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| 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 located at the 3' end of the plus strand rather than near the 5' end . In place of the normal location of gene 7 is gene H, whose product is necessary for normal phage development, but not necessary for in vitro genomic packaging and replication . Genomic packaging is dependent upon the activity of an NTPase motor protein, P4 . P4 was purified from cell extracts and was found to form hexamers with little NTPase activity until associated with inner core particles . Labeling studies of in vitro packaging of phi8 RNA do not show serial dependence; however, studies involving in vitro packaging for the formation of live virus indicate that packaging is stringent . Studies with the acquisition of chimeric segments in live virus indicate that phi8 does package RNA in the order s/m/l . The inner core of bacteriophage phi8 differs from that of its relatives in the Cystoviridae in that the major structural protein P1 is able to interact with the host cell membrane to effect penetration of the inner core into the cell. Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5046 - 51 Epub 2003 Apr 18. Endonuclease cleavage of blocked replication forks: An indirect pathway of DNA damage from antitumor drug-topoisomerase complexes; Hong G et al.; The cytotoxicity of several important antitumor drugs depends on formation of the covalent topoisomerase-DNA cleavage complex . However, cellular processes such as DNA replication are necessary to convert the cleavage complex into a cytotoxic lesion, but the molecular mechanism of this conversion and the precise nature of the cytotoxic lesion are unknown . Using a bacteriophage T4 model system, we have previously shown that antitumor drug-induced cleavage complexes block replication forks in vivo . In this report, we show that these blocked forks can be cleaved by T4 endonuclease VII to create overt DNA breaks . The accumulation of blocked forks increased in endonuclease VII-deficient infections, suggesting that endonuclease cleavage contributes to fork processing in vivo . Furthermore, purified endonuclease VII cleaved the blocked forks in vitro close to the branch points . These results suggest that an indirect pathway of branched-DNA cleavage contributes to the cytotoxicity of antitumor drugs that target DNA topoisomerases. J Biol Chem, 2003 Jul 11, 278(28), 25435 - 47 Epub 2003 Apr 16. Function and assembly of the bacteriophage T4 DNA replication complex: interactions of the T4 polymerase with various model DNA constructs; Delagoutte E et al.; Complexes formed between DNA polymerase and genomic DNA at the replication fork are key elements of the replication machinery . We used sedimentation velocity, fluorescence anisotropy, and surface plasmon resonance to measure the binding interactions between bacteriophage T4 DNA polymerase (gp43) and various model DNA constructs . These results provide quantitative insight into how this replication polymerase performs template-directed 5' --> 3' DNA synthesis and how this function is coordinated with the activities of the other proteins of the replication complex . We find that short (single- and double-stranded) DNA molecules bind a single gp43 polymerase in a nonspecific (overlap) binding mode with moderate affinity (Kd approximately 150 nm) and a binding site size of approximately 10 nucleotides for single-stranded DNA and approximately 13 bp for double-stranded DNA . In contrast, gp43 binds in a site-specific (nonoverlap) mode and significantly more tightly (Kd approximately 5 nm) to DNA constructs carrying a primer-template junction, with the polymerase covering approximately 5 nucleotides downstream and approximately 6-7 bp upstream of the 3'-primer terminus . The rate of this specific binding interaction is close to diffusion-controlled . The affinity of gp43 for the primer-template junction is modulated specifically by dNTP substrates, with the next "correct" dNTP strengthening the interaction and an incorrect dNTP weakening the observed binding . These results are discussed in terms of the individual steps of the polymerase-catalyzed single nucleotide addition cycle and the replication complex assembly process . We suggest that changes in the kinetics and thermodynamics of these steps by auxiliary replication proteins constitute a basic mechanism for protein coupling within the replication complex. J Biol Chem, 2003 Jul 4, 278(27), 25247 - 55 Epub 2003 Apr 14. Properties of bacteriophage T4 proteins deficient in replication repair; Kadyrov FA et al.; An epistasis group of mutations engendering increased sensitivity to diverse DNA-damaging agents was described previously in bacteriophage T4 . These mutations are alleles of genes 32 and 41, which, respectively, encode a single-stranded DNA-binding protein (gp32) and the replicative DNA helicase (gp41) . The mechanism by which the lethality of DNA damage is mitigated is unknown but seems not to involve the direct reversal of damage, excision repair, conventional recombination repair, or translesion synthesis . Here we explore the hypothesis that the mechanism involves a switch in DNA primer extension from the cognate template to an alternative template, the just-synthesized daughter strand of the other parental strand . The activities of the mutant proteins are reduced about 2-fold (for gp32) or 4-fold (for gp41) in replication complexes catalyzing coordinated synthesis of leading and lagging strands, in binding single-stranded DNA, promoting DNA annealing, and promoting branch migration . In striking contrast, the mutant proteins are strongly impaired in promoting template switching, thus supporting the hypothesis of survival by template switching. Mutat Res, 2003 Apr 20, 536(1-2), 1 - 6 On the origin of unusual mutations recovered from the lacI transgenic assay; de Boer JG; Amongst approximately 25,000 mutants recovered from tissues of the lacI mouse and rat transgenic mutation assay, we identified seven mutants that carry changes that are unlike the majority of mutations that are normally recovered in these systems . The recovered mutants feature replacements and insertions of sequences that originate in the animal's genome, in the bacteriophage lambda construct that harbors the lacI gene, and in the genome of the E . coli plating host . These mutants demonstrate that mutations resulting from diverse mechanisms, in addition to the normal point mutations, can be recovered . In addition, the data indicate that such mutations may often not be of animal origin. J Biol Chem, 2003 Jun 27, 278(26), 23762 - 72 Epub 2003 Apr 11. A covalent linkage between the gene 5 DNA polymerase of bacteriophage T7 and Escherichia coli thioredoxin, the processivity factor: fate of thioredoxin during DNA synthesis; Johnson DE et al.; Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin . The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template . We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins . The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT) . However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA . gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity . However, gp5-S-S-trx has an identical processivity and rate of elongation when E . coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA . Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA template . However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA. Biochemistry, 2003 Apr 15, 42(14), 4253 - 64 DNA-stimulated assembly of oligomeric bacteriophage 434 repressor: evidence for cooperative binding by recruitment; Ciubotaru M et al.; Typical of many transcriptional regulatory proteins, the lambdoid bacteriophage repressors bind cooperatively to multiple sites on DNA . This cooperative binding is essential for establishment and maintenance of phage lysogeny . In the phage, two repressor homodimers, one bound at each of the adjacent operator sites, interact to form the tetramer that is necessary for the cooperative binding of the repressor . Bacteriophage 434 repressor does not form tetramers in the absence of DNA, and the mechanism by which the tetramer assembles on the two adjacent sites is unknown . Hence DNA binding may stimulate the repressor to form tetramers and formation of a repressor oligomer (> or = 3 monomers) on a single DNA sites may precede multisite binding . Consistent with these ideas, a complex containing three repressor molecules readily assembles on a single operator (O(R)1) site . Mutations that inhibit cooperative tetramer binding to the adjacent O(R)1 and O(R)2 sites also block formation of this complex . Together with other evidence, these findings show that the complex that forms on a single site assembles using the same interface as does the tetramer assembled on adjacent operator sites . Adding additional O(R)1 DNA dissociates the oligomeric repressor-DNA complexes into dimeric repressor-O(R)1 complexes . In contrast, adding O(R)2 to these complexes results in the formation of a repressor oligomer containing an O(R)2 and an O(R)1 site . The observation that a repressor oligomer bound to two O(R)1 sites is less stable than the one formed between repressor dimers bound to O(R)1 and O(R)2 implies that DNA allosterically influences the structure of the 434 repressor . Together these findings suggest that an O(R)1-bound repressor may cooperatively help repressor bind to O(R)2 by recruiting an additional repressor molecule from solution that subsequently occupies O(R)2. J Bacteriol, 2003 Apr, 185(8), 2653 - 66 Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination; Bankhead TM et al.; Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites . The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur . We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q) . We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions . Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination . The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways . The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways . We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway . Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR. Biophys J, 2003 Apr, 84(4), 2585 - 92 Penton release from P22 heat-expanded capsids suggests importance of stabilizing penton-hexon interactions during capsid maturation; Teschke CM et al.; Bacteriophage assembly frequently begins with the formation of a precursor capsid that serves as a DNA packaging machine . The DNA packaging is accompanied by a morphogenesis of the small round precursor capsid into a large polyhedral DNA-containing mature phage . In vitro, this transformation can be induced by heat or chemical treatment of P22 procapsids . In this work, we examine bacteriophage P22 morphogenesis by comparing three-dimensional structures of capsids expanded both in vitro by heat treatment and in vivo by DNA packaging . The heat-expanded capsid reveals a structure that is virtually the same as the in vivo expanded capsid except that the pentons, normally present at the icosahedral fivefold positions, have been released . The similarities of these two capsid structures suggest that the mechanism of heat expansion is similar to in vivo expansion . The loss of the pentons further suggests the necessity of specific penton-hexon interactions during expansion . We propose a model whereby the penton-hexon interactions are stabilized through interactions of DNA, coat protein, and other minor proteins . When considered in the context of other studies using chemical or heat treatment of capsids, our study indicates that penton release may be a common trend among double-stranded DNA containing viruses. J Virol Methods, 2003 Apr, 109(1), 99 - 101 Comparison of polyvinylidene fluoride and polyether sulfone membranes in filtering viral suspensions; Moce-Llivina L et al.; Low-protein-binding membranes with a pore size of 0.22 microm are used to filter aqueous solutions containing viruses . Virus adsorption to the membranes is avoided if they are made of polyvinylidene fluoride (PVDF) or if they are made of cellulose esters saturated with beef extract . Recently, a new kind of membrane filter made of polyether sulfone (PES) has become available commercially . The manufacturers claim that such membranes allow the filtration of greater volumes of sample than those made of PVDF . We compared the filtration rate and volume that could be filtered before clogging for these two membranes . The bacteriophage and enterovirus counts were then compared in sewage after filtration through the two membranes . There were no differences in virus recovery after filtration, but PES membranes allowed a higher filtration rate and clogged more slowly . The use of PES membranes is recommended. Biochemistry, 2003 Apr 8, 42(13), 3777 - 86 In vitro studies of transcript initiation by Escherichia coli RNA polymerase . 1 . RNA chain initiation, abortive initiation, and promoter escape at three bacteriophage promoters; Hsu LM et al.; RNA chain initiation and promoter escape is the latter stage of transcription initiation . This stage is characterized by several well-defined biochemical events: synthesis and release of short RNA products ranging 2 to 15 nucleotides in length, release of the sigma subunit from the enzyme-promoter complex, and initial translocation of the polymerase away from the promoter . In this paper, we report the use of a steady-state transcription assay with {gamma-(32)P}ATP labeling to subject the RNA chain initiation-promoter escape reaction to quantitative analysis . The specific parameters we follow to describe the chain initiation-promoter escape process include the abortive and productive rates, the abortive probability, the abortive:productive ratio, and the maximal size of the abortive product . In this study, we measure these parameters for three bacteriophage promoters transcribed by Escherichia coli RNA polymerase: T7 A1, T5 N25, and T5 N25(antiDSR) . Our studies show that all three promoters form substantial amounts of abortive products under all conditions we tested . However, each of the promoters shows distinct differences from the others when the various parameters are compared . At 100 microM NTP, in a 10 min reaction, the abortive and productive yields are 87 and 13%, respectively, for T7 A1; 97 and 3%, respectively, for T5 N25; and 99.4 and 0.6%, respectively, for T5 N25(antiDSR) . These values correspond to approximately 7, 32, and 165 abortive transcripts per productive transcript for the three promoters, respectively . The yield of most of the abortive products is not affected by the elevated concentration of the NTP substrate corresponding to the next template-specified nucleotide; hence, abortive products are not normally formed through a simple process of "kinetic competition" . Instead, formation of abortive products appears to be determined by intrinsic DNA signals embedded in the promoter recognition region and the initial transcribed sequence region of each promoter. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Sep, 16(3), 223 - 5 {Screening and application of human-derived HBsAg bacteriophage single chain antibody in clinical diagnosis}; Zhong Y et al.; OBJECTIVE: To identify human single chain Fv antibody (ScFv) against hepatitis B viral surface antigen . METHODS: The recombinant phages were panned by HBsAg which was coated in a microtiter plate, after five rounds of biopanning, 56 phage clones were identified specific to HBsAg . The specificity of ScFv was evaluated by ELISA and immunohistochemistry, respectively . RESULTS: The data of HB sAg-ScFv DNA digestion and DNA sequencing showed that the ScFv gene is composed of 750 bp . ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis B surface antigen has a specific combination character with hepatitis B surface antigen of different sources and paraffin-embedded patients tissue specimens, it did not react with normal liver tissue and HCV . CONCLUSIONS: The application of HBsAg specific ScFv in immunohistochemistry was successfully achieved. EMBO J, 2003 Apr 1, 22(7), 1461 - 6 The OmpL porin does not modulate redox potential in the periplasmic space of Escherichia coli; Sardesai AA et al.; The Escherichia coli DsbA protein is the major oxidative catalyst in the periplasm . Dartigalongue et al . (EMBO J., 19, 5980-5988, 2000) reported that null mutations in the ompL gene of E.coli fully suppress all phenotypes associated with dsbA mutants, i.e . sensitivity to the reducing agent dithiothreitol (DTT) and the antibiotic benzylpenicillin, lack of motility, reduced alkaline phosphatase activity and mucoidy . They showed that OmpL is a porin and hypothesized that ompL null mutations exert their suppressive effect by preventing efflux of a putative oxidizing-reducing compound into the medium . We have repeated these experiments using two different ompL null alleles in at least three different E.coli K-12 genetic backgrounds and have failed to reproduce any of the ompL suppressive effects noted above . Also, we show that, contrary to earlier results, ompL null mutations alone do not result in partial DTT sensitivity or partial motility, nor do they appreciably affect bacterial growth rates or block propagation of the male-specific bacteriophage M13 . Thus, our findings clearly demonstrate that ompL plays no perceptible role in modulating redox potential in the periplasm of E.coli. Biochim Biophys Acta, 2003 Apr 1, 1611(1-2), 5 - 15 Protein-lipid interactions of bacteriophage M13 major coat protein; Stopar D et al.; During the past years, remarkable progress has been made in our understanding of the replication cycle of bacteriophage M13 and the molecular details that enable phage proteins to navigate in the complex environment of the host cell . With new developments in molecular membrane biology in combination with spectroscopic techniques, we are now in a position to ask how phages carry out this delicate process on a molecular level, and what sort of protein-lipid and protein-protein interactions are involved . In this review we will focus on the molecular details of the protein-protein and protein-lipid interactions of the major coat protein (gp8) that may play a role during the infection of Escherichia coli by bacteriophage M13. Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3826 - 31 Epub 2003 Mar 24. A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase; Logan DT et al.; A Zn(Cys)(4) center has been found in the C-terminal region of the crystal structure of the anaerobic class III ribonucleotide reductase (RNR) from bacteriophage T4 . The metal center is structurally related to the zinc ribbon motif and to rubredoxin and rubrerythrin . Mutant enzymes of the homologous RNR from Escherichia coli, in which the coordinating cysteines, conserved in almost all known class III RNR sequences, have been mutated into alanines, are shown to be inactive as the result of their inability to generate the catalytically essential glycyl radical . The possible roles of the metal center are discussed in relationship to the currently proposed reaction mechanism for generation of the glycyl radical in class III RNRs. Nucleic Acids Res, 2003 Apr 1, 31(7), 1984 - 94 3'-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-{4-oxo-4-(3-pyridyl)butyl}guanine; Park S et al.; Tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a chemical carcinogen thought to be involved in the initiation of lung cancer in smokers . NNK is metabolically activated to methylating and pyridyloxobutylating species that form promutagenic adducts with DNA nucleobases, e.g . O(6)-{4-oxo-4-(3-pyridyl)butyl}guanine (O(6)-POB-dG) . O(6)-POB-dG is a strongly mispairing DNA lesion capable of inducing both G-->A and G-->T base changes, suggesting its importance in NNK mutagenesis and carcinogenesis . Our earlier investigations have identified the ability of O(6)-POB-dG to hinder DNA digestion by snake venom phosphodiesterase (SVPDE), a 3'-exonuclease commonly used for DNA ladder sequencing and as a model enzyme to test nuclease sensitivity of anti-sense oligonucleotide drugs . We now extend our investigation to three other enzymes possessing 3'-exonuclease activity: bacteriophage T4 DNA polymerase, Escherichia coli DNA polymerase I, and E.coli exonuclease III . Our results indicate that, unlike SVPDE, 3'-exonuclease activities of these three enzymes are not blocked by O(6)-POB-dG lesion . Conformational analysis and molecular dynamics simulations of DNA containing O(6)-POB-dG suggest that the observed resistance of the O(6)-POB-dG lesion to SVPDE-catalyzed hydrolysis may result from the structural changes in the DNA strand induced by the O(6)-POB group, including C3'-endo sugar puckering and the loss of stacking interaction between the pyridyloxobutylated guanine and its flanking bases . In contrast, O(6)-methylguanine lesion used as a control does not induce similar structural changes in DNA and does not prevent its digestion by SVPDE. Eur J Biochem, 2003 Apr, 270(7), 1458 - 65 Preventing nondesired RNA-primed RNA extension catalyzed by T7 RNA polymerase; Nacheva GA et al.; The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated . These templates consisted of 17 nucleotide-long sequences under the control of the minimal bacteriophage T7 promoter and represented all possible combinations of nucleotides at positions +8, +10 and +11 . Two clearly distinct types of template were identified, which produced the range of transcription patterns observed: (a) those that yielded 17-nucleotide-long RNA as the only detectable run-off product (only 15% of the total), and (b) templates that in addition to the expected full-length RNA, produced other products longer than 17 nucleotides . Self-complementarity analysis of the expected run-off transcripts showed that those obtained from the first type of template were able to form stable intermolecular duplexes with non-base-paired 3'-ends . However, the second type of template yielded RNAs able to generate energetically favorable intermolecular duplexes with 3'-end complementarity, therefore yielding an RNA-primed RNA-template . The gel-purified 17-nucleotide-long RNAs transcribed from the latter yielded longer products when incubated under in vitro transcription conditions in the absence of a DNA template . No extension was observed when assaying the 17-nucleotide RNA products resulting from the first type of template . We observed that just a single nucleotide change within the DNA template could convert the RNA product from an RNA-primed template into a nonextendible dimer thus leading to a drastic switch of the 17-nucleotide product yield from less than 10% to 100% . Further, two type B DNA templates were extended by two nucleotides at the 3'-end, to produce RNA transcripts theoretically unable to form 3'-end base-paired duplexes . The full-length products of these modified DNA templates were found to be nonextendible by T7 RNA polymerase under the standard in vitro transcription conditions. Biochemistry, 2003 Apr 1, 42(12), 3437 - 45 Structural roles of subunit cysteines in the folding and assembly of the DNA packaging machine (portal) of bacteriophage P22; Rodriguez-Casado A et al.; The DNA packaging machine (portal assembly) of bacteriophage P22 is constructed from 12 copies of a multidomain 725-residue subunit comprising a complex alpha/beta fold . The portal subunit contains four cysteines (Cys 153, Cys 173, Cys 283, and Cys 516), which produce distinctive Raman markers in the spectral interval 2500-2600 cm(-1) originating from S-H bond-stretching vibrations diagnostic of S-H...X hydrogen-bonding interactions . The Raman spectrum is unique in the capability to characterize cysteine sulfhydryl interactions in proteins and shows that portal cysteine environments are significantly altered by assembly (Rodriguez-Casado et al . (2001) Biochemistry 40, 13583-13591) . We have employed site-directed mutagenesis, size-exclusion chromatography, and Raman difference spectroscopy to characterize the roles of portal cysteines in subunit folding and dodecamer assembly . The stability of the portal monomer is severely reduced by a Cys --> Ser point mutation introduced at either residue 173 or 516 . In the case of C516S, the destabilized monomer still forms portal rings, as visualized by negative-stain electron microscopy, whereas portal ring formation cannot be detected for C173S, which forms aberrant aggregates . The C283S mutant is a hyperstable monomer that is defective in portal ring formation . Interestingly, Cys 283 is suggested by secondary structure homology with the phi29 portal to be within a domain involved in DNA translocation . Conversely, the phenotype of the C153S mutant is close to that of the wild-type protein, implying that the sulfhydryl moiety of Cys 153 is not essential to formation of the native subunit fold and productive assembly dynamics . The present results demonstrate that cysteines of the P22 portal protein span a wide range of sulfhydryl hydrogen-bonding strengths in the wild-type assembly, that three of the four sulfhydryls play key roles in portal protein stability and assembly kinetics, and that substitution of a mutant seryl interaction (O-H...X) for a wild-type cysteinyl interaction (S-H...X) can either stabilize or destabilize the native fold depending upon sequence context. DNA Seq, 2002 Dec, 13(6), 321 - 31 Cloning and characterization of a Helicobacter bizzozeronii urease gene cluster; Zhu J et al.; The urease gene cluster from Helicobacter bizzozeronii was cloned and sequenced . A genomic library was constructed in a lambda-ZAPII vector using TSP5091-digested H . bizzozeronii chromosomal DNA . Four overlapping recombinant bacteriophages carrying the H . bizzozeronii urease genes were identified by using a fragment of H . bizzozeronii ureB as a probe . Sequence analysis of two clones (pHB1 and pHB3) revealed seven open reading frames encoding proteins with predicted masses of 26.5, 60.3, 21.7, 19.5, 28.6, 21.7 and 29.6 kDa representing the structural genes, Urease A and B and its accessory genes, urease I, E, F, G and H, respectively . In addition, three open reading frames upstream of the ureA gene encoding a putative tRNA transferase, a putative Glucose inhibited division protein B (GidB) and a protein with unknown function were also identified . A clone (pHB5) containing a complete urease gene cluster was constructed . The homologue analysis revealed that UreA polypeptide exhibited 64-90% identity to that of Helicobacter heilmanii, Helicobacter felis, Helicobacter pylori, Helicobacter mustelae and Helicobacter hepaticus . UreB polypeptides exhibited 76.8-96% identity to that of H . heilmanii, H . felis, H . pylori, H . mustelae and H . hepaticus . The UreI, E, F, G and H also showed 44-86% identity to that of H . pylori . Among these accessory genes, UreE had a lowest percentage identity to that of H . pylori. Protein Expr Purif, 2003 Mar, 28(1), 86 - 92 High-level synthesis of Johnson grass mosaic virus coat protein in Escherichia coli and its auto-assembly to form virus-like particles; Saini M et al.; The coat protein (CP) of Johnson grass mosaic virus (JGMV) auto-assembles to form virus-like particles (VLPs) and hence could be useful for presenting small peptides to the immune system . We are therefore attempting to synthesize JGMV CP in large amounts in Escherichia coli . The JGMV CP-encoding DNA, cloned under the bacteriophage T7 promoter, showed only low levels of CP synthesis in E . coli . The predicted secondary structure of the CP mRNA showed that its translational initiation codon was part of a stable hairpin-loop structure . The initiation codon could be relieved of the hairpin-loop structure by substitution of three neighboring nucleotides . This resulted in a single amino acid change at the N-terminus of the protein . The modified RNA translated very efficiently, resulting in at least 16-fold higher CP accumulation in E . coli . The N-terminal amino acid substitution did not affect CP folding, as it auto-assembled in E . coli to form VLPs. J Biol Chem, 2003 Jun 6, 278(23), 21276 - 85 Epub 2003 Mar 20. Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins; Chastain PD 2nd et al.; Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins . Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections . In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy . Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed . The tails are fully duplex except for one to three single-stranded DNA segments close to the fork . This pattern reflects Okazaki fragments stopped at different stages in their synthesis . Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail . In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome . The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins") . These bobbins constitute a major portion of the mass of the full replication complex. J Struct Biol, 2003 Mar, 141(3), 179 - 88 Models of bacteriophage DNA packaging motors; Serwer P; An ATP-dependent motor drives a DNA genome into a bacteriophage capsid during morphogenesis of double-stranded DNA bacteriophages both in vivo and in vitro . The DNA molecule enters the capsid through a channel in the center of a symmetric protein ring called a connector . Mechanisms in two classes have been proposed for this motor: (1) An ATP-driven rotating connector pulls a DNA molecule via serial power strokes . (2) The connector rectifies DNA motion that is either thermal, biased thermal, or oscillating electrical field-induced (motor-ratchet hypothesis) . Mechanisms in the first class have previously been proposed to explain the detailed structure of DNA packaging motors . The present study demonstrates that the motor-ratchet hypothesis also explains the current data, including data in the following categories: biochemical genetics, energetics, structure, and packaging dynamics. Clin Pharmacokinet, 2003, 42(4), 315 - 25 Pharmacokinetic principles of bacteriophage therapy; Payne RJ et al.; Use of bacteriophage to control bacterial infections, including antibiotic-resistant infections, shows increasing therapeutic promise . Effective bacteriophage therapy requires awareness of various novel kinetic phenomena not known in conventional drug treatments . Kinetic theory predicts that timing of treatment could be critical, with the strange possibility that inoculations given too early could be less effective or fail completely . Another paradoxical result is that adjuvant use of an antibiotic can sometimes diminish the efficacy of phage therapy . For a simple kinetic model, mathematical formulae predict the values of critical density thresholds and critical time points, given as functions of independently measurable biological parameters . Understanding such formulae is important for interpreting data and guiding experimental design . Tailoring pharmacokinetic models for specific systems needs to become standard practice in future studies. Otolaryngol Head Neck Surg, 2003 Mar, 128(3), 364 - 71 Gene discovery using a human vestibular schwannoma cDNA library constructed from a patient with neurofibromatosis type 2 (NF2); Halum SL et al.; BACKGROUND: Despite a strong association of schwannomin/merlin gene mutations with vestibular schwannoma formation, the regulatory mechanisms and biologic pathways involved are still largely unknown . The hypothesis of this study is that the genesis and growth characteristics of neurofibromatosis type 2 (NF2)-associated vestibular schwannomas are determined by genetic alterations that vary in gene transcript expression; this transcript expression includes oncogenic gene products that may be identified by construction and sequencing of a cDNA library from NF2-associated vestibular schwannoma . METHODS: Approximately 3 mL of fresh tumor was obtained during resection of a 4-cm vestibular schwannoma from a patient with NF2 . Poly(A)(+) mRNA was isolated, synthesized into double-stranded cDNA, and unidirectionally inserted into Uni-Zap XR (Stratagene, La Jolla, CA) bacteriophage vectors . Bacteriophage vectors containing cDNA inserts were processed into phagemids according to Uni-Zap XR protocol, and inserted vectors were sequenced and analyzed using BLAST software (National Institutes of Health, Bethesda, MD) with GenBank, EMBL, DDBJ, and PBD databases . RESULTS: The cDNA library contained 2.4 million primary plaques . Inserts averaged 1.8 kilobases (kb) in length, with a range of 0.8 to 3.0 kb . BLAST multidatabase comparison of the sequence data obtained from 50 randomly selected clones yielded identification of 13 sequences representing known human genes and 17 sequences representing cloned sequences with unknown function . Three clones represented sequences not previously described in vestibular schwannomas but strongly implicated in oncogenesis within other tissues . CONCLUSIONS: These data have implications for understanding the molecular mechanisms of vestibular schwannoma tumor biology . Identified genes may provide future diagnostic/prognostic markers and therapeutic targets. Biophys Chem, 2003, 100(1-3), 341 - 50 Role of an N(cap) residue in determining the stability and operator-binding affinity of Arc repressor; Anderson TA et al.; The Arc repressor of bacteriophage P22 is a member of the ribbon-helix-helix family of transcription factors . Ser32 is a solvent-exposed position that serves a structural role as the N(cap) residue of alpha-helix B of Arc, but also serves a functional role because its side chain is packed close to the sugar-phosphate DNA backbone in the repressor-operator complex . The tolerance of this N(cap) position to amino-acid substitutions was probed by determining the repressor activity in vivo, the thermal stability and the operator-binding activity in vitro of a set of 13 mutant proteins . The stability of position-32 Arc variants, except for Cys32, correlated well with the frequencies observed for the corresponding residues at N(cap) positions in alpha-helices of other proteins . Cysteine was quite stabilizing at the helix-B N(cap) position in Arc, but surprisingly was the least frequent N(cap) residue in the protein database . This latter finding may reflect a hyper-reactivity of N(cap) cysteines, which makes them prone to chemical modification . In general, only Arc variants with small, uncharged residues at position 32 were active in vivo or showed strong operator binding in vitro . Based upon the results presented here, revised sequence alignments of the MetJ and NikR subfamilies with Arc and other ribbon-helix-helix proteins are proposed. J Bacteriol, 2003 Apr, 185(7), 2296 - 305 Evidence for horizontal transfer of the EcoT38I restriction-modification gene to chromosomal DNA by the P2 phage and diversity of defective P2 prophages in Escherichia coli TH38 strains; Kita K et al.; A DNA fragment carrying the genes coding for a novel EcoT38I restriction endonuclease (R.EcoT38I) and EcoT38I methyltransferase (M.EcoT38I), which recognize G(A/G)GC(C/T)C, was cloned from the chromosomal DNA of Escherichia coli TH38 . The endonuclease and methyltransferase genes were in a head-to-head orientation and were separated by a 330-nucleotide intergenic region . A third gene, the C.EcoT38I gene, was found in the intergenic region, partially overlapping the R.EcoT38I gene . The gene product, C.EcoT38I, acted as both a positive regulator of R.EcoT38I gene expression and a negative regulator of M.EcoT38I gene expression . M.EcoT38I purified from recombinant E . coli cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence . R.EcoT38I was purified from E . coli HB101 expressing M.EcoT38I and formed a homodimer . The EcoT38I restriction (R)-modification (M) system (R-M system) was found to be inserted between the A and Q genes of defective bacteriophage P2, which was lysogenized in the chromosome at locI, one of the P2 phage attachment sites observed in both E . coli K-12 MG1655 and TH38 chromosomal DNAs . Ten strains of E . coli TH38 were examined for the presence of the EcoT38I R-M gene on the P2 prophage . Conventional PCR analysis and assaying of R activity demonstrated that all strains carried a single copy of the EcoT38I R-M gene and expressed R activity but that diversity of excision in the ogr, D, H, I, and J genes in the defective P2 prophage had arisen. J Bacteriol, 2003 Apr, 185(7), 2187 - 93 The IntP C-terminal segment is not required for excision of bacteriophage Mx8 from the Myxococcus xanthus chromosome; Tojo N et al.; During lysogenization of myxophage Mx8, phage DNA can be integrated into the attB site of the Myxococcus xanthus chromosome through site-specific recombination . We previously demonstrated that the Mx8 attP site is located within the coding sequence of the Mx8 intP gene . Hence, the integration of Mx8 into the M . xanthus chromosome results in the conversion of the 112-amino-acid C-terminal segment of the IntP protein into a 13-amino-acid C-terminal segment of a new protein, IntR . To examine whether IntR is active for Mx8 excision, we have constructed a series of plasmids carrying various lengths of the intP-attP or intR-attR regions as well as the lacZ gene . The integrated Mx8 was excised at a high frequency, indicating that IntR is active for the excision . For Mx8 excision, a gene designated xis was shown to be required in addition to intR. Water Sci Technol, 2003, 47(3), 179 - 84 Assessing UV reactor performance for treatment of finished water; Bukhari Z et al.; Recently, use of low levels of medium- and low-pressure ultraviolet light for successful inactivation of Cryptosporidium parvum oocysts has generated tremendous excitement in the water industry . Accurate delivery of the target dose, lamp performance, sensor stability and impact of water characteristics are some factors that could impact disinfection efficacy, in turn influencing decisions on application of this technology . To this end, American Water Systems, the largest investor owned water utility in the US, has responded to some of these challenges by ascertaining the long-term feasibility of applying UV for treatment of finished water . A 4 x 1 UV reactor with a 12 inch (0.3 m) diameter was installed after granular activated carbon filtration and was operated with a finished water flow rate of 600 gpm (2,700 L/min) . Over a 12-month period, various chemical (THM, HAA, UV254, DOC, TOC, metals, nitrate, nitrites) and physical measurements (lamp voltage, current, sensor measurements) were monitored to define their impact (if any) on the operation of the reactor . MS2 bacteriophage challenge studies were conducted with various lamp configurations and lamp age . These inactivation data demonstrated high levels of correlation with controlled bench scale inactivation data . For C . parvum oocysts, bench scale studies were performed with a modified in vitro infectivity assay using HCT-8 cells, an enhanced infectivity protocol and with either immunofluorescence or quantitative PCR based detection . While both assays indicated increasing infections levels of HCT-8 cells with increasing oocyst inocula, UV treatment of oocysts produced markedly different infectivity responses . Based on the data generated in this study, one in vitro infectivity assay was selected to demonstrate > 3 logs inactivation with low UV doses (5 mJ/cm(20-10 mJ/cm2). EMBO Rep, 2003 Mar, 4(3), 284 - 9 RNA chaperone activity of the Sm-like Hfq protein; Moll I et al.; The Escherichia coli Sm-like host factor I (Hfq) protein is thought to function in post-transcriptional regulation by modulating the function of small regulatory RNAs . Hfq also interferes with ribosome binding on E . coli ompA messenger RNA, indicating that Hfq also interacts with mRNAs . In this study, we have used stimulation of group I intron splicing in vivo and a modified in vitro toeprinting assay to determine whether Hfq acts as an RNA chaperone . Hfq was able to rescue an RNA 'folding trap' in a splicing defective T4 bacteriophage td gene in vivo . Enzymatic analysis showed that Hfq affects the accessibility of the ompA start codon, as well as other bases within the ribosome-binding site, explaining its negative effect on ribosome binding . We also show that the Hfq-induced structural changes in ompA mRNA are maintained after proteolytic digestion of the protein, which classifies Hfq as an RNA chaperone. J Mol Biol, 2003 Mar 28, 327(3), 571 - 8 Kinetic regulation of single DNA molecule denaturation by T4 gene 32 protein structural domains; Pant K et al.; Bacteriophage T4 gene 32 protein (gp32) specifically binds single-stranded DNA, a property essential for its role in DNA replication, recombination, and repair . Although on a thermodynamic basis, single-stranded DNA binding proteins should lower the thermal melting temperature of double-stranded DNA (dsDNA), gp32 does not . Using single molecule force spectroscopy, we show for the first time that gp32 is capable of slowly destabilizing natural dsDNA . Direct measurements of single DNA molecule denaturation and renaturation kinetics in the presence of gp32 and its proteolytic fragments reveal three types of kinetic behavior, attributable to specific protein structural domains, which regulate gp32's helix-destabilizing capabilities . Whereas the full-length protein exhibits very slow denaturation kinetics, a truncate lacking the acidic C-domain exhibits much faster kinetics . This may reflect a steric blockage of the DNA binding site and/or a conformational change associated with this domain . Additional removal of the N-domain, which is needed for binding cooperativity, further increases the DNA denaturation rate, suggesting that both of these domains are critical to the regulation of gp32's helix-destabilization capabilities . This regulation is potentially biologically significant because uncontrolled helix-destabilization would be lethal to the cell . We also obtain equilibrium measurements of the helix-coil transition free energy in the presence of these proteins for the first time. EMBO J, 2003 Mar 17, 22(6), 1255 - 62 Structure of a viral DNA gatekeeper at 10 A resolution by cryo-electron microscopy; Orlova EV et al.; In tailed bacteriophages and herpes viruses, the viral DNA is packaged through the portal protein channel . Channel closure is essential to prevent DNA release after packaging . Here we present the connector structure from bacteriophage SPP1 using cryo-electron microscopy and single particle analysis . The multiprotein complex comprises the portal protein gp6 and the head completion proteins gp15 and gp16 . Although we show that gp6 in the connector has a fold similar to that of the isolated portal protein, we observe conformational changes in the region of gp6 exposed to the DNA-packaging ATPase and to gp15 . This reorganization does not cause closure of the channel . The connector channel traverses the full height of gp6 and gp15, but it is closed by gp16 at the bottom of the complex . Gp16 acts as a valve whose closure prevents DNA leakage, while its opening is required for DNA release upon interaction of the virus with its host. J Mol Biol, 2003 Mar 21, 327(2), 413 - 29 Mechanism of inhibition of site-specific recombination by the Holliday junction-trapping peptide WKHYNY: insights into phage lambda integrase-mediated strand exchange; Cassell GD et al.; Holliday junctions are central intermediates in site-specific recombination reactions mediated by tyrosine recombinases . Because these intermediates are extremely transient, only artificially assembled Holliday junctions have been available for study . We have recently identified hexapeptides that cause the accumulation of natural Holliday junctions of bacteriophage lambda Integrase (Int)-mediated reactions . We now show that one of these peptides acts after the first DNA cleavage event to stabilize protein-bound junctions and to prevent their resolution . The peptide acts before the step affected by site affinity (saf) mutations in the core region, in agreement with a model that the peptide stabilizes the products of strand exchange (i.e . Holliday junctions) while saf mutations reduce ligation of exchanged strands.Strand exchange events leading to Holliday junctions in phage lambda integration and excision are asymmetric, presumably because interactions between Int and some of its core-binding sites determine the order of strand cleavage . We have compared the structure of Holliday junctions in one unidirectional and in two bidirectional Int-mediated pathways and show that the strand cleavage steps are much more symmetric in the bidirectional pathways . Thus Int-DNA interactions which determine the order of top and bottom strand cleavage and exchange are unique in each recombination pathway. J Mol Biol, 2003 Mar 21, 327(2), 335 - 46 Twelve new MotA-dependent middle promoters of bacteriophage T4: consensus sequence revised; Truncaite L et al.; Bacteriophage T4 middle-mode transcription requires Escherichia coli RNA polymerase, phage-encoded transcriptional activator MotA and co-activator AsiA that form a complex at a middle promoter DNA . T4 middle promoters have been defined by a consensus sequence deduced from the list of 14 middle promoters identified in earlier studies . To date, 33 middle promoters have been mapped on the T4 genome . Of these, 12 contain differences even at the highly conserved positions of the consensus sequence . In the T4 prereplicative gene cluster between genes e and rpbA, we have identified 12 new middle promoters, most of which contain differences from the consensus sequence deduced previously . Analysis of base conservation in the different sequence positions of new middle promoters, as well as those identified previously, revealed some new features of middle T4 promoters . We propose to define these promoters by a MotA box (a/t)(a/t)(a/t)TGCTTtA centred at the position -30, the sequence TAtaAT centred at -10 relative to the transcriptional start site, and the spacer region of 12(+/-1) base-pairs between them. Med Immunol . 2003 Feb 14;2(1):2. New insights into the possible role of bacteriophages in host defense and disease; Gorski A et al.; BACKGROUND: While the ability of bacteriophages to kill bacteria is well known and has been used in some centers to combat antibiotics - resistant infections, our knowledge about phage interactions with mammalian cells is very limited and phages have been believed to have no intrinsic tropism for those cells . PRESENTATION OF THE HYPOTHESIS: At least some phages (e.g., T4 coliphage) express Lys-Arg-Gly (KGD) sequence which binds beta3 integrins (primarily alphaIIbbeta3) . Therefore, phages could bind beta3+ cells (platelets, monocytes, some lymphocytes and some neoplastic cells) and downregulate activities of those cells by inhibiting integrin functions . TESTING THE HYPOTHESIS: Binding of KGD+ phages to beta3 integrin+ cells may be detected using standard techniques involving phage - mediated bacterial lysis and plaque formation . Furthermore, the binding may be visualized by electron microscopy and fluorescence using labelled phages . Binding specificity can be confirmed with the aid of specific blocking peptides and monoclonal antibodies . In vivo effects of phage - cell interactions may be assessed by examining the possible biological effects of beta3 blockade (e.g., anti-metastatic activity) . IMPLICATION OF THE HYPOTHESIS: If, indeed, phages can modify functions of beta3+ cells (platelets, monocytes, lymphocytes, cancer cells) they could be important biological response modifiers regulating migration and activities of those cells . Such novel understanding of their role could open novel perspectives in their potential use in treatment of cardiovascular and autoimmune disease, graft rejection and cancer. Structure (Camb), 2003 Mar, 11(3), 339 - 46 Collagen stabilization at atomic level: crystal structure of designed (GlyProPro)10foldon; Stetefeld J et al.; In a designed fusion protein the trimeric domain foldon from bacteriophage T4 fibritin was connected to the C terminus of the collagen model peptide (GlyProPro)(10) by a short Gly-Ser linker to facilitate formation of the three-stranded collagen triple helix . Crystal structure analysis at 2.6 A resolution revealed conformational changes within the interface of both domains compared with the structure of the isolated molecules . A striking feature is an angle of 62.5 degrees between the symmetry axis of the foldon trimer and the axis of the triple helix . The melting temperature of (GlyProPro)(10) in the designed fusion protein (GlyProPro)(10)foldon is higher than that of isolated (GlyProPro)(10,) which suggests an entropic stabilization compensating for the destabilization at the interface. Structure (Camb), 2003 Mar, 11(3), 309 - 22 The receptor binding protein P2 of PRD1, a virus targeting antibiotic-resistant bacteria, has a novel fold suggesting multiple functions; Xu L et al.; Bacteriophage PRD1 is unusual, with an internal lipid membrane, but has striking resemblances to adenovirus that include receptor binding spikes . The PRD1 vertex complex contains P2, a 590 residue monomer that binds to receptors on antibiotic-resistant strains of E . coli and so is the functional counterpart to adenovirus fiber . P2 structures from two crystal forms, at 2.2 and 2.4 A resolution, reveal an elongated club-shaped molecule with a novel beta propeller "head" showing pseudo-6-fold symmetry . An extended loop with another novel fold forms a long "tail" containing a protruding proline-rich "fin." The head and fin structures are well suited to recognition and attachment, and the tail is likely to trigger the processes of vertex disassembly, membrane tube formation, and subsequent DNA injection. Mol Microbiol, 2003 Mar, 47(6), 1669 - 79 SeqA-mediated stimulation of a promoter activity by facilitating functions of a transcription activator; Slominska M et al.; It was demonstrated recently that the SeqA protein, a main negative regulator of Escherichia coli chromosome replication initiation, is also a specific transcription factor . SeqA specifically activates the bacteriophage lambda pR promoter while revealing no significant effect on the activity of another lambda promoter, pL . Here, we demonstrate that lysogenization by bacteriophage lambda is impaired in E . coli seqA mutants . Genetic analysis demonstrated that CII-mediated activation of the phage pI and paQ promoters, which are required for efficient lysogenization, is less efficient in the absence of seqA function . This was confirmed in in vitro transcription assays . Interestingly, SeqA stimulated CII-dependent transcription from pI and paQ when it was added to the reaction mixture before CII, although having little effect if added after a preincubation of CII with the DNA template . This SeqA-mediated stimulation was absolutely dependent on DNA methylation, as no effects of this protein were observed when using unmethylated DNA templates . Also, no effects of SeqA on transcription from pI and paQ were observed in the absence of CII . Binding of SeqA to templates containing the tested promoters occurs at GATC sequences located downstream of promoters, as revealed by electron microscopic studies . In contrast to pI and paQ, the activity of the third CII-dependent promoter, pE, devoid of neighbouring downstream GATC sequences, was not affected by SeqA both in vivo and in vitro . We conclude that SeqA stimulates transcription from pI and paQ promoters in co-operation with CII by facilitating functions of this transcription activator, most probably by allowing more efficient binding of CII to the promoter region. Biosci Biotechnol Biochem, 2003 Jan, 67(1), 198 - 202 Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages; Iida Y et al.; Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages . Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes . This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes. J Magn Reson, 2003 Feb, 160(2), 85 - 90 Dynamic nuclear polarization at 9T using a novel 250GHz gyrotron microwave source; Bajaj VS et al.; In this communication, we report enhancements of nuclear spin polarization by dynamic nuclear polarization (DNP) in static and spinning solids at a magnetic field strength of 9T (250 GHz for g=2 electrons, 380 MHz for 1H) . In these experiments, 1H enhancements of up to 170+/-50 have been observed in 1-13C-glycine dispersed in a 60:40 glycerol/water matrix at temperatures of 20K; in addition, we have observed significant enhancements in 15N spectra of unoriented pf1-bacteriophage . Finally, enhancements of approximately 17 have been obtained in two-dimensional 13C-13C chemical shift correlation spectra of the amino acid U-13C, 15N-proline during magic angle spinning (MAS), demonstrating the stability of the DNP experiment for sustained acquisition and for quantitative experiments incorporating dipolar recoupling . In all cases, we have exploited the thermal mixing DNP mechanism with the nitroxide radical 4-amino-TEMPO as the paramagnetic dopant . These are the highest frequency DNP experiments performed to date and indicate that significant signal enhancements can be realized using the thermal mixing mechanism even at elevated magnetic fields . In large measure, this is due to the high microwave power output of the 250 GHz gyrotron oscillator used in these experiments. J Mol Biol, 2003 Mar 14, 327(1), 1 - 6 A second symmetry mismatch at the portal vertex of bacteriophage T7: 8-fold symmetry in the procapsid core; Cerritelli ME et al.; Like other bacteriophages, T7 has a singular vertex that is the site of a symmetry mismatch involving the portal/connector protein, a 12-fold ring at the vertex site which is also a 5-fold axis for the icosahedral capsid . In the mature virion, a 6-fold-symmetric tail extends outwards from the connector . T7 also has a cylindrical "core" that assembles on the inner surface of the connector during procapsid formation, is retained in the mature virion, and is required for infectivity . We have investigated the core structure by cryo-electron microscopy and image analysis of procapsids and find that it observes 8-fold symmetry . Stoichiometry data indicate that its major constituent is an octamer of gp15. J Biol Chem, 2003 May 16, 278(20), 17601 - 8 Epub 2003 Feb 27. Structure-function analysis of T4 RNA ligase 2; Yin S et al.; Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a polynucleotide ligase family that includes the trypanosome RNA-editing ligases and putative RNA ligases encoded by eukaryotic viruses and archaea . Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining . We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of ligase adenylylation and phosphodiester bond formation . The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates . Our mutagenesis results underscore a shared active site architecture in Rnl2-like ligases, DNA ligases, and mRNA capping enzymes . They also highlight two essential signature residues, Glu(34) and Asn(40), that flank the active site lysine nucleophile (Lys(35)) and are unique to the Rnl2-like ligase family. Bioinformatics, 2003 Mar 1, 19(4), 483 - 9 Estimating the diversity of peptide populations from limited sequence data; Makowski L et al.; MOTIVATION: Combinatorial libraries of peptides such as those displayed on the surface of a bacteriophage particle have become widely used tools for characterizing protein-protein and protein-small molecule interactions . The quality of a library frequently depends on its completeness, or diversity-the proportion of possible sequences actually present in the library . The diversity of these libraries is frequently quoted on the basis of phage titers that provide little information about their completeness . RESULTS: Here, an analytical expression for diversity is introduced and a method for estimating the diversity of a peptide library from the sequences of a limited number of the members of the library is demonstrated . The diversities of a number of computationally constructed and actual peptide libraries are estimated using this method. Mol Biotechnol, 2003 Jan, 23(1), 73 - 81 The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection; Babon JJ et al.; Mutation and polymorphism detection is of increasing importance in the field of molecular genetics . This is reflected by the plethora of chemical, enzymatic, and physically based methods of mutation detection . The ideal method would detect mutations in large fragments of DNA and position them to single base-pair (bp) accuracy . Few methods are able to quickly screen kilobase lengths of DNA and position the mutation at the same time . The Enzyme Mismatch Cleavage (EMC) method of mutation detection is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp . This method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and, more recently, a second bacteriophage resolvase, T7 endonuclease I . The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and mutant DNA . Digestion fragments indicate the presence, and the position, of any mutations . The method is robust and reliable and much faster and cheaper than sequencing . These attributes have resulted in its increasing use in the field of mutation detection. Biophys J, 2003 Mar, 84(3), 1969 - 76 Orientation and interactions of an essential tryptophan (Trp-38) in the capsid subunit of Pf3 filamentous virus; Tsuboi M et al.; The filamentous bacteriophage Pf3 consists of a covalently closed DNA single strand of 5833 nucleotides sheathed by approximately 2500 copies of a 44-residue capsid subunit . The capsid subunit contains a single tryptophan residue (Trp-38), which is located within the basic C-terminal sequence (-RWIKAQFF) and is essential for virion assembly in vivo . Polarized Raman microspectroscopy has been employed to determine the orientation of the Trp-38 side chain in the native virus structure . The polarized Raman measurements show that the plane of the indolyl ring is tilted by 17 degrees from the virion axis and that the indolyl pseudo-twofold axis is inclined at 46 degrees to the virion axis . Using the presently determined orientation of the indolyl ring and side-chain torsion angles, chi(1) (N-C(alpha)-C(beta)-C(gamma)) and chi(2,1) (C(alpha)-C(beta)-C(gamma)-C(delta1)), we propose a detailed molecular model for the local structure of Trp-38 in the Pf3 virion . The present Pf3 model is consistent with previously reported Raman, ultraviolet-resonance Raman and fluorescence results suggesting an unusual environment for Trp-38 in the virion assembly, probably involving an intrasubunit cation-pi interaction between the guanidinium moiety of Arg-37 and the indolyl moiety of Trp-38 . Such a C-terminal Trp-38/Arg-37 interaction may be important for the stabilization of a subunit conformation that is required for binding to the single-stranded DNA genome during virion assembly. Biophys J, 2003 Mar, 84(3), 1616 - 27 Forces and pressures in DNA packaging and release from viral capsids; Tzlil S et al.; In a previous communication (Kindt et al., 2001) we reported preliminary results of Brownian dynamics simulation and analytical theory which address the packaging and ejection forces involving DNA in bacteriophage capsids . In the present work we provide a systematic formulation of the underlying theory, featuring the energetic and structural aspects of the strongly confined DNA . The free energy of the DNA chain is expressed as a sum of contributions from its encapsidated and released portions, each expressed as a sum of bending and interstrand energies but subjected to different boundary conditions . The equilibrium structure and energy of the capsid-confined and free chain portions are determined, for each ejected length, by variational minimization of the free energy with respect to their shape profiles and interaxial spacings . Numerical results are derived for a model system mimicking the lambda-phage . We find that the fully encapsidated genome is highly compressed and strongly bent, forming a spool-like condensate, storing enormous elastic energy . The elastic stress is rapidly released during the first stage of DNA injection, indicating the large force (tens of pico Newtons) needed to complete the (inverse) loading process . The second injection stage sets in when approximately 1/3 of the genome has been released, and the interaxial distance has nearly reached its equilibrium value (corresponding to that of a relaxed torus in solution); concomitantly the encapsidated genome begins a gradual morphological transformation from a spool to a torus . We also calculate the loading force, the average pressure on the capsid's walls, and the anisotropic pressure profile within the capsid . The results are interpreted in terms of the (competing) bending and interaction components of the packing energy, and are shown to be in good agreement with available experimental data. J Immunol Methods, 2003 Mar 1, 274(1-2), 115 - 27 Bispecific monoclonal antibodies against a viral and an enzyme: utilities in ultrasensitive virus ELISA and phage display technology; Liu F et al.; A quadroma (hybrid-hybridoma) secreting bispecific antibodies with one paratope specific for M13 bacteriophage coat protein and another paratope specific for alkaline phosphatase (AP) was developed by electro-fusion of the two parental hybridomas and selected by a fluorescence activated cell sorter (FACS) . The anti-phage M13/anti-AP bsMAbs were purified from anti-phage M13 monospecific MAb by a novel affinity method using Mimetic Blue A6XL as immune complexes with AP . The purified bsMAbs with potentially every molecule uniformly bound with AP generated an immuno-probe with the theoretical highest specificity . An ultrasensitive sandwich ELISA for detecting viruses was developed by using this bsMAb coupled with an amplified ELISA procedure . The sensitivity of the assay was increased 1000 times compared with conventional ELISA to achieve detection of 100 phage particles which is approximately 2.3 fg of phage coat protein . This type of bsMAb probe and ELISA format can be used to design new body fluid assays for viral load of HIV, hepatitis and other human pathogens as rapid and inexpensive alternatives to the PCR based method . This unique bispecific probe also allowed rapid and sensitive detection of bound M13/fd phage clones while panning for specific phages displaying peptide mimics against an antigen from a phage display peptide library . Furthermore, we demonstrate the principle virus purification using bsMAb as affinity ligand with a mild phosphate buffer elution . The results indicate that bsMAb could be used to develop affinity chromatography for purifying highly contagious and pathogenic viruses avoiding procedures employing prolonged high-speed centrifugation. Environ Mol Mutagen, 2003, 41(2), 121 - 5 Development of a microplate assay for the detection of single plaque-forming units of bacteriophage PhiX174 in crude lysates; Slattery SD et al.; Mice containing the PhiX174 am3 transgene can be used for measuring in vivo mutation; however, the single burst analysis method used for distinguishing in vivo mutations from mutations generated during sample processing is labor-intensive . A liquid microplate assay was developed that detects a single mutant plaque-forming unit (PFU) of PhiX174 bacterial virus in the presence of excess nonmutant virus . The assay is based on inhibiting reduction of the tetrazolium dye, MTS, by bacterial cells selective for mutant virus . The assay is performed with crude lysates of infected bacteria and is as accurate as scoring viral plaques on a bacterial lawn . This microplate assay may have application in increasing throughput of the single burst analysis of PhiX174 in transgenic mouse mutation assays . Published 2003 Wiley-Liss, Inc. Mol Microbiol, 2003 Mar, 47(5), 1225 - 37 Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus; Figge RM et al.; In Caulobacter crescentus the partitioning proteins ParA and ParB operate a molecular switch that couples chromosome partitioning to cytokinesis . Homologues of these proteins have been shown to be important for the stable inheritance of F-plasmids and the prophage form of bacteriophage P1 . In C . crescentus, ParB binds to sequences adjacent to the origin of replication and is required for the initiation of cell division . Additionally, ParB influences the nucleotide-bound state of ParA by acting as a nucleotide exchange factor . Here we have performed a genetic analysis of the chromosome partitioning protein ParB . We show that C . crescentus ParB, like its plasmid homologues, is composed of three domains: a carboxyl-terminal dimerization domain; a central DNA-binding, helix-turn-helix domain; and an amino-terminal domain required for the interaction with ParA . In vivo expression of amino-terminally deleted parB alleles has a dominant lethal effect resulting in the inhibition of cell division . Fluorescent in situ hybridization experiments indicate that this phenotype is not caused by a chromosome partitioning defect, but by the reversal of the amounts of ATP- versus ADP- bound ParA inside the cell . We present evidence suggesting that amino-terminally truncated and full-length, wild-type ParB form heterodimers which fail to interact with ParA, thereby reversing the intracellular ParA-ATP to ParA-ADP ratio . We hypothesize that the amino-terminus of ParB is required to regulate the nucleotide exchange of ParA which, in turn, regulates the initiation of cell division. Intervirology, 2002, 45(4-6), 371 - 80 RNA bacteriophage capsid-mediated drug delivery and epitope presentation; Brown WL et al.; OBJECTIVE: To use our knowledge of the three-dimensional structure and self-assembly mechanism of RNA bacteriophage capsids to develop novel virus-like particles (VLPs) for drug delivery and epitope presentation . METHODS: Site-directed mutagenesis of a recombinant MS2 coat protein expression construct has been used to generate translational fusions encompassing short epitope sequences . These chimeric proteins still self-assemble in vivo into T = 3 shells with the foreign epitope in an accessible location . Covalent conjugation has also been used to generate RNA stem-loops attached to the toxin, ricin A chain, or to nucleotide-based drugs, that are still capable of stimulating self-assembly of the capsid in vitro . These packaged drugs can then be directed to specific cells in culture by further covalent decoration of the capsids with targeting molecules . RESULTS: Chimeric VLPs are strongly immunogenic when carrying either B or T cell epitopes, the latter generating cytokine profiles consistent with memory responses . Immune responses to the underlying phage epitopes appear to be proportional to the area of the phage surface accessible . Phage shells effectively protect nucleic acid-based drugs and, for the toxin construct, make cell-specific delivery systems with LD50 values in culture sub-nanomolar . CONCLUSION: VLP technology has potential for therapeutic and prophylactic intervention in disease. Biochemistry (Mosc), 2002 Dec, 67(12), 1366 - 70 Engineering of bacteriophage T4 tail sheath protein; Efimov AV et al.; Gene product 18 (gp18, 659 amino acids) forms bacteriophage T4 contractile tail sheath . Recombinant protein assembles into different length polysheaths during expression in the cell, which complicates the preparation of protein crystals for its spatial structure determination . To design soluble monomeric gp18 mutants unable to form polysheaths and useful for crystallization, we have used Bal31 nuclease for generation deletions inside gene 18 encoding the Ile507-Gly530 region . Small deletions in the region of Ile507-Ile522 do not affect the protein assembly into polysheaths . Protein synthesis termination occurs because of reading frame failure in the location of deletions . Some fragments of gp18 containing short pseudo-accidental sequence in the C-terminal, while being soluble, have lost the ability for polysheath assembly . For the first time we succeeded in obtaining crystals of a soluble gp18 fragment containing 510 amino acids which, according to trypsin resistance, is similar to native protein monomer. Neuroreport, 2003 Feb 10, 14(2), 265 - 8 Screening and characterization of human single-chain Fv antibody against beta-amyloid peptide 40; Cai J et al.; Expression of single-chain variable fragment antibody on the surface of filamentous bacteriophage is widely used to make antibodies with pre-defined specificities . Using direct selection on solid phase-bound amyloid-beta 40 peptide, which is a main pathogenic feature of Alzheimer's disease, we obtained single-chain Fv antibody with specific binding activity . The binding epitope of the antibody is located between amino acids 1 and 16 of the peptide antigen . DNA sequencing showed that the gene coding for the single chain Fv antibody consists of 768 bp, and the complementarity-determining regions were deduced. Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2345 - 50 Epub 2003 Feb 21. Retroevolution of lambda Cro toward a stable monomer; LeFevre KR et al.; The Cro protein from bacteriophage lambda has a dimeric alpha+beta fold that evolved from an ancestral all-alpha monomer . The sequence mutations responsible for this dramatic structural evolution are unknown . Here we use analysis of sequence alignments to show that Ala-33, a small side chain in the hydrophobic "ball-and-socket" dimer interface of lambda Cro, was a much larger tryptophan side chain at a previous point in evolution . The retroevolutionary lambda Cro-A33W mutant shows a 10-fold reduction in dimerization affinity relative to the wild type as well as a large increase in monomer thermal stability (Delta T(m) > 10 degrees C), apparently due to partial filling of the hydrophobic socket from within the same monomer . An additional mutation in the dimer interface, F58D, almost completely abolishes detectable dimerization while maintaining the high monomer stability . The secondary structure content of the monomerized versions of lambda Cro is similar to that of the wild-type protein, and the tertiary structure of the monomer appears relatively well defined . These results (i) support a model in which the ball-and-socket dimer interface of lambda Cro was created by altered volume mutations within a limited branch of the Cro lineage and (ii) suggest the possibility that the evolution of the alpha+beta dimer from an all-alpha monomer proceeded through an alpha+beta monomer intermediate. Protein Expr Purif, 2003 Feb, 27(2), 220 - 8 Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli; Seddi R et al.; Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli . When the protein was expressed under the control of bacteriophage T7 promoter in E . coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively high level in the cytoplasm (4 mg/L culture medium) . Although the recombinant protein essentially accumulated as inclusion bodies, as much as 30% of the fusion protein was recovered in a soluble form at low growth temperature and could therefore be purified to homogeneity in a single-step procedure by metal-affinity chromatography . The recombinant precursor form of bovine carboxypeptidase A was recognized by a monoclonal antibody directed against purified bovine pancreatic carboxypeptidase A . Moreover, upon tryptic activation it gave rise to an enzyme, the N-terminal sequence, molecular size,and specific activity of which were comparable to those of the enzyme derived from the native precursor purified from bovine pancreas . Protein Expr Purif, 2003 Feb, 27(2), 195 - 201 Purification and characterization of the deoxynucleoside monophosphate kinase of bacteriophage T5; Mikoulinskaia GV et al.; Deoxynucleoside monophosphate kinase (dNMP kinase) of bacteriophage T5 (EC 2.7.4.13) was purified to apparent homogeneity from phage-infected Escherichia coli cells . Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel showed that the enzyme has a molecular mass of about 29 kDa . The molecular mass of dNMP kinase estimated by analytical equilibrium ultracentrifugation turned out to be 29.14 +/- 3.03 kDa . These data suggest that the enzyme exists in solution as a monomer . The isoelectric point of dNMP kinase was found to be 4.2 . The N-terminal amino acid sequence, comprising 21 amino acids, was determined to be VLVGLHGEAGSGKDGVAKLII . A comparison of this amino acid sequence and those of known enzymes with a similar function suggests the presence of a nucleotide-binding site in the sequenced region . Life Sci Space Res, 1977, 15, 295 - 8 Apollo-Soyuz Test Project on biorhythm of zone-forming fungi: preliminary work; Akoev IG et al.; The purpose of the experiment was to study general and local effects of space flight factors on the rhythm of cellular activity and on the morphological and genetic properties of biological objects . The Pushchino strain, Actinomyces levoris Kras 17-225A-IBFM, isolated at the Institute of Biological Physics, Moscow, was chosen as the main biological object . Under appropriate conditions it gives distinct and continuous rings of spore formation reflecting its intrinsic high degree of synchronism in changing its reproduction forms seen with the unaided eye as transparent rings (vegetative growth) alternate with convex white rings (spore-formation growth) . As an additional test object, a film culture of bacteriophage T4Br+ developed at the institute was used . The strains were placed together in one bioblock together with plastic detectors for detecting nuclear particles . The film culture of bacteriophage enabled us to amplify the area of registration of local radiation effects by studying the genetic effects of these: frequency of mutations, induced radiation, their spectrum, subsequent revertability under the action of chemical mutagens with known mechanisms of action on DNA molecules. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 538 - 40 Epub 2003 Feb 21. Crystallization of the membrane-containing bacteriophage PRD1 in quartz capillaries by vapour diffusion; Cockburn JJ et al.; Crystals of bacteriophage PRD1, a virus containing an internal lipid bilayer, have been grown in thin-walled quartz capillary tubes by vapour diffusion as a means of eliminating mechanical handling of the crystals during data collection . It has been found that the addition of polyethylene glycol 20 000 (PEG 20K) to the mother liquor that bathes the crystals allows far higher resolution diffraction intensities to be observed . Growing and treating the crystals in this way has produced a small number of crystals which are particularly amenable to X-ray diffraction analysis. Nature, 2003 Feb 20, 421(6925), 859 - 63 Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA; Aas PA et al.; Repair of DNA damage is essential for maintaining genome integrity, and repair deficiencies in mammals are associated with cancer, neurological disease and developmental defects . Alkylation damage in DNA is repaired by at least three different mechanisms, including damage reversal by oxidative demethylation of 1-methyladenine and 3-methylcytosine by Escherichia coli AlkB . By contrast, little is known about consequences and cellular handling of alkylation damage to RNA . Here we show that two human AlkB homologues, hABH2 and hABH3, also are oxidative DNA demethylases and that AlkB and hABH3, but not hABH2, also repair RNA . Whereas AlkB and hABH3 prefer single-stranded nucleic acids, hABH2 acts more efficiently on double-stranded DNA . In addition, AlkB and hABH3 expressed in E . coli reactivate methylated RNA bacteriophage MS2 in vivo, illustrating the biological relevance of this repair activity and establishing RNA repair as a potentially important defence mechanism in living cells . The different catalytic properties and the different subnuclear localization patterns shown by the human homologues indicate that hABH2 and hABH3 have distinct roles in the cellular response to alkylation damage. J Soc Gynecol Investig, 2003 Feb, 10(2), 67 - 73 Cloning and tissue expression of the tissue prothrombinase Fgl-2 in the Sprague-Dawley rat; Rychlik DF et al.; OBJECTIVE: To sequence and characterize the expression of the prothrombinase Fgl-2 in the Sprague-Dawley rat . METHODS: Reverse-transcriptase polymerase chain reaction was performed on RNA from spontaneously cycling adult pregnant Sprague-Dawley rats by using specific Fgl-2 primers . The resulting amplicon was also used to screen a rat spleen bacteriophage library and to probe a Northern blot of various tissues . The rat Fgl-2 amino acid sequence was compared with the known sequences in mouse and human . RESULTS: Fgl-2-specific amplicon bands were observed in the rat brain, kidney, liver, ovary, spleen, and gestational day 22 and postpartum uterus . The rat Fgl-2 cDNA and amino acid sequence were found to be homologous with those of human (86% and 74%, respectively) and mouse (91% and 87%, respectively) . Northern blotting demonstrated two different-sized transcripts (1.3 and 3.4 kb), and expression was observed in the cervix, heart, liver, ovary, and nongestational and gestational day 22 myometrium . CONCLUSION: Thrombin is classically generated from the cleavage of the proenzyme prothrombin by activated factors V and X . In tissues thrombin appears to be generated by a novel prothrombinase Fgl-2 (fibrinogen-like protein) whose activity is stimulated by proinflammatory mediators . Fgl-2 provides the mechanistic coupling between proinflammatory cytokines and the generation of active thrombin independent of the coagulation cascade . Our studies confirmed the expression of Fgl-2 mRNA in several rat tissues, including the pregnant uterus, where it could play a key role in the initiation of parturition especially in response to local or systemic infection. Protein Sci, 2003 Mar, 12(3), 620 - 6 Protein folding coupled to DNA binding in the catalytic domain of bacteriophage lambda integrase detected by mass spectrometry; Kamadurai HB et al.; Bacteriophage lambda integrase (lambda-Int) is the prototypical member of a large family of enzymes that catalyze site-specific DNA recombination via single-strand cleavage and the formation of a Holliday junction intermediate . Crystallographic and biochemical evidence indicate that substantial conformational change (i.e., folding) in the catalytic domain of the protein is required for substrate recognition and catalysis . We have examined the solution conformation of the catalytic domain (C170) in the absence and presence of a cognate "half-site" DNA oligonucleotide by electrospray ionization mass spectrometry, and circular dichroism and fluorescence spectroscopy . The distribution of ions in the positive ion electrospray mass spectrum of the free protein reveals the presence of three distinct species in solution, one corresponding to the folded protein, one to the unfolded protein, and one to a dimer . In the presence of DNA, ions are observed only for the protein-DNA complex and the folded form of the free protein . We therefore conclude that DNA binding stabilizes the global fold of the protein in a manner that is consistent with folding-coupled target recognition as a mechanism to control site-specific recombination . Furthermore, we find that inspection of the charge state distribution of ions in electrospray mass spectra provides a quick and effective means to identify conformational heterogeneity of proteins in solution and to investigate dynamic protein-nucleic acid interactions. Protein Sci, 2003 Mar, 12(3), 403 - 11 Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints; Marassi FM et al.; A solid-state NMR approach for simultaneous resonance assignment and three-dimensional structure determination of a membrane protein in lipid bilayers is described . The approach is based on the scattering, hence the descriptor "shotgun," of (15)N-labeled amino acids throughout the protein sequence (and the resulting NMR spectra) . The samples are obtained by protein expression in bacteria grown on media in which one type of amino acid is labeled and the others are not . Shotgun NMR short-circuits the laborious and time-consuming process of obtaining complete sequential assignments prior to the calculation of a protein structure from the NMR data by taking advantage of the orientational information inherent to the spectra of aligned proteins . As a result, it is possible to simultaneously assign resonances and measure orientational restraints for structure determination . A total of five two-dimensional (1)H/(15)N PISEMA (polarization inversion spin exchange at the magic angle) spectra, from one uniformly and four selectively (15)N-labeled samples, were sufficient to determine the structure of the membrane-bound form of the 50-residue major pVIII coat protein of fd filamentous bacteriophage . Pisa (polarity index slat angle) wheels are an essential element in the process, which starts with the simultaneous assignment of resonances and the assembly of isolated polypeptide segments, and culminates in the complete three-dimensional structure of the protein with atomic resolution . The principles are also applicable to weakly aligned proteins studied by solution NMR spectroscopy . {The structure we determined for the membrane-bound form of the Fd bacteriophage pVIII coat protein has been deposited in the Protein Data Bank as PDB file 1MZT.} J Bacteriol, 2003 Mar, 185(5), 1693 - 700 P15 and P3, the tail completion proteins of bacteriophage T4, both form hexameric rings; Zhao L et al.; Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail . gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube . In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism . Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains . Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube . The differential roles between gp15 and gp3 upon completion of the tail are discussed. DNA Cell Biol, 2003 Jan, 22(1), 11 - 8 Processing of filamentous bacteriophage virions in antigen-presenting cells targets both HLA class I and class II peptide loading compartments; Gaubin M et al.; Virions of filamentous bacteriophage fd are capable of displaying multiple copies of peptide epitopes and generating powerful immune responses to them . To investigate the antigen processing mechanisms in human B cell lines used as antigen presenting cells, the major coat protein (pVIII) in intact virions was fluorescently labeled, and its localization in various intracellular compartments was followed using confocal microscopy . We show that the virions were taken up and processed to yield peptides that reach both the major histocompatibility complex (MHC) class II compartment and the endoplasmic reticulum . Moreover, when exposed to bacteriophages displaying a cytotoxic T lymphocyte (CTL) epitope from the reverse transcriptase of human immunodeficiency virus type-1 (HIV-1), B cells were lysed by specific cytotoxic lymphocytes . This confirms that filamentous bacteriophage virions are capable of being taken up and processed efficiently by MHC class I and class II pathways, even in nonprofessional antigen presenting cells . These remarkable features explain, at least in part, the unexpected ability of virions displaying foreign T-cell epitopes to prime strong T-helper-dependent CTL responses . These findings have important implications for the development of peptide-based vaccines, using filamentous bacteriophage virions as scaffolds. J Virol, 2003 Mar, 77(5), 2972 - 80 Multitasking in replication is common among geminiviruses; Preiss W et al.; Geminiviruses package single-stranded circular DNA and replicate via double-stranded DNA intermediates . During the past decade, increasing evidence has led to the general acceptance that their replication follows a rolling-circle replication mechanism like bacteriophages with single-stranded DNA . In a recent study, we showed that this is also true for Abutilon mosaic geminivirus (AbMV), but that this particular virus may also use a recombination-dependent replication (RDR) route in analogy to T4 phages . Because AbMV is a special case, since it has been propagated on ornamental plants for more than a hundred years, it was interesting to determine whether RDR is common among other geminiviruses . We analyzed geminiviruses from different genera and geographic origins by using BND cellulose chromatography in combination with an improved high resolution two-dimensional gel electrophoresis, and we conclude that multitasking in replication is widespread, at least for African cassava mosaic, Beet curly top, Tomato golden mosaic, and Tomato yellow leaf curl virus. J Mol Biol, 2003 Feb 21, 326(3), 679 - 90 Interaction of T4 AsiA with its target sites in the RNA polymerase sigma70 subunit leads to distinct and opposite effects on transcription; Minakhin L et al.; Bacteriophage T4 AsiA is a homodimeric protein that orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of Escherichia coli RNA polymerase holoenzyme (Esigma(70)) and preventing promoter complex formation on most E.coli and early T4 promoters . In addition, Esigma(70)AsiA, but not Esigma(70), is a substrate of transcription activation by T4-encoded DNA-binding protein MotA, a co-activator of transcription from middle viral promoters . The molecular determinants of sigma(70)-AsiA interaction necessary for transcription inhibition reside in the sigma(70) conserved region 4.2, which recognizes the -35 promoter consensus element . The molecular determinants of sigma(70)-AsiA interaction necessary for MotA-dependent transcription activation have not been identified . Here, we show that in the absence of sigma(70) region 4.2, AsiA interacts with sigma(70) conserved region 4.1 and activates transcription in a MotA-independent manner . Further, we show that the AsiA dimer must dissociate to interact with either region 4.2 or region 4.1 of sigma(70) . We propose that MotA may co-activate transcription by restricting AsiA binding to sigma(70) region 4.1. J Biol Chem, 2003 Apr 25, 278(17), 15261 - 71 Epub 2003 Feb 07. Activation of the RegB endoribonuclease by the S1 ribosomal protein is due to cooperation between the S1 four C-terminal modules in a substrate-dependant manner; Bisaglia M et al.; The RegB protein, encoded by the T4 bacteriophage genome, is a ribonuclease involved in the inactivation of the phage early messenger RNAs . Its in vitro activity is very low but can be enhanced up to 100-fold in the presence of the ribosomal protein S1 . The latter is made of six repeats of a conserved module found in many other proteins of RNA metabolism . Considering the difference between its size (556 amino acids) and that of several RegB substrates (10 nucleotides), we wondered whether all six modules are necessary for RegB activation . We studied the influence of twelve S1 fragments on the cleavage efficiency of three short substrates . RegB activation requires the cooperation of different sets of modules depending on the substrates . Two RNAs are quite well cleaved in the presence of the fragment formed by the fourth and fifth modules, whereas the third requires the presence of the four C-terminal domains . However, NMR interaction experiments showed that, despite these differences, the interactions of the substrates with either the bi- or tetra-modules are similar, suggesting a common interaction surface . In the case of the tetra-module the interactions involve all four domains, raising the question of the spatial organization of this region. Adv Space Res, 2002, 30(6), 1533 - 8 Stability of nucleic acid under the effect of UV radiation; Ronto G et al.; Nucleic acids (combined with protein molecules) are essential constituents of the living systems playing an important role in the early evolution of life as well . A specific feature of these molecules has been found and directly confirmed recently: under the influence of short-wavelength UV radiation bipyrimidine photoproducts (cyclobutane dimers and 6-4 bipyrimidines) are induced and the reversion of them can be provoked by the same photons . However, reversion is preferred by the shorter wavelengths . With increasing ratio of the longer wavelength components of the radiation (using artificial UV sources and solar light on the Earth's surface) the impact of the reversible photoproducts in the harmful biological effect decreases and other photoproducts are dominant . Assuming the photoinduced reactions (dimerisation and reversion) are statistical events, during the irradiation the chance for a number of nucleoprotein molecules to survive the radiation damage can be reality . The theoretical and experimental basis of these assumptions will be discussed in the case of bacteriophage T7 nucleoprotein . c2002 COSPAR . Published by Elsevier Science Ltd . All rights reserved. J Mol Evol, 2003 Feb, 56(2), 162 - 8 Can an arbitrary sequence evolve towards acquiring a biological function? Hayashi Y, Sakata H, Makino Y, Urabe I, Yomo T. To explore the possibility that an arbitrary sequence can evolve towards acquiring functional role when fused with other pre-existing protein modules, we replaced the D2 domain of the fd-tet phage genome with the soluble random polypeptide RP3-42 . The replacement yielded an fd-RP defective phage that is six-order magnitude lower infectivity than the wild-type fd-tet phage . The evolvability of RP3-42 was investigated through iterative mutation and selection . Each generation consists of a maximum of ten arbitrarily chosen clones, whereby the clone with highest infectivity was selected to be the parent clone of the generation that followed . The experimental evolution attested that, from an initial single random sequence, there will be selectable variation in a property of interest and that the property in question was able to improve over several generations . fd-7, the clone with highest infectivity at the end of the experimental evolution, showed a 240-fold increase in infectivity as compared to its origin, fd-RP . Analysis by phage ELISA using anti-M13 antibody and anti-T7 antibody revealed that about 37-fold increase in the infectivity of fd-7 was attributed to the changes in the molecular property of the single polypeptide that replaced the D2 domain of the g3p protein . This study therefore exemplifies the process of a random polypeptide generating a functional role in rejuvenating the infectivity of a defective bacteriophage when fused to some preexisting protein modules, indicating that an arbitrary sequence can evolve toward acquiring a functional role . Overall, this study could herald the conception of new perspective regarding primordial polypeptides in the field of molecular evolution. Virology, 2003 Jan 20, 305(2), 276 - 87 Biochemical characterization of bacteriophage lambda genome packaging in vitro; Yang Q et al.; Bacteriophage lambda has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA packaging at the molecular level . Limited in vitro packaging efficiency has hampered progress toward this end, however . It has been suggested that limited packaging efficiency is related to poor activity of purified procapsids . We describe the construction of a vector that expresses lambda procapsids with a yield that is 40-fold greater than existing systems . Consistent with previous studies, packaging of a mature lambda genome is very inefficient in vitro, with only 4% of the input procapsids utilized . Concatemeric DNA is the preferred packaging substrate in vivo, and procapsids interact with a nucleoprotein complex known as complex I to initiate genome packaging . When complex I is used as a packaging substrate in vitro, capsid utilization is extremely efficient, and 40% of the input DNA is packaged . Finally, we provide evidence for a packaging-stimulated ATPase activity, and kinetically characterize this reaction quantifying the energetic cost of DNA packaging in bacteriophage lambda. Appl Environ Microbiol, 2003 Feb, 69(2), 1059 - 66 Comparison of Shiga toxin production by hemolytic-uremic syndrome-associated and bovine-associated Shiga toxin-producing Escherichia coli isolates; Ritchie JM et al.; There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens . The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood . Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection . Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates . Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC . In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains . Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC . In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC . Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC . However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC . These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics. Curr Microbiol, 2003 Mar, 46(3), 224 - 7 Effects of lysogeny of Shiga toxin 2-encoding bacteriophages on pulsed-field gel electrophoresis fragment pattern of Escherichia coli K-12; Iguchi A et al.; Escherichia coli K-12 lysogens of three different Shiga toxin 2 (Stx2)-encoding bacteriophages were examined for variability in their pulsed-field gel electrophoresis (PFGE) fragment patterns . The PFGE fragment patterns could be classified into three types (i.e., PFGE types B, C, and D) . For the PFGE type D, a 255-kbp fragment present in the original K-12 strain was apparently shifted by the size of Stx 2-encoding phage genomic DNA (ca . 65 kbp) to the position at 320 kbp . In contrast, the types B and C showed the above fragment shift plus further 6- and 10-fragment differences, respectively, from the original K-12 strain . The evidence suggests that even a single genetic event like lysogeny can cause marked genotypic modification of the host strain. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1999, 17(3), 143 - 5 {Cloning and sequence analysis of histidine-rich protein-II gene fragment of Plasmodium falciparum yunnan strain}; Xiao J et al.; AIM: To determine the nucleotide sequence of the partial gene of the histidine-rich protein II (HRP II) the Plasmodium falciparum PFD-3/YN and find out the differences of the HRP II gene sequence between this isolate and other isolates . METHODS: The histidine-rich protein-II gene fragment was amplified by polymerase chain reaction and cloned into M13 bacteriophage . M13-HRP II single strand DNAs of three positive clones were extracted, respectively . The nucleotide sequence of the HRP II gene fragment was determined by the dideoxy chain termination method . Using PCGENE software to compare and analyze the HRP II gene sequence among the different isolates . RESULTS: Different degrees of diversity of the HRP II gene sequences were found among Plasmodium falciparum PFD-3/YN(from China) and two other isolates (7G8 from Brazil and D10 from Gambia) . The HRP II in the three isolates exhibited 70.3% homology in amino acid sequences and 68.6% homology in the nucleic acid sequences . CONCLUSION: There were differences in HRP II gene sequence among the Plasmodium falciparum PFD-3/YN and two other isolates (7G8 and D10). J Bacteriol, 2003 Feb, 185(4), 1475 - 7 Corrected sequence of the bacteriophage p22 genome; Pedulla ML et al.; We report the first accurate genome sequence for bacteriophage P22, correcting a 0.14% error rate in previously determined sequences . DNA sequencing technology is now good enough that genomes of important model systems like P22 can be sequenced with essentially 100% accuracy with minimal investment of time and resources. Sheng Wu Gong Cheng Xue Bao, 2002 Sep, 18(5), 531 - 5 {Progress in the study of the structure and function of Cre recombinase}; Wang LX et al.; The Cre recombinase, an integrase from bacteriophage P1, catalyzes site-specific recombination between 34-bp repeats termed loxP sites, in the absence of any additional cofactors and energy . Mediated by Cre recombinase, specific DNA fragments can be excised, inversed or integrated depending on the orientation or position of loxP sites in vitro or in vivo . Because of its simplicity and high efficiency, Cre/loxP site-specific recombination system has been widely used in gene deletion and function identification, gene site-specific integration, gene trapping and chromosome engineering . It has been used as a useful tool for DNA recombination in transgenic yeast, plants, insects and mammals . Here progress in the study of the structure and function of Cre recombinase is discussed. J Mol Biol, 2003 Feb 14, 326(2), 435 - 51 Examination of the role of the clamp-loader and ATP hydrolysis in the formation of the bacteriophage T4 polymerase holoenzyme; Trakselis MA et al.; Transient kinetic analyses further support the role of the clamp-loader in bacteriophage T4 as a catalyst which loads the clamp onto DNA through the sequential hydrolysis of two molecules of ATP before and after addition of DNA . Additional rapid-quench and pulse-chase experiments have documented this stoichiometry . The events of ATP hydrolysis have been related to the opening/closing of the clamp protein through fluorescence resonance energy transfer (FRET) . In the absence of a hydrolysable form of ATP, the distance across the subunit interface of the clamp does not increase as measured by intramolecular FRET, suggesting gp45 cannot be loaded onto DNA . Therefore, ATP hydrolysis by the clamp-loader appears to open the clamp wide enough to encircle DNA easily . Two additional molecules of ATP then are hydrolyzed to close the clamp onto DNA . The presence of an intermolecular FRET signal indicated that the dissociation of the clamp-loader from this complex occurred after guiding the polymerase onto the correct face of the clamp bound to DNA . The final holoenzyme complex consists of the clamp, DNA, and the polymerase . Although this sequential assembly mechanism can be generally applied to most other replication systems studied to date, the specifics of ATP utilization seem to vary across replication systems. Res Microbiol, 2002 Dec, 153(10), 639 - 46 DcrA and dcrB Escherichia coli genes can control DNA injection by phages specific for BtuB and FhuA receptors; Samsonov VV et al.; We had previously shown that the Escherichia coli proteins DcrA (SdaC) and DcrB, located, respectively, in the inner membrane and periplasm, are involved in the early development of virulent bacteriophage C1, which recognises BtuB as an outer membrane receptor . In the present work it is demonstrated that the DcrA and DcrB proteins, coordinately with another outer membrane receptor protein, FhuA, are also involved in an early stage of development of a newly isolated virulent phage, C6 . In both cases, DcrA and DcrB probably are required in the second stage of phage adsorption-the DNA injection process . This means that DcrA and DcrB proteins can participate in phage DNA transport pathways in cooperation with different outer membrane receptors, including FhuA and BtuB . The increased sensitivity of bacterial cells to SDS following C1 and C6 adsorption suggests that adsorption by these phages triggers the opening of diffusion channels through the outer membrane . Our results also indirectly demonstrate that the DcrA and DcrB proteins participate in the opening or formation of these channels. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2002 Sep, 19(3), 435 - 9 {Cloning and expression of heavy chain variable region genes against telomerase protein hTERT}; Zhang H et al.; Single domain antibodies against telomerase protein hTERT were prepared by technique of displayed on the surface of recombinant bacterio phages . Total RNA of spleen lymphocytes were extracted from mice immunized recombinant hTERT and transcripted to cDNA . First-strand cDNA was used as a template, heavy chain variable region genes against hTERT were amplified with VHfor and VHback primers by PCR technique . Amplification reaction yielded a fragment about 350 base pairs in length . Amplified cDNA were cloned into the vehide of bacteriophage PCANTAB 5E, the phagemid containing VH gene transformed into competent E . coli TG1, in the presence of helper phage M13K07, VH-g3 fusion proteins were display on the surface of recombinant phages . The phage carrying VH genes that encode binding activities could be detected directly with DOT BLOT . Single domain antibodies were generated successfully and had binding activities with hTERT . Results suggest that phage display technique be a new way of making antibodies . VH genes were cloned successfully, which could provide possibility for futher preparing single-chain antibodies(ScFv) anti-hTERT. J Biol Chem, 2003 Apr 11, 278(15), 12634 - 44 Epub 2003 Jan 29. Proteolytic processing and oligomerization of bacteriophage-derived endosialidases; Muhlenhoff M et al.; Bacteriophages infecting the neuroinvasive pathogen Escherichia coli K1 require an endosialidase to penetrate the polysialic acid capsule of the host . Sequence information is available for the endosialidases endoNE, endoNF, and endoN63D of the K1-specific phages phi K1E, phi K1F, and 63D, respectively . The cloned sequences share a highly conserved catalytic domain but differ in the length of the N- and C-terminal parts . Although the expression of active recombinant enzyme succeeded in the case of endoNE, it failed for endoNF . Protein alignments of all three endosialidase sequences gave rise to the assumption that inactivity of the cloned endoNF is caused by a C-terminal truncation . By reinvestigation of the respective gene locus in the phi K1F genome, we identified an extended open reading frame of 3195 bp, encoding a 119-kDa protein . Full-length endoNF contains the C-terminal domain conserved in all endosialidases, which may act as an intramolecular chaperone . Comparative studies carried out with endoNE and endoNF demonstrate that endosialidases are proteolytically processed, releasing the C-terminal domain . Using a mutational approach in combination with protein analytical techniques we demonstrate that (i) the C-terminal domain is a common feature of endosialidases and other tail fiber proteins; (ii) the integrity of the C-terminal domain and its presence in the nascent protein are crucial for the formation of active enzymes; (iii) proteolytic processing is not essential for enzymatic activity; and (iv) functional folding is a prerequisite for trimerization of endoNF. Am J Trop Med Hyg, 2003 Jan, 68(1), 61 - 4 Generation and characterization of cDNA clones from Sarcoptes scabiei var . hominis for an expressed sequence tag library: identification of homologues of house dust mite allergens; Fischer K et al.; Molecular studies on scabies, a disease of considerable human and veterinary significance, have been limited because of the difficulty of obtaining the causative organism Sarcoptes scabiei, the "itch mite." We have used skin from the bedding of crusted scabies patients as a source of mites for the construction of libraries of cDNAs from S . scabiei var . hominis in the bacteriophage lambda vector lambdaZAP express . Sequences of 145 clones established that the libraries predominantly contain sequences from S . scabiei, enabling a major sequencing program to begin . Among those sequenced to date, cDNAs encoding S . scabiei homologues of 3 house dust mite allergens-the M-177 apolipoprotein, glutathione S-transferase, and paramyosin--were identified . The availability of cDNA libraries from S . scabiei var . hominis and S . scabiei var . vulpes and the emerging public sequence databases from both opens up new possibilities in scabies research. BMC Struct Biol . 2003 Jan 28;3(1):1. Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases; Iyer LM et al.; BACKGROUND: The eukaryotic RNA-dependent RNA polymerase (RDRP) is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing . This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria . No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery . RESULTS: Using extensive sequence profile searches, we identified bacteriophage homologs of the eukaryotic RDRP . The comparison of the eukaryotic RDRP and their homologs from bacteriophages led to the delineation of the conserved portion of these enzymes, which is predicted to harbor the catalytic site . Further, detailed sequence comparison, aided by examination of the crystal structure of the DNA-dependent RNA polymerase (DDRP), showed that the RDRP and the beta' subunit of DDRP (and its orthologs in archaea and eukaryotes) contain a conserved double-psi beta-barrel (DPBB) domain . This DPBB domain contains the signature motif DbDGD (b is a bulky residue), which is conserved in all RDRPs and DDRPs and contributes to catalysis via a coordinated divalent cation . Apart from the DPBB domain, no similarity was detected between RDRP and DDRP, which leaves open two scenarios for the origin of RDRP: i) RDRP evolved at the onset of the evolution of eukaryotes via a duplication of the DDRP beta' subunit followed by dramatic divergence that obliterated the sequence similarity outside the core catalytic domain and ii) the primordial RDRP, which consisted primarily of the DPBB domain, evolved from a common ancestor with the DDRP at a very early stage of evolution, during the RNA world era . The latter hypothesis implies that RDRP had been subsequently eliminated from cellular life forms and might have been reintroduced into the eukaryotic genomes through a bacteriophage . Sequence and structure analysis of the DDRP led to further insights into the evolution of RNA polymerases . In addition to the beta' subunit, beta subunit of DDRP also contains a DPBB domain, which is, however, distorted by large inserts and does not harbor a counterpart of the DbDGD motif . The DPBB domains of the two DDRP subunits together form the catalytic cleft, with the domain from the beta' subunit supplying the metal-coordinating DbDGD motif and the one from the beta subunit providing two lysine residues involved in catalysis . Given that the two DPBB domains of DDRP contribute completely different sets of active residues to the catalytic center, it is hypothesized that the ultimate ancestor of RNA polymerases functioned as a homodimer of a generic, RNA-binding DPBB domain . This ancestral protein probably did not have catalytic activity and served as a cofactor for a ribozyme RNA polymerase . Subsequent evolution of DDRP and RDRP involved accretion of distinct sets of additional domains . In the DDRPs, these included a RNA-binding Zn-ribbon, an AT-hook-like module and a sandwich-barrel hybrid motif (SBHM) domain . Further, lineage-specific accretion of SBHM domains and other, DDRP-specific domains is observed in bacterial DDRPs . In contrast, the orthologs of the beta' subunit in archaea and eukaryotes contains a four-stranded alpha + beta domain that is shared with the alpha-subunit of bacterial DDRP, eukaryotic DDRP subunit RBP11, translation factor eIF1 and type II topoisomerases . The additional domains of the RDRPs remain to be characterized . CONCLUSIONS: Eukaryotic RNA-dependent RNA polymerases share the catalytic double-psi beta-barrel domain, containing a signature metal-coordinating motif, with the universally conserved beta' subunit of DNA-dependent RNA polymerases . Beyond this core catalytic domain, the two classes of RNA polymerases do not have common domains, suggesting early divergence from a common ancestor, with subsequent independent domain accretion . The beta-subunit of DDRP contains another, highly diverged DPBB domain . The presence of two distinct DPBB domains in two subunits of DDRP is compatible with the hypothesis that the ith the hypothesis that the ultimate ancestor of RNA polymerases was a RNA-binding DPBB domain that had no catalytic activity but rather functioned as a homodimeric cofactor for a ribozyme polymerase. J Virol, 2003 Feb, 77(4), 2436 - 44 Baculovirus alkaline nuclease possesses a 5'-->3' exonuclease activity and associates with the DNA-binding protein LEF-3; Mikhailov VS et al.; Alkaline nuclease (AN) of the Autographa californica multiple-capsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133) was expressed in recombinant baculovirus as a His(6)-tagged fusion and purified by sequential chromatography on Ni-NTA-agarose, DEAE-Toyopearl, and heparin-Sepharose . At all stages of purification, AcMNPV AN was found to copurify with a 44-kDa polypeptide which was identified as the baculovirus single-stranded DNA (ssDNA)-binding (SSB) protein, LEF-3 . Sedimentation analysis in glycerol gradients of highly purified samples suggested that AN and LEF-3 are associated in a complex (designated *AN/L3), predominantly as heterodimers, although oligomeric forms containing both proteins were evident . In reactions with single- or double-stranded 62-mer oligonucleotides that were labeled with (32)P at the 5' or 3' ends, *AN/L3 carried out exonucleolytic hydrolysis of both substrates exclusively in a 5'-->3' direction . Saturation of ssDNA with an excess of LEF-3 prior to the addition of *AN/L3 resulted in a marked decrease in the rate of ssDNA hydrolysis . This suggests that excess LEF-3 may protect ssDNA from digestion by a AN-LEF-3 complex, thus regulating its activity in infected cells . The association of baculovirus AN with the viral SSB LEF-3 and the 5'-->3' exonuclease activity of this complex suggests that AN and LEF-3 may participate in homologous recombination of the baculovirus genome in a manner similar to that of exonuclease (Redalpha) and DNA-binding protein (Redbeta) of the Red-mediated homologous recombination system of bacteriophage lambda. Biopolymers, 2003 Feb, 68(2), 250 - 64 Operator recognition by the phage 434 cI repressor: MD simulations of free and bound 50-bp DNA reveal important differences between the OR1 and OR2 sites; Hartmann B et al.; Using molecular dynamics simulations in explicit solvent, we investigated the behavior of a 50-bp DNA sequence containing the 434 bacteriophage operators OR1 and OR2 separated by an 8-bp spacer . Two simulations of 1 ns each were carried out, with DNA alone and with DNA complexed to dimers of the R1-69 DNA binding domain of the phage 434 cI repressor protein at the OR1 and OR2 sites . Strong correlations among average structural parameters are observed between our simulations and available experimental data for the bound OR1/OR2 subsites . In the free state, some differences appear between the three relevant fragments (OR1, the spacer, and OR2) . Unbound OR1 exhibits a large, shallow major groove into which the base atoms protrude and is also bent toward the major groove . This structure is maintained because structural fluctuations are weak . Unbound OR2 resembles canonical B-DNA although the structural parameters show greater fluctuations, essentially due to a malleable step (the innermost CpA/TpG), absent in OR1 . Complexation with the proteins slightly alters the base positions but strongly modifies the sugar and backbone motions . The most crucial repressor effects are changes in the flexibility of the OR1/OR2 sites . Structural fluctuations are enhanced for OR1, conferring a favorable energetic contribution to the OR1 binding, whereas they are reduced for OR2 . Therefore, both structural and dynamic properties of DNA suggest OR1 is the most attractive site for the repressor, which may explain the different binding association constants observed for the OR1 and OR2 sites . Finally, we also investigated the impact of the protein on the DNA backbone dynamics and find that direct or indirect interactions facilitate the DNA structural variations required for achieving complementarity with the protein . Eur J Immunol, 2003 Feb, 33(2), 314 - 25 Transgenic mice with hematopoietic and lymphoid specific expression of Cre; de Boer J et al.; Bacteriophage P1 Cre/loxP based systems can be used to manipulate the genomes ofmice in vivo and in vitro, allowing the generation of tissue-specific conditional mutants . We have generated mouse lines expressing Cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hCD2 promoter and locus control region (LCR) . The R26R-EYFP Cre reporter mouse line was used to determine the pattern of Cre expression in each line and enabled the assessment of Cre activity at a single-cell level . Analysis showed that the vav promoter elements were able to direct Cre-mediated recombination in all cells of the hematopoietic system . The hCD2 promoter and LCR on the other hand were able to drive Cre-mediated recombination only in T cells and B cells, but not in other hematopoietic