J Microsc, 2005 Jan, 217(Pt 1), 83 - 92 Single-particle visualization of assembly: I . Dimerization in a planar zone; Wang H et al.; Summary Single-particle fluorescence microscopy of association/dissociation is required for analysis of biological assembly reactions . Toward achieving this goal, Wang et al . (J . Microsc., 2004, 213, 101-109) used molten agarose to concentrate thermally diffusing particles in a thin zone of solution next to the surface of a coverglass (plane of concentration) . The present study details the first real-time, single-particle analysis of the association/dissociation of thermally diffusing particles in the plane of concentration . The test particles were procapsids of bacteriophage lambda (radius = 31 nm) . Quantification of thermal motion was developed and used to determine whether co-diffusing particles were bound to each other . The data are explained by (1) the presence of a molten agarose-generated barrier that is 93-155 nm from the coverglass surface, and (2) non-random orientation of procapsid dimers in the plane of concentration. Nucleic Acids Res . 2005 Jan 14;33(1):e10. Covalent antibody display--an in vitro antibody-DNA library selection system; Reiersen H et al.; The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone . This enzyme has now been exploited as a new in vitro display tool for antibody fragments . We have constructed genetic fusions of P2A with single-chain antibodies (scFvs) . Linear DNA of these fusion proteins were processed in an in vitro coupled transcription-translation mixture of Escherichia coli S30 lysate . Complexes of scFv-P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning . We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes . This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA-protein complexes. Chem Res Toxicol, 2005 Jan 17, 18(1), 12 - 18 Urea Lesion Formation in DNA as a Consequence of 7,8-Dihydro-8-oxoguanine Oxidation and Hydrolysis Provides a Potent Source of Point Mutations; Henderson PT et al.; The DNA oxidation product 7,8-dihydro-8-oxoguanine (8-oxoG) forms several mutagenic oxidation products, including a metastable oxaluric acid (Oa) derivative . We report here that a synthetic oligonucleotide containing Oa hydrolyzes under simulated "in vivo" conditions to form a mutagenic urea (Ua) lesion . Using the Oa 2'-deoxyribonucleoside as a model, the hydrolysis rate depended strongly upon the concentrations of bicarbonate and divalent magnesium . In buffered solutions containing physiologically relevant levels of these species, the half-life of Oa nucleoside was approximately 40 h at 37 degrees C . The mutagenic properties of Ua in DNA were investigated using a M13mp7L2 bacteriophage genome containing Ua at a specific site . Transfection of the lesion-containing genome into wild-type AB1157 Escherichia coli allowed determination of the mutation frequency and DNA polymerase bypass efficiency from the resulting progeny phage . Ua was bypassed with an efficiency of 11% as compared to a guanine control and caused a 99% G-->T mutation frequency, assuming the lesion originated from G, which is at least an order of magnitude higher than the mutation frequency of 8-oxoG under the same conditions . SOS induction of bypass DNA polymerase(s) in the bacteria prior to transfection caused the mutation frequency and type to shift to 43% G-->T, 46% G-->C, and 10% G-->A mutations . We suggest that Ua is instructional, meaning that the shape of the lesion and its interactions with DNA polymerases influence which nucleotide is inserted opposite the lesion during replication and that the instructional nature of the lesion is modulated by the size of the binding pocket of the DNA polymerase . Replication past Ua, when formed by hydrolysis of the 8-oxoG oxidation product Oa, denotes a pathway that nearly quantitatively generates point mutations in vivo. Anal Chem, 2005 Jan 15, 77(2), 652 - 7 Electrochemical Phagemid Assay for the Specific Detection of Bacteria Using Escherichia coli TG-1 and the M13KO7 Phagemid in a Model System; Neufeld T et al.; We describe a reporter phagemid system for the specific amperometric detection of bacteria . We constructed a phagemid a bacteriophage containing a bacterial plasmid using the M13KO7 helper phage and a commercial plasmid, pFLAG-ATS-BAP, which contains a gene encoding for a reporter enzyme, alkaline phosphatase . In the bacteria, the enzyme reacts with the substrate, p-aminophenyl phosphate, in the periplamic space that separates the outer plasma membrane from the cell wall . Thus, the activity of the reporter enzyme can be measured directly in an electrochemical cell without further treatment . The product of the enzymatic activity, p-aminophenol, diffuses out and is oxidized at the working electrode with an applied potential of 220 mV vs the reference electrode Ag/AgCl . The lower detection limit was 1 cfu/mL E . coli TG1 in less than 3 h in a very specific manner . The use of plasmid alkaline phosphatase as the reporter increased the sensitivity by 10-fold over our earlier electrochemical lytic phage method . Such a system can be used for the rapid detection of any strain of bacteria using the appropriate bacteriophage and reporter gene. Dev Biol (Basel), 2004, 118, 129 - 31 PDA Virus Filter Task Force update; Sofer G; The PDA Virus Filter Task Force has made significant progress in producing a method that will standardize the nomenclature for virus-removal filters that remove large viruses . A method for preparation of the model bacteriophage, PR 772, has also been produced and will shortly undergo a feasibility study . A technical report on the use of virus removal filters is also being prepared. Dev Biol (Basel), 2004, 118, 89 - 98 Use of bacteriophages as surrogates for mammalian viruses; McAlister M et al.; The threat of viral contamination is common to all processes using biological products of animal or human origin . Therefore, demonstration of virus clearance (i.e . validation of virus removal and/or inactivation steps) is of utmost importance to the biopharmaceutical industries . Ultimately, virus clearance studies should show that any virus removal/inactivation stage incorporated into the manufacturing process not only removes or inactivates known viruses that may be conceivably present (e.g . from cell banks and source materials), but also other viruses that may be introduced adventitiously (e.g . by addition of supplements downstream of the manufacturing process) . In this paper, we outline the shared properties of mammalian viruses and similar sized bacteriophages, and factors that may influence the virus clearance process . We also present test data from filtration studies, showing similar titre reductions for both types of virus . We propose that well-characterised bacteriophage, such as PP7 and PR772 can be used as models for mammalian viruses if the virus removal mechanism is based on size exclusion. Nucleic Acids Res, 2005 Jan 07, 33(1), 126 - 34 Print 2005. A precise DNA bend angle is essential for the function of the phage phi29 transcriptional regulator; Perez-Lago L et al.; Bacteriophage phi29 protein p4 is essential for the regulation of the switch from early to late phage transcription . The protein binds to two regions of the phage genome located between the regulated promoters . Each region contains two inverted repeats separated by 1 bp . We used circular permutation assays to study the topology of the DNA upon binding of the protein and found that p4 induced the same extent of bending independent of the topology of the binding region . In addition, the results revealed that the p4-induced bending is not dependent on the affinity to the binding site but is intrinsic to p4 binding . Independent binding sites were identified through the characterization of the minimal sequence required for p4 binding . The protein has different affinity for each of its binding sites, with those overlapping the A2c and A2b promoter cores (sites 1 and 3), having the highest affinity . The functionality of the p4 binding sites and the contribution of p4-mediated promoter restructuring in transcription regulation is discussed. Protein Expr Purif, 2005 Feb, 39(2), 247 - 253 Expression and purification of histidine-tagged bacteriophage T7 DNA polymerase; Schlicke M et al.; The formation of inclusion bodies is a frequent consequence of high-level production of foreign protein in the cytoplasm of Escherichia coli . This phenomenon is also observed with bacteriophage T7 gene 5 protein, the phage-encoded subunit of T7 DNA polymerase, if expression is based on the T5 promoter/lac operator transcription-translation system present in a vector with ColE1 origin of replication . To avoid tedious procedures for recovering protein from insoluble aggregates, we studied the expression of T7 gene 5 protein using a series of E . coli strains, and optimized the yield of soluble, histidine-tagged (His-tagged) protein by varying the respective growth conditions (temperature, amount of inducer isopropyl-beta-d-thiogalactopyranoside, and presence of organic osmolytes) . Although the expression levels in three different strains (BL21, SG13009, and XL1-Blue) were almost comparable with a given set of growth conditions, the yields of soluble protein differed markedly . The largest quantities of soluble, His-tagged T7 gene 5 protein were achieved using "cloning strain" XL1-Blue which benefitted significantly from the presence of sorbitol and glycine betaine-in contrast to the expression strains BL21 and SG13009 . Purification of His-tagged T7 gene 5 protein was achieved using single-step metal-affinity chromatography that yielded large amounts of highly active polymerase (97% homogeneity) . The application of this expression/purification approach represents not only a useful method to purify large quantities of T7 DNA polymerase for structural investigations but also, provides a fast and efficient protocol for the parallel purification of T7 DNA polymerase variants, e.g., for automated screenings or directed evolution experiments. Biochemistry, 2005 Jan 18, 44(2), 666 - 74 Chemically modified DNA substrates implicate the importance of electrostatic interactions for DNA unwinding by dda helicase; Eoff RL et al.; Helicase-catalyzed