBS challenge, E . coli challenge followed by injection of heat-killed or active bacteriophage immediately after challenge or at 24 or 48 h after challenge . In both studies the E . coli challenge consisted of injecting 10(4) cfu into the thoracic air sac . Treatment of this severe E . coli infection with the bacteriophage aerosol spray significantly reduced mortality from 50 to 20% when given immediately after the challenge but had little treatment efficacy when administered 24 or 48 h after challenge . The i.m . injection of bacteriophage significantly reduced mortality from 53 to 17%, 46 to 10%, and 44 to 20% when given immediately, 24, or 48 h after challenge, respectively . Only a few birds sprayed with bacteriophage had detectable bacteriophage in their blood with an average of 96 pfu/mL 1 h after bacteriophage administration, and no bacteriophage was detected 24 and 48 h after bacteriophage administration . All birds injected i.m . with bacteriophage had detectable levels of bacteriophage in their blood at levels of 10(4) pfu/mL of blood up to 6 h after bacteriophage administration, and four of the five birds had detectable bacteriophage in their blood at an average level of 70 pfu/mL of blood 24 h after bacteriophage administration . The relative inefficiency of the spray treatment to the i.m . injection treatment may be due to the inability to get bacteriophage into the blood at high concentrations when the birds are sprayed versus the consistent high titers achieved with the i.m . injection of bacteriophage . These data provide support to the concept that bacteriophage may be an effective alternative to antibiotics in animal production when they are administered in a way that delivers high titers of the bacteriophage to the critical site of the bacterial infection. J Bacteriol, 2003 Aug, 185(15), 4609 - 14 Identification of upstream sequences essential for activation of a bacteriophage P2 late promoter; Christie GE et al.; We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, P(F), which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters . All mutants were assayed for activation by P4 delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for delta binding . The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription . A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif . The mutational scan also led to identification of the -35 region of the promoter . Introduction of a sigma(70) -35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by delta . These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor sigma(70) contacts and helps to recruit RNA polymerase holoenzyme. J Bacteriol, 2003 Aug, 185(15), 4572 - 7 Isolation and analysis of mutants of double-stranded-RNA bacteriophage phi6 with altered packaging specificity; Qiao J et al.; The genomes of bacteriophage phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented double-stranded RNA genomes into preformed polyhedral structures called procapsids or inner cores . This packaging requires hydrolysis of nucleoside triphosphates and takes place in the order S-M-L . Packaging is dependent on unique sequences of about 200 nucleotides near the 5' ends of plus strand transcripts of the three genomic segments . Changes in the pac sequences lead to loss of packaging ability but can be suppressed by second-site changes in RNA or amino acid changes in protein P1, the major structural protein of the procapsid . It appears that P1 is the determinant of the RNA binding sites, and it is suggested that the binding sites overlap or are conformational changes of the same domains. J Bacteriol, 2003 Aug, 185(15), 4558 - 63 Phase variation in the phage growth limitation system of Streptomyces coelicolor A3(2); Sumby P et al.; The phase-variable phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) is an unusual bacteriophage resistance mechanism that confers protection against the temperate phage phiC31 and homoimmune relatives . Pgl is subject to phase variation, and data presented here show that this is at least partially due to expansion and contraction of a polyguanine tract present within the putative adenine-specific DNA methyltransferase gene, pglX . Furthermore, the pglX paralogue SC6G9.02, here renamed pglS, was shown to be able to interfere with the Pgl phenotype, suggesting that PglS could provide an alternative activity to that conferred by PglX. Nucleic Acids Res, 2003 Jul 15, 31(14), 3918 - 28 Bacteriophage P1 Ban protein is a hexameric DNA helicase that interacts with and substitutes for Escherichia coli DnaB; Lemonnier M et al.; Since the ban gene of bacteriophage P1 suppresses a number of conditionally lethal dnaB mutations in Escherichia coli, it was assumed that Ban protein is a DNA helicase (DnaB analogue) that can substitute for DnaB in the host replication machinery . We isolated and sequenced the ban gene, purified the product, and analysed the function of Ban protein in vitro and in vivo . Ban hydrolyses ATP, unwinds DNA and forms hexamers in the presence of ATP and magnesium ions . Since all existing conditionally lethal dnaB strains bear DnaB proteins that may interfere with the protein under study, we constructed a dnaB null strain by using a genetic set-up designed to provoke the conditional loss of the entire dnaB gene from E.coli cells . This novel tool was used to show that Ban restores the viability of cells that completely lack DnaB at 30 degrees C, but not at 42 degrees C . Surprisingly, growth was restored by the dnaB252 mutation at a temperature that is restrictive for ban and dnaB252 taken separately . This indicates that Ban and DnaB are able to interact in vivo . Complementary to these results, we demonstrate the formation of DnaB-Ban hetero-oligomers in vitro by ion exchange chromatography . We discuss the interaction of bacterial proteins and their phage-encoded analogues to fulfil functions that are essential to phage and host growth. J Biol Chem, 2003 Sep 19, 278(38), 36905 - 15 Epub 2003 Jul 08. Identification of Cre residues involved in synapsis, isomerization, and catalysis; Lee L et al.; The Cre protein of bacteriophage P1 is a tyrosine recombinase and catalyzes recombination via formation of a covalent protein-DNA complex and a Holliday junction intermediate . Several co-crystal structures of Cre bound to its target lox site have provided novel insights into its biochemical activities . We have used these structures to guide the mutagenesis of several Cre residues that contact the lox spacer region and/or are involved in intersubunit protein-protein interactions . None of the mutant proteins had significant defects in DNA binding, DNA bending, or strand-specific initiation of recombination . We have identified novel functions of several amino acids that are involved in three aspects of the Cre reaction . 1) Single mutation of several NH2-terminal basic residues that contact the spacer region of loxP caused the accumulation of Holliday junction (HJ) intermediates but only a modest impairment of recombination . These residues may be involved in the isomerization of the Holliday intermediate . 2) We identified three new residues (Arg-118, Lys-122, and Glu-129) that are involved in synapsis . Cre R118A, K122A, and E129Q were catalytically competent . 3) Mutations E129R, Q133H, and K201A inactivated catalysis by the protein . The function of these Cre residues in recombination is discussed. Arch Histol Cytol, 2003 May, 66(2), 175 - 81 Atomic force microscopy analysis of rolling circle amplification of plasmid DNA; Mizuta R et al.; Rolling circle amplification (RCA) of plasmid DNA using random hexamers and bacteriophage phi29 DNA polymerase is an increasingly applied technique for amplifying template DNA for DNA sequencing . We analyzed this RCA reaction at a single-molecular level by atomic force microscopy (AFM) and found that multibranched amplified products containing tandem repeats of a circle unit are formed within 1 h . We also used the RCA product of a GFP expression vector for the protein expression in cells, and found that the crude RCA product from one bacterial colony is sufficient for the GFP expression . Thus, the RCA reaction is useful in amplifying DNA for both DNA sequencing and protein expression. Arch Microbiol, 2003 Sep, 180(3), 161 - 8 Epub 2003 Jul 04. 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; Szalewska-Palasz A et al.; 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 overexpressing nus genes on bacteriophage lambda development in an E . coli rpoA341 host was examined . In addition, the effect of overproduction of the N protein in these genetic backgrounds was assessed . Analogous bacterial strains were employed to measure the efficiency of the antitermination process using the lacZ reporter gene under control of the lambda p(R) promoter, and containing the phage nutR region and the t( R1) terminator between the promoter and lacZ . The experimental results suggest interactions between components of the N-antitermination complex, which have been established biochemically, as well as additional functional relationships within the complex . Furthermore, the results indicate that amino acid substitution in the alpha subunit C-terminal domain encoded by the rpoA341 mutation may specifically disrupt the function of the NusA and NusE proteins . During this analysis, it was also found that the E . coli nusA1 mutant exhibits a conditional lethal phenotype. J Mol Biol, 2003 Jul 11, 330(3), 485 - 92 Thermodynamics of DNA packaging inside a viral capsid: the role of DNA intrinsic thickness; Marenduzzo D et al.; We characterize the equilibrium thermodynamics of a thick polymer confined in a spherical region of space . This is used to gain insight into the DNA packaging process . The experimental reference system for the present study is the recent characterization of the loading process of the genome inside the phi29 bacteriophage capsid . Our emphasis is on the modelling of double-stranded DNA as a flexible thick polymer (tube) instead of a beads-and-springs chain . By using finite-size scaling to extrapolate our results to genome lengths appropriate for phi29, we find that the thickness-induced force may account for up to half the one measured experimentally at high packing densities . An analogous agreement is found for the total work that has to be spent in the packaging process . Remarkably, such agreement can be obtained in the absence of any tunable parameters and is a mere consequence of the DNA thickness . Furthermore, we provide a quantitative estimate of how the persistence length of a polymer depends on its thickness . The expression accounts for the significant difference in the persistence lengths of single and double-stranded DNA (again with the sole input of their respective sections and natural nucleotide/base-pair spacing). Appl Environ Microbiol, 2003 Jul, 69(7), 3975 - 8 Reduction of Norwalk virus, poliovirus 1, and bacteriophage MS2 by ozone disinfection of water; Shin GA et al.; Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission . In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays . Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR . Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5 degrees C for up to 5 min . Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log(10) within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods . Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone . Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Proteins, 2003 Aug 1, 52(2), 272 - 82 Identification of the domains for DNA binding and transactivation function of C protein from bacteriophage Mu; Paul BD et al.; The C protein, a middle gene product of bacteriophage Mu, is the determinant of the transition from middle to late gene expression . C activates transcription from four late gene promoters, P(lys), P(I), P(P), and P(mom) by binding to a site overlapping their -35 elements . Site-specific, high-affinity binding of C to its recognition sequence results in both axial and torsional distortion of DNA at P(mom), which appears to play a role in recruitment of RNA polymerase to the promoter for mom gene transactivation . To identify the regions of C protein important for its function, deletion and site-directed mutagenesis were carried out . We demonstrate here that a helix-turn-helix (HTH) motif located toward the carboxy terminal end of the protein is the DNA-binding domain and amino acid residues involved in transactivation overlap the HTH motif . Mutagenesis studies also aided in the identification of the region important for dimerization . Structure-based sequence alignment and molecular modeling in conjunction with mutational analysis suggest that the HTH motif is part of a three-helix bundle, with remarkable similarity to paired (prd), a developmental regulatory protein from Drosophila . Additional key residues identified in the model to be crucial for C protein structure and DNA binding were shown to be important by mutagenesis . These results provide a structural framework for C function and insight into the mechanism of transactivation at the mom promoter . Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1238 - 40 Epub 2003 Jun 27. Crystallization and preliminary X-ray crystallographic analysis of the excisionase-DNA complex from bacteriophage lambda; Sam MD et al.; 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 cooperatively interacting with the lambda integrase (Int) protein . In an attempt to understand how Xis controls the directionality of bacteriophage lambda recombination, co-crystals of the DNA-binding domain of Xis in complex with its binding site within the P-arm of the phage have been obtained using the hanging-drop vapor-diffusion method . Using sodium acetate as a precipitating reagent, the Xis-DNA complex crystallizes in space group C2, with unit-cell parameters a = 80.2, b = 72.7, c = 38.8 A, beta = 104.1 degrees . These crystals diffract beyond 1.5 A resolution and are well suited for structural analysis using X-ray crystallography. FEBS Lett, 2003 Jul 10, 546(2-3), 167 - 72 The diverse spectrum of sliding clamp interacting proteins; Vivona JB et al.; DNA polymerase sliding clamps are a family of ring-shaped proteins that play essential roles in DNA metabolism . The proteins from the three domains of life, Bacteria, Archaea and Eukarya, as well as those from bacteriophages and viruses, were shown to interact with a large number of cellular factors and to influence their activity . In the last several years a large number of such proteins have been identified and studied . Here the various proteins that have been shown to interact with the sliding clamps of Bacteria, Archaea and Eukarya are summarized. DNA Cell Biol, 2003 Apr, 22(4), 261 - 91 Type III intermediate filament proteins interact with four-way junction DNA and facilitate its cleavage by the junction-resolving enzyme T7 endonuclease I; Li G et al.; The isolation from proliferating mouse and human embryo fibroblasts of SDS-stable crosslinkage products of vimentin with DNA fragments containing inverted repeats capable of cruciform formation under superhelical stress and the competitive effect of a synthetic Holliday junction on the binding of cytoplasmic intermediate filament (cIF) proteins to supercoiled DNA prompted a detailed investigation of the proteins' capacity to associate with four-way junction DNA and to influence its processing by junction-resolving endonucleases . Electrophoretic mobility shift analysis of reaction products obtained from vimentin and Holliday junctions under varying ionic conditions revealed efficient complex formation of the filament protein not only with the unstacked, square-planar configuration of the junctions but also with their coaxially stacked X-conformation . Glial fibrillary acidic protein (GFAP) was less efficient and desmin virtually inactive in complex formation . Electron microscopy showed binding of vimentin tetramers or octamers almost exclusively to the branchpoint of the Holliday junctions under physiological ionic conditions . Even at several hundredfold molar excess, sequence-related single- and double-stranded DNAs were unable to chase Holliday junctions from their complexes with vimentin . Vimentin also stimulated bacteriophage T7 endonuclease I in introducing single-strand cuts diametrically across the branchpoint and thus in the resolution of the Holliday junctions . This effect is very likely due to vimentin-induced structural distortion of the branchpoint, as suggested by the results of hydroxyl radical footprinting of Holliday junctions in the absence and the presence of vimentin . Moreover, vimentin, and to a lesser extent GFAP and desmin, interacted with the cruciform structures of inverted repeats inserted into a supercoiled vector plasmid, thereby changing their configuration via branch migration and sensibilizing them to processing by T7 endonuclease I . This refers to both plasmid relaxation caused by unilateral scission and, particularly, linearization via bilateral scission at primary and cIF protein-induced secondary cruciform branchpoints that were identified by T7 endonuclease I footprinting . cIF proteins share these activities with a variety of other architectural proteins interacting with and structurally modulating four-way DNA junctions . In view of the known and hypothetical functions of four-way DNA junctions and associated protein factors in DNA metabolism, cIF proteins as complementary nuclear matrix proteins may play important roles in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription, with special emphasis on both the preservation and evolution of the genome. Eur J Biochem, 2003 Jul, 270(13), 2789 - 95 Selection of peptides inhibiting a beta-lactamase-like activity; Yribarren AS et al.; A library of random peptide sequences was used to select peptides that inhibit an anti-idiotypic catalytic Ig, immunoglobulin (IgG) 9G4H9, with a beta-lactamase-like activity . This library displays cyclic heptapeptides on the surface of bacteriophages and represents a collection of up to 4.5 x 109 peptides . The first selection step aimed at enriching the library in species that bind to the whole Ig molecule . The second step was to discriminate peptides that bind to part of the molecule other than the active site . Selected peptides were then screened by surface plasmon resonance analysis . Those displaying measurable Kd values were assayed for their ability to inhibit the catalytic Ig. Protein Expr Purif, 2003 Jul, 30(1), 26 - 31 Construction of an overproducing strain, purification, and biochemical characterization of the 6His-Eco29kI restriction endonuclease; Nikitin D et al.; We constructed a strain of Escherichia coli overproducing 6His-tagged Eco29kI by placing the coding sequence under control of a strong bacteriophage T5 promoter . The yield of 6His-Eco29kI restriction endonuclease expression could be increased to about 20% of the total cellular protein, but inclusion bodies formed consisting of insoluble 6His-Eco29kI protein . We developed a fast and effective protocol for purification of the homogeneous enzyme from both soluble and insoluble fractions and established their identity by catalytic activity assay . The isolated enzymes were tested for recognition specificity and optimal reaction conditions as a function of NaCl and KCl concentrations, temperature, and pH compared with the native Eco29kI restriction endonuclease . The 6His-tagged enzyme retained the specificity of the native protein but had an altered optimum of its catalytic reaction. J Virol Methods, 2003 Jul, 111(1), 29 - 36 Enhanced genetic rescue of negative-strand RNA viruses: use of an MVA-T7 RNA polymerase vector and DNA replication inhibitors; Kovacs GR et al.; A modified cDNA rescue system that improves recovery of recombinant nonsegmented, negative-strand RNA viruses from cloned DNAs is described . Rescue systems based on vaccinia virus-T7 RNA polymerase vectors have been used to derive many negative-strand viruses; however, some strains can be recalcitrant to rescue possibly because of the simultaneous replication of the vaccinia virus-T7 vector . Our goal was