cell types . Furthermore, in the appropriate tissues, deletion of the floxed target was complete in all cells, thereby excluding the possibility of variegated expression of the Cre transgene . Both of these Cre-transgenic lines will be useful in generating tissue-specific gene deletions within all the cells of hematopoietic or lymphoid tissues. Water Environ Res, 2002 Nov-Dec, 74(6), 516 - 20 The effect of storage and lag time on MS2 bacteriophage susceptibility to ultraviolet radiation; Jolis D; The susceptibility of MS2 bacteriophage suspensions to UV radiation was assessed using a collimated beam technique . Storage of MS2 bacteriophage cultures at 4 degrees C resulted in a decrease in phage susceptibility to UV radiation over time . After 18 days, the level of MS2 bacteriophage inactivation achieved for the range of UV doses tested decreased by 0.7 to 1.1 logs, but remained constant after that point . Changes in the protein coat of the bacteriophage, a decrease in viability over time, and an increase in coagulation may have played a role in the observed susceptibility decrease . A 2-hour lag time between the preparation of the MS2 suspension and the irradiation test also resulted in a decrease in phage susceptibility. Nat Struct Biol, 2003 Feb, 10(2), 131 - 5 Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions; Jiang W et al.; Bacteriophage P22 is a prototypical biological machine used for studying protein complex assembly and capsid maturation . Using cryo-EM, we solved the structures of P22 before and after the capsid maturation at 8.5 A and 9.5 A resolutions, respectively . These structures allowed visualization of alpha-helices and beta-sheets from which the capsid protein fold is derived . The capsid fold is similar to that of the coat protein of HK97 bacteriophage . The cryo-EM shows that a large conformational change of the P22 capsid during maturation transition involves not only the domain movement of individual subunits, but also refolding of the capsid protein. Lett Appl Microbiol, 2003, 36(2), 92 - 6 Levels of male-specific RNA bacteriophage and Escherichia coli in molluscan bivalve shellfish from commercial harvesting areas; Dore WJ et al.; AIMS: Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas . However, it has been demonstrated that E . coli is an inadequate indicator of the viral risk associated with shellfish . An alternative indicator organism, male-specific RNA (FRNA) bacteriophage has been proposed for this role . This study compared the distribution of E . coli and FRNA bacteriophage in shellfish harvesting areas . METHODS AND RESULTS: A total of 608 shellfish samples from 49 shellfish harvesting areas were analysed for E . coli and FRNA bacteriophage using standard published methods . The geometric mean concentration of FRNA bacteriophage in all samples was over three times greater than that of E . coli (1800 and 538 counts/100 g for FRNA bacteriophage and E . coli, respectively) . In contrast to E . coli, FRNA bacteriophage concentrations were strongly influenced by season with a geometric mean count of 4503 PFU/100 g in the winter (October-March) compared with 910 PFU/100 g in the summer (April-September) . CONCLUSIONS: FRNA bacteriophage were present in shellfish at higher concentrations than E . coli . Elevated levels of FRNA bacteriophage observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in the UK . Levels of FRNA bacteriophage found in many shellfish from category B harvesting areas would not be eliminated by conventional treatment processes . SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study will inform future proposals to introduce FRNA bacteriophage as an indicator of the viral risk associated with shellfish. Mol Gen Mikrobiol Virusol, 2002, (4), 26 - 31 {Z-form of intraphage DNA}; Ulanov BP et al.; The bacteriophage lambda gt10 DNA containing an insertion of 20 pairs of GC-bases capable of being arranged as Z-form was cloned . Two independent methodological approaches based on the main properties of Z-form were used to study the shape of the insertion: formation of transition bridges composed of unpaired nucleotides between left-rotating Z-forms and right-rotating B-forms of helix (j-domain) and high immunogenic activity of Z-form . O-beta-diethylaminoethylhydroxyamine (OHA), an analogue of hydroxylamine, is capable of reacting specifically with unpaired cytidines . In this work this modification was used to inhibit the process of restriction at BamH1-site adjacent to (gc)10 insertion, that N-Methyl-bis(2-chloethyl) amine (MBCA) is capable of fixing the Z-form of the insertion in situ . Fixed Z-form is conserved even after DNA has been isolated from bacteriophage, thereby providing an opportunity of its identification by anti-Z-antibodies . It was shown that from 4 to 6% of the total number of insertions are in the Z-form . The hypothesis of significant role of Z-form in the process of packing of DNA molecules in capsid is put forward. Genesis, 2003 Feb, 35(2), 94 - 9 Odontoblast-specific expression of cre recombinase successfully deletes gene segments flanked by loxP sites in mouse teeth; Sreenath TL et al.; Embryonic or neonatal lethality of mice with targeted disruption of critical genes preclude them from further characterization of specific roles of these genes during postnatal development and aging . In order to study the molecular roles of such genes in teeth, we generated transgenic mouse lines expressing bacteriophage Cre recombinase under the control of the mouse dentin sialophosphoprotein (dspp) gene promoter . The expression of Cre recombinase protein was mainly detected in the nucleus of the odontoblasts . The efficiency of Cre activity was analyzed by crossing the Dspp-Cre mice with ROSA26 reporter (R26R) mice . The offspring with both genotypes have shown specific deletion of intervening sequences flanked by loxP sites upstream of the reporter gene, thereby facilitating the expression of the beta-galactosidase (beta-gal) gene in the teeth . The activity of beta-gal was initially observed in the odontoblasts of 1-day-old mice and increased with tooth development . Almost all of the odontoblasts have shown lacZ activity by 3 weeks of age . We could not detect Cre recombinase activity in any other cells, including ameloblasts . These studies indicate that the Dspp-Cre transgenic mice will be valuable to generate odontoblast-specific gene knockout mice so as to gain insight into the molecular roles of critical genes in the odontoblasts during dentinogenesis. J Bacteriol, 2003 Feb, 185(3), 983 - 90 Escherichia coli endoribonucleases involved in cleavage of bacteriophage T4 mRNAs; Otsuka Y et al.; The dmd mutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA . Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon . Other cleavages are independent of translation . In the present study, by introducing plasmids carrying various soc alleles, we could detect cleavages of soc RNA in uninfected cells identical to those found in dmd mutant-infected cells . We isolated five Escherichia coli mutant strains in which the dmd mutant was able to grow . One of these strains completely suppressed the dmd mutant-specific cleavages of soc RNA . The loci of the E . coli mutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing the dmd mutant-specific mRNA degradation is attributable to a novel RNase . In addition, we present evidence that 5'-truncated soc RNA, a stable form in T4-infected cells regardless of the presence of a dmd mutation, is generated by RNase E. World J Gastroenterol, 2003 Feb, 9(2), 304 - 8 A novel hepatitis B virus mutant with A-to-G at nt551 in the surface antigen gene; Chen HB et al.; AIM: Hepatitis B surface antigen (HBsAg) mutant of hepatitis B virus (HBV) is one of the important factors that result in immune escape and cause failure of immunization . In this study we reported and characterized a novel HBV mutant with A-to-G at nt551 and intended to provide theoretical data for prevention of HBV infection in China . METHODS: A methodology comprising polymerase chain reaction (PCR) amplifying, M13 bacteriophage cloning and nucleotide sequencing was used to analyze the sera of the pediatric patient who was hepatitis B (HB) immune failure . Expression plasmids containing the mutant S gene and a wild-type (adr) S gene were constructed respectively and the recombinant HBsAg were expressed in COS-7 cells under the regulation of SV40 early promoter . The recombinant proteins were investigated for their immunological reactivity with different monoclonal antibodies (mAb) against "a" determinant and vaccine-raised human neutralizing antibodies . RESULTS: It was found that there was a new point mutation at nt551 of the HBV (adr) genome from A to G, leading to a substitution of methionine (Met) to valine (Val) at position 133 in the "a" determinant of HBsAg . Compared to the wild-type HBsAg, the binding activity of the mutant HBsAg to mAbs (A6, A11 and S17) and to vaccine-raised human anti-hepatitis B surface antibody (anti-HBs) decreased significantly . CONCLUSION: According to the facts that the patient has been immunized with HB vaccine and that the serum is anti-HBs positive and HBsAg negative, and based on the nucleotide sequence analysis of the mutant HBV S gene and its alteration of antigenicity, the HBV is considered to be a new vaccine-induced immune escape mutant different from the known ones. J Mol Biol, 2003 Jan 31, 325(5), 827 - 41 Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma70 subunit of E . coli RNA polymerase and its role as a transcriptional inhibitor and co-activator; Pal D et al.; Bacteriophage T4 AsiA, a protein of 90 amino acid residues, binds to the sigma(70) subunit of Escherichia coli RNA polymerase and inhibits host or T4 early transcription or, together with the T4 MotA protein, activates T4 middle transcription . To investigate which regions within AsiA are involved in forming a complex with sigma(70) and in providing transcriptional functions we generated random mutations throughout AsiA and targeted mutations within the C-terminal region . We tested mutant proteins for their ability to complement the growth of T4 asiA am phage under non-suppressing conditions, to inhibit E . coli growth, to interact with sigma(70) region 4 in a two-hybrid assay, to bind to sigma(70) in a native protein gel, and to inhibit or activate transcription in vitro using a T4 middle promoter that is active with RNA polymerase alone, is inhibited by AsiA, and is activated by MotA/AsiA . We find that substitutions within the N-terminal half of AsiA, at amino acid residues V14, L18, and I40, rendered the protein defective for binding to sigma(70) . These residues reside at the monomer-monomer interface in recent NMR structures of the AsiA dimer . In contrast, AsiA missing the C-terminal 44 amino acid residues interacted well with sigma(70) region 4 in the two-hybrid assay, and AsiA missing the C-terminal 17 amino acid residues (Delta74-90) bound to sigma(70) and was fully competent in standard in vitro transcription assays . However, the presence of the C-terminal region delayed formation of transcriptionally competent species when the AsiA/polymerase complex was pre-incubated with the promoter in the absence of MotA . Our results suggest that amino acid residues within the N-terminal half of AsiA are involved in forming or maintaining the AsiA/sigma(70) complex . The C-terminal region of AsiA, while not absolutely required for inhibition or co-activation, aids inhibition by slowing the formation of transcription complexes between a promoter and the AsiA/polymerase complex. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Sep, 12(3), 213 - 6 {Functional phage display of human interferon alpha lc/86D}; Ma X et al.; We have successfully displayed human interferon-alpha lc/86D(IFN-alpha lc/86D) on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E) . The IFN-alpha lc/86D cDNA was fused to a DNA sequence encoding the amino-terminal domain of pIII, a minor coat protein exposed at one end of the phage . Fusion gene was packaged into phagemid particles upon superinfection with M13K07 helper phage . Expression of IFN-alpha lc/86D was verified by its reactivity with IFN-alpha l specific neutralizing antibodies and the phage displayed IFN-alpha lc86D was found to possess antiviral biological activity on VSV challenged WISH cells comparable to that of human recombinant IFN-alpha lb . These results demonstrate that IFN-alpha lc/86D can be displayed on phage in a correctly folded and functionally active form, and this system can be applicable to the sorting of a large repertoire of phage-displayed IFN-alpha lc/86D variants. Life Sci Space Res, 1973, 11, 225 - 31 Estimation of the biological danger of the very high energy component of space radiation; Akoev IG et al.; In modelling the action of the high energy component of space radiation in a space ship, the secondary radiation resulting from the interaction of 76 GeV protons with a target was used . The radiation flow consisted of neutrons, mesons of different kinds and charges, protons and gamma-quanta of wide energy spectrum . We studied the influence of radiation on the survival of E . coli B and T4Br+ bacteriophage, on the growth, dry weight and survival of Vicia faba, on the frequency of chromosome aberrations, and number of cells with abnormal mitoses, on the rate of post-irradiation recovery according to these characteristics, and also on the yield of the r-mutants of T4Br+ bacteriophage, their distribution and biochemical identification . The probability of strong interactions with intra-nuclear cascade processes was minor . Their action on the background of the main mass of radiation with an RBE of less than 1 could be considerably masked . Nevertheless, the RBE of combined secondary radiation was sufficiently greater than 137Cs radiation; from approximate curves, it was from 1.2 to 4.0 times as great and in single experimental points it was more than 5 . The spectrum of mutation was also different. Curr Microbiol, 2003 Feb, 46(2), 88 - 93 Complementation of bacteriophage induction and recombination defects in Escherichia coli RecA(-) mutants by expression of the cloned T4 bacteriophage uvsX gene; Kuhl SA et al.; Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination . Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect . The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion . These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E . coli RecA(-) mutants with a complete recA deletion . These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present. Mol Microbiol, 2003 Jan, 47(2), 397 - 409 DNA gyrase requirements distinguish the alternate pathways of Mu transposition; Sokolsky TD et al.; The MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms . The first integration event following infection occurs through a non-replicative mechanism . In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome . We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase-inhibiting drug and several distinct gyrase mutants . These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection . In contrast, gyrase inhibition causes severe defects in replicative transposition . In two of the mutants, as well as in drug-treated cells, replicative transposition is almost completely blocked . Experiments probing for formation of MuA-DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination . The findings establish that DNA structure-based signals are used differently for integrative and replicative transposition . We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA /architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle. Appl Environ Microbiol, 2003 Jan, 69(1), 170 - 6 Coevolution of bacteriophage PP01 and Escherichia coli O157:H7 in continuous culture; Mizoguchi K et al.; The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture . Following the addition of bacteriophage PP01, E . coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h(-1) and by 3 orders of magnitude at a lower dilution rate (0.327 h(-1)) . However, the appearance of a series of phage-resistant E . coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture . The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared . Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E . coli O157:H7 to escape PP01 infection . One of the escape mutants of E . coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h(-1) and persisted until the end of the experiment (approximately 200 h) . Mu mutant cells could coexist with bacteriophage PP01 in batch culture . Concentrations of the Mu cells and bacteriophage PP01 increased together . The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture . Thus, coevolution of phage and E . coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture. Viral Immunol, 2002, 15(4), 627 - 43 Chimeric bacteriophage fr virus-like particles harboring the immunodominant C-terminal region of hamster polyomavirus VP1 induce a strong VP1-specific antibody response in rabbits and mice; Voronkova T et al.; The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids . Screening of sera from HaPyV-infected papilloma-bearing and papilloma-free hamsters demonstrated the immunodominant features of all three capsid proteins . For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384 . The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions . Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1 . The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1. Vestn Oftalmol, 2002 Nov-Dec, 118(6), 38 - 40 {Effect of bacteriophage on the lipid peroxidation process and antioxidant protective enzymes in experimental uveitis}; Karimova MKh et al.; Experimental uveitis features distinct hyperlipoperoxidation in damaged eye tissues, blood serum and the liver . The activity of antioxidant defense (AOD) enzymes decreases in tissues and blood of experimental animals whereas catalase compensatorily activates in hepatic tissue . Experimental therapy of uveitis with gentamycin and bacteriophage results in reducing hyperlipoperoxidation, increased activity of AOD enzymes but no complete normalization is observed . This manifested in preservation of inflammations to a certain degree. Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 18 - 21 {Action of Spirulina platensis on bacterial viruses}; Gorobets OB et al.; The impact of the biomass of the blue-green microalga (cyanobacterium) S . platensis on bacteriophage T4 (bacterial virus) has been evaluated . The study revealed that the addition of S . platensis biomass into the agar nutrient medium, followed by sterilization with 2% chloroform and thermal treatment, produced an inhibiting or stimulating effect on the reproduction of the bacteriophage in Escherichia coli B cells, depending on the concentration of S . platensis and the multiplicity of phage infection, as well as on the fact whether the microalgae were added during the first cycle of the development of the virus . The reproduction of the bacteriophage in E . coli B was influenced by the method and duration of the sterilization of the nutrient medium with S . platensis. J Virol, 2003 Jan, 77(2), 1195 - 203 Two distinct mechanisms ensure transcriptional polarity in double-stranded RNA bacteriophages; Yang H et al.; In most double-stranded RNA (dsRNA) viruses, RNA transcription occurs inside a polymerase (Pol) complex particle, which contains an RNA-dependent RNA Pol subunit as a minor component . Only plus- but not minus-sense copies of genomic segments are produced during this reaction . In the case of phi6, a dsRNA bacteriophage from the Cystoviridae family, isolated Pol synthesizes predominantly plus strands using virus-specific dsRNAs in vitro, thus suggesting that Pol template preferences determine the transcriptional polarity . Here, we dissect transcription reactions catalyzed by Pol complexes and Pol subunits of two other cystoviruses, phi8 and phi13 . While both Pol complexes synthesize exclusively plus strands over a wide range of conditions, isolated Pol subunits can be stimulated by Mn(2+) to produce minus-sense copies on phi13 dsRNA templates . Importantly, all three Pol subunits become more prone to the native-like plus-strand synthesis when the dsRNA templates (including phi13 dsRNA) are activated by denaturation before the reaction . Based on these and earlier observations, we propose a model of transcriptional polarity in Cystoviridae controlled on two independent levels: Pol affinity to plus-strand initiation sites and accessibility of these sites to the Pol in a single-stranded form. J Biol Chem, 2003 Mar 7, 278(10), 7829 - 33 Epub 2002 Dec 24. Bacteriophage T4 Dam DNA-(N6-adenine)-methyltransferase . Processivity and orientation to the methylation target; Zinoviev VV et al.; We carried out steady state and pre-steady state (burst) kinetic analyses of the bacteriophage T4 Dam DNA-(N(6)-adenine)-methyltransferase (MTase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to Ade in oligonucleotide duplexes containing one or two specific GATC sites with different combinations of methylated and unmodified targets . We compared the results for ligated 40-mer duplexes with those of the mixtures of the two unligated duplexes used to generate the 40-mers . The salient results are as follows: (i) T4 Dam MTase modifies 40-mer duplexes in a processive fashion . (ii) During processive movement, T4 Dam rapidly exchanges product S-adenosyl-l-homocysteine (AdoHcy) for substrate AdoMet without dissociating from the DNA duplex . (iii) T4 Dam processivity is consistent with an ordered bi-bi mechanism AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow . However, in contrast to the steady state, here DNA(Me) upward arrow signifies departure from a methylated site GMTC upward arrow without physically dissociating from the DNA . (iv) Following methyl transfer at one site and linear diffusion to a hemimethylated site, a reconstituted T4 Dam-AdoMet complex rapidly reorients itself to the (productive) unmethylated strand . T4 Dam-AdoHcy cannot reorient at an enzymatically created GMTC site . (v) The inhibition potential of fully methylated sites 5'-GMTC/5'-GMTC is much lower for a long DNA molecule compared with short single-site duplexes. J Biol Chem, 2003 Feb 28, 278(9), 7247 - 56 Epub 2002 Dec 20. The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7; Hyland EM et al.; Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival . Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA . However, there is no experimental evidence to support this hypothesis . Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein . This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain . Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides . A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA . A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein . The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Delta26C mediates the reaction more effectively than does wild-type . Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently . With the exception of gp2.5-Delta26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase. Mol Microbiol, 2003 Jan, 47(1), 171 - 82 Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C-terminal domain; Mukhopadhyay B et al.; Rapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease-hypersensitive repressors (Vir) with an altered C-terminal domain (CTD) . Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild-type repressor CTD modulate Vir-induced degradation by Escherichia coli ClpXP protease and DNA binding by the N-terminal DNA-binding domain (DBD) . Although deletions of the repressor C-terminus resulted in both resistance to ClpXP protease and suppression of a temperature-sensitive DBD mutation (cts62), some cysteine-replacement mutations in the CTD elicited only one of the two phenotypes . Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild-type repressor, and at least two mutant repressors protected Vir from proteolysis . One protease-resistant mutant became susceptible to Vir-induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease-sensitive conformation . Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor . The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding. Arch Virol, 2002 Dec, 147(12), 2419 - 29 Remarkable morphological diversity of viruses and virus-like particles in hot terrestrial environments; Rachel R et al.; Electron microscopic studies of the viruses in two hot springs (85 degrees C, pH 1.5-2.0, and 75-93 degrees C, pH 6.5) in Yellowstone National Park revealed particles with twelve different morphotypes . This diversity encompassed known viruses of hyperthermophilic archaea, filamentous Lipothrixviridae, rod-shaped Rudiviridae, and spindle-shaped Fuselloviridae, and novel morphotypes previously not observed in nature . Two virus types resembled head-and-tail bacteriophages from the families Siphoviridae and Podoviridae, and constituted the first observation of these viruses in a hydrothermal environment . Viral hosts in the acidic spring were members of the hyperthermophilic archaeal genus Acidianus. Arch Virol, 2002 Dec, 147(12), 2261 - 79 RNA-protein interactions in spherical viruses; Bink HH et al.; The three-dimensional structure of many non-enveloped spherical RNA viruses has been determined in great detail, mainly using X-ray crystallography . Great insight in the structure of the protein capsid has been obtained, but much less information is available about the secondary and tertiary structure of the RNA in situ, due to a number of methodological problems . In this paper the current knowledge about RNA-protein interactions and the folding of the RNA is reviewed, with a special emphasis on the plant virus Turnip yellow mosaic virus . A major characteristic of many spherical RNA viruses appears to be the positioning of A-type double helical segments of 7-9 basepairs at icosahedral symmetry axes, probably interacting via its phosphates with basic amino acid residues of the coat protein in a sequence-independent manner . It is only in the case of the RNA bacteriophages that we know in atomic detail how an RNA hairpin interacts with the coat protein. Biophys Chem, 2002 Dec 10, 101-102, 475 - 84 Investigation of viral DNA packaging using molecular mechanics models; Arsuaga J et al.; A simple molecular mechanics model has been used to investigate optimal spool-like packing conformations of double-stranded DNA molecules in viral capsids with icosahedral symmetry . The model represents an elastic segmented chain by using one pseudoatom for each ten basepairs (roughly one turn of the DNA double helix) . Force constants for the various terms in the energy function were chosen to approximate known physical properties, and a radial restraint was used to confine the DNA into a sphere with a volume corresponding to that of a typical bacteriophage capsid . When the DNA fills 90% of the spherical volume, optimal packaging is obtained for coaxially spooled models, but this result does not hold when the void volume is larger . When only 60% of the spherical volume is filled with DNA, the lowest energy structure has two layers, with a coiled core packed at an angle to an outer coaxially spooled shell . This relieves bending strain associated with tight curvature near the poles in a model with 100% coaxial spooling . Interestingly, the supercoiling density of these models is very similar to typical values observed in plasmids in bacterial cells . Potential applications of the methodology are also discussed . Biophys Chem, 2002 Dec 10, 101-102, 43 - 56 A helix initiation signal in T4 lysozyme identified by polyalanine mutagenesis; Zhang XJ et al.; To better understand the relation between sequence and structure, and in an attempt to simplify the protein folding problem, a series of alanine substitutions was introduced into bacteriophage T4 lysozyme . In contrast to previous studies in this system, which were restricted to single alpha-helices, the present analysis included a helix-turn-helix region, a loop-helix region, and two alpha-helices that were well separated in the three-dimensional structure . It was shown previously that T4 lysozyme is very tolerant of alanine substitutions within alpha-helices, especially at solvent-exposed sites . The present study shows that the protein is also tolerant of such substitutions in turn and loop regions, although less than in helices . The results confirm that the structural information in the amino acid sequence is highly redundant . For example, the protein with the sequence 127AAAAAALAAAAWAAA141 folds normally, has melting temperature only 0.8 degrees C lower than wildtype, and has a crystal structure that is also very similar to wildtype . Polyalanine substitutions within turns or loops can, however, lead to differences in structure and in folding . In one example the triple substitution K35A/S36A/P37A caused this region of the molecule to change to a more helical conformation . In a second case the mutant with the sequence 34AAAAALAAAKAALAAA49, which spans a loop-helix region, had a dramatically altered thermal unfolding transition, suggesting that this region may tend to form a single, uninterrupted, helix . Substitution of Ala38 in the above construct with aspartic acid caused the unfolding to be more like wildtype, suggesting that residue 38, which is at a helix-capping position in the wildtype structure, provides an initiation signal that is essential in the polyalanine mutant for the correct formation of alpha-helix 39-50 . In a typical protein, the information that codes for the 3D structure is presumably distributed over many amino acids . The present results suggest that in simplified sequences the key folding information may be restricted to a subset of critical residues, and so be more readily accessible to experimental analysis . Biochemistry, 2002 Dec 24, 41(51), 15404 - 9 Mn2+ is a native metal ion activator for bacteriophage lambda protein phosphatase; Reiter TA et al.; Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11 . lambdaPP can be activated several-fold by various divalent metal ions, with Mn(2+) and Ni(2+) providing the most significant activation . Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP in vivo . In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of lambdaPP . Escherichia coli cells overexpressing lambdaPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express lambdaPP . Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate . EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn(2+) and dinuclear {(Mn(2+))(2)} species bound to lambdaPP . Spin quantitation of EPR spectra of intact E . coli cells overexpressing lambdaPP indicates the presence of approximately 40 microM mononuclear Mn(2+)-lambdaPP and 60 microM {(Mn(2+))(2)}-lambdaPP . The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-{(Mn(2+))(2)} metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E . coli. Plant Physiol, 2002 Dec, 130(4), 1686 - 96 Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization; Yim YS et al.; Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73 . High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda . The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences . The use of libraries from multiple enzymes increased the chance of recovering each region of the genome . Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map . All of the clones used as hybridization probes detected at least three BACs . Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones . This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 17043 - 8 Epub 2002 Dec 12. Genomic diversity of enterohemorrhagic Escherichia coli O157 revealed by whole genome PCR scanning; Ohnishi M et al.; Enterohemorrhagic Escherichia coli O157 is one of the leading worldwide public health concerns, causing large outbreaks of hemorrhagic colitis as well as numerous small outbreaks and sporadic cases . The variability of restriction enzyme-digestion patterns of O157 genomes, which is widely used to distinguish strains in the molecular epidemiology of O157 infections, suggests the presence of some genomic diversity among the strains . Based on the complete genome sequence of O157 Sakai, we analyzed the whole genome structures of eight O157 strains displaying diverse XbaI-digestion patterns by a systematic PCR analysis that we have named whole genome PCR scanning . This analysis identified not only the O157-specific sequences that are highly conserved among the strains, but also revealed an unexpectedly high degree of genomic diversity . In particular, prophages, including Shiga toxin-transducing phages, exhibited extensive structural and positional diversity, implying that variation of bacteriophages is a major factor in generating genomic diversity among the O157 lineage. J Biol Chem, 2003 Feb 21, 278(8), 6251 - 7 Epub 2002 Dec 11. A novel S100 target conformation is revealed by the solution structure of the Ca2+-S100B-TRTK-12 complex; McClintock KA et al.; The Alzheimer-linked neural protein S100B is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner . Upon binding calcium, a conformational change occurs in S100B exposing a hydrophobic surface for target protein interactions . The synthetic peptide TRTK-12 (TRTKIDWNKILS), derived from random bacteriophage library screening, bears sequence similarity to several intermediate filament proteins and has the highest calcium-dependent affinity of any target molecule for S100B to date (K(d) <1 microm) . In this work, the three-dimensional structure of the Ca(2+)-S100B-TRTK-12 complex has been determined by NMR spectroscopy . The structure reveals an extended, contiguous hydrophobic surface is formed on Ca(2+)-S100B for target interaction . The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp(7), and interacts with multiple hydrophobic contacts in helices III and IV of Ca(2+)-S100B . This interaction is strikingly different from the alpha-helical structures found for other S100 target peptides . By using the TRTK-12 interaction as a guide, in combination with other available S100 target structures, a recognition site on helix I is identified that may act in concert with the TRTK-12-binding site from helices III and IV . This would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for S100B target protein interactions. Biochemistry, 2002 Dec 17, 41(50), 14820 - 30 Affinity and sequence specificity of DNA binding and site selection for primer synthesis by Escherichia coli primase; Khopde S et al.; Primase is an essential DNA replication enzyme in Escherichia coli and responsible for primer synthesis during lagging strand DNA replication . Although the interaction of primase with single-stranded DNA plays an important role in primer RNA and Okazaki fragment synthesis, the mechanism of DNA binding and site selection for primer synthesis remains unknown . We have analyzed the energetics of DNA binding and the mechanism of site selection for the initiation of primer RNA synthesis on the lagging strand of the replication fork . Quantitative analysis of DNA binding by primase was carried out using a number of oligonucleotide sequences: oligo(dT)(25) and a 30 bp oligonucleotide derived from bacteriophage G4 origin (G4ori-wt) . Primase bound both sequences with moderate affinity (K(d) = 1.2-1.4 x 10(-)(7) M); however, binding was stronger for G4ori-wt . G4ori-wt contained a CTG trinucleotide, which is a preferred site for initiation of primer synthesis . Analysis of DNA binding isotherms derived from primase binding to the oligonucleotide sequences by fluorescence anisotropy indicated that primase bound to DNA as a dimer, and this finding was further substantiated by electrophoretic mobility shift assays (EMSAs) and UV cross-linking of the primase-DNA complex . Dissection of the energetics involved in the primase-DNA interaction revealed a higher affinity of primase for DNA sequences containing the CTG triplet . This sequence preference of primase may likely be responsible for the initiation of primer synthesis in the CTG triplet sites in the E . coli lagging strand as well as in the origin of replication of bacteriophage G4. J Mol Biol, 2003 Jan 3, 325(1), 85 - 97 phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is required for a stable and functional interaction with the terminal protein; Rodriguez I et al.; Bacteriophage phi29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication . By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx(2)h motif, and a glutamate belonging to the Exo III motif . Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of phi29 DNA polymerase . Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished . As a consequence, very few initiation products were obtained, and essentially no transition products were detected . Interestingly, phi29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules . These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps. J Mol Biol, 2003 Jan 3, 325(1), 11 - 24 Structural studies of bacteriophage alpha3 assembly; Bernal RA et al.; Bacteriophage alpha3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include phiX174 . These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein . The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein . Assembly proceeds via a 108S empty procapsid that requires the external D and internal B scaffolding proteins for its formation.The alpha3 "open" procapsid structural intermediate was determined to 15A resolution by cryo-electron microscopy (cryo-EM) . Unlike the phiX174 "closed" procapsid and the infectious virion, the alpha3 open procapsid has 30A wide pores at the 3-fold vertices and 20A wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein . The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging . Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid . Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30A wide pores.The structure of the alpha3 mature virion was solved to 3.5A resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure . The main differences between the alpha3 and phiX174 virion structures are in the spike and the DNA-binding proteins . The alpha3 pentameric spikes have a rotation of 3.5 degrees compared to those of phiX174 . The alpha3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the phiX174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein . The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in alpha3 compared to phiX174, allowing the building of about 10% of the ribose-phosphate backbone. J Mol Biol, 2002 Dec 13, 324(5), 1003 - 14 Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12; Inman KG et al.; The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276: TRTKIDWNKILS) . This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL) . As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding . The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B-TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding . However, helical TRTK-12 in the S100B-peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100-peptide complexes . The three-dimensional structure of the S100B-TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B-peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity . A comparison of the S100B-TRTK peptide structure to the structures of apo- and Ca(2+)-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca(2+)-bound S100B as necessary to bind the TRTK-12 peptide. J Biochem Mol Biol, 2002 Nov 30, 35(6), 637 - 41 Identification of bacteriophage K11 genomic promoters for K11 RNA polymerase; Han KG et al.; Only one natural promoter that interacts with bacteriophage K11 RNA polymerase has so far been identified . To identify more, in the present study restriction fragments of the phage genome were individually assayed for transcription activity in vitro . The K11 genome was digested with two 4-bp-recognizing restriction enzymes, and the fragments cloned in pUC119 were assayed with purified K11 RNA polymerase . Eight K11 promoterbearing fragments were isolated and sequenced . We report that the nine K11 promoter sequences (including the one previously identified) were highly homologous from -17 to +4, relative to the initiation site at +1 . Interestingly, five had -10G and -8A, while the other four had -10A and -8C . The consensus sequences with the natural -10G/-8A and -10A/-8C, and their variants with -10G/-8C and -10A/-8A, showed nearly equal transcription activity, suggesting residues at -10 and -8 do not regulate promoter activity . Using hybridization methods, physical positions of the cloned promoter-bearing sequences were mapped on SalIand KpnI-restriction maps of the K11 genome . The flanking sequences of six cloned K11 promoters were found to be orthologous with T7 or T3 genomic sequences. Int J Biochem Cell Biol, 2003 Jan, 35(1), 16 - 21 The tail lysozyme complex of bacteriophage T4; Arisaka F et al.; The tail baseplate of bacteriophage T4 contains a structurally essential, three-domain protein encoded by gene 5 in which the middle domain possesses lysozyme activity . The gene 5 product (gp5) undergoes post-translational cleavage, allowing the resultant N-terminal domain (gp5*) to assemble into the baseplate as a trimer . The lysozyme activity of the undissociated cleaved gp5 is inhibited until infection has been initiated, when the C-terminal portion of the molecule is detached and the rest of the molecule dissociates into monomers . The 3D structure of the undissociated cleaved gp5, complexed with gp27 (another component of the baseplate), shows that it is a cell-puncturing device that functions to penetrate the outer cell membrane and to locally dissolve the periplasmic cell wall. J Biol Chem, 2003 Feb 14, 278(7), 4618 - 27 Epub 2002 Dec 03. Isolation and characterization of T4 bacteriophage gp17 terminase, a large subunit multimer with enhanced ATPase activity; Baumann RG et al.; Phage T4 terminase is a two-subunit enzyme that binds to the prohead portal protein and cuts and packages a headful of concatameric DNA . To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein . Following cleavage and release of gp17 from chitin, two additional column steps completed purification . The purification yielded (i) homogeneous soluble gp17 highly active in in vitro DNA packaging ( approximately 10% efficiency, >10(8) phage/ml of extract); (ii) gp17 lacking endonuclease and contaminating protease activities; and (iii) a DNA-independent ATPase activity stimulated >100-fold by the terminase small subunit, gp16 (18 kDa), and modestly by portal gp20 and single-stranded binding protein gp32 multimers . Analyses revealed a preparation of highly active and slightly active gp17 forms, and the latter could be removed by immunoprecipitation using antiserum raised against a denatured form of the gp17 protein, leaving a terminase with the increased specific activity (approximately 400 ATPs/gp17 monomer/min) required for DNA packaging . Analysis of gp17 complexes separated from gp16 on glycerol gradients showed that a prolonged enhanced ATPase activity persisted after exposure to gp16, suggesting that constant interaction of the two proteins may not be required during packaging. J Mol Biol, 2002 Dec 6, 324(4), 791 - 805 Regulation of directionality in bacteriophage lambda site-specific recombination: structure of the Xis protein; Sam MD et al.; Upon induction of a bacteriophage lambda lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host . A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision . Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function . Xis adopts an unusual "winged"-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single alpha-helix and loop structure ("wing"), respectively . The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA . We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase-Xis-DNA complex required for phage excision. J Mol Biol, 2002 Dec 6, 324(4), 775 - 89 Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase; Sarkar D et al.; The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites . The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region . Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes . We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites . However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein . We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively . To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region . We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites . Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination. Plasmid, 2002 Nov, 48(3), 202 - 12 When phage, plasmids, and transposons collide: genomic islands, and conjugative- and mobilizable-transposons as a mosaic continuum; Osborn AM et al.; Plasmids and bacteriophage represent the classical vectors for gene transfer within the horizontal gene pool . However, the more recent discovery of an increasing array of other mobile genetic elements (MGE) including genomic islands (GIs), conjugative transposons (CTns), and mobilizable transposons (MTns) which each integrate within the chromosome, offer an increasingly diverse assemblage contributing to bacterial adaptation and evolution . Molecular characterisation of these elements has revealed that they are comprised of functional modules derived from phage, plasmids, and transposons, and further that these modules are combined to generate a continuum of mosaic MGE . In particular, they are comprised of any one of three distinct types of recombinase, together with plasmid-derived transfer and mobilisation gene functions . This review highlights both the similarities and distinctions between these integrating transferable elements resulting from combination of the MGE toolbox. Plasmid, 2002 Nov, 48(3), 174 - 8 The P1 plasmid in action: time-lapse photomicroscopy reveals some unexpected aspects of plasmid partition; Li Y et al.; The prophage of bacteriophage P1 is a low copy number plasmid in Escherichia coli and is segregated to daughter cells by an active partition system . The dynamics of the partition process have now been successfully followed by time-lapse photomicroscopy . The process appears to be fundamentally different from that previously inferred from statistical analysis of fixed cells . A focus containing several plasmid copies is captured at the cell center . Immediately before cell division, the copies eject bi-directionally along the long axis of the cell . Cell division traps one or more plasmid copies in each daughter cell . These copies are free to move, associate, and disassociate . Later, they are captured to the new cell center to re-start the cycle . Studies with mutants suggest that the ability to segregate accurately at a very late stage in the cell cycle is dependent on a novel ability of the plasmid to control cell division . Should segregation be delayed, cell division is also delayed until segregation is successfully completed. Biochemistry (Mosc), 2002 Oct, 67(10), 1124 - 35 Structural-functional analysis of bacteriophage T7 RNA polymerase; Tunitskaya VL et al.; This review summarizes our results of the structural and functional studies of bacteriophage T7 DNA-dependent RNA polymerase (T7 RNAP) . Particular features of this enzyme (the single-subunit composition, relatively low molecular weight) make it the most convenient model for investigating the physicochemical aspects of transcription . The review discusses the main properties of T7 RNAP, interaction between the enzyme and promoter, principle stages of T7-transcription, and also the results of structural and functional studies by affinity modification and both random and site-directed mutagenesis techniques. J Biol Chem, 2003 Feb 7, 278(6), 3876 - 81 Epub 2002 Nov 27. Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69; Sun S et al.; The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis . DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome . To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering . We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions . Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action . We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable . The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction. Biochimie, 2002 Sep, 84(9), 925 - 44 RNA loop-loop interactions as dynamic functional motifs; Brunel C et al.; RNA loop-loop interactions are frequently used to trigger initial recognition between two RNA molecules . In this review, we present selected well-documented cases that illustrate the diversity of biological processes using RNA loop-loop recognition properties . The first one is related to natural antisense RNAs that play a variety of regulatory functions in bacteria and their extra-chromosomal elements . The second one concerns the dimerization of HIV-1 genomic RNA, which is responsible for the encapsidation of a diploid RNA genome . The third one concerns RNA interactions involving double-loop interactions . These are used by the bicoid mRNA to form dimers, a property that appears to be important for mRNA localization in drosophila embryo, and by bacteriophage phi29 pRNA which forms hexamers that participate in the translocation of the DNA genome through the portal vertex of the capsid . Despite the high diversity of systems and mechanisms, some common features can be highlighted . (1) Efficient recognition requires rapid bi-molecular binding rates, regardless of the RNA pairing scheme . (2) The initial recognition is favored by particular conformations of the loops enabling a proper presentation of nucleotides (generally a restricted number) that initiate the recognition process . (3) The fate of the initial reversible loop-loop complex is dictated by both functional and structural constraints . RNA structures have evolved either to "freeze" the initial complex, or to convert it into a more stable one, which involves propagation of intermolecular interactions along topologically feasible pathways . Stabilization of the initial complex may also be assisted by proteins and/or formation of additional contacts. Genetics, 2002 Nov, 162(3), 1045 - 53 Scanning mutagenesis identifies amino acid residues essential for the in vivo activity of the Escherichia coli DnaJ (Hsp40) J-domain; Genevaux P et al.; The DnaJ (Hsp40) cochaperone regulates the DnaK (Hsp70) chaperone by accelerating ATP hydrolysis in a cycle closely linked to substrate binding and release . The J-domain, the signature motif of the Hsp40 family, orchestrates interaction with the DnaK ATPase domain . We studied the J-domain by creating 42 mutant E . coli DnaJ variants and examining their phenotypes in various separate in vivo assays, namely, bacterial growth at low and high temperatures, motility, and propagation of bacteriophage lambda . Most mutants studied behaved like wild type in all assays . In addition to the (33)HisProAsp(35) (HPD) tripeptide found in all known functional J-domains, our study uncovered three new single substitution mutations (Y25A, K26A, and F47A) that totally abolish J-domain function . Furthermore, two glycine substitution mutants in an exposed flexible loop (R36G, N37G) showed partial loss of J-domain function alone and complete loss of function as a triple (RNQ-GGG) mutant coupled with the phenotypically silent Q38G . Interestingly, all the essential residues map to a small region on the same solvent-exposed face of the J-domain . Engineered mutations in the corresponding residues of the human Hdj1 J-domain grafted in E . coli DnaJ also resulted in loss of function, suggesting an evolutionarily conserved interaction surface . We propose that these clustered residues impart critical sequence determinants necessary for J-domain catalytic activity and reversible contact interface with the DnaK ATPase domain. Genetics, 2002 Nov, 162(3), 1019 - 30 Coordination of DNA ends during double-strand-break repair in bacteriophage T4; Stohr BA et al.; The extensive chromosome replication (ECR) model of double-strand-break repair (DSBR) proposes that each end of a double-strand break (DSB) is repaired independently by initiating extensive semiconservative DNA replication after strand invasion into homologous template DNA . In contrast, several other DSBR models propose that the two ends of a break are repaired in a coordinated manner using a single repair template with only limited DNA synthesis . We have developed plasmid and chromosomal recombinational repair assays to assess coordination of the broken ends during DSBR in bacteriophage T4 . Results from the plasmid assay demonstrate that the two ends of a DSB can be repaired independently using homologous regions on two different plasmids and that extensive replication is triggered in the process . These findings are consistent with the ECR model of DSBR . However, results from the chromosomal assay imply that the two ends of a DSB utilize the same homologous repair template even when many potential templates are present, suggesting coordination of the broken ends during chromosomal repair . This result is consistent with several coordinated models of DSBR, including a modified version of the ECR model. Genetics, 2002 Nov, 162(3), 1003 - 18 Dissecting the fidelity of bacteriophage RB69 DNA polymerase: site-specific modulation of fidelity by polymerase accessory proteins; Bebenek A et al.; Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3' exonuclease . Crystal structures have been determined for this enzyme with and without DNA substrates . We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol(+) Exo(+)) enzyme, an exonuclease-deficient mutator variant (Pol(+) Exo(-)), mutator variants with substitutions at Tyr(567) in the polymerase active site (Pol(M) Exo(+)), and the double mutator Pol(M) Exo(-) . Comparing the mutational spectra of the Pol(+) Exo(-) and Pol(+) Exo(+) enzymes revealed the patterns and efficiencies of proofreading, while Tyr(567) was identified as an important determinant of base-selection fidelity . Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro . Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different . Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others. Phys Rev Lett . 