disruption of double-stranded nucleic acid is vital to DNA replication, recombination, and repair in all forms of life . The relative influence of specific chemical interactions between helicase and the substrate over a series of multistep catalytic events is still being defined . To this end, three modified DNA oligonucleotides were designed to serve as substrates for the bacteriophage T4 helicase, Dda . A 5'-DNA-PNA-DNA-3' chimera was synthesized, thereby, conferring both a loss of charge and altering the conformational flexibility of the oligonucleotide . The second modified oligonucleotide possessed a single methylphosphonate replacement on the phosphate backbone, creating a gap in the charge distribution of the substrate . The third modification introduced an abasic site into the oligonucleotide sequence . This abasic site retains the charge distribution of the normal DNA substrate yet alters the conformational flexibility of the oligonucleotide . The loss of a base also serves to disrupt the hydrogen-bonding lattice, the intramolecular base-stacking interactions, as well as the intermolecular base-stacking interactions between aromatic amino acid side chains and the substrate . Our results indicate that a gap in the charge distribution along the backbone of the substrate has a more pronounced effect upon helicase-catalyzed unwinding than does the loss of a single base . While all three substrates exhibited some degree of inhibition, analysis of both pre-steady-state and excess enzyme experiments places a greater value upon the electrostatic interactions between helicase and the substrate. Appl Environ Microbiol, 2005 Jan, 71(1), 480 - 6 Nearly Identical Bacteriophage Structural Gene Sequences Are Widely Distributed in both Marine and Freshwater Environments; Short CM et al.; Primers were designed to amplify a 592-bp region within a conserved structural gene (g20) found in some cyanophages . The goal was to use this gene as a proxy to infer genetic richness in natural cyanophage communities and to determine if sequences were more similar in similar environments . Gene products were amplified from samples from the Gulf of Mexico, the Arctic, Southern, and Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of ca . 3,246 m in the Chuckchi Sea . Amplicons were separated by denaturing gradient gel electrophoresis, and selected bands were sequenced . Phylogenetic analysis revealed four previously unknown groups of g20 clusters, two of which were entirely found in freshwater . Also, sequences with >99% identities were recovered from environments that differed greatly in temperature and salinity . For example, nearly identical sequences were recovered from the Gulf of Mexico, the Southern Pacific Ocean, an Arctic freshwater cyanobacterial mat, and Lake Constance, Germany . These results imply that closely related hosts and the viruses infecting them are distributed widely across environments or that horizontal gene exchange occurs among phage communities from very different environments . Moreover, the amplification of g20 products from deep in the cyanobacterium-sparse Chuckchi Sea suggests that this primer set targets bacteriophages other than those infecting cyanobacteria. Arch Biochem Biophys, 2005 Feb 15, 434(2), 352 - 7 Asymmetric binding of membrane proteins to GroEL; Sun J et al.; The interaction of GroEL with non-native soluble proteins has been studied intensively and structure-function relationships have been established in considerable detail . Recently, we found that GroEL is also able to bind membrane proteins in the absence of detergents and deliver them to liposomes in a biologically active state . Here, we report that three well-studied membrane proteins (bacteriorhodopsin, LacY, and the bacteriophage lambda holin) bind asymmetrically to tetradecameric GroEL . Each of the membrane proteins was visualized in one of the center cavities of GroEL using single particle analysis. Microb Cell Fact . 2005 Jan 7;4(1):3 {Epub ahead of print} Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase; Vethanayagam JG et al.; BACKGROUND: Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production . Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production . Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure . RESULTS: We demonstrated that decreases in protein overproduction levels are not due to significant plasmid loss nor to mutations arising on the plasmid, but instead largely are attributable to chromosomal mutations that diminish the level of functional T7 RNA polymerase, resulting in decreased expression from the plasmid . Isolation of plasmid DNA from non-expressing strains and reintroduction of the plasmid into a T7 RNA polymerase-producing strain such as BL21(lDE3) reproducibly restored high level protein production . CONCLUSIONS: Our results suggest that a major contributing factor to decreased expression levels in T7 based systems is chromosomal mutation resulting in loss of functional T7 RNA polymerase . Consistent with this hypothesis, we found that optimal protein overproduction was obtained reproducibly from T7 promoters using freshly transformed cells that had not been subjected to outgrowth during which mutations could accumulate. Am J Transplant, 2005 Jan, 5(1), 50 - 7 Rituximab Inhibits the In Vivo Primary and Secondary Antibody Response to a Neoantigen, Bacteriophage phiX174; Bearden CM et al.; The response to primary immunization in patients treated with Rituximab (RIT) is not clear . We studied the in vivo antibody response of chronic renal failure (CRF) patients to the neoantigen bacteriophage phiX174 given alone or after ablation with RIT . Eighteen CRF subjects received two immunizations with phiX174 separated by 6 weeks . Nine subjects received a single dose of RIT . The intensity and immunoglobulin isotype of the antibody response (K(v)) were measured post-infusion . In addition, three subjects previously immunized and treated with RIT underwent a third and fourth immunization with phiX174 and a tetanus control 2 years later . RIT significantly decreased peak K(v) responses when compared to both historic non-CRF controls and to CRF subjects . CRF itself decreased peak K(v) responses compared to non-CRF controls . Percent-ratio of anti-phage IgM to IgG was significantly decreased in RIT treated subjects . One of three subjects treated with RIT was found to have developed a partial B cell tolerance to phiX174 administration 2 years later . RIT decreases antibody production and isotype switching to neoantigens and might be useful to prevent antibody response to therapeutic drugs and to newly transplanted organs. Proc Natl Acad Sci U S A, 2005 Jan 11, 102(2), 390 - 5 Epub 2005 Jan 03. Experimental evolution of conflict mediation between genomes; Sachs JL et al.; Transitions to new levels of biological complexity often require cooperation among component individuals, but individual selection among those components may favor a selfishness that thwarts the evolution of cooperation . Biological systems with elements of cooperation and conflict are especially challenging to understand because the very direction of evolution is indeterminate and cannot be predicted without knowing which types of selfish mutations and interactions can arise . Here, we investigated the evolution of two bacteriophages (f1 and IKe) experimentally forced to obey a life cycle with elements of cooperation and conflict, whose outcome could have ranged from extinction of the population (due to selection of selfish elements) to extreme cooperation . Our results show the de novo evolution of a conflict mediation system that facilitates cooperation . Specifically, the two phages evolved to copackage their genomes into one protein coat, ensuring cotransmission with each other and virtually eliminating conflict . Thereafter, IKe evolved such extreme genome reduction that it lost the ability to make its own virions independent of f1 . Our results parallel a variety of conflict mediation mechanisms existing in nature: evolution of reduced genomes in symbionts, cotransmission of partners, and obligate coexistence between cooperating species. Virology, 2005 Jan 20, 331(2), 325 - 37 Complete genomic nucleotide sequence and analysis of the temperate bacteriophage VWB; Van Dessel W et al.; The entire double-stranded DNA genome of the Streptomyces venezuelae bacteriophage VWB was sequenced and analyzed . Its size is 49,220 bp with an overall molar G + C content of 71.2 mol% . Sixty-one potential open reading frames were identified and annotated using several complementary bioinformatics tools . Clusters of functionally related putative genes were defined, supporting a refined version of the modular theory of phage evolution. J Photochem Photobiol B, 2005 Jan 14, 78(1), 53 - 60 Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23; Stortelder A et al.; The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated . The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera . Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting . A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation . In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues . A model for the origin of the observed lifetimes of 0.032+/-0.003, 0.39+/-0.06, 2.1+/-0.1 and 6.8+/-0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23. J Immunol Methods, 2004 Dec, 295(1-2), 149 - 60 Epub 2004 Nov 14. Construction of antibody mimics from a noncatalytic enzyme-detection of polysialic acid; Jokilammi A et al.; We have used a conceptually novel way to construct antibody mimics based on the binding of a noncatalytic enzyme to its substrate . Bacteriophage-derived endosialidase cleaves polysialic acid (polySia), an important oncofetal and bacterial antigen, which is poorly immunogenic . We fused to green fluorescent protein (GFP) a catalytically inactive endosialidase known to bind but not degrade polysialic acid . The fusion protein is a convenient single-step reagent in fluorescence microscopy, binding assays and immunoblots . It efficiently and specifically detected polysialic acid in developing brain, neuroblastoma cells and bacteria causing meningitis . Enzyme-substrate interactions represent an unexploited source of molecular recognition events . Some of these could be used in designing well-defined substitute antibodies for the study of target molecules which are difficult to purify, available in low quantities, are unstable or have poor immunogenity. Biochemistry (Mosc), 2004 Nov, 69(11), 1213 - 8 Diversity of Structure and Function of DNA Polymerase (gp43) of T4-Related Bacteriophages; Petrov VM et al.; The replication DNA polymerase (gp43) of the bacteriophage T4 is a member of the pol B family of DNA polymerases, which are found in all divisions of life in the biosphere . The enzyme is a modularly organized protein that has several activities in one polypeptide chain (~900 amino acid residues) . These include two catalytic functions, POL (polymerase) and EXO (3 -exonuclease), and specific binding activities to DNA, the mRNA for gp43, deoxyribonucleotides (dNTPs), and other T4 replication proteins . The gene for this multifunctional enzyme (gene 43) has been preserved in evolution of the diverse group of T4-like phages in nature, but has diverged in sequence, organization, and specificity of the binding functions of the gene product . We describe here examples of T4-like phages where DNA rearrangements have created split forms of gene 43 consisting of two cistrons instead of one . These gene 43 variants specify separate gp43A (N-terminal) and gp43B (C-terminal) subunits of a split form of gp43 . Compared to the monocistronic form, the interruption in contiguity of the gene 43 reading frame maps in a highly diverged sequence separating the code for essential components of two major modules of this pol B enzyme, the FINGERS and PALM domains, which contain the dNTP binding pocket and POL catalytic residues of the enzyme . We discuss the biological implications of these gp43 splits and compare them to other types of pol B splits in nature . Our studies suggest that DNA mobile elements may allow genetic information for pol B modules to be exchanged between organisms. Biochemistry (Mosc), 2004 Nov, 69(11), 1190 - 202 Molecular architecture of bacteriophage t4; Mesyanzhinov VV et al.; In studying bacteriophage T4--one of the basic models of molecular biology for several decades--there has come a Renaissance, and this virus is now actively used as object of structural biology . The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography . Three-dimensional reconstruction of the infection device--one of the most complex multiprotein components--has been developed on the basis of cryo-electron microscopy images . The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines. Biosens Bioelectron, 2005 Feb 15, 20(8), 1491 - 8 On-chip PCR amplification of very long templates using immobilized primers on glassy surfaces; Nickisch-Rosenegk M et al.; In this paper we describe a novel method for visualizing very long DNA fragments (for example >6kb) which are difficult to spot with commonly used arrayers or capillary samplers with very small nanoliter volumes, using directly bound primers on "on-chip" polymerase chain reaction (PCR) . We have used the genomes of the M13 bacteriophage (7.2kb) the human mitochondrion (16.5kb) as examples of long DNA templates to test the PCR and were able to elicit robust reactivity . Over 75% of the immobilized primers could be elongated to their fullest extent . In addition we were able to elicit the PCR reaction with double stranded templates in which one primer was immobilized and the other suspended in the reaction solution . These synthesized PCR products were visualized by either confocal microarray scanning or fluorescence microscopy using Cy5-dye fluorescence of the modified free primer, or the fluorescence of intercalating dyes. Biochem Biophys Res Commun, 1976 Dec 20, 73(4), 1083 - 93 On the possible modulating role of the isoleucine AUA-codon in bacteriophage MS2 RNA; Jou WM et al.; A set of MS2 mutants were shown to have an additional silent mutation met --> ile at position 108 of the coat protein . As transitions are more frequent than transversions one would have expected an AUA codon in this position in the mutant RNAs . As the AUA codon is one of the best candidates for a modulation role in the control of translation in E . coli, the presence of this AUA in the gene for the protein made in major amounts upon viral infection would impose serious doubt on the theory of modulation . We have directly proven by minifinger-printing of mutant RNA and further analysis of the relevant spots that, in fact, the isoleucine residue at position 108 of the coat protein gene is specified by the non-rate-limiting AUU codon, in agreement with a modulation type of control of protein synthesis. Biochem Biophys Res Commun, 1976 Dec 20, 73(4), 953 - 9 A physiological role for tRNA nucleotidyltransferase during bacteriophage infection; Morse JW et al.; Bacteriophage infection of E . coli cells deficient in the enzyme tRNA nucleotidyltransferase (cca mutants) resulted in greatly decreased production of viable progeny phage compared to wild type cells . This decrease amounted to as much as 90% in the case of T-even bacteriophages, and 50-65% for T-odd bacteriophages . However, infection by the RNA phages, Qbeta and f2, was unaffected by the cca mutation . Examination of T4 infection of cca hosts indicated that phage development proceeded normally, that near-normal numbers of progeny particles were formed, but that most of these particles were non-viable . Possible functions for E . coli tRNA nucleotidyltransferase during bacteriophage infection are discussed. BMC Microbiol . 2004 Dec 22;4(1):48 {Epub ahead of print} A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system; Greub G et al.; BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs) . RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed . This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats . Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element . Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region . Thus, this region largely fulfills the criteria of GIs . The G+C content analysis shows that several modules compose this GI . Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer . A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation . These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit . CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system . This is the first hint of a putative conjugative system in chlamydiae . Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles . Such a conjugative system might be involved in DNA transfer between internalized bacteria . Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria . It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole . Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity . In future, conjugative systems might be developed as genetic tools for Chlamydiales. Poult Sci, 2004 Dec, 83(12), 1944 - 7 Therapeutic efficacy of bacteriophage and Baytril (enrofloxacin) individually and in combination to treat colibacillosis in broilers; Huff WE et al.; A study was conducted to evaluate the therapeutic efficacy of bacteriophage and the antibiotic enrofloxacin individually and in combination to treat colibacillosis . The experimental design was a 2 x 2 x 2 factorial with 8 treatments and 4 replicate pens of 10 birds . The treatments were 1) control, 2) unchallenged birds treated with bacteriophage, 3) enrofloxacin, or 4) the combination; 5) birds challenged with Escherichia coli, and birds challenged with E . coli and treated with 6) bacteriophage, 7) enrofloxacin, or 8) the combination of bacteriophage and enrofloxacin . Birds in the E . coli challenged treatments were challenged at 7 d of age by injecting 10(4) cfu of E . coli into the thoracic air sac . The antibiotic treatment was initiated immediately after the birds were challenged and consisted of 50 ppm enrofloxacin in the drinking water for 7 consecutive days . The bacteriophage treatment consisted of a single intramuscular injection of 2 different bacteriophage (10(9) pfu) administered immediately after the E . coli challenge . Mortality in the birds challenged with E . coli and untreated was 68%, and the bacteriophage and enrofloxacin treatments significantly decreased mortality to 15 and 3%, respectively . There was total protection in birds that received both the bacteriophage and enrofloxacin representing a significant synergy . The decrease in mortality with enrofloxacin (3%) was significantly better than the decrease in mortality with bacteriophage (15%) . Airsacculitis lesion scores and lesion incidence in surviving birds were significantly less in the enrofloxacin treatment compared with the bacteriophage treatment . Both bacteriophage and enrofloxacin provided effective treatments of colibacillosis, and the synergy between these 2 treatments suggests that bacteriophage combined with antibiotic treatment has significant value. Biophys J . 2004 Dec 21; {Epub ahead of print} {lambda} Repressor Oligomerization Kinetics at High Concentrations using Fluorescence Correlation Spectroscopy in Zero Mode Waveguides; Samiee KT et al.; Fluorescence Correlation Spectroscopy (FCS) has demonstrated its utility for measuring transport properties and kinetics at low fluorophore concentrations . In this article, we demonstrate that simple optical nanostructures, known as Zero Mode Waveguides, can be used to significantly reduce the FCS observation volume . This, in turn, allows FCS to be applied to solutions with significantly higher fluorophore concentrations . We derive an empirical FCS model accounting for one dimensional diffusion in a finite tube with a simple exponential observation profile . This technique is used to measure the oligomerization of the bacteriophage lambda repressor protein at micromolar concentrations . The results agree with previous studies utilizing conventional techniques . Additionally, we demonstrate that the Zero Mode Waveguides can be used to assay biological activity by measuring changes in diffusion constant as a result of ligand binding. Mol Biol (Mosk), 2004 Nov-Dec, 38(6), 1059 - 66 {Photoaffinity modification of bacteriophage T7 DNA-dependent RNA polymerase by the reaction product containing the azido derivative of UTP}; Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination; Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USAThere are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means . Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome . We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes. J Appl Microbiol, 2005, 98(1), 203 - 9 Survival of viruses on fresh produce, using MS2 as a surrogate for norovirus; Dawson DJ et al.; Abstract d.j . dawson, a . paish, l.m . staffell, i.j . seymour and h . appleton . 2004.Aims: To study the survival and removal of viruses from fresh fruit and vegetables using the bacteriophage MS2 as a potential surrogate for noroviruses . Method and Results: Survival of MS2 in buffer and on fresh produce was studied at 4, 8 and 22 degrees C . At 4 and 8 degrees C a reduction of <1 log(10) was observed after 50 days in buffer; however a reduction in excess of 1 log(10) occurred within 9 days at 22 degrees C . Similar results were obtained with fresh produce with virus survival times exceeding the shelf life of the produce . In washing experiments, using a chlorine wash (100 ppm), in all but one case <1.5 log(10) MS2 bacteriophage was removed from fruit and vegetables . The mean across all produce types was 0.89 log(10) . With potable water, reduction was lower (0.3 log mean across all produce types) . Conclusions: MS2 survived for prolonged periods, both in buffer and on fresh produce, at temperatures relevant to chilled foods . It was not removed effectively by chlorine washing . Significance and Impact of the Study: Bacteriophage MS2 has been evaluated as a potential surrogate for noroviruses on fresh produce . Experimental results together with current knowledge of norovirus resistance and survival indicate that MS2 could be used as an effective surrogate in future evaluations. Nat Struct Mol Biol . 2004 Dec 19; {Epub ahead of print} Crystal structure of the polysialic acid-degrading endosialidase of bacteriophage K1F; Stummeyer K et al.; Phages infecting the polysialic acid (polySia)-encapsulated human pathogen Escherichia coli K1 are equipped with capsule-degrading tailspikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia . As polySia also promotes cellular plasticity and tumor metastasis in vertebrates, endosialidases are widely applied in polySia-related neurosciences and cancer research . Here we report the crystal structures of endosialidase NF and its complex with oligomeric sialic acid . The structure NF, which reveals three distinct domains, indicates that the unique polySia specificity evolved from a combination of structural elements characteristic of exosialidases and bacteriophage tailspike proteins . The endosialidase assembles into a catalytic trimer stabilized by a triple beta-helix . Its active site differs markedly from that of exosialidases, indicating an endosialidase-specific substrate-binding mode and catalytic mechanism . Residues essential for endosialidase activity were identified by structure-based mutational analysis. Biotechnol Lett, 2004 Nov, 26(22), 1695 - 700 Drastically lowering the titer of waterborne bacteriophage PRD1 by exposure to immobilized hydrophobic polycations; Gelman F et al.; Decrease in the titer of bacteriophage PRD1 (a model of animal adenoviruses) in aqueous solutions caused by the presence of systematically chemically derivatized surfaces was kinetically investigated . The greatest loss of infectivity - up to a 4-log reduction in the titer - was observed with immobilized hydrophobic polyethylenimine-based and dendrimer-based polycations. Plant Mol Biol, 2004 Jul, 55(4), 491 - 500 PVX-Cre-mediated marker gene elimination from transgenic plants; Kopertekh L et al.; Cre recombinase gene from bacteriophage P1 was transiently expressed by a Potato Virus X (PVX)-based vector in transgenic lox -target Nicotiana benthamiana plants to remove the selectable marker gene . The target construct consisted of two directly oriented lox sites flanking a bar gene located between a gfp coding region and an upstream CaMV 35S promoter . The Cre-mediated excision of intervening sequence placed the gfp coding region under the transcriptional control of the CaMV 35S promoter . GFP activity was observed in PVX-Cre systemically infected leaves, regenerants from PVX-Cre infected explants and T1 progeny of these regenerants . PVX-Cre was removed efficiently from the regenerants by adding the nucleoside analogue ribavirin to the culture medium . Molecular data proved a correlation between gfp expression and precise site-specific excision of the bar gene in all examined transgenic lines . The frequency of recombination expressed as a percentage of regenerated plants exhibiting marker gene excision varied from 48% to 82% . These results demonstrate that a plant virus vector can be used efficiently to express cre recombinase in vivo providing an alternative method for the production of transgenic plants without marker genes. Nucleic Acids Res . 2004 Dec 15;32(22):e182. Selection of genomic sequences that bind tightly to Ff gene 5 protein: primer-free genomic SELEX; Wen JD et al.; Single-stranded DNA or RNA libraries used in SELEX experiments usually include primer-annealing sequences for PCR amplification . In genomic SELEX, these fixed sequences may form base pairs with the central genomic fragments and interfere with the binding of target molecules to the genomic sequences . In this study, a method has been developed to circumvent these artificial effects . Primer-annealing sequences are removed from the genomic library before selection with the target protein and are then regenerated to allow amplification of the selected genomic fragments . A key step in the regeneration of primer-annealing sequences is to employ thermal cycles of hybridization-extension, using the sequences from unselected pools as templates . The genomic library was derived from the bacteriophage fd, and the gene 5 protein (g5p) from the phage was used as a target protein . After four rounds of primer-free genomic SELEX, most cloned sequences overlapped at a segment within gene 6 of the viral genome . This sequence segment was pyrimidine-rich and contained no stable secondary structures . Compared with a neighboring genomic fragment, a representative sequence from the family of selected sequences had about 23-fold higher g5p-binding affinity . Results from primer-free genomic SELEX were compared with the results from two other genomic SELEX protocols. Sichuan Da Xue Xue Bao Yi Xue Ban, 2003 Jan, 34(1), 16 - 9 {Construction and screening of TsF cDNA library specific for AchR}; Li W et al.; OBJECTIVE: To construct the T suppress factor (TsF) cDNA library specific for AchR and screen out the positive clones of TsF . METHODS: The full-length cDNA was synthesized by the reverse transcripting from the TsF PolyA+mRNA, which had been extracted from the AchR specific suppressor lines (ARSL) . The cDNA library was obtained using Lambda gt11 vector . Finally, this cDNA library was screened by Western blotting using Analysis of this cDNA library showed that titer of the monoclonal antibody specific for TCR-achain . RESULTS: recombinant bacteriophage was 1.4 x 10(7) pfu/ml, the rate of positive recombinant was 86.9% . The recombinant bacteriophage DNA were digested by EcoRI and electrophoresis analysis found that the main size of insertion was 0.8 to approximately 4.0 kb . As a result of screening, 27 positive clones were obtained . The positive recombinant bacteriophage DNA were digested by EcoRI, and electrophoresis analysis found that the size of insertion was 1 kb . Conclusion The results suggested that this cDNA library is available, the recombination phages can express TsF specific for AchR effectively and could be used to prepare the products of gene engineering of TsF. FEMS Microbiol Lett, 2004 Dec 15, 241(2), 171 - 7 The coexistence of Escherichia coli serotype O157:H7 and its specific bacteriophage in continuous culture; Fischer CR et al.; For the development of phage therapy, systematic understanding mechanisms of bacteriophage resistance will be required . We describe a new strain of Escherichia coli O157:H7, named Mu(L), which stably co-exists with the O157:H7-specific lytic bacteriophage PP01 . Chemostat cultures of E . coli O157:H7 infected with PP01 showed unchanging cell concentration, but phage concentrations which increased by approximately 10(8) PFU mL(-1) . However, the latent period, burst size, and growth rate of Mu(L) were the same as in a PP01-susceptible strain . The binding rate of PP01 to the cell surface was diminished 8.5-fold in Mu(L) . By observation of the binding of fluorescently labeled O157:H7-specific phage to individual Mu(L) cells, we found that clonal Mu(L) cultures were heterogeneous in their ability to bind bacteriophage . 15% of the Mu(L) population was completely resistant to PP01 infection . Mu(L) also co-existed with bacteriophages unrelated to PP01 . Broad-range phage resistance by clonal heterogeneity represents a new class of bacteria-phage interactions. BMC Mol Biol . 2004 Dec 13;5(1):22 {Epub ahead of print} Chromosomal duplications and cointegrates generated by the bacteriophage lambda Red system in Escherichia coli K-12; Poteete AR et al.; BACKGROUND: An Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage lambda Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome . Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E . coli chromosome to generate a chloramphenicol-resistant Lac- recombinant . The dsDNA was delivered into the cell as part of the chromosome of a non-replicating lambda vector, from which it was released by the action of a restriction endonuclease in the infected cell . This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants . RESULTS: A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions . (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation . (3) The Lac+ recombinants were unstable, segregating Lac- progeny . (4) A tetracycline-resistance marker in a site of the phage chromosome distant from cat was not frequently co-inherited with cat . (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants . In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red . (7) The linked tetracycline-resistance marker was frequently co-inherited in this case . CONCLUSIONS: The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome . When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant. Microbiol Mol Biol Rev, 2004 Dec, 68(4), 796 - 813 Little lambda, who made thee? Gottesman ME, Weisberg RA. The study of the bacteriophage lambda has been critical to the discipline of molecular biology . It was the source of key discoveries in the mechanisms of, among other processes, gene regulation, recombination, and transcription initiation and termination . We trace here the events surrounding these findings and draw on the recollections of the participants . We show how a particular atmosphere of interactions among creative scientists yielded spectacular insights into how living things work. Nucleic Acids Res . 