to engineer a system where replication of the vaccinia virus-T7 vector could be blocked, yet allow for sufficient T7 RNA polymerase expression to enable genetic rescue . To that end, a recombinant modified vaccinia virus Ankara (MVA) was engineered that contained the bacteriophage T7 gene-1 under the control of a strong early promoter that would enable T7 RNA polymerase expression in the absence of MVA DNA replication . The new T7 helper, MVAGKT7, was then utilized successfully for the genetic rescue of a measles virus minigenome and full-length cDNAs, in the presence of DNA synthesis inhibitors . In addition to blocking completely MVAGKT7 replication, AraC treatment was found to enhance minigenome-encoded gene expression and the efficiency of measles virus rescue. Biochemistry, 2003 Jul 1, 42(25), 7717 - 26 Characterization of the interactions between the bacteriophage T4 AsiA protein and RNA polymerase; Simeonov MF et al.; The anti-sigma factor AsiA effects a change in promoter specificity of the Escherichia coli RNA polymerase via interactions with two conserved regions of the sigma(70) subunit, denoted 4.1 and 4.2 . Free AsiA is a symmetrical homodimer . Here, we show that AsiA is monomeric when bound to sigma(70) and that a subset of the residues that contribute to the homodimer interface also contributes to the interface with sigma(70) . AsiA interacts primarily with C-terminal sections of regions 4.1 and 4.2, which show remarkable sequence similarity . An AsiA monomer can simultaneously, and apparently cooperatively, bind both isolated regions 4.1 and 4.2 at preferred, distinct subsites, whereas region 4.1 alone or region 4.2 alone can interact with either subsite . These results suggest structural and functional plasticity in the interaction of AsiA with sigma(70) and support the notion of discrete roles for regions 4.1 and 4.2 in transcription regulation by AsiA . Furthermore, we show that AsiA inhibits recognition of the -35 consensus promoter element by region 4 of sigma(70) indirectly, as the residues on region 4 responsible for AsiA binding are distinct from those involved in DNA binding . Finally, we show that AsiA must directly disrupt the interaction of region 4 with the RNA polymerase beta subunit flap domain, resulting in a distance change between region 2 and region 4 of sigma(70) . Thus, a new paradigm for transcription regulation by AsiA is emerging, whereby the distance between the DNA binding domains in sigma(70) is regulated, and promoter recognition specificity is modulated, by mediating the interactions of the sigma region 4 with the beta subunit flap domain. Ann Clin Lab Sci, 2003 Spring, 33(2), 142 - 8 Selective detection of autoimmune antibodies to single- and double-stranded DNA by enzyme immunoassay; Makowski GS et al.; Presence in serum of anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA) is one of the diagnostic criteria for systemic lupus erythematosus . Anti-single stranded DNA antibodies (anti-ssDNA) also occur, but their clinical significance is unclear . Use of enzyme immunoassay (EIA) kits that are specific for anti-dsDNA is desirable but problematic, since preparation of dsDNA of high integrity is difficult (ie, regions of ssDNA may persist) . This study evaluated an EIA kit that uses low Mw plasmid/bacteriophage DNA as a nucleic acid source . Anti-dsDNA results were compared to results obtained by an anti-ssDNA EIA and an immunofluorescent assay (IFA) that uses Crithidia luciliae (specific for anti-dsDNA) . Consecutive serum samples (n=139, 88% female) submitted to the clinical laboratory for anti-dsDNA analysis were evaluated . EIA precision was determined at three levels . Intra-assay precision {mean +/- SD (CV)} for anti-dsDNA: 36 +/- 3.5 IU/ml (9.8%); 98 +/- 4.4 (4.5%); and 245 +/- 5.8 (2.4%); and anti-ssDNA . 40 +/- 1.4 U/ml (3.5%); 190 +/- 4.8 (2.5%); and 283 +/- 10.3 (3.7%) (n=8) . Inter-assay precision for anti-dsDNA: 36 +/- 6.3 IU/ml (17.5%); 90 +/- 5.9 (6.6%); and 207 +/- 20.9 (10.1%); and anti-ssDNA: 48 +/- 8.2 U/ml (17.0%); 193 +/- 12.7 (6.6%); and 263 +/- 21.5 (8.2%) (n=8) . Linearity was assessed with (a) high dsDNA/high ssDNA and (b) low dsDNA/high ssDNA samples (no high dsDNA/low ssDNA samples were identified) . Linearity (>200 IU/ml) was found for both sample types with a correlation coefficient (r) of 0.995-0.999 . Anti-dsDNA immunoreactivity was not apparent with the low dsDNA/high ssDNA sample . More patients were positive for anti-ssDNA (54%), compared to anti-dsDNA (17%) . IFA confirmation (Crithidia) indicated a relative sensitivity and specificity of 94.1% and 93.4% for the anti-dsDNA EIA . IFA positivity correlated with increased anti-dsDNA level: 20% (30-60 IU/ml); 70% (61-200 IU/ml); and 89% (>200 IU/ml) . Of specimens that were anti-ssDNA positive and anti-dsDNA negative (n=51), only one was IFA positive . When IFA was compared to an anti-dsDNA EIA kit that used high Mw calf thymus DNA, lower relative specificity (88.2%) and sensitivity (72.6%) was obtained . Anti-ssDNA was found in many false positive specimens (87%). Mol Biol (Mosk), 2003 May-Jun, 37(3), 556 - 60 {Immunogenetic properties of peptides mimicking a human immunodeficiency virus gp41 (HIV-1) epitope recognized by virus-neutralizing antibody 2F5}; Tumanova OIu et al.; Phage display was used to obtain peptides mimicking a HIV-1 gp41 conserved epitope recognized by virus-neutralizing monoclonal antibodies (MCA) 2F5 . Rabbits and mice were immunized with the peptides exposed on the surface of filamentous bacteriophages . Antibodies to gp41 were detected in the sera of immunized animals . The virus-neutralizing activity of the sera was examined. Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8119 - 23 Epub 2003 Jun 18. The RNA-protein complex: direct probing of the interfacial recognition dynamics and its correlation with biological functions; Xia T et al.; 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 . Specifically, we examined the influence of structural changes in four peptides on the femtosecond dynamics of boxB RNA (2-aminopurine labeled in different positions), through mutations of critical residues of N peptide (residues 1-22) . We then compare their in vivo (Escherichia coli) transcription antitermination activities with the dynamics . The results demonstrate that the RNA-peptide complexes adopt essentially two dynamical conformations with the time scale for interfacial interaction in the two structures being vastly different, 1 ps for the stacked structure and nanosecond for the unstacked one; only the weighted average of the two is detected in NMR by nuclear Overhauser effect experiments . Strikingly, the amplitude of the observed ultrafast dynamics depends on the identity of the amino acid residues that are one helical turn away from Trp-18 in the peptides and is correlated with the level of biological function of their respective full-length proteins. Cell Mol Biol Lett, 2003, 8(2), 305 - 10 The optimal eukaryotic signal for translation initiation from non-AUG codons, present upstream of bacteriophage lambda P cistron, is inactive in Escherichia coli; Wrobel B et al.; 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 upstream the P cistron) is inactive in Escherichia coli . Comparative sequence analysis confirms that such a signal is unlikely to be important for P synthesis . Instead, a weak Shine-Dalgarno sequence may be present upstream the P cistron, and be active in the absence of O gene expression. J Bacteriol, 2003 Jul, 185(13), 3795 - 803 Identification and mutational analysis of bacteriophage PRD1 holin protein P35; Rydman PS et al.; 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, missense, and insertion mutations in PRD1 gene XXXV were analyzed . These studies support the overall conclusion that the charged amino acids at the protein C terminus are involved in the timing of host cell lysis. Di Yi Jun Yi Da Xue Xue Bao, 2003 Jun, 23(6), 538 - 41, 545 {Construction and identification of the cDNA phage expression library for human colorectal cancer antigens}; Liu YH et al.; OBJECTIVE: To construct a cDNA phage expression library for human colorectal carcinoma antigens . METHODS: After the total RNA was extracted from human colorectal cancer tissues, the single-strand and double-strand cDNA were synthesized through reverse transcriptase PCR and long-distance PCR, with the cDNA fragments smaller than 500 bp removed and the remaining cDNA combined with the right and left arms of dephosphorylated lambdaTriplEx2 phage vector . The recombinant phage were then packaged in vitro by MaxPlax Packaging extract, and a small portion of the packaged phage was used to infect E.coli XL1-Blue . Titer measurement was performed so as to determine the capacity of the library . SfiI restriction endonucleases was used to cut the recombined phage DNA in order to identify the size of inserted cDNA . RESULTS: The constructed cDNA phage expression library for human colorectal cancer antigens consisted of 2.39 x 10(6) pfu/ml bacteriophages with a recombination rate of 97.5% and the length of the inserted cDNA fragment ranged from 600 to 4,000 bp with an average of 1,400 bp . CONCLUSION: The cDNA phage expression library of human colorectal cancer antigens is successfully constructed to meet the currently recognized standards, and can be well applicable in screening cDNA-cloned genes of human colorectal cancer-associated antigens by immunoscreening. J Environ Qual, 2003 May-Jun, 32(3), 816 - 23 Virus retention and transport as influenced by different forms of soil organic matter; Zhuang J et al.; Organic materials are widespread in natural soil and aquatic environments . Their effect on virus transport is very important in assessing the risk for contamination of ground water by viruses . This study aimed to determine how different forms (mineral-associated and dissolved) of natural organic matter influence the retention and transport of two bacteriophages (MS-2 and phiX174) in two porous media (a sand and a soil) . We found that mineral-associated organic matter significantly promoted the transport of one virus (MS-2) but not the other (phiX174) in a phosphate-buffered saline solution . Similarly, MS-2 was retained less in sand columns with increasing concentrations of dissolved humic acid, while little effect was observed for phiX174 under the same conditions . The two viruses have different surface properties and thus exhibited different reactivity to the metal oxides present on sand particles and were affected differently by organic matter . Because the organic matter used in the study was negatively charged and hydrophilic, blocking of virus sorption sites and increasing of virus-medium electrostatic repulsion arising from modification of the sand and virus surface by organic matter are probably responsible for the facilitated transport . For dissolved humic acid, its competition for sorption sites with viruses was an additional mechanism involved . This study suggests that the effect of organic matter varied depending on the organic material properties and the type of viruses involved . As a general trend, the effect of organic matter was dominated by electrostatic rather than hydrophobic interactions. J Virol, 2003 Jul, 77(13), 7425 - 33 The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro; Reuven NB et al.; 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-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redbeta synaptase component of the phage lambda recombinase . Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda . Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products . The optimal conditions for strand exchange were 1 mM MgCl(2), 40 mM NaCl, and pH 7.5 . Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end . Nuclease-defective UL12 could not support this reaction . These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism. Ultramicroscopy, 2003 Oct-Nov, 97(1-4), 249 - 55 Imaging stretched single DNA molecules by pulsed-force-mode atomic force microscopy; Kwak KJ et al.; The effect of a surface water layer on DNA strands deposited on a substrate was studied by atomic force microscopy (AFM) . DNA molecules were deposited and stretched on chemically modified glass coverslips by a molecular combing method . Lambda bacteriophage DNA molecules were aligned on the organosilane-modified substrate surfaces by chemical and physical adsorption during the molecular combing . The combed DNA molecules were observed in humidity-controlled air and in aqueous solutions by pulsed-force-mode AFM (PFM-AFM) . Chemical modification of cantilevers with an Au-coated tip by organothiol compounds was also applied to DNA observation . Mapping adhesive forces in aqueous media was useful to discriminate chemically the DNA strands from the substrate surface . The results suggest that PFM-AFM can be used widely to image the stretched DNA molecules on the silane-modified substrates. Res Microbiol, 2003 May, 154(4), 277 - 82 Prophage insertion sites; Campbell A; Insertion of viral DNA into host chromosomes is an ancient process essential for propagation in the proviral form . Many present-day bacteriophages insert at specific sites on the host chromosome . Insertion by two coliphage families (lambdoid and P4-like) is compared . For both families, insertion sites frequently lie within tRNA genes . The lambdoid phages insert at anticodon loops, whereas the p4-like phages insert in the TpsiC loops downstream from them . The association of both groups with tRNA genes suggests that the primordial insertion site of both groups may have been within a tRNA gene . The integrase proteins used in phage insertion may have originated at that stage, with subsequent diversification of specificity. Res Microbiol, 2003 May, 154(4), 253 - 7 Bacteriophages with tails: chasing their origins and evolution; Hendrix RW et al.; Comparative genomic analysis of the tailed bacteriophages shows that they are genetically mosaic with respect to each other, implying that horizontal exchange of sequences is an important component of their evolution . Horizontal exchange occurs intensively among closely related phages but also at reduced frequency across the entire population of tailed phages . It results in exchange of homologous functions, exchange of analogous but non-homologous functions as with the prophage integrases, and introduction of novel functions into the genome as with the morons . Extrapolation of these processes back in evolutionary time leads to a speculative model for the origins and early evolution of phages. Res Microbiol, 2003 May, 154(4), 245 - 51 Bacteriophage observations and evolution; Ackermann HW; Bacteriophages are classified into one order and 13 families . Over 5100 phages have been examined in the electron microscope since 1959 . At least 4950 phages (96%) are tailed . They constitute the order Caudovirales and three families . Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages) . Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses . Bacteriophages occur in over 140 bacterial or archaeal genera . Their distribution reflects their origin and bacterial phylogeny . Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent . Tailed phages appear as monophyletic and as the oldest known virus group. Can J Microbiol, 2003 Mar, 49(3), 225 - 9 Bacteriophage lambda repressor allelic modulation of the Rex exclusion phenotype; Slavcev RA et al.; 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 participation of a genetic element mapping outside of cI-rexA-rexB. J Biol Chem, 2003 Aug 15, 278(33), 31210 - 7 Epub 2003 Jun 05. Effect of mutations in the C-terminal domain of Mu B on DNA binding and interactions with Mu A transposase; Coros CJ et al.; Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B . Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function . The recent NMR structure of the C-terminal domain of Mu B (Mu B223-312) revealed that there is a patch of positively charged residues on the solvent-exposed surface . This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223-312 peptide . We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223- 312) . To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein . The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity . Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A . Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase. Mol Microbiol, 2003 Jun, 48(6), 1621 - 31 Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes; Hansen AM et al.; The stringent starvation protein A (SspA), an Escherichia coli RNA polymerase (RNAP)-associated protein, has been reported to be essential for lytic growth of bacteriophage P1 . Unlike P1 early promoters, P1 late promoters are not recognized by RNAP alone . A phage-encoded early protein, Lpa (late promoter activator protein, formerly called gp10), has been shown to be required for P1 late transcription in vivo . Here, we demonstrate that SspA is a transcription activator for P1 late genes . Our results indicated that Lpa is not limiting in an sspA mutant . However, the transcription of P1 late genes was deficient in an sspA mutant in vivo . We demonstrated that SspA/Lpa are required for transcription activation of the P1 late promoter Ps in vitro . In addition, SspA and Lpa were shown to facilitate the binding of RNAP to Ps late promoter DNA . Activation of late transcription by SspA/Lpa was dependent on holoenzyme containing sigma70 but not sigmaS, indicating that the two activators discriminate between the two forms of the holoenzyme . Furthermore, P1 early gene expression was downregulated in the wild-type background, whereas it persisted in the sspA mutant background, indicating that SspA/Lpa mediate the transcriptional switch from the early to the late genes during P1 lytic growth . Thus, this work provides the first evidence for a function of the E . coli RNAP-associated protein SspA. Biosci Biotechnol Biochem, 2003 Apr, 67(4), 869 - 76 Contributions of polysaccharide and lipid regions of lipopolysaccharide to the recognition by spike G protein of bacteriophage phi X174; Kawaura T et al.; A histidine-tagged G protein of bacteriophage phi X174 (HisG) bound strongly with lipopolysaccharide (LPS) of Escherichia coli C, one of a phi X174-sensitive Ra strain . The dissociation constant, Kd, was measured to be 0.16 +/- 0.04 microM by fluorometric titration . HisG showed slightly less affinity to LPSs of the insensitive Rc and Rd 2 strains having shorter R-core polysaccharide sequences than that of the sensitive Ra strains . The difference between the two types of LPS was demonstrated by CD spectra; LPSs of the sensitive strains increased the signal intensity for beta-sheet, while the insensitive strains decreased it . The chemically degraded LPS derivatives lacking a hydrophobic lipid region showed much less affinity to HisG, indicating the importance of the lipid region of LPS for strong binding with HisG . On the other hand, since the degraded derivatives increased the intensity of CD spectra, the polysaccharide region is thought to contribute to the conformation change of the protein. Nat Struct Biol, 2003 Jul, 10(7), 572 - 6 Bacteriophage phi29 scaffolding protein gp7 before and after prohead assembly; Morais MC et al.; Three-dimensional structures of the double-stranded DNA bacteriophage phi29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 A, respectively . Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail . The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis . DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release . A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells . The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers. Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 6946 - 51 Epub 2003 May 30. Viral assembly of oriented quantum dot nanowires; Mao C et al.; The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires . To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals . The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus . The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence . ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid . Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their {001} perpendicular to the viral surface . Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires . Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid . This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure. Antonie Van Leeuwenhoek, 2003, 83(4), 305 - 15 Bacterial host strains that support replication of somatic coliphages; Muniesa M et al.; Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality . Their potential replication in the water environment is considered a drawback for their use as indicators . However, the contribution of replication outside the gut to the total numbers has never been quantified . It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment . We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests) . Only a 3.02% of the tests showed sensitivity . Additionally, six environmental strains were used as hosts to count phages in sewage and seawater . Phages isolated on these strains were used to infect strain WG5 . The environmental strains detected 1 log10 fewer phages than strain WG5 in sewage and seawater . The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples . Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E . coli . We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible. Antonie Van Leeuwenhoek, 2003, 83(3), 223 - 9 New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break; Jung SO et al.; PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range . Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay . Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days . In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared . The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1% . Recently, the complete PM2 DNA genome sequence of 10,079 bp was published . The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease . The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report. Nat Rev Genet, 2003 Jun, 4(6), 471 - 7 The future of bacteriophage biology; Campbell A; After an illustrious history as one of the primary tools that established the foundations of molecular biology, bacteriophage research is now undergoing a renaissance in which the primary focus is on the phages themselves rather than the molecular mechanisms that they explain . Studies of the evolution of phages and their role in natural ecosystems are flourishing . Practical questions, such as how to use phages to combat human diseases that are caused by bacteria, how to eradicate phage pests in the food industry and what role they have in the causation of human diseases, are receiving increased attention . Phages are also useful in the deeper exploration of basic molecular and biophysical questions. Nucleic Acids Res, 2003 Jun 1, 31(11), 2751 - 8 The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active; Saida F et al.; The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes . The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease . This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated . Here, we show, using 31P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2',3'-phosphodiester product . In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB . The activity of these mutants was characterized both in vivo and in vitro . In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by 1H/15N NMR correlation spectroscopy . The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure . Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues . The effect of the in vivo selected R52L mutation is also described and discussed. Biophys J, 2003 Jun, 84(6), 3894 - 903 Structural studies of MS2 bacteriophage virus particle disassembly by nuclear magnetic resonance relaxation measurements; Anobom CD et al.; In this article we studied, by nuclear magnetic resonance relaxation measurements, the disassembly of a virus particle-the MS2 bacteriophage . MS2 is one of the single-stranded RNA bacteriophages that infect Escherichia coli . At pH 4.5, the phage turns to a metastable state, as is indicated by an increase in the observed nuclear magnetic resonance signal intensity upon decreasing the pH from 7.0 to 4.5 . Steady-state fluorescence and circular dichroism spectra at pH 4.5 show that the difference in conformation and secondary structure is not pronounced if compared with the phage at pH 7.0 . At pH 4.5, two-dimensional (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum shows approximately 40 crosspeaks, corresponding to the most mobile residues of MS2 coat protein at pH 4.5 . The (15)N linewidth is approximately 30 Hz, which is consistent with an intermediate with a rotational relaxation time of 100 ns . The average spin lattice relaxation time (T(1)) of the mobile residues was measured at different temperatures, clearly distinguishing between the dimer and the equilibrium intermediate . The results show, for the first time, the presence of intermediates in the process of dissociation of the MS2 bacteriophage. Biophys J, 2003 Jun, 84(6), 3583 - 93 Molecular dynamics simulations of peptides and proteins with amplified collective motions; Zhang Z et al.; We present a novel method that uses the collective modes obtained with a coarse-grained model/anisotropic network model to guide the atomic-level simulations . Based on this model, local collective modes can be calculated according to a single configuration in the conformational space of the protein . In the molecular dynamics simulations, the motions along the slowest few modes are coupled to a higher temperature by the weak coupling method to amplify the collective motions . This amplified-collective-motion (ACM) method is applied to two test systems . One is an S-peptide analog . We realized the refolding of the denatured peptide in eight simulations out of 10 using the method . The other system is bacteriophage T4 lysozyme . Much more extensive domain motions between the N-terminal and C-terminal domain of T4 lysozyme are observed in the ACM simulation compared to a conventional simulation . The ACM method allows for extensive sampling in conformational space while still restricting the sampled configurations within low energy areas . The method can be applied in both explicit and implicit solvent simulations, and may be further applied to important biological problems, such as long timescale functional motions, protein folding/unfolding, and structure prediction. J Mol Biol, 2003 Jun 6, 329(3), 423 - 39 Dynamics and DNA substrate recognition by the catalytic domain of lambda integrase; Subramaniam S et al.; 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 domain of lambda-Int (C170) obtained in the absence of DNA revealed the tyrosine nucleophile at the protein's C terminus to be located on a beta-hairpin far from the other conserved catalytic residues and adjacent to a disordered loop . This observation suggested that a conformational change in the C terminus of the protein was required to generate the active site in cis, or alternatively, that the active site could be completed in trans by donation of the tyrosine nucleophile from a neighboring molecule in the recombining synapse . We used NMR spectroscopy together with limited proteolysis to examine the dynamics of the lambda-Int catalytic domain in the presence and absence of DNA half-site substrates with the goal of characterizing the expected conformational change . Although the C terminus is indeed flexible in the absence of DNA, we find that conformational changes in the tyrosine-containing beta-hairpin are not coupled to DNA binding . To gain structural insights into C170/DNA complexes, we took advantage of mechanistic conservation with Cre and Flp recombinases to model C170 in half-site and tetrameric Holliday junction complexes . Although the models do not reveal the nature of the conformational change required for cis cleavage, they are consistent with much of the available experimental data and provide new insights into the how trans complementation could be accommodated. J Biol Chem, 2003 Aug 8, 278(32), 29454 - 62 Epub 2003 May 24. Mutational analysis of bacteriophage T4 RNA ligase 1 . Different functional groups are required for the nucleotidyl transfer and phosphodiester bond formation steps of the ligation reaction; Wang LK et al.; T4 RNA ligase 1 (Rnl1) exemplifies an ATP-dependent RNA ligase family that includes fungal tRNA ligase (Trl1) and a putative baculovirus RNA ligase . Rnl1 acts via a covalent enzyme-AMP intermediate generated by attack of Lys-99 N zeta on the alpha phosphorus of ATP . Mutation of Lys-99 abolishes ligase activity . Here we tested the effects of alanine mutations at 19 conserved positions in Rnl1 and thereby identified 9 new residues essential for ligase activity: Arg-54, Lys-75, Phe-77, Gly-102, Lys-119, Glu-227, Gly-228, Lys-240, and Lys-242 . Seven of the essential residues are located within counterparts of conserved nucleotidyltransferase motifs I (99KEDG102), Ia (118SK119), IV (227EGYVA231), and V (238HFKIK242) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligase 2 . Three other essential residues, Arg-54, Lys-75 and Phe-77, are located upstream of the AMP attachment site within a conserved domain unique to the Rnl1-like ligase family . We infer a shared evolutionary history and active site architecture in Rnl1 (a tRNA repair enzyme) and Trl1 (a tRNA splicing enzyme) . We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Rnl1 adenylylation (step 1) and phosphodiester bond formation (step 3) . Lys-75, Lys-240, and Lys-242 were found to be essential for step 1 and overall ligation of 5'-phosphorylated RNA but not for phosphodiester bond formation . These results suggest that the composition of the Rnl1 active site is different during steps 1 and 3 . Mutations at Arg-54 and Lys-119 abolished the overall RNA ligation reaction without affecting steps 1 and 3 . Arg-54 and Lys-119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway. J Biol Chem, 2003 Aug 8, 278(32), 29538 - 45 Epub 2003 May 24. The carboxyl-terminal domain of bacteriophage T7 single-stranded DNA-binding protein modulates DNA binding and interaction with T7 DNA polymerase; He ZG et al.; Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5) . Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions . A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue . gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5 . gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase . A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta 26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase . gp2.5-Delta 26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration . The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase. Infect Immun, 2003 Jun, 71(6), 3409 - 18 Immunity profiles of wild-type and recombinant shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens; Allison HE et al.; 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 . Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles . Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain . The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted . Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host . Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages . The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage lambda, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E . coli serotypes. J Protein Chem, 2003 Feb, 22(2), 193 - 204 Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen; Landon LA et al.; Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells . Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries . These bound to TF antigen on carcinoma cells but were of low affinity and solubility . We hypothesized that peptide amino acid sequence changes would result in increased affinity and solubility, which would translate into improved carcinoma cell binding and increased inhibition of aggregation . The new peptides were more soluble and exhibited up to fivefold increase in affinity (Kd approximately equal to 60 nM) . They bound cultured human breast and prostate carcinoma cells at low concentrations, whereas the earlier peptides did not . Moreover, the new peptides were potent inhibitors of homotypic aggregation . The maturated peptides will have expanded applications in basic studies of the TF antigen and particular utility as clinical carcinoma-targeting agents. Ann Ist Super Sanita, 2002, 38(4), 401 - 10 {Use of phage libraries for the in vitro production of recombinant monoclonal antibodies of predetermined specificity}; Flego M et al.; The biotechnological generation of monoclonal antibodies of predetermined specificity has traditionally involved the production of hybridomas obtained by somatic cellular fusion of splenocytes from immunized animals with myeloma cell lines bearing selectable markers . Now, monoclonal antibodies could be genetically engineered thus bypassing all the natural systems for making antibodies . Filamentous bacteriophages provides a means to display and select large single chain fragments variable (scFv) repertoires created by cloning the natural rearranged V-immunoglobulin genes or introducing predetermined level of randomization into germline V-gene segments . In this article we demonstrated that by using a well characterized scFv phage synthetic library it is possible to generate in vitro recombinant human monoclonal antibodies directed to a large array of antigens showing different molecular weights, conformations and origins. RNA, 2003 Jun, 9(6), 663 - 76 Structural mimicry in the phage phi21 N peptide-boxB RNA complex; Cilley CD et al.; We determined the solution structure of a 22-amino-acid peptide from the amino-terminal domain of the bacteriophage phi21 N protein in complex with its cognate 24-mer boxB RNA hairpin using heteronuclear magnetic resonance spectroscopy . The N peptide binds as an alpha-helix and interacts predominately with the major groove side of the 5' half of the boxB RNA stem-loop . This binding interface is defined by surface complementarity of polar and nonpolar interactions, and little sequence-specific recognition . The phi21 boxB loop (CUAACC) has hydrogen bond and backbone torsions typical of the "U-turn" motif, as well as base stacking of the last 4 nt, and a hydrogen bonded C:C pair closing the loop . The exposed face of the phi21 boxB loop, in complex with the N peptide, is strikingly similar to the GNRA tetraloop-like folds of the related lambda and P22 bacteriophage N peptide-boxB RNA complexes . The N peptide-boxB complexes of the various phage, while individually distinct, provide similar structural features for interactions with the Escherichia coli host factors to enable antitermination. Genes Dev, 2003 May 15, 17(10), 1281 - 92 RNA polymerase mutations that impair conversion to a termination-resistant complex by Q antiterminator proteins; Santangelo TJ et al.; 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 are essential for Q modification . A specific conjecture is that Q modification stabilizes interactions of RNAP with the DNA/RNA hybrid and optimizes alignment of the nucleic acids in the catalytic site . Such changes would inhibit the activity of the RNA hairpin of an intrinsic terminator to disrupt the 5'-terminal bases of the hybrid and remove the RNA 3' terminus from the active site. Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6458 - 63 Epub 2003 May 15. Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy; Zeri AC et al.; The atomic resolution structure of fd coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles differs from that previously determined by x-ray fiber diffraction . Most notably, the 50-residue protein is not a single curved helix, but rather is a nearly ideal straight helix between residues 7 and 38, where there is a distinct kink, and then a straight helix with a different orientation between residues 39 and 49 . Residues 1-5 have been shown to be mobile and unstructured, and proline 6 terminates the helix . The structure of the coat protein in virus particles, in combination with the structure of the membrane-bound form of the same protein in bilayers, also recently determined by solid-state NMR spectroscopy, provides insight into the viral assembly process . In addition to their roles in molecular biology and biotechnology, the filamentous bacteriophages continue to serve as model systems for the development of experimental methods for determining the structures of proteins in biological supramolecular assemblies . New NMR results include the complete sequential assignment of the two-dimensional polarization inversion spin-exchange at the magic angle spectrum of a uniformly 15N-labeled 50-residue protein in a 1.6 x 107 Da particle in solution, and the calculation of the three-dimensional structure of the protein from orientational restraints with an accuracy equivalent to an rms deviation of approximately 1A. J Biol Chem, 2003 Aug 1, 278(31), 29098 - 105 Epub 2003 May 14. A single-stranded DNA-binding protein of bacteriophage T7 defective in DNA annealing; Rezende LF et al.; The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair . In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function . We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity . Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5 . Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA . A carboxyl-terminal-deleted protein, gene 2.5 protein-Delta26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein . The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein . Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy. J Virol, 2003 Jun, 77(11), 6314 - 21 The unique vertex of bacterial virus PRD1 is connected to the viral internal membrane; Stromsten NJ et al.; Icosahedral double-stranded DNA (dsDNA) bacterial viruses are known to package their genomes into preformed procapsids via a unique portal vertex . Bacteriophage PRD1 differs from the more commonly known icosahedral dsDNA phages in that it contains an internal lipid membrane . The packaging of PRD1 is known to proceed via preformed empty capsids . Now, a unique vertex has been shown to exist in PRD1 . We show in this study that this unique vertex extends to the virus internal membrane via two integral membrane proteins, P20 and P22 . These small membrane proteins are necessary for the binding of the putative packaging ATPase P9, via another capsid protein, P6, to the virus particle. Transgenic Res, 2003 Apr, 12(2), 191 - 201 Kidney-specific activity of the bovine uromodulin promoter; Kim HT et al.; A 10-kilobase (kb) lambda bacteriophage bovine genomic clone containing 5.4 kb of the 5'-flanking region, exons, and introns of bovine uromodulin gene was isolated . Transgenic mice containing 3.9 kb of the bovine uromodulin promoter and a lacZ reporter gene were generated by pronuclear microinjection . RT-PCR and northern blot analyses of transgene expression in various tissues of founder and F1 mice showed that the transgene was expressed exclusively in the kidney . In situ hybridization and histochemistry for lacZ demonstrated that transgene expression was restricted to tubule epithelial cells of the loop of Henle in the kidney . Stepwise 5' deletion analysis revealed that transfection of luciferase reporter constructs fused to various proximal 5'-flanking regions of the bovine uromodulin gene markedly increased luciferase activity in mouse renal epithelial cells but not in mesenchymal cells and that the most critical cis elements of the uromodulin gene are located within the 600 bp upstream region. J Immunol Methods, 2003 May 1, 276(1-2), 135 - 41 Peptabodies as tools to test ligands isolated from phage-displayed peptide libraries; Clement G et al.; We have previously isolated filamentous bacteriophages, expressing linear hexa-peptides and homing to bone marrow endothelium (BME), by panning in vivo of a phage-displayed peptide library in mice . Here, we used peptabody fusion proteins to test the binding capacity of the hexa-peptide SSLTTG to BME cells in vitro . To display this motif in a multimeric form, as originally presented on the bacteriophage, we expressed it N-terminally as a fusion with the peptabody