2002 Nov 4;89(19):198102 . Epub 2002 Oct 17. Insulating behavior of lambda-DNA on the micron scale; Zhang Y et al.; We have investigated the electrical conductivity of lambda-DNA using DNA covalently bonded to Au electrodes . Thiol-modified dTTP was incorporated into the "sticky" ends of bacteriophage lambda-DNA using DNA polymerase . Two-probe measurements on such molecules provide a hard lower bound for the resistivity rho>10(6)Omega cm at bias potentials up to 20 V, in conflict with recent claims of moderate to high conductivity . By direct imaging, we show that the molecules are present after the measurements . We stress the importance of eliminating salt residues in these measurements. Genes Genet Syst, 2002 Oct, 77(5), 301 - 8 Role of Escherichia coli Hfq in late-gene silencing of bacteriophage T4 dmd mutant; Ueno H et al.; When the dmd gene of bacteriophage T4 is mutated, many T4 late genes are post-transcriptionally silenced because of rapid mRNA degradation . Here we show that the host hfq gene is involved in the rapid mRNA degradation in a dmd mutant . A disruption of the hfq gene caused weak but significant effects on the stability of late-gene mRNA, the late-gene expression and the growth of a dmd mutant . By probing with the soc gene, we found that disruption of the hfq gene impaired the translation-independent mRNA degradation, one of two mechanisms promoting rapid mRNA degradation . We also showed that purified Hfq protein bound stoichiometrically to soc RNA . These results strongly suggest that the hfq gene has a stimulatory role in dmd mutant-specific mRNA degradation. Protein Sci, 2002 Dec, 11(12), 2899 - 908 Modifying specificity of antidigoxin antibodies using insertional mutagenesis; Krykbaev RA et al.; Certain antibodies (Abs) elicited using the cardiac glycoside digoxin (digoxigenin tridigitoxoside) bind preferentially to analogs that differ from digoxin by substitutions on the cardenolide rings, the lactone, or by the presence or absence of attached sugars . Antibody 26-10 binds equally well to digoxin and digitoxin, which differ only by the presence in the former and the absence in the latter of an hydroxyl group at C12 . Other antidigoxin Abs, however, can distinguish between these ligands by three orders of magnitude in binding . Inspection of the structure of Fab 26-10 complexed with digoxin shows a gap in complementarity in the region between the digoxin O12 and LCDR3 . We proposed that insertions in LCDR3 might result in Abs that bind digitoxin preferentially . We produced libraries of mutants displayed on bacteriophage which were randomized at LCDR3 and contained LCDR3 insertions . Mutants were selected by panning against digoxin and analogs . The mutants bound digitoxin preferentially up to 47-fold greater than digoxin . The mutants that bound well to digitoxin demonstrated a consensus sequence including the substitution of Trp at position L:94 . Using site-directed mutagenesis, the binding to digitoxin was shown to be maximized by the combination of an insertion and L:Trp94 mutation, moving the L 94 side chain closer to digoxin . We also selected mutants that bound preferentially to gitoxin, which, like digitoxin, lacks the 12-hydroxyl, increasing relative binding to gitoxin up to 600-fold compared to the unmutated Ab 26-10. J Mol Biol, 2002 Nov 22, 324(2), 297 - 307 E.coli cell-cycle regulation by bacteriophage lambda; Sergueev K et al.; We re-examined the old but surprising claim of Kourilsky and Knapp that transient expression of genes located downstream of the p(L) promoter of bacteriophage lambda can induce cell-cycle synchrony in a population of Escherichia coli cells . Although we were unable to reproduce a lasting synchrony, a cessation of division, followed by one or two fairly synchronous cell divisions was observed . This line up of the cell cycle was found to be due to two genetically separable events: a temporary block of cell division and, at the same time, a block to the initiation of new rounds of DNA replication . These blocks then release after about one mass doubling so that chromosome replication and cell division occur during a short time interval in all the cells in the population . The cell division block is a result of the transient expression of the lambda kil gene . The block to initiation of DNA replication requires a region that we term bin (blocks initiation) immediately upstream of the xis gene . The region consists of ea22 and ea8.5 and two small open reading frames (ORFs) that flank them . Deletion-substitution mutagenesis suggests that all four ORFs may be required for the initiation block . The ability of the phage to modify two aspects of the host cell cycle presumably reflects a stratagem that provides the phage with an advantage for lysogeny or lytic growth. Virology, 2002 Oct 25, 302(2), 433 - 44 Structural analysis of vaccinia virus DIs strain: application as a new replication-deficient viral vector; Ishii K et al.; DIs is a restrictive host range mutant of vaccinia virus strain DIE that grows well only in chick embryo fibroblast cells but is unable to grow in most mammalian cells . In this study, we identified one major deletion (15.4 kbp) which results in the loss of 19 putative open reading frames in the left end of the genome . We then established a system to express foreign genes by inserting them into the deleted region of DIs . We constructed rDIs to express the bacteriophage T7 polymerase (T7pol) gene and showed the expression in various mammalian cell lines by reporter luciferase gene expression under the T7 promoter . We also expressed the full-length human immunodeficiency virus (HIV)-1 NL432 gag gene . The expressed gag gene product induced high levels of cytotoxic T lymphocytes in immunized mice . These data suggest that DIs is useful as an efficient, transient replication-deficient viral vector. Res Microbiol, 2002 Oct, 153(8), 511 - 8 Characterization of the cts4 repressor mutation in transposable bacteriophage Mu; Rousseau P et al.; Mucts4 was isolated more than 30 years ago and was the first available thermoinducible derivative of transposable phage Mu . We have characterized the cts4 mutation and the corresponding mutant protein . Contrary to previously characterized thermoinducible Mu prophages (e.g., Mucts62), Mucts4 lysogenizes at reduced frequency even at 30 degrees C . The cts4 mutation (Leu129Val) was located in this central repressor region . The cts4 protein was thermosensitive for operator DNA binding in vitro . Temperature-dependent changes in protein-protein cross-linking patterns in the absence of DNA were detected for purified wild type, cts62 and cts4 repressor proteins . The cts4 protein exhibited a subtly different electrophoretic profile, which became more marked at higher temperatures, from both the wild type and cts62 . In addition the cts4 repressor generated a significantly different pattern of binding to DNA fragments carrying the early operator region . Consistent with the predicted involvement of the central leucine-rich region of the Mu repressor in the formation of multimeric forms, the cts4 mutation thus appeared to affect protein-protein interactions. Res Microbiol, 2002 Oct, 153(8), 493 - 501 Breaking free: "protein antibiotics" and phage lysis; Bernhardt TG et al.; Bacteriophages must destroy the bacterial cell wall to lyse their host and release their progeny into the environment . There are at least two distinct mechanisms by which phages destroy the cell wall . Bacteriophages with large genomes use a holin-endolysin system, while bacteriophages with small genomes encode a single lysis protein . Three unrelated single protein lysis systems are known and these proteins will be the focus of the review . Recent results indicate that at least two of these proteins inhibit cell wall synthesis and are thus the phage analogs of antibiotics like penicillin. Biochem Biophys Res Commun, 2002 Nov 22, 299(1), 57 - 61 Non-AUG translation initiation of mRNA encoding plastid-targeted phage-type RNA polymerase in Nicotiana sylvestris; Kobayashi Y et al.; A third nuclear gene encoding a bacteriophage T7-type RNA polymerase, NsRpoT-C, was isolated and characterized from Nicotiana sylvestris . The gene, NsRpoT-C, consists of 21 exons and 20 introns and encodes a polypeptide of 977 amino acid residues . The predicted NsRpoT-C protein shows the highest identity (72% amino acid identity) with Arabidopsis thaliana RpoT;3 which is a plastid-targeted protein . Surprisingly, comparison of the deduced amino acid sequence of NsRpoT-C with that of A . thaliana RpoT;3 predicted that the NsRpoT-C starts at a CUG triplet, a rare translation initiation codon . Transient expression assays in protoplasts from tobacco leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-C encodes a targeting signal directing the protein into chloroplasts . This strongly suggests that NsRpoT-C functions as an RNA polymerase transcribing plastid-encoded genes . We have designated this protein NsRpoTp. J Exp Bot, 2002 Dec, 53(379), 2341 - 9 Plastid genes transcribed by the nucleus-encoded plastid RNA polymerase show increased transcript accumulation in transgenic plants expressing a chloroplast-localized phage T7 RNA polymerase; Magee AM et al.; A gene fusion encoding a plastid-targeted bacteriophage T7 RNA polymerase (T7RNAP) under the transcriptional control of the light-regulated promoter and the plastid-targeting signals of a ribulose-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene was introduced into the nuclear genome of Nicotiana tabacum (tobacco) . Immunoblot analysis, in vitro transcription assays and protease treatment of isolated chloroplasts revealed that T7RNAP activity was localized within chloroplasts . RNA gel blot analyses showed a substantial increase in transcript abundance for several plastid genes that are normally transcribed by the nucleus-encoded plastid RNA polymerase (NEP) including rpoC1, rpl33, rps18, rps12, and clpP . By contrast, no significant changes were observed in the levels of psbD, 16SrDNA, and ndhA transcripts . These results suggest a possible direct or indirect T7RNAP-mediated enhancement of transcription of a subset of plastid genes that contain NEP promoters . Despite these alterations in plastid transcript levels, the plants showed no visible abberant phenotype. Annu Rev Genet, 2002, 36, 361 - 88 Epub 2002 Jun 11. Genetic engineering using homologous recombination; Court DL et al.; In the past few years, in vivo technologies have emerged that, due to their efficiency and simplicity, may one day replace standard genetic engineering techniques . Constructs can be made on plasmids or directly on the Escherichia coli chromosome from PCR products or synthetic oligonucleotides by homologous recombination . This is possible because bacteriophage-encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 50 base pairs . This technology, termed recombineering, is providing new ways to modify genes and segments of the chromosome . This review describes not only recombineering and its applications, but also summarizes homologous recombination in E . coli and early uses of homologous recombination to modify the bacterial chromosome . Finally, based on the premise that phage-mediated recombination functions act at replication forks, specific molecular models are proposed. J Biol Chem, 2003 Jan 31, 278(5), 3145 - 52 Epub 2002 Nov 09. Protein-protein interactions in the bacteriophage T4 replisome . The leading strand holoenzyme is physically linked to the lagging strand holoenzyme and the primosome; Ishmael FT et al.; The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA . This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer . These units interact further to form the complete replisome . The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome . The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit . The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes . Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex . The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation. Biochim Biophys Acta, 2002 Dec 12, 1579(2-3), 196 - 202 The Shiga-toxin VT2-encoding bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome; De Greve H et al.; The plaque-forming VT2-encoding lambdoid bacteriophage varphi297 was isolated from a Belgian clinical Escherichia coli O157:H7 isolate . PCR walking, starting from the int gene of phage varphi297, demonstrated that the varphi297 prophage integrated in the yecE gene of a lysogenic E . coli K12 strain . This integration site, in E . coli K12 and in the original clinical O157:H7 isolate, was confirmed by PCR using primers flanking this site . The excisionase protein of phage varphi297 is identical to the excisionase of VT1-encoding phage VT1-Sakai, while the integrases, which are 82% identical, show significant sequence divergence in the central and C-terminal region . This can explain the different integration sites of both prophages . The activity of the integrase was proven by its ability to mediate the integration of a suicide plasmid, carrying the attachment site of varphi297, at the appropriate position in the E . coli chromosome. J Bacteriol, 2002 Dec, 184(23), 6522 - 31 Programmed translational frameshift in the bacteriophage P2 FETUD tail gene operon; Christie GE et al.; The major structural components of the P2 contractile tail are encoded in the FETUD tail gene operon . The sequences of genes F(I) and F(II), encoding the major tail sheath and tail tube proteins, have been reported previously (L . M . Temple, S . L . Forsburg, R . Calendar, and G . E . Christie, Virology 181:353-358, 1991) . Sequence analysis of the remainder of this operon and the locations of amber mutations Eam30, Tam5, Tam64, Tam215, Uam25, Uam77, Uam92, and Dam6 and missense mutation Ets55 identified the coding regions for genes E, T, U, and D, completing the sequence determination of the P2 genome . Inspection of the DNA sequence revealed a new open reading frame overlapping the end of the essential tail gene E . Lack of an apparent translation initiation site and identification of a putative sequence for a programmed translational frameshift within the E gene suggested that this new reading frame (E') might be translated as an extension of gene E, following a -1 translational frameshift . Complementation analysis demonstrated that E' was essential for P2 lytic growth . Analysis of fusion polypeptides verified that this reading frame was translated as a -1 frameshift extension of gpE, with a frequency of approximately 10% . The arrangement of these two genes within the tail gene cluster of phage P2 and their coupling via a translational frameshift appears to be conserved among P2-related phages . This arrangement shows a striking parallel to the organization in the tail gene cluster of phage lambda, despite a lack of amino acid sequence similarity between the tail gene products of these phage families. Biochimie, 2002 May-Jun, 84(5-6), 413 - 21 Analysis of the Escherichia coli Tol-Pal and TonB systems by periplasmic production of Tol, TonB, colicin, or phage capsid soluble domains; Bouveret E et al.; The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the periplasm of Escherichia coli and between them and colicins or g3p protein of filamentous bacteriophages . Domains of colicins or periplasmic soluble domains of Tol or TonB proteins can be artificially addressed to the periplasm of bacteria by fusing them to a signal sequence from an exported protein . These domains interact specifically in the periplasm with the Tol or TonB complexes and disturb their function, which can be directly detected by the appearance of specific tol or tonB phenotypes . This technique can be used to detect new interactions, to characterize them biochemically and to map them or to induce tol or tonB phenotypes to study the functions of these two complexes. Nature, 2002 Nov 7, 420(6911), 43 - 50 Epub 2002 Oct 09. Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution; Tahirov TH et al.; The single-subunit bacteriophage T7 RNA polymerase carries out the transcription cycle in an identical manner to that of bacterial and eukaryotic multisubunit enzymes . Here we report the crystal structure of a T7 RNA polymerase elongation complex, which shows that incorporation of an 8-base-pair RNA-DNA hybrid into the active site of the enzyme induces a marked rearrangement of the amino-terminal domain . This rearrangement involves alternative folding of about 130 residues and a marked reorientation (about 130 degrees rotation) of a stable core subdomain, resulting in a structure that provides elements required for stable transcription elongation . A wide opening on the enzyme surface that is probably an RNA exit pathway is formed, and the RNA-DNA hybrid is completely buried in a newly formed, deep protein cavity . Binding of 10 base pairs of downstream DNA is stabilized mostly by long-distance electrostatic interactions . The structure implies plausible mechanisms for the various phases of the transcription cycle, and reveals important structural similarities with the multisubunit RNA polymerases. J Mol Biol, 2002 Nov 15, 324(1), 17 - 34 Protein-protein and protein-DNA interactions of sigma70 region 4 involved in transcription activation by lambdacI; Nickels BE et al.; The cI protein of bacteriophage lambda (lambdacI) activates transcription from promoter P(RM) through an acidic patch on the surface of its DNA-binding domain . Genetic evidence suggests that this acidic patch stimulates transcription from P(RM) through contact with the C-terminal domain (region 4) of the sigma(70) subunit of Escherichia coli RNA polymerase . Here, we identify two basic residues in region 4 of sigma(70) that are critical for lambdacI-mediated activation of transcription from P(RM) . On the basis of structural modeling, we propose that one of these sigma(70) residues, K593, facilitates the interaction between lambdacI and region 4 of sigma(70) by inducing a bend in the DNA upstream of the -35 element, whereas the other, R588, interacts directly with a critical acidic residue within the activating patch of lambdacI . Residue R588 of sigma(70) has been shown to play an important role in promoter recognition; our findings suggest that the R588 side-chain has a dual function at P(RM), facilitating the interaction of region 4 with the promoter -35 element and participating directly in the protein-protein interaction with lambdacI. Water Res, 2002 Oct, 36(17), 4235 - 42 Fluorescent dye labeled bacteriophages--a new tracer for the investigation of viral transport in porous media: 2 . Studies of deep-bed filtration; Gitis V et al.; Viral transport in deep-bed sand filters was studied by a new method that enables rapid and simple quantitation of labeled viruses . The residence time distribution (RTD) of viruses in the bed was compared to the RTD of a fluorescein dye under conditions that simulate a filter run . The characteristics of the RTD curves for the free dye and the labeled bacteriophages followed very different trends during the filter run . While the retention time of free dye was practically independent of the filtration stage, the average retention time of the labeled bacteriophage depended in a non-linear way on filtration time . Average virus retention time as well as virus-removal efficiency were minimal at the ripening stage, increased during the operational stage and then decreased again towards the turbidity breakthrough stage . This complex trend reflects two opposing mechanisms that dominate the behavior of the filter . During the ripening stage the accumulation of the kaolin-alum material in the filter increases the adsorption surface area and retards virus mobility . After sufficient kaolin-alum deposit is accumulated in the filter, aging and densification of the alum deposit induces size exclusion phenomenon giving faster apparent mobility of viruses in the filter bed. Genes Genet Syst, 2002 Aug, 77(4), 219 - 25 Scarce adenylation in bacteriophage T4 mRNAs; Yonesaki T; The degradation of mRNA is crucial for the rapid shift of bacteriophage T4 gene expression from early to late . The present study was conducted to investigate whether T4 mRNA is polyadenylated or not, because polyadenylation is known to facilitate the degradation of mRNA in Escherichia coli cells . Total RNA extracted from T4-infected cells was subjected to self-circularization or intermolecular ligation by T4 RNA ligase, and a region containing the 3'-5' junction was amplified by RT-PCR . Cloning and sequencing as well as the length distribution of amplified DNA fragments revealed no adenines at the 3'-ends of uvsY and soc RNAs . The present result suggests that T4 mRNA is not significantly adenylated. J Vet Med Sci, 2002 Oct, 64(10), 927 - 31 Long-term excretion of Shiga toxin-producing Escherichia coli (STEC) and experimental infection of a sheep with O157; Kim S et al.; To investigate a long-term shedding of Shiga toxin (Stx)-producing Escherichia coli (STEC) from sheep, a fifteen-month study for STEC isolation from a sheep, which had yielded STEC before, was attempted . The sheep continued to shed STEC and 39 STEC were isolated . The number of STEC in the feces was estimated at 1.7 x 10(3) per gram . In addition, although Stx1-negative O157 and stx2-encoding bacteriophage were experimentally infected to the sheep, Stx-positive O157 or Stx2- producing bacterial cells were not detected . The genetical and biochemical characterization of those 39 STEC strains showed that all STEC strains produced Shiga toxin 1 (Stx1) and were divided into three classes (I to III) . From phylogenetic analysis of their amino acid sequences, class-I STEC was classified as group 1 comprising mainly human STEC, and classes II/III were as group 2 comprising sheep STEC . Our results suggest that STEC easily colonized in sheep and that the sheep continued to shed STEC, showing that sheep might be an important reservoir for human STEC infection. Trends Microbiol, 2002 Nov, 10(11), 521 - 9 Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved; Boyd EF et al.; There are common themes among bacteriophage-encoded virulence factors, which include the well-characterized bacterial toxins and proteins that alter antigenicity as well as several new classes of bacteriophage-encoded proteins such as superantigens, effectors translocated by a type III secretion system, and proteins required for intracellular survival and host cell attachment . These virulence factors are encoded by a diversity of bacteriophages, members of the viral families Siphoviridae, Podoviridae, Myoviridae and Inoviridae, with some bacteriophages having characteristics of more than one virus family . The location of virulence genes within the bacteriophage genomes is non-random and consistent with an origin via imprecise prophage excision or as either transferable cassettes or integral components of the bacteriophage genome. J Mol Biol, 2002 Nov 1, 323(4), 685 - 700 Crp1p, a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae; Rass U et al.; A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity . Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified . They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1) . Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site . Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity . The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background . This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p . The DNA-binding domain of Crp1p was mapped to positions 120-141 . This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs) . As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4. Nat Rev Mol Cell Biol, 2002 Nov, 3(11), 826 - 35 Motors and switches: AAA+ machines within the replisome; Davey MJ et al.; Clamp loaders are required to load the ring-shaped clamps that tether replicative DNA polymerases onto DNA . Recently solved crystal structures, along with a series of biochemical studies, have provided a detailed understanding of the clamp loading reaction . In particular, studies of the Escherichia coli clamp loader--an AAA+ machine--have provided insights into the architecture of clamp loaders from eukaryotes, bacteriophage T4 and archaea . Other AAA+ proteins are also involved in the initiation of DNA replication, and studies of the E . coli clamp loader indicate mechanisms by which these proteins might function. Bioorg Med Chem, 2002 Dec, 10(12), 4057 - 65 A cell-penetrating peptide from a novel pVII-pIX phage-displayed random peptide library; Gao C et al.; A novel random peptide library was constructed using a phage-display format on the coat proteins pVII and pIX of filamentous bacteriophage . Panning against B-lymphocyte WI-L2 cells yielded one unique peptide-phage, denoted CHL8, that specifically bound to and penetrated the cells . Studies of each peptide derived from CHL8, denoted pep7 and pep9, established that only pep7 mediated the observed activity and only as a homodimer . Peptide libraries displayed on pVII-pIX should serve as a novel source of bioactive ligands for a variety of applications. Neurosci Res, 2002 Nov, 44(3), 255 - 65 Identification of brain proteins that interact with 2-methylnorharman . An analog of the parkinsonian-inducing toxin, MPP+; Gearhart DA et al.; N-Methylated beta-carbolines, including 2-methylnorharman, are structural and functional analogs of the parkinsonian-inducing toxin, MPP+ . We are investigating N-methylated beta-carbolines, including 2-methylnorharman, as possible etiologic factors in the pathogenesis of Parkinson's disease . The cellular targets of N-methylated beta-carboline-mediated cytotoxicity are unknown; therefore, we used the T7Select Phage Display System in a novel approach to identify brain proteins that bind to 2-methylnorharman . We incubated (biopanned) immobilized 2-methylnorharman with a phage display cDNA library that expressed a library of human brain proteins on the surface of bacteriophage T7 . We washed off unbound phage, amplified the phage that were bound to 2-methylnorharman, and enriched for toxin-interacting phage by repeating the biopanning and amplification steps . The cDNA sequences from the toxin-interacting phage were used to derive the amino acid sequences of the phage-displayed proteins . Five of the six 2-methylnorharman-interacting proteins may have relevance to Parkinson's disease: alpha-tubulin, paraoxonase, dorfin, fatty acid binding protein, and platelet-activating factor acetylhydrolase . Dorfin has sequence homology with parkin, which is interesting because mutations in the parkin gene associate with early-onset Parkinson's disease . Our findings are the basis for future studies aimed at determining whether 2-methylnorharman affects the function of these specific proteins in vitro and in vivo. Poult Sci, 2002 Oct, 81(10), 1486 - 91 Prevention of Escherichia coli infection in broiler chickens with a bacteriophage aerosol spray; Huff WE et al.; Bacteriophage to an Escherichia coli isolate that is pathogenic in poultry were isolated from municipal sewer treatment facilities or poultry processing plants . Three studies were conducted to determine the efficacy of aerosol administration of bacteriophage to prevent an E . coli respiratory infection in broiler chickens . In all three studies the experimental design consisted of nine treatments with three replicate pens of 10 birds . Three treatments were not challenged with E . coli and consisted of unsprayed birds, birds sprayed with a diluent control, and birds sprayed with a combination of two bacteriophages . Six treatments were challenged with E . coli by injecting 10(4) cfu into the thoracic air sac when birds were 7, 8, or 10 d of age after being sprayed at 7 d of age with either a diluent control or a combination of two bacteriophages . In Studies 1 and 2, BW at 2 wk of age of all the birds challenged with E . coli, regardless of spray treatment, were decreased significantly from the unchallenged controls, except in Study 2 for the birds sprayed with bacteriophage and challenged at 10 d of age . There was a significant decrease in mortality in Studies 1 and 2 when the birds were challenged with E . coli immediately after bacteriophage administration and in Study 2 in birds challenged at 10 d of age . In Study 3 a suspected pre-existing E . coli infection resulted in mortality in the unchallenged, unsprayed controls, and in the diluent sprayed controls of 20 and 27%, respectively . The mortality in the unchallenged bacteriophage sprayed birds was 3%, representing a significant decrease . Mortality in Study 3 was significantly decreased in the bacteriophage-sprayed birds challenged with E . coli immediately or 1 d later but not 3 d after bacteriophage administration . The decrease in BW at 2 wk of age in challenged birds indicates that bacteriophage treatment did not provide complete protection; however, in all three studies mortality was significantly decreased, indicating that aerosol spray of bacteriophage may be practical for administration of bacteriophage and may provide an alternative to the use of antibiotics in poultry production. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14758 - 63 Epub 2002 Oct 31. The structure and evolution of the major capsid protein of a large, lipid-containing DNA virus; Nandhagopal N et al.; Paramecium bursaria Chlorella virus type 1 (PBCV-1) is a very large, icosahedral virus containing an internal membrane enclosed within a glycoprotein coat consisting of pseudohexagonal arrays of trimeric capsomers . Each capsomer is composed of three molecules of the major capsid protein, Vp54, the 2.0-A resolution structure of which is reported here . Four N-linked and two O-linked glycosylation sites were identified . The N-linked sites are associated with nonstandard amino acid motifs as a result of glycosylation by virus-encoded enzymes . Each monomer of the trimeric structure consists of two eight-stranded, antiparallel beta-barrel, "jelly-roll" domains related by a pseudo-sixfold rotation . The fold of the monomer and the pseudo-sixfold symmetry of the capsomer resembles that of the major coat proteins in the double-stranded DNA bacteriophage PRD1 and the double-stranded DNA human adenoviruses, as well as the viral proteins VP2-VP3 of picornaviruses . The structural similarities among these diverse groups of viruses, whose hosts include bacteria, unicellular eukaryotes, plants, and mammals, make it probable that their capsid proteins have evolved from a common ancestor that had already acquired a pseudo-sixfold organization . The trimeric capsid protein structure was used to produce a quasi-atomic model of the 1,900-A diameter PBCV-1 outer shell, based on fitting of the Vp54 crystal structure into a three-dimensional cryoelectron microscopy image reconstruction of the virus. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14722 - 7 Epub 2002 Oct 31. Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor; Nanduri B et al.; Helicases are molecular motor enzymes that unwind and translocate nucleic acids . One of the central questions regarding helicase activity is whether the process of coupling ATP hydrolysis to DNA unwinding requires an oligomeric form of the enzyme . We have applied a pre-steady-state kinetics approach to address this question with the bacteriophage T4 Dda helicase . If a helicase can function as a monomer, then the burst amplitude in the pre-steady state might be similar to the concentration of enzyme, whereas if the helicase required oligomerization, then the amplitude would be significantly less than the enzyme concentration . DNA unwinding of an oligonucleotide substrate was conducted by using a Kintek rapid quench-flow instrument . The substrate consisted of 12 bp adjacent to 12 nucleotides of single-stranded DNA . Dda (4 nM) was incubated with substrate (16 nM) in buffer, and the unwinding reaction was initiated by the addition of ATP (5 mM) and Mg(2+) (10 mM) . The reaction was stopped by the addition of 400 mM EDTA . Product formation exhibited biphasic kinetics, and the data were fit to the equation for a single exponential followed by a steady state . The amplitude of the first phase was 3.5 +/- 0.2 nM, consistent with a monomeric helicase . The burst amplitude of product formation was measured over a range of enzyme and substrate concentrations and remained consistent with a functional monomer . Thus, Dda can rapidly unwind oligonucleotide substrates as a monomer, indicating that the functional molecular motor component of a helicase can reside within a single polypeptide. EMBO J, 2002 Nov 1, 21(21), 5815 - 23 The phage N4 virion RNA polymerase catalytic domain is related to single-subunit RNA polymerases; Kazmierczak KM et al.; In vitro, bacteriophage N4 virion RNA polymerase (vRNAP) recognizes in vivo sites of transcription initiation on single-stranded templates . N4 vRNAP promoters are comprised of a hairpin structure and conserved sequences . Here, we show that vRNAP consists of a single 3500 amino acid polypeptide, and we define and characterize a transcriptionally active 1106 amino acid domain (mini-vRNAP) . Biochemical and genetic characterization of this domain indicates that, despite its peculiar promoter specificity and lack of extensive sequence similarity to other DNA-dependent RNA polymerases, mini-vRNAP is related to the family of T7-like RNA polymerases. J Clin Microbiol, 2002 Nov, 40(11), 4313 - 6 Rapid assessment of phenotypic resistance to protease inhibitors in human immunodeficiency virus type 1 group O; Rodes B et al.; A bacteriophage lambda-based method was used to investigate the development of resistance to protease inhibitors (PI) in one subject infected with human immunodeficiency virus (HIV) type 1 group O who underwent multiple treatment regimens over a period of 4 years . A reduction in the susceptibility to indinavir of 6-fold and a reduction in the susceptibility to saquinavir of 24-fold were recognized after long exposure to these drugs with respect to baseline . The emergence of PI resistance corresponded to the selection of amino acid changes L10V, G48M, F53L, I54V, and L90M at the protease . The results were concordant with those obtained by a drug susceptibility assay with primary HIV isolates. J Clin Microbiol, 2002 Nov, 40(11), 4010 - 4 Molecular detection and seroepidemiology of the Chlamydia pneumoniae bacteriophage (PhiCpn1); Karunakaran KP et al.; Recent whole-genome analysis has demonstrated limited genetic variation in Chlamydia pneumoniae, with one strain (AR39) containing a 4,524 nucleotide single-stranded DNA bacteriophage, PhiCpn1 . Using PCR, reverse transcription (RT)-PCR, and Western blotting, we confirmed the presence and functional expression of PhiCpn1 in C . pneumoniae strain AR39 and its absence in strain CWL029 . Six additional epidemiologically distinct clinical isolates of C . pneumoniae also did not contain PhiCpn1 . We generated recombinant viral protein 1 (Vp1) from PhiCpn1 in Escherichia coli and showed that Vp1 antigen is highly immunogenic in mice and that murine antisera readily recognize native Vp1 from C . pneumoniae strain AR39 elementary bodies (EB) . We developed an enzyme-linked immunosorbent assay (ELISA) to measure antibodies to recombinant Vp1 in human sera collected from 32 patients with abdominal aortic aneurysm (AAA) and 40 controls . Among the 72 subjects, 61 had C . pneumoniae EB antibodies shown by ELISA . Antibodies to Vp1 were found in 39 of the 61 (64%) seropositive individuals and were significantly correlated with AAA (adjusted odds ratio, 13.9; 95% confidence interval, 1.1 to 175) . Our studies indicate that phage-containing strains of C . pneumoniae are uncommonly found by isolation but may commonly infect individuals with vascular disease. Mol Cell, 2002 Sep, 10(3), 611 - 22 The sigma(70) subunit of RNA polymerase is contacted by the (lambda)Q antiterminator during early elongation; Nickels BE et al.; The Q protein of bacteriophage lambda is a transcription antiterminator that modifies the elongation properties of E . coli RNA polymerase (RNAP) . To do this, DNA-bound (lambda)Q must first engage a paused elongation complex . Here we show that this engagement of (lambda)Q with RNAP involves an interaction between (lambda)Q and sigma(70), demonstrating that sigma(70) can be a target of regulation during elongation . Furthermore, we provide evidence that this interaction between (lambda)Q and sigma(70) stabilizes a conformation of RNAP that requires the disengagement of a segment of sigma(70) from the core enzyme . Recent structure-based models posit that the transition from the initiation to the elongation phase of transcription involves the staged displacement of sigma(70) from the RNAP core . Our findings provide support for this proposal. J Struct Biol, 2002 Aug, 139(2), 103 - 12 Diffraction quality crystals of PRD1, a 66-MDa dsDNA virus with an internal membrane; Bamford JK et al.; It has proved difficult to obtain well diffracting single crystals of macromolecular complexes rich in lipid . We report here the path that has led to crystals of the bacteriophage PRD1, a particle containing approximately 2,000 protein subunits from 18 different protein species, around 10 of which are integral membrane proteins associated with a host-derived lipid bilayer of some 12,500 lipid molecules . These crystals are capable of diffracting X-rays to Bragg spacings below 4A . It is hoped that some lessons learned from PRD1 will be applicable to other lipidic systems and that these crystals will allow, as a proof of principle, the determination of the structure of the virus in terms of a detailed atomic model. J Invest Dermatol, 2002 Oct, 119(4), 865 - 9 Selection of mimotopes of the cell surface adhesion molecule Mel-CAM from a random pVIII-28aa phage peptide library; Hafner C et al.; The cell surface adhesion molecule Mel-CAM is highly expressed in advanced primary and metastatic melanoma . Mel-CAM was first described as an integral membrane glycoprotein of malignant melanoma cells . The murine monoclonal antibody MAd18-5D7 recognizes an epitope of the extracellular domain of Mel-CAM and is able to enhance Mel-CAM mediated adhesion of melanoma cells in aggregation assays . For the characterization of peptides that antigenically mimic surface-exposed areas of Mel-CAM we screened a newly constructed random pVIII-28aa bacteriophage peptide library against MAd18-5D7 . After three panning rounds a population of phages binding to MAd18-5D7 was enriched . Peptides expressed on the surface of these phages were then tested for their specificity for the antibody's antigen binding site . DNA sequences coding for two specific peptide ligands were determined . One of the deduced amino acid sequences showed similarity to a portion of the sequence of the third immunoglobulin-like extracellular domain of Mel-CAM . Both peptides blocked the interaction of MAd18-5D7 with Mel-CAM present in a MelJuSo melanoma cell line lysate . Phage displayed as well as synthetic peptides inhibited in a dose-dependent manner the binding of MAd18-5D7 to recombinant Mel-CAM in enzyme-linked immunosorbent assay experiments . No such inhibition was observed using a panel of other anti-Mel-CAM antibodies . Our results clearly indicate that these 28mer peptides are structural equivalents of the MAd18-5D7 epitope of Mel-CAM and that they will be useful tools for further in vitro and in vivo studies of Mel-CAM mediated cell-cell interaction. J Contam Hydrol, 2002 Oct, 58(3-4), 243 - 59 Column experiments to study nonlinear removal of bacteriophages by passage through saturated dune sand; Schijven JF et al.; In a recent field study on dune recharge, bacteriophages MS2 and PRD1 were found to be removed 3 log10 over the first 2.4 m and only 5 log10 over the next 27 m . To understand the causes of this nonlinear removal, column experiments were carried out under conditions similar to the field: same recharge water, temperature (5 +/- 3 degrees C) and pore water velocity (1.5 m day(-1)) . Soil samples were taken along a streamline between the recharge canal and the first monitoring well . Bacteriophage phiX174 was included for comparison . The high initial removal in the field was found not to be due to heterogeneity of phage suspensions but to soil heterogeneity . Phage removal rates correlated strongly positively with soil organic carbon content, and relatively strongly positively with silt content and the presence of ferric oxyhydroxides . Soil organic carbon content, silt content and the presence of ferric oxyhydroxides were found to decrease exponentially with travel distance . Removal rates of phiX174 were found to be 3-10 times higher than those of MS2 and PRD1 due to the lower electrostatic repulsion that the less negatively charged phiX174 experiences . It is suggested that the high initial removal in the field is due to the presence of favorable sites for attachment formed by ferric oxyhydroxides that decrease exponentially with travel distance . Similar removal rates may be found at both laboratory and field scale . However, due to local variations at field scale detailed knowledge on soil heterogeneity may be needed to enable a reliable prediction of removal. Genetics, 2002 Oct, 162(2), 543 - 56 Focused genetic recombination of bacteriophage t4 initiated by double-strand breaks; Shcherbakov V et al.; A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segCDelta strain of bacteriophage T4 was developed . The ets1, a 66-bp DNA fragment of phage T2L containing the cleavage site for the T4 SegC site-specific endonuclease, was inserted into the proximal part of the T4 rIIB gene . Under segC(+) conditions, the ets1 behaves as a recombination hotspot . Crosses of the ets1 against rII markers located to the left and to the right of ets1 gave similar results, thus demonstrating the equal and symmetrical initiation of recombination by either part of the broken chromosome . Frequency/distance relationships were studied in a series of two- and three-factor crosses with other rIIB and rIIA mutants (all segC(+)) separated from ets1 by 12-2100 bp . The observed relationships were readily interpretable in terms of the modified splice/patch coupling model . The advantages of this localized or focused recombination over that distributed along the chromosome, as a model for studying the recombination-replication pathway in T4 in vivo, are discussed. Biotechniques, 2002 Oct, 33(4), 806 - 10, 812 Bacteriophage gene targeting vectors generated by transplacement; Aoyama C et al.; A rate-determining step in gene targeting is the generation of the targeting vector . We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction . Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination . We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement . Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage . In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible. J Virol, 2002 Nov, 76(22), 11770 - 4 Altered DNA mutation spectrum in aflatoxin b1-treated transgenic mice that express the hepatitis B virus x protein; Madden CR et al.; Humans chronically infected with hepatitis B virus (HBV) are at further risk of liver cancer upon exposure to dietary aflatoxin B1 (AFB1), a carcinogenic product of the mold Aspergillus flavus . For the present study, we utilized double-transgenic mice (ATX mice) that express the HBV X protein (HBx) and possess a bacteriophage lambda transgene to evaluate the in vivo effect of HBx expression on AFB1-induced DNA mutations . The expression of HBx correlated with a 24% increase in mutation frequency overall and an approximately twofold increase in the incidence of G/C-to-T/A transversion mutations following AFB1 exposure . These results are consistent with a model in which expression of HBx during chronic HBV infection may contribute to the development of hepatocellular carcinoma following exposure to environmental carcinogens. Acta Virol, 2002, 46(2), 69 - 74 Cloning of pac gene and its flanking regions using mini-Mu bacteriophage; Vizvariova M et al.; An in vitro method for cloning and mapping Escherichia coli genes by means of mini-Mu phage and its application to the penicillin G acylase (pac) gene of E . coli PAC2 strain with its flanking regions is described . The gene was marked by insertion of a fragment bearing kanamycin resistance (Km(r)) . Most of Km(r) clones obtained from mini-Mu transductants contained the whole pac gene with its flanking regions . Localization of pac gene to 98.5 min of E . coli PAC2 chromosome was confirmed by an in vivo P1 phage transduction. Acta Virol, 2002, 46(2), 57 - 62 Mutations in bacteriophage T4 genome; Switala-Jelen K et al.; Bacteriophage (phage) T4 belonging to T-even phages is one of the best known phages with a completely deciphered genome sequence . As a model of living systems, T4 phage has many technical advantages . It can be very easily grown in large quantities, manipulated by classical genetics, and engineered by site-directed mutagenesis . Many substances have been first tested for mutagenicity in T-even phages . The results of these tests were very often applicable to higher organisms due to similar mechanisms of mutagenesis . T4 phage is also important in phage therapy, which represents an alternative treatment of bacterial infections since the bacterial resistance to antibiotics has become a serious medical problem . The site-directed mutagenesis is a method that enables to introduce mutations which can influence phage affinity to bacteria and can be a practical technique for enriching phage collections and for widening specificity of phages for new bacterial strains now insensitive to phage therapy. Sheng Wu Gong Cheng Xue Bao, 2002 Jul, 18(4), 497 - 500 {Expression of cre gene in Escherichia coli and bioassay its expression product}; Wang LX et al.; The Cre recombinase from bacteriophage P1 can recognize specific DNA sequences, cleave DNA at specific target sites, and then ligate it to the cleaved DNA of a second site . In this study, cre gene was cloned into the pGEM-T Easy vector via PCR procedure . Then the cre gene was inserted into an expression vector pET-29a and expressed in E . coli BL21 (DE3) . A 38 kD soluble protein was expressed and named CRE . CRE was purified by DEAE-52 chromatography . Bioassay of the partially purified product showed that CRE can cleave the plasmid pGLGFP which contains two loxP site with the same direction. Nucleic Acids Res, 2002 Oct 15, 30(20), 4387 - 97 Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates; Kadyrov FA et al.; Replication complexes were reconstituted using the eight purified bacteriophage T4 replication proteins and synthetic circular 70-, 120- or 240-nt DNA substrates annealed to a leading-strand primer . To differentiate leading strands from lagging strands, the circular parts of the substrates lacked dCMP; thus, no dCTP was required for leading-strand synthesis and no dGTP for lagging-strand synthesis . The size of the substrates was crucial, the longer substrates supporting much more DNA synthesis . Leading and lagging strands were synthesized in a coupled manner . Specifically targeting leading-strand synthesis by decreasing the concentration of dGTP decreased the rate of extension of leading strands . However, blocking lagging-strand synthesis by lowering the dCTP concentration, by omitting dCTP altogether, by adding ddCTP, or with a single abasic site had no immediate effect on the rate of extension of leading strands. Environ Sci Technol, 2002 Oct 1, 36(19), 4017 - 24 Effect of surfactants on the survival and sorption of viruses; Chattopadhyay D et al.; There is an increasing concern about the protection of groundwater from contamination by enteric viruses and the prevention of outbreaks of waterborne diseases . Knowledge of survivability and transport of viruses from their point of origin is necessary to determine their potential effects on the neighboring groundwater systems . The distribution of virus is, in turn, dependent on the physical and chemical compositions of the surrounding soil and subsurface systems . For the present study, we have determined the effects of different surfactants (cationic, anionic, nonionic, and biological) and natural organic matter (NOM) on bacteriophages . Results indicated that surfactants and NOM adversely affect phage survival in binary systems, with surfactants being the most harmful . Studies with ternary systems also showed that the presence of surfactants reduced sorption of phages on sorbents either by occupying available sorption sites on the sorbent material or by displacing the sorbed phages from the sorbent surface . Water contact angles of the selected phages and different sorbent surfaces have been measured . Experimental data demonstrated that the sorption of hydrophobic viruses was favored by hydrophobic sorbents, while the sorption of hydrophilic viruses was favored by hydrophilic sorbents. J Biol Chem, 2002 Dec 27, 277(52), 50643 - 53 Epub 2002 Oct 12. Essential amino acid residues in the single-stranded DNA-binding protein of bacteriophage T7 . Identification of the dimer interface; Rezende LF et al.; Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein . T7 phage with gene 2.5 deleted can grow only on Escherichia coli cells that express gene 2.5 from a plasmid . This complementation assay was used to screen for lethal mutations in gene 2.5 . By screening a library of randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethal in vivo . The location of these essential residues within the three-dimensional structure of gene 2.5 protein assists in the identification of motifs in the protein . In this study we show that a subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure . Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the wild-type gene 2.5 protein exists as a dimer . The basis of the lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase. Arch Virol, 2002 Oct, 147(10), 2025 - 37 Rapid pathotyping of Newcastle disease virus using a single-chain Fv displayed on phage against the C-terminal end of the F2 polypeptide; Li Y et al.; Filamentous bacteriophage display technology has been used to generate specific antibody fragments for differentiating virulent and avirulent Newcastle disease virus . A single-chain Fv fragment to the motif (112)RRQ(114), present at the F2 C-terminal end of many virulent Newcastle disease virus isolates, was isolated from a phage display library derived from a rabbit immunized with a peptide conjugate . An ELISA evaluation was carried out to test its ability to differentiate between 11 avirulent and 34 virulent NDV isolates . The antibody fragment reacted with 25/28 virulent viruses with the putative motif (112)RRQ(114) . The three exceptions were viruses with an arginine instead of glycine, at position 110 of the fusion protein, just preceding the cleavage site . Five of six virulent isolates, whose predicted motif was different from that usually found in virulent strains, also tested negative . However, the antibody did react with one isolate with the motif (112)KRQ(114) . There was no apparent reactivity with any of the avirulent isolates tested . We conclude that this antibody may, in the future, be a useful aid for the pathotyping of NDV isolates. J Struct Biol, 2002 Jul, 139(1), 46 - 54 Preliminary crystallographic analysis of the bacteriophage P22 portal protein; Cingolani G et al.; Portal proteins are components of large oligomeric dsDNA pumps connecting the icosahedral capsid of tailed bacteriophages to the tail . Prior to the tail attachment, dsDNA is actively pumped through a central cavity formed by the subunits . We have studied the portal protein of bacteriophage P22, which is the largest connector characterized among the tailed bacteriophages . The molecular weight of the monomer is 82.7 kDa, and it spontaneously assembles into an oligomeric structure of approximately 1.0 MDa . Here we present a preliminary biochemical and crystallographic characterization of this large macromolecular complex . The main difficulties related to the crystallization of P22 portal protein lay in the intrinsic dynamic nature of the portal oligomer . Recombinant connectors assembled from portal monomers expressed in Escherichia coli form rings of different stoichiometry in solution, which cannot be separated on the basis of their size . To overcome this intrinsic heterogeneity we devised a biochemical purification that separates different ring populations on the basis of their charge . Small ordered crystals were grown from drops containing a high concentration of the kosmotropic agent tert-butanol and used for data collection . A preliminary crystallographic analysis to 7.0-A resolution revealed that the P22 portal protein crystallized in space group I4 with unit cell dimensions a=b=409.4A, c=260.4A . This unit cell contains a total of eight connectors . Analysis of the noncrystallographic symmetry by the self-rotation function unambiguously confirmed that bacteriophage P22 portal protein is a dodecamer with a periodicity of 30 degrees . The cryo-EM reconstruction of the dodecahedral bacteriophage T3 portal protein will be used as a model to initiate phase extension and structure determination. Curr Protein Pept Sci, 2000 Sep, 1(2), 155 - 69 Phage display of antibody fragments; Pini A et al.; In recent years, phage display of peptides and proteins has become a very popular method in oncology, immunology, protein engineering and ligand-receptor studies among others . Antibody fragments, as Fabs or single chain Fv, have been among the first proteins to be displayed on the surface of a filamentous bacteriophage with a procedure initially described in 1990 by McCafferty et al . (Nature, 348, 552-554) . From that time, molecular biology techniques have allowed the creation of large repertoires of antibody fragments from antibody V genes, bypassing hybrydoma technology and even immunisation . A large number of phage antibody libraries, from which molecules of the desired functional properties can be rapidly selected, have been built and distributed in many laboratories world-wide . Antibody fragments recovered from phage libraries generally show moderate binding strength; with different systems of biopanning binders can be obtained with dissociation constant ranged between 10-(5) to 10-(8) M . Nevertheless, antibody fragments can be furtherly modified to improve affinity or avidity, respectively by mutating crucial residues of complementarity determining regions or by increasing the number of binding sites making dimeric, trimeric or multimeric molecules . Here, we summarise the latest progress in this field, with particular reference to applications of scFv in the diagnosis and therapy of solid tumours and in the molecular mimicry of viral antigens and membrane receptors . In fact, the production of artificial protein epitopes by phage antibodies is becoming a valid system to overcome problems caused by difficult cloning and low expression of particular recombinant proteins. Mol Microbiol, 2002 Sep, 45(6), 1631 - 46 The DNA site utilized by bacteriophage P22 for initiation of DNA packaging; Wu H et al.; Virion proteins recognize their cognate nucleic acid for encapsidation into virions through recognition of a specific nucleotide sequence contained within that nucleic acid . Viruses like bacteriophage P22, which have partially circularly permuted, double-stranded virion DNAs, encapsidate DNA through processive series of packaging events in which DNA is recognized for packaging only once at the beginning of the series . Thus a single DNA recognition event programmes the encapsidation of multiple virion chromosomes . The protein product of P22 gene 3, a terminase component, is thought to be responsible for this recognition . The site on the P22 genome that is recognized by the gene 3 protein to initiate packaging series is called the pac site . We report here a strategy for assaying pac site activity in vivo, and the utilization of this system to identify and characterize the site genetically . It is an asymmetric site that spans 22 basepairs and is located near the centre of P22 gene 3. Anal Chem, 2002 Sep 15, 74(18), 4653 - 61 Dissociation of multiple protein ion charge states following a single gas-phase purification and concentration procedure; He M et al.; The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone . This approach is illustrated using off-resonance collisional activation of the {M + 8H}8+ to {M + 6H}6+ precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the {M + 8H}8+ charge state . This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the {M + 12H}12+ to {M + 5H}5+ ions . Gas-phase ion/ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra . The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages . Maximum structural information is obtained by fragmentation of the intermediate-charge states. Appl Environ Microbiol, 2002 Oct, 68(10), 4979 - 85 Excretion of human beta-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli; Jeong KJ et al.; Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation . From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E . coli . The ompF gene of E . coli BL21(DE3) was first knocked out by using the red operon of bacteriophage lambda to construct E . coli MBEL-BL101 . For the excretion of human beta-endorphin as a model protein, the beta-endorphin gene was fused to the C terminus of the E . coli ompF gene by using a linker containing the Factor Xa recognition site . To develop a fed-batch culture condition that allows efficient production of OmpF-beta-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined . Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h) . Under this condition, up to 5.6 g of OmpF-beta-endorphin fusion protein per liter was excreted into culture medium . The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield beta-endorphin, which was finally purified by reverse-phase chromatography . From 2.7 liters of culture supernatant, 545.4 mg of beta-endorphin was obtained.
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