2004 Dec;32(21):e174. Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h; Chiu J et al.; Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution) . The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube . The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products . Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid . The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions . In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5 . The overall efficiency for obtaining the desired product was >95%. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2004 Dec, 98(6), 692 - 7 Application of laser capture microdissection to phage display peptide library screening; Lu H et al.; OBJECTIVE: When identifying important regulatory genes using methods such as phage display peptide library screening it is critical to select such peptides from cells and tissues in their native state . Here, we report a novel approach to screen tumors using phage display and laser capture microdissection (LCM) . STUDY DESIGN: A phage peptide library was screened directly on fresh oral tumor tissue, such that specifically bound peptides were selected from fresh tumor cells in the native tissue state . Tissue processing conditions were modified to ensure the survival of the bacteriophage . RESULTS: Our results demonstrate that live phage-peptide conjugates can be recovered from laser capture microdissected cells in a form suitable for additional cycles of amplification . CONCLUSION: Thus, LCM will be a valuable adjunct to phage display studies. Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2368 - 70 Epub 2004 Dec. Crystallization and preliminary X-ray diffraction analysis of bacteriophage varphi12 packaging factor P7; Kainov DE et al.; Bacteriophage varphi12 protein P7 is a structural component of the polymerase complex and ensures stable packaging of the genomic RNA . varphi12 P7 has been cloned, purified and crystallized . Crystals belong to space group P3(2)21, with unit-cell parameters a = 75.7, b = 75.7, c = 45.2 A, alpha = 90, beta = 90, gamma = 120 degrees , and diffract beyond 2.0 A . Multiple anomalous dispersion data have been collected from crystals of selenomethionylated P7 . Mass spectroscopy showed proteolysis of the crystallized protein and a truncated form, P7DeltaC, gave crystals of similar morphology . Cross-linking experiments implicated the N-terminal domain of P7 as being essential for dimerization. J Mol Biol, 2005 Jan 21, 345(3), 475 - 85 Non-equivalent interactions between amino-terminal domains of neighboring lambda integrase protomers direct Holliday junction resolution; Lee SY et al.; The bacteriophage lambda site-specific recombinase (Int), in contrast to other family members such as Cre and Flp, has an amino-terminal domain that binds "arm-type" DNA sequences different and distant from those involved in strand exchange . This defining feature of the heterobivalent recombinases confers a directionality and regulation that is unique among all recombination pathways . We show that the amino-terminal domain is not a simple "accessory" element, as originally thought, but rather is incorporated into the core of the recombination mechanism, where it is well positioned to exert its profound effects . The results reveal an unexpected pattern of intermolecular interactions between the amino-terminal domain of one protomer and the linker region of its neighbor within the tetrameric Int complex and provide insights into those features distinguishing an "active" from an "inactive" pair of Ints during Holliday junction resolution. Curr Protein Pept Sci, 2004 Dec, 5(6), 487 - 96 Strategies for the construction and use of peptide and antibody libraries displayed on phages; Pini A et al.; Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery . A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning . The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure . This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide . The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity . Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use . Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries . Particular attention is paid to advanced strategies for the construction, preservation and panning. Curr Gene Ther, 2004 Dec, 4(4), 385 - 408 Herpes simplex virus type 1 amplicons and their hybrid virus partners, EBV, AAV, and retrovirus; Oehmig A et al.; HSV-1 amplicons can accommodate foreign DNA of any size up to 150 kbp . Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, or multiple transgenes can be inserted in a modular fashion . HSV-1 amplicon vectors deliver DNA efficiently into the cell nucleus as an extrachromosomal, non-replicating circular concatenate, which is rapidly diluted, at least in dividing cells . Consequently, transgene expression is lost within days to weeks in dividing cells, but may be retained for months in non-dividing cells . In contrast, vectors based on Epstein-Barr virus, adeno-associated virus, or retroviruses can mediate long-term transgene expression, as vector DNA is retained by episomal replication or chromosomal integration . Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, thereby conserving the large transgene capacity of HSV-1, and genetic elements from other viruses that confer genetic stability to the vector DNA within transduced cells . Additional strategies to sustain genetic material in infected cells include the incorporation of recombinases from different bacteriophages or transposable elements of the Tc1/mariner family in the amplicon vector . Moreover, modification of the HSV-1 virion itself offers a myriad of possibilities to improve gene delivery by targeting specific cell populations or transporting foreign proteins, such as Cre recombinase or the adeno-associated virus Rep protein, which can control the fate and expression of the therapeutic transgene. J Bacteriol, 2004 Dec, 186(24), 8401 - 6 Phage phi29 proteins p1 and p17 are required for efficient binding of architectural protein p6 to viral DNA in vivo; Gonzalez-Huici V et al.; Bacteriophage phi29 protein p6 is a viral architectural protein, which binds along the whole linear phi29 DNA in vivo and is involved in initiation of DNA replication and transcription control . Protein p1 is a membrane-associated viral protein, proposed to attach the viral genome to the cell membrane . Protein p17 is involved in pulling phi29 DNA into the cell during the injection process . We have used chromatin immunoprecipitation and real-time PCR to analyze in vivo p6 binding to DNA in cells infected with phi29 sus1 or sus17 mutants; in both cases p6 binding is significantly decreased all along phi29 DNA . phi29 DNA is topologically constrained in vivo, and p6 binding is highly increased in the presence of novobiocin, a gyrase inhibitor that produces a loss of DNA negative superhelicity . Here we show that, in cells infected with phi29 sus1 or sus17 mutants, the increase of p6 binding by novobiocin is even higher than in cells containing p1 and p17, alleviating the p6 binding deficiency . Therefore, proteins p1 and p17 could be required to restrain the proper topology of phi29 DNA, which would explain the impaired DNA replication observed in cells infected with sus1 or sus17 mutants. J Bacteriol, 2004 Dec, 186(24), 8363 - 9 In vivo bypass of chaperone by extended coiled-coil motif in T4 tail fiber; Qu Y et al.; The distal-half tail fiber of bacteriophage T4 is made of three gene products: trimeric gp36 and gp37 and monomeric gp35 . Chaperone P38 is normally required for folding gp37 peptides into a P37 trimer; however, a temperature-sensitive mutation in T4 (ts3813) that suppresses this requirement at 30 degrees C but not at 42 degrees C was found in gene 37 (R . J . Bishop and W . B . Wood, Virology 72:244-254, 1976) . Sequencing of the temperature-sensitive mutant revealed a 21-bp duplication of wild-type gene 37 inserted into its C-terminal portion (S . Hashemolhosseini et al., J . Mol . Biol . 241:524-533, 1994) . We noticed that the 21-amino-acid segment encompassing this duplication in the ts3813 mutant has a sequence typical of a coiled coil and hypothesized that its extension would relieve the temperature sensitivity of the ts3813 mutation . To test our hypothesis, we crossed the T4 ts3813 mutant with a plasmid encoding an engineered pentaheptad coiled coil . Each of the six mutants that we examined retained two amber mutations in gene 38 and had a different coiled-coil sequence varying from three to five heptads . While the sequences varied, all maintained the heptad-repeating coiled-coil motif and produced plaques at up to 50 degrees C . This finding strongly suggests that the coiled-coil motif is a critical factor in the folding of gp37 . The presence of a terminal coiled-coil-like sequence in the tail fiber genes of 17 additional T-even phages implies the conservation of this mechanism . The increased melting temperature should be useful for "clamps" to initiate the folding of trimeric beta-helices in vitro and as an in vivo screen to identify, sequence, and characterize trimeric coiled coils. J Bacteriol, 2004 Dec, 186(24), 8287 - 94 Isolation of Escherichia coli bacteriophages from the stool of pediatric diarrhea patients in Bangladesh; Chibani-Chennoufi S et al.; A 3-week coliphage survey was conducted in stool samples from 140 Bangladeshi children hospitalized with severe diarrhea . On the Escherichia coli indicator strain K803, all but one phage isolate had 170-kb genomes and the morphology of T4 phage . In spot tests, the individual T4-like