cartilage oligomeric matrix assembly protein (COMP) pentamerization domain, either alone or followed by the N1 domain of the pIII phage coat protein . Binding of the peptabody constructs to the mouse BME cell line STR-10 was investigated by immunofluorescence using anti-COMP antibodies . Only peptabody fusion proteins co-expressing pIII-N1 exhibited binding to STR-10, regardless of the presence or absence of SSLTTG . These results indicate that the phage coat protein pIII-N1 domain is the principle determinant responsible for the binding of filamentous bacteriophages to cells of the reticulo-endothelial system (RES) . Peptabodies expressing pIII-N1 did not bind to the osteoblast-like cell line MC3T3-E1, indicating that binding is mediated by receptors specifically expressed by BME cells in vivo . Polyinosinic acid (poly-I) was able to inhibit binding of bacteriophages and pIII-N1 expressing peptabodies to STR-10, confirming our previous studies showing that bacteriophages bind to scavenger receptors (SR) expressed by BME cells . In summary, the present study shows the usefulness of peptabodies as a general tool to test the binding capacity of peptide ligands identified by phage display. New Microbiol, 2003 Apr, 26(2), 163 - 8 Various morphological aspects of Escherichia coli lysis by RNA bacteriophage MS2 observed by transmission and scanning electron microscopes; Nishihara T; Escherichia coli cell lysis profiles induced by the cloned lysis gene from RNA bacteriophages MS2 were presented . Transmission electron micrographs showed that ballooning structures appeared on the cell surfaces and the others were leaking materials through the cell wall . Harsh rupture of the host cell was also observed . Scanning electron micrographs revealed many extruded structures from the cells . For the scanning electron microscopy, plastic sheets coated with calf dermis collagen and SEMPORE filters were successfully used to collect the samples and for various chemical treatments . The lysing cells by bacteriophage MS2 lysis gene observed in transmission and scanning electron micrographs showed various morphological aspects of the intermediates in the lysing process. Photochem Photobiol, 2003 Apr, 77(4), 397 - 404 Ultraviolet (280-400 nm)-induced DNA damage in the eggs and larvae of Calanus finmarchicus G . (Copepoda) and Atlantic cod (Gadus morhua); Browman HI et al.; In previous work, we evaluated the effects of ultraviolet (UV = 280-400 nm) radiation on the early life stages of a planktonic Calanoid copepod (Calanus finmarchicus Gunnerus) and of Atlantic cod (Gadus morhua) . Both are key species in North Atlantic food webs . To further describe the potential impacts of UV exposure on the early life stages of these two species, we measured the wavelength-specific DNA damage (cyclobutane pyrimidine dimer {CPD} formation per megabase of DNA) induced under controlled experimental exposure to UV radiation . UV-induced DNA damage in C . finmarchicus and cod eggs was highest in the UV-B exposure treatments . Under the same spectral exposures, CPD loads in C . finmarchicus eggs were higher than those in cod eggs, and for both C . finmarchicus and cod embryos, CPD loads were generally lower in eggs than in larvae . Biological weighting functions (BWF) and exposure response curves that explain most of the variability in CPD production were derived from these data . Comparison of the BWF revealed significant differences in sensitivity to UV-B: C . finmarchicus is more sensitive than cod, and larvae are more sensitive than eggs . This is consistent with the raw CPD values . Shapes of the BWF were similar to each other and to a quantitative action spectrum for damage to T7 bacteriophage DNA that is unshielded by cellular material . The strong similarities in the shapes of the weighting functions are not consistent with photoprotection by UV-absorbing compounds, which would generate features in BWF corresponding to absorption bands . The BWF reported in this study were applied to assess the mortality that would result from accumulation of a given CPD load: for both C . finmarchicus and cod eggs, an increased load of 10 CPD Mb(-1) of DNA due to UV exposure would result in approximately 10% mortality. Biol Proced Online, 2003, 5, 78 - 89 Epub 2003 Mar 24. Use of Site-Specifically Tethered Chemical Nucleases to Study Macromolecular Reactions; Mukherjee S et al.; During a complex macromolecular reaction multiple changes in molecular conformation and interactions with ligands may occur . X-ray crystallography may provide only a limited set of snapshots of these changes . Solution methods can augment such structural information to provide a more complete picture of a macromolecular reaction . We analyzed the changes in protein conformation and protein:nucleic acid interactions which occur during transcription initiation by using a chemical nuclease tethered to cysteines introduced site-specifically into the RNA polymerase of bacteriophage T7 (T7 RNAP) . Changes in cleavage patterns as the polymerase steps through transcription reveal a series of structural transitions which mediate transcription initiation . Cleavage by tethered chemical nucleases is seen to be a powerful method for revealing the conformational dynamics of macromolecular reactions, and has certain advantages over cross-linking or energy transfer approaches. J Biol Chem, 2003 Jul 11, 278(28), 26102 - 10 Epub 2003 May 02. Identification and characterization of BCL-3-binding protein: implications for transcription and DNA repair or recombination; Watanabe N et al.; A putative oncogene bcl-3 was originally identified and cloned at the breakpoint in the recurring chromosome translocation t(14;19) found in some cases of B cell chronic lymphocytic leukemia . Studies of bcl-3-deficient mice demonstrated a critical role for bcl-3 in the development of a normal immune response and the formation of germinal centers in secondary lymphoid organs . However, the molecular mechanism that underlies B cell leukemogenesis and the knockout mouse phenotype remains unclear . Here we have identified and characterized BCL-3-binding protein (B3BP) as a protein interacting specifically with the bcl-3 gene product (BCL-3) by a yeast two-hybrid screen . We found that B3BP associates with not only BCL-3 but also p300/CBP histone acetyltransferases . The N-terminal region of B3BP that contains the ATP-binding site is important for the interaction with BCL-3 and p300/CBP . Homology searches indicate that the ATP-binding region of B3BP, which contains a typical Walker-type ATP-binding P-loop, most resembles that of 2',3'-cyclic nucleotide 3'-phosphodiesterase of mammals and polynucleotide kinase of T4 bacteriophage . In fact B3BP shows intrinsic ATP binding and hydrolyzing activity . Furthermore, we demonstrated that B3BP is a 5'-polynucleotide kinase . We also found a small MutS-related domain, which is thought to be involved in the DNA repair or recombination reaction, in the C-terminal region of B3BP, and it shows nicking endonuclease activity . These observations might help to gain new insights into the function of BCL-3 and p300/CBP, especially the coupling of transcription with repair or recombination. J Bacteriol, 2003 May, 185(10), 3076 - 80 The Escherichia coli Fis protein stimulates bacteriophage lambda integrative recombination in vitro; Esposito D et al.; 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 Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro . In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly . In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination . These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell. J Mol Biol, 2003 May 9, 328(4), 821 - 33 Structure and location of gene product 8 in the bacteriophage T4 baseplate; Leiman PG et al.; Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells . In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail . The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process . Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined . A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich . The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer . Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures . The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex . The gp8 dimers were found to be located in the upper part of the baseplate outer rim . About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10 . With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure. J Mol Biol, 2003 May 9, 328(4), 791 - 804 Conserved intermediates on the assembly pathway of double-stranded RNA bacteriophages; Kainov DE et al.; Double-stranded RNA (dsRNA) viruses are complex RNA processing machines that sequentially perform packaging, replication and transcription of their genomes . In order to characterize the assembly intermediates of such a machine we have developed an efficient in vitro assembly system for the procapsid of bacteriophage phi8 . The major structural protein P1 is a stable and soluble tetramer . Three tetramers associate with a P2 monomer (RNA-dependent RNA polymerase) to form the nucleation complex . This complex is further stabilized by a P4 hexamer (packaging motor) . Further assembly proceeds via rapid addition of individual building blocks . The incorporation of the packaging and replication machinery is under kinetic control . The in vitro assembled procapsids perform packaging, replication and transcription of viral RNA . Comparison with another dsRNA phage, phi6, indicates conservation of key assembly intermediates in the absence of sequence homology and suggests that a general assembly mechanism for the dsRNA virus lineage may exist. J Mol Biol, 2003 May 16, 328(5), 1027 - 45 Evaluating the effects of enhanced processivity and metal ions on translesion DNA replication catalyzed by the bacteriophage T4 DNA polymerase; Reineks EZ et al.; The fidelity of DNA replication is achieved in a multiplicative process encompassing nucleobase selection and insertion, removal of misinserted nucleotides by exonuclease activity, and enzyme