phages infected up to 27 out of 40 diarrhea-associated E . coli, representing 22 O serotypes and various virulence factors; only five of them were not infected by any of these new phages . A combination of diagnostic PCR based on g32 (DNA binding) and g23 (major capsid protein) and Southern hybridization revealed that half were T-even phages sensu strictu, while the other half were pseudo-T-even or even more distantly related T4-like phages that failed to cross-hybridize with T4 or between each other . Nineteen percent of the acute stool samples yielded T4-like phages, and the prevalence was lower in convalescent stool samples . T4-like phages were also isolated from environmental and sewage water, but with low frequency and low titers . On the enteropathogenic E . coli strain O127:K63, 14% of the patients yielded phage, all of which were members of the phage family Siphoviridae with 50-kb genomes, showing the morphology of Jersey- and beta-4 like phages and narrow lytic patterns on E . coli O serotypes . Three siphovirus types could be differentiated by lack of cross-hybridization . Only a few stool samples were positive on both indicator strains . Phages with closely related restriction patterns and, in the case of T4-like phages, identical g23 gene sequences were isolated from different patients, suggesting epidemiological links between the patients. Structure (Camb), 2004 Dec, 12(12), 2221 - 31 Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E . coli DNA polymerase III; Derose EF et al.; DNA polymerase III, the main replicative polymerase of E . coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions . It was recently discovered that E . coli bacteriophage P1 encodes a theta homolog, named HOT . The (1)H-(15)N HSQC spectrum of HOT exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a determination of the HOT solution structure by NMR . The structure contains three alpha helices, as reported previously for theta; however, the folding topology of the two proteins is very different . Residual dipolar coupling measurements on labeled theta support the conclusion that it is structurally homologous with HOT . As judged by CD measurements, the melting temperature of HOT was 62 degrees C, compared to 56 degrees C for theta, consistent with other data suggesting greater thermal stability of the HOT protein. Genome Biol . 2004;5(12):357 . Epub 2004. Why genomics is more than genomes; Lawrence JG; A report on the 2004 meeting on Molecular Genetics of Bacteria and Bacteriophages, Cold Spring Harbor, USA, 25-29 August 2004. Proc Natl Acad Sci U S A, 2004 Dec 14, 101(50), 17365 - 70 Epub 2004 Dec 01. The bacteriophage T4 late-transcription coactivator gp33 binds the flap domain of Escherichia coli RNA polymerase; Nechaev S et al.; Transcription of bacteriophage T4 late genes requires concomitant DNA replication . T4 late promoters, which consist of a single 8-bp -10 motif, are recognized by a holoenzyme containing Escherichia coli RNA polymerase core and the T4-encoded promoter specificity subunit, gp55 . Initiation of transcription at these promoters by gp55-holoenzyme is inefficient, but is greatly activated by the DNA-loaded DNA polymerase sliding clamp, gp45, and the coactivator, gp33 . We report that gp33 attaches to the flap domain of the Escherichia coli RNA polymerase beta-subunit and that this interaction is essential for activation . The beta-flap also mediates recognition of -35 promoter motifs by binding to sigma(70) domain 4 . The results suggest that gp33 is an analogue of sigma(70) domain 4 and that gp55 and gp33 together constitute two parts of the T4 late sigma . We propose a model for the role of the gp45 sliding clamp in activation of T4 late-gene transcription. Mol Cell, 2004 Dec 3, 16(5), 673 - 85 Does common architecture reveal a viral lineage spanning all three domains of life? Benson SD, Bamford JK, Bamford DH, Burnett RM. Our discovery that the major coat protein of bacteriophage PRD1 resembles that of human adenovirus raised the unexpected possibility that viruses infecting bacteria could be related by evolution to those infecting animal hosts . We first review the development of this idea . We then describe how we have used structure-based modeling to show that several other viruses with no detectable sequence similarity are likely to have coats constructed from similar proteins-the "double-barrel trimer." There is evidence that the group includes a diversity of viruses infecting very different hosts in all three domains of life: Eukarya; Bacteria; and Archaea that diverged billions of years ago . The current classification of viruses obscures such similarities . We propose that the occurrence of a double-barrel trimer coat protein in an icosahedral dsDNA virus with large facets, irrespective of its host, is a very strong indicator of its membership in a lineage of viruses with a common ancestor. J Mol Biol, 2005 Jan 14, 345(2), 375 - 86 The N terminus of the head protein of T4 bacteriophage directs proteins to the GroEL chaperonin; Snyder L et al.; The head protein of T4 bacteriophage requires the GroEL chaperonin for its insertion into a growing T4 head . Hundreds of thousands of copies of this protein must pass through the chaperonin in a limited time later in infection, indicating that the protein must use GroEL very efficiently and may contain sequences that bind tightly to GroEL . We show that green fluorescent protein (GFP) fused to the N terminus of the head protein can fold at temperatures higher than those at which the GFP protein can fold well by itself . We present evidence that this folding is promoted by the strong binding of N-terminal head protein sequences to GroEL . This binding is so strong that some fusion proteins can apparently deplete the cell of the GroEL needed for other cellular functions, altering the cellular membranes and slowing growth. J Pept Sci, 2004 Nov, 10(11), 648 - 58 Cyclic peptides selected by phage display mimic the natural epitope recognized by a monoclonal anti-colicin A antibody; Coulon S et al.; A 10-mer random peptide library displayed on filamentous bacteriophage was used to determine the molecular basis of the interaction between the monoclonal anti-colicin A antibody 1C11 and its cognate epitope . Previous studies established that the putative epitope recognized by 1C11 antibody is composed of amino acid residues 19-25 (RGSGPEP) of colicin A . Using the phage display technique it was confirmed that the epitope of 1C11 antibody was indeed restricted to residues 19-25 and the consensus motif RXXXPEP was identified . Shorter consensus sequences (RXXPEP, RXXEP, KXXEP) were also selected . It was also demonstrated that the disulfide bond found in one group of the selected peptides was crucial for 1C11 antibody recognition . It was shown that cyclization of the peptides by disulfide bond formation could result in a structure that mimics the natural epitope of colicin A. Virology, 2004 Dec 20, 330(2), 493 - 500 Efficient bunyavirus rescue from cloned cDNA; Lowen AC et al.; Bunyaviruses are trisegmented, negative-sense RNA viruses . Previously, we described a rescue system to recover infectious Bunyamwera virus (genus Orthobunyavirus) entirely from cloned cDNA (Bridgen, A . and Elliott, R.M . (1996) Proc . Nat . Acad . Sci . USA 93, 15400-15404) utilizing a recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase to drive intracellular transcription of transfected T7 promoter-containing plasmids . Here we report efforts to improve the efficiency of the system by comparing different methods of providing T7 polymerase . We found that a BHK-derived cell line BSR-T7/5 that constitutively expresses T7 RNA polymerase supported efficient and reproducible recovery of Bunyamwera virus, routinely generating >10(7) pfu per rescue experiment . Furthermore, we show that the virus can be recovered from transfecting cells with just three plasmids that express full-length antigenome viral RNAs, greatly simplifying the procedure . We suggest that this procedure should be applicable to viruses in other genera of the family Bunyaviridae and perhaps also to arenaviruses. J Virol, 2004 Dec, 78(24), 14057 - 61 Genetic screen for monitoring severe acute respiratory syndrome coronavirus 3C-like protease; Parera M et al.; A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome . Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes . Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease . This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch . A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor . The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct . Upon infection of Escherichia coli cells containing the two plasmids encoding the cI . SCoV P1/P2-cro and the beta-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease . This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors. Biopolymers, 2005 Jan, 77(1), 9 - 17 Binding of histone H1 to DNA is described by an allosteric model; Mamoon NM et al.; Equilibrium binding data were analyzed to characterize the interaction of the linker histone H1 degrees with unmodified T4 phage DNA . Data were cast into the Scatchard-type plot described by McGhee and von Hippel and fit to their eponymous model for nonspecific binding of ligand to DNA . The data were not fit by the simple McGhee-von Hippel model, nor fit satisfactorily by the inclusion of a cooperativity parameter . Instead, the interaction appeared to be well described by Crothers' allosteric model, in which the higher affinity of the protein for one conformational form of the DNA drives an allosteric transition of the DNA to the conformational form with higher affinity (form 2) . At 214 mM Na(+), the observed affinity K for an isolated site on unmodified T4 bacteriophage DNA in the form 2 conformation is 4.5 x 10(7) M(-1) . The binding constant for an isolated site on DNA in the conformation with lower affinity, form 1, appears to be about 10-fold lower . Binding affinity is dependent on ion concentration: the magnitude of K is about 10-fold higher at 14 mM (5.9 x 10(8) M(-1) for form 2 DNA) than at 214 mM Na(+) concentration . (c) 2004 Wiley Periodicals, Inc . Biopolymers, 2005. Infect Immun, 2004 Dec, 72(12), 7322 - 5 Bacteriophage MAV1 is not associated with virulence of Mycoplasma arthritidis; Clapper B et al.; Previous studies demonstrated that Mycoplasma arthritidis strain 158 acquired a high degree of virulence upon lysogenization with bacteriophage MAV1 . In the present study, the association between MAV1 and virulence was reexamined by creating new lysogens of 158 and of a relatively avirulent mutant, strain 158-1 . In the absence of lysogenization, 158 was more virulent than expected . The virulence of 158 and 158-1 did not increase upon lysogenization . A major antigenic difference between 158 and 158-1 was identified that is unrelated to MAV1 and could account for the difference in virulence. Infect Immun, 2004 Dec, 72(12), 7131 - 9 Diversity and host range of Shiga toxin-encoding phage; Gamage SD et al.; Shiga toxin 2 (Stx2) from the foodborne pathogen Escherichia coli O157:H7 is encoded on a temperate bacteriophage . Toxin-encoding phages from C600::933W and from six clinical E . coli O157:H7 isolates were characterized for PCR polymorphisms, phage morphology, toxin production, and lytic and lysogenic infection profiles on O157 and non-O157 serotype E . coli . The phages were found to be highly variable, and even phages isolated from strains with identical pulsed-field gel electrophoresis profiles differed . Examination of cross-plaquing and lysogeny profiles further substantiated that each phage is distinct; reciprocal patterns of susceptibility and resistance were not observed and it was not possible to define immunity groups . The interaction between Shiga toxin-encoding phage and intestinal E . coli was examined . Lytic infection was assessed by examining Shiga toxin production following overnight incubation with phage . While not common, lytic infection was observed, with a more-than-1,000-fold increase in Stx2 seen in one case, demonstrating that commensal E . coli cells can amplify Shiga toxin if they are susceptible to infection by the Shiga toxin-encoding phages . Antibiotic-resistant derivatives of the Stx2-encoding phages were used to examine lysogeny . Different phages were found to lysogenize different strains of intestinal E . coli . Lysogeny was found to occur more commonly than lytic infection . The presence of a diverse population of Shiga toxin-encoding phages may increase the pathogenic fitness of E . coli O157:H7. Infect Immun, 2004 Dec, 72(12), 7030 - 9 First-time isolation and characterization of a bacteriophage encoding the Shiga toxin 2c variant, which is globally spread in strains of Escherichia coli O157; Strauch E et al.; A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E . coli K-12 laboratory strains C600 and MG1655 . Production of Stx2c was found in the wild-type E . coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin . Phage 2851 is the first reported viable bacteriophage which carries an stx(2c) gene . Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda . Sequence analysis of an 8.4-kb region flanking the stx(2c) gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages . Phage 2851 showed lysis of E . coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages . Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E . coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E . coli O157 strains . Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E . coli O157 strains producing Stx2c . The phage 2851 q and o genes were frequently detected in Stx2c-producing E . coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E . coli O157 that were isolated in different locations and time periods. Protein Sci, 2004 Dec, 13(12), 3298 - 313 Probabilistic cross-link analysis and experiment planning for high-throughput elucidation of protein structure; Ye X et al.; Emerging high-throughput techniques for the characterization of protein and protein-complex structures yield noisy data with sparse information content, placing a significant burden on computation to properly interpret the experimental data . One such technique uses cross-linking (chemical or by cysteine oxidation) to confirm or select among proposed structural models (e.g., from fold recognition, ab initio prediction, or docking) by testing the consistency between cross-linking data and model geometry . This paper develops a probabilistic framework for analyzing the information content in cross-linking experiments, accounting for anticipated experimental error . This framework supports a mechanism for planning experiments to optimize the information gained . We evaluate potential experiment plans using explicit trade-offs among key properties of practical importance: discriminability, coverage, balance, ambiguity, and cost . We devise a greedy algorithm that considers those properties and, from a large number of combinatorial possibilities, rapidly selects sets of experiments expected to discriminate pairs of models efficiently . In an application to residue-specific chemical cross-linking, we demonstrate the ability of our approach to plan experiments effectively involving combinations of cross-linkers and introduced mutations . We also describe an experiment plan for the bacteriophage lambda Tfa chaperone protein in which we plan dicysteine mutants for discriminating threading models by disulfide formation . Preliminary results from a subset of the planned experiments are consistent and demonstrate the practicality of planning . Our methods provide the experimenter with a valuable tool (available from the authors) for understanding and optimizing cross-linking experiments. Biophys J . 2004 Nov 19; {Epub ahead of print} Forces During Bacteriophage DNA Packaging and Ejection; Purohit PK et al.; The conjunction of insights from structural biology, solution biochemistry, genetics and single molecule biophysics has provided a renewed impetus for the construction of quantitative models of biological processes . One area that has been a beneficiary of these experimental techniques is the study of viruses . In this paper we describe how the insights obtained from such experiments can be utilized to construct physical models of processes in the viral life cycle . We focus on dsDNA bacteriophages and show that the bending elasticity of DNA and its electrostatics in solution can be combined to determine the forces experienced during packaging and ejection of the viral genome . Furthermore, we quantitatively analyze the effect of fluid viscosity and capsid expansion on the forces experienced during packaging . Finally, we present a model for DNA ejection from bacteriophages based on the hypothesis that the energy stored in the tightly packed genome within the capsid leads to its forceful ejection . The predictions of our model can be tested through experiments in vitro where DNA ejection is inhibited by the application of external osmotic pressure. Mol Cell, 2004 Nov 19, 16(4), 609 - 18 Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29; Kamtekar S et al.; The DNA polymerase from phage phi29 is a B family polymerase that initiates replication using a protein as a primer, attaching the first nucleotide of the phage genome to the hydroxyl of a specific serine of the priming protein . The crystal structure of phi29 DNA polymerase determined at 2.2 A resolution provides explanations for its extraordinary processivity and strand displacement activities . Homology modeling suggests that downstream template DNA passes through a tunnel prior to entering the polymerase active site . This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands . Members of the B family of DNA polymerases that use protein primers contain two sequence insertions: one forms a domain not previously observed in polymerases, while the second resembles the specificity loop of T7 RNA polymerase . The high processivity of phi29 DNA polymerase may be explained by its topological encirclement of both the downstream template and the upstream duplex DNA. J Med Virol, 2005 Jan, 75(1), 147 - 52 Identification of epitopes in the nucleocapsid protein of Nipah virus using a linear phage-displayed random peptide library; Eshaghi M et al.; A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus . The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein . A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein . The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region . Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein . The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests . Biophys J . 2004 Nov 12; {Epub ahead of print} DNA EJECTION FROM BACTERIOPHAGE T5: ANALYSIS OF THE KINETICS AND ENERGETICS; de Frutos M et al.; DNA ejection from bacteriophage T5 can be passively driven in vitro by the interaction with its specific host receptor . Light scattering was used to determine the physical parameters associated with this process . By studying the ejection kinetics at different temperatures, we demonstrate that an activation energy of the order of 70 kBT must be overcome to allow the complete DNA ejection . A complex shape of the kinetics was found whatever the temperature . This shape may be actually understood using a phenomenological model based on a multistep process . Passing from one stage to another requires the mentioned thermal activation of pressurized DNA inside the capsids . Both effects contribute to shorten or to lengthen the pause time between the different stages explaining why the T5 DNA ejection is so slow compared to other types of phage. J Virol Methods, 2004 Dec 15, 122(2), 141 - 5 A natural vaccinia virus promoter with exceptional capacity to direct protein synthesis; Liu X et al.; A survey of vaccinia virus promoters, through a reporter gene approach, has identified the viral I1L promoter as having exceptional activity . The I1L promoter exhibited over 10 times the activity of other vaccinia promoters and even rivaled the activity of the bacteriophage T7 promoter in the hybrid vaccinia/T7 expression system . The I1L promoter had high activity in both transient transfection experiments and in the context of recombinant viruses . The I1L promoter should be useful for high-level protein synthesis and poxvirus studies in general. BMC Cancer . 2004 Nov 12;4(1):78. Identification of a panel of tumor-associated antigens from breast carcinoma cell lines, solid tumors and testis cDNA libraries displayed on lambda phage; Pavoni E et al.; BACKGROUND: Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy . Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX) . METHODS: Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed . The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D . Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer . RESULTS: A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified . Three of these antigens (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to normal breast . A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27) . CONCLUSIONS: Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease . The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis. J Biol Chem, 2005 Jan 14, 280(2), 1165 - 78 Epub 2004 Nov 08. Kinetics of Nucleotide Incorporation Opposite DNA Bulky Guanine N2 Adducts by Processive Bacteriophage T7 DNA Polymerase (Exonuclease-) and HIV-1 Reverse Transcriptase; Zang H et al.; Six oligonucleotides with carcinogen derivatives bound at the N2 atom of deoxyguanosine were prepared, including adducts derived from butadiene, acrolein, crotonaldehyde, and styrene, and examined for effects on the replicative enzymes bacteriophage DNA polymerase T7(-) (T7(-)) and HIV-1 reverse transcriptase for comparison with previous work on smaller DNA adducts . All of these adducts strongly blocked dCTP incorporation opposite the adducts . dATP was preferentially incorporated opposite the acrolein and crotonaldehyde adducts, and dTTP incorporation was preferred at the butadiene- and styrene-derived adducts . Steady-state kinetic analysis indicated that the reduced catalytic efficiency with adducted DNA involved both an increased K(m) and attenuated k(cat) . Fluorescence estimates of K(d) and pre-steady-state kinetic measurements of k(off) showed no significantly decreased affinity of T7(-) with the adducted oligonucleotides or the dNTP . Pre-steady-state kinetics showed no burst phase kinetics for dNTP incorporation with any of the modified oligonucleotides . These results indicate that phosphodiester bond formation or a conformational change of the enzyme(*) DNA complex is rate-limiting instead of the step involving release of the oligonucleotide . Thio elemental effects for dNTP incorporation were generally relatively small but variable, indicating that the presence of adducts may sometimes make phosphodiester bond formation rate-limiting but not always. Proc Natl Acad Sci U S A, 2004 Nov 16, 101(46), 16186 - 91 Epub 2004 Nov 16. Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis; Dutta S et al.; The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF) . Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication . dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion . dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G --> T transversions . We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position . Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct . The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation . In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations. Carcinogenesis . 2004 Nov 4; {Epub ahead of print} Peptides specific to the galectin-3 carbohydrate recognition domain inhibit metastasis-associated cancer cell adhesion1; Zou J et al.; Intravascular cancer cell adhesion plays a significant role in the metastatic process . Studies indicate that galectin-3, a member of the galectin family of soluble animal lectins, is involved in carbohydrate-mediated metastatic cell heterotypic (between carcinoma cells and endothelium) and homotypic (between carcinoma cells) adhesion via interactions with the tumor-specific Thomsen-Friedenreich glycoantigen (TFAg) . We hypothesized that blocking the galectin-3 carbohydrate recognition domain with synthetic peptides would significantly reduce metastasis-associated carcinoma cell adhesion . To test this hypothesis, we identified peptide antagonists of galectin-3 carbohydrate recognition domain using combinatorial bacteriophage display technology . The peptides bound with high affinity to purified, recombinant galectin-3 protein (Kd congruent with17 approximately 80 nM) and to cell surface galectin-3 . Experiments with a series of recombinant serially truncated galectin-3 mutants indicated that the peptides bound the carbohydrate recognition domain of galectin-3 . Furthermore, the peptides did not bind the carbohydrate recognition domain of other galectins and plant lectins . Synthetic galectin-3 carbohydrate recognition domain-specific peptides blocked the interaction between galectin-3 and TFAg, and significantly inhibited rolling and stable heterotypic adhesion of human MDA-MB-435 breast carcinoma cells to endothelial cells in flow, as well as homotypic tumor cell aggregation . These results demonstrate that carbohydrate-mediated metastasis-associated tumor cell adhesion could be inhibited efficiently with short synthetic peptides, which do not mimic naturally occurring glycoepitopes, yet bind to the galectin-3 carbohydrate recognition domain with high affinity and specificity. Biosystems, 2004 Nov, 77(1-3), 151 - 61 Artificial life simulation of self-assembly in bacteriophage by movable finite automata; Shirayama M et al.; This paper presents a model which is based on biological research using the movable finite automata (MFA) on a self-assembly of T4 phage, and exhibits the results of artificial life simulation . In the previous work, Thompson and Goel {Artificial Life, Addison Weley, 1989, pp . 317-340; Biosystems 18 (1985) 23; J . Theor . Biol . 131 (1988) 351} presented the movable finite automata (MFA) which has a capability of moving on finite automata, and simulated on a computer . They were represented individual rectangular boxes, however, the results of simulation was different from real T4 phage . We propose the sphere model as a protein structure, and simulate the self-assembly of the entire structure of the T4 phage on a computer. Nature, 2004 Nov 4, 432(7013), 122 - 5 Membrane structure and interactions with protein and DNA in bacteriophage PRD1; Cockburn JJ et al.; Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment . Biological membranes and their associated proteins present considerable difficulties for structural analysis . Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described . The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus . The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper . Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA . The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets . The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell . In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit . In addition, the lipid headgroups show a surprising degree of order. Nature, 2004 Nov 4, 432(7013), 68 - 74 Insights into assembly from structural analysis of bacteriophage PRD1; Abrescia NG et al.; The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution . Here we describe the structure and location of proteins P3, P16, P30 and P31 . Different structural proteins seem to have specialist roles in controlling virus assembly . The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together . Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16 . The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses. Mol Microbiol, 2004 Nov, 54(4), 1036 - 50 A second-site suppressor of a folding defect functions via interactions with a chaperone network to improve folding and assembly in vivo; Parent KN et al.; Single amino acid substitutions in a protein can cause misfolding and aggregation to occur . Protein misfolding can be rescued by second-site amino acid substitutions called suppressor substitutions (su), commonly through stabilizing the native state of the protein or by increasing the rate of folding . Here we report evidence that su substitutions that rescue bacteriophage P22 temperature-sensitive-folding (tsf) coat protein variants function in a novel way . The ability of tsf:su coat proteins to fold and assemble under a variety of cellular conditions was determined by monitoring levels of phage production . The tsf:su coat proteins were found to more effectively utilize P22 scaffolding protein, an assembly chaperone, as compared with their tsf parents . Phage-infected cells were radioactively labelled to quantify the associations between coat protein variants and folding and assembly chaperones . Phage carrying the tsf:su coat proteins induced more GroEL and GroES, and increased formation of protein:chaperone complexes as compared with their tsf parents . We propose that the su substitutions result in coat proteins that are more assembly competent in vivo because of a chaperone-driven kinetic partitioning between aggregation-prone intermediates and the final assembled state . Through more proficient use of this chaperone network, the su substitutions exhibit a novel means of suppression of a folding defect. Biochemistry, 2004 Nov 9, 43(44), 13972 - 80 Membrane assembly of M13 major coat protein: evidence for a structural adaptation in the hinge region and a tilted transmembrane domain; Spruijt RB et al.; New insights into the low-resolution structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy . New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC) . Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe . It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane . From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal . As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region . It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix . On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus . From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process. J Bacteriol, 2004 Nov, 186(22), 7659 - 69 Purification and characterization of the repressor of the shiga toxin-encoding bacteriophage 933W: DNA binding, gene regulation, and autocleavage; Koudelka AP et al.; The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains . The stx genes are expressed only during lytic growth of these temperate bacteriophages . We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein . Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (OR) . Also typical, when bound at OR, 933W repressor functions as an activator at