dissociation from primer/templates that are misaligned due to mispairing . In this study, we have evaluated the effect of altering these kinetic processes on the dynamics of translesion DNA replication using the bacteriophage T4 replication apparatus as a model system . The effect of enhancing the processivity of the T4 DNA polymerase, gp43, on translesion DNA replication was evaluated using a defined in vitro assay system . While the T4 replicase (gp43 in complex with gp45) can perform efficient, processive replication using unmodified DNA, the T4 replicase cannot extend beyond an abasic site . This indicates that enhancing the processivity of gp43 does not increase unambiguously its ability to perform translesion DNA replication . Surprisingly, the replicase composed of an exonuclease-deficient mutant of gp43 was unable to extend beyond the abasic DNA lesion, thus indicating that molecular processes involved in DNA polymerization activity play the predominant role in preventing extension beyond the non-coding DNA lesion . Although neither T4 replicase complex could extend beyond the lesion, there were measurable differences in the stability of each complex at the DNA lesion . Specifically, the exonuclease-deficient replicase dissociates at a rate constant, k(off), of 1.1s(-1) while the wild-type replicase remains more stably associated at the site of DNA damage by virtue of a slower measured rate constant (k(off) 0.009s(-1)) . The increased lifetime of the wild-type replicase suggests that idle turnover, the partitioning of the replicase from its polymerase to its exonuclease active site, may play an important role in maintaining fidelity . Further attempts to perturb the fidelity of the T4 replicase by substituting Mn(2+) for Mg(2+) did not significantly enhance DNA synthesis beyond the abasic DNA lesion . The results of these studies are interpreted with respect to current structural information of gp43 alone and complexed with gp45. Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5956 - 61 Epub 2003 Apr 28. Parkin, a gene implicated in autosomal recessive juvenile parkinsonism, is a candidate tumor suppressor gene on chromosome 6q25-q27; Cesari R et al.; In an effort to identify tumor suppressor gene(s) associated with the frequent loss of heterozygosity observed on chromosome 6q25-q27, we constructed a contig derived from the sequences of bacterial artificial chromosomeP1 bacteriophage artificial chromosome clones defined by the genetic interval D6S1581-D6S1579-D6S305-D6S1599-D6S1008 . Sequence analysis of this contig found it to contain eight known genes, including the complete genomic structure of the Parkin gene . Loss of heterozygosity (LOH) analysis of 40 malignant breast and ovarian tumors identified a common minimal region of loss, including the markers D6S305 (50%) and D6S1599 (32%) . Both loci exhibited the highest frequencies of LOH in this study and are each located within the Parkin genomic structure . Whereas mutation analysis revealed no missense substitutions, expression of the Parkin gene appeared to be down-regulated or absent in the tumor biopsies and tumor cell lines examined . In addition, the identification of two truncating deletions in 3 of 20 ovarian tumor samples, as well as homozygous deletion of exon 2 in the lung adenocarcinoma cell lines Calu-3 and H-1573, supports the hypothesis that hemizygous or homozygous deletions are responsible for the abnormal expression of Parkin in these samples . These data suggest that the LOH observed at chromosome 6q25-q26 may contribute to the initiation andor progression of cancer by inactivating or reducing the expression of the Parkin gene . Because Parkin maps to FRA6E, one of the most active common fragile sites in the human genome, it represents another example of a large tumor suppressor gene, like FHIT and WWOX, located at a common fragile site. Protein Sci, 2003 May, 12(5), 1126 - 30 Stability of monomeric Cro variants: Isoenergetic transformation of a type I' to a type II' beta-hairpin by single amino acid replacements; Mollah AK et al.; 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 determined from guanidine hydrochloride range from 2.8 to 3.3 kcal/mole for the three proteins . Thermal denaturation yields van't Hoff unfolding enthalpies of 36 to 40 kcal /mole at midpoint temperatures in the range of 53 to 58 degrees C . Extrapolation of the thermal denaturation free energies with heat capacities of 400 to 600 cal/mole deg gives good agreement with the parameters determined in denaturant titrations . As predicted from statistical surveys of amino acid replacements in beta-hairpins, energetic barriers to transformation from a type I' turn (DG) to a type II' turn (GT) can be quite small. Mol Genet Genomics, 2003 Apr, 269(1), 40 - 8 Epub 2003 Mar 05. Stationary phase-like properties of the bacteriophage lambda Rex exclusion phenotype; Slavcev RA et al.; 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 multicopy plasmids expressing rexA-rexB: cells were about two-fold contracted in length, expressed membrane-bound and detached flagella, were insensitive to infection by a variety of phages and clumped extensively; in addition, cultures of these cells were odorous . Our observations support the hypothesis that the Rex system can cause a stationary-phase-like response that protects the host against infection by T4 rII. J Virol Methods, 2003 May, 109(2), 203 - 7 Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template; Kothapalli R et al.; In order to identify the reverse transcriptase activity in sera and conditioned media from peripheral blood mononuclear cells (PBMCs) of large granular lymphocyte leukemia patients product enhanced reverse transcriptase activity (PERT) assays were performed using bacteriophage MS2 RNA as a template . All samples obtained from conditioned media of virus-infected cell lines as well as PBMCs of lymphocytic leukemia patients and normal healthy individuals tested positive with this assay . Therefore the validity of the assay was questioned . Careful evaluation of the assay revealed that some of the essential reagents used, such as Taq DNA polymerase and RNase inhibitor contained indigenous amplifiable DNA . DNase I treatment of Taq DNA polymerase before PCR reduced the product significantly . Moreover, no false positive results were observed when encephalomyocarditis virus RNA was used instead of MS2 RNA as the template . These results suggest a need for caution when using bacteriophage MS2 RNA as the template in PERT assays to confirm the presence of retroviral infection or for identification of novel retroviruses. Antimicrob Agents Chemother, 2003 May, 47(5), 1760 - 5 Genetic screen for monitoring hepatitis C virus NS3 serine protease activity; Martinez MA et al.; 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-galactosidase (betagal)-HCV NS3(2-181)/4(21-34) protease construct . Upon infection of Escherichia coli cells containing the two plasmids encoding the cI.HCV5AB-cro and the betagal-HCV NS3(2-181)/4(21-34) protease constructs, lambda phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS3(2-181)/4(21-34) protease . This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine protease, and it has the potential to be used for screening specific inhibitors. Acta Microbiol Pol, 2002, 51(4), 379 - 85 Identification of a second gene-27 product (27bis) of bacteriophage T4, and its involvement in regulatation of expression of gene 51; Nieradko J; The 27 gene of bacteriophage T4 has been shown of encode two proteins of 44 and 39 kilodaltons (designated 27-44 and 27-39 bis, respectively) as a result of independent translational initiation at two different start codons within the same reading frame . The first product is the structural component of the viral baseplate . The latter with molecular weight 39 kDa probably plays significant role in regulation of expression of gene 51. Biosens Bioelectron, 2003 May, 18(5-6), 755 - 63 Real time device for biosensing: design of a bacteriophage model using love acoustic waves; Tamarin O et al.; Love wave sensors (ST-cut quartz substrate with interdigital transducers, SiO(2) guiding layer and sensitive coating) have been receiving a great deal of attention for a few years . Indeed, the wave coupled in a guiding layer confers a high gravimetric sensitivity and the shear horizontal (SH) polarization allows to work in liquid media . In this paper, an analytical method is proposed to calculate the Love wave phase velocity and the gravimetric sensitivity for a complete multilayer structure . This allows us to optimize the Love wave devices design in order to improve their gravimetric sensitivity in liquid media . As a model for virus or bacteria detection in liquids (drinking or bathing water, food em leader ) we design a model using M13 bacteriophage . The first step is the anti-M13 (AM13) monoclonal antibody grafting, on the device surface (SiO(2)) . The second step is an immunoreaction in between the M13 bacteriophage and the AM13 antibody . The Love wave device allows to detect in real time the graft of the AM13 sensitive coating, as well as the immobilization of the M13 bacteriophages . With a pH change, the M13 bacteriophages can be removed from the sensor surface, in order to be numerated as plaque forming unit (pfu) . Results on the sensitivity of Love waves are compared with similar immunological works with bulk acoustic wave devices, and demonstrate the high potentialities of Love waves sensors. Virology, 2003 Apr 10, 308(2), 354 - 61 Unique properties of the inner core of bacteriophage phi8, a virus with a segmented dsRNA genome; Sun Y et al.; The inner core of bacteriophage phi8 is capable of packaging and replicating the plus strands of the RNA genomic segments of the virus in vitro . The particles composed of proteins P1, P2, P4, and P7 can be assembled in cells of E . coli that carry plasmids with cDNA copies of genomic segment L . The gene arrangement on segment L was found to differ from that of other cystoviruses in that the gene for the ortholog of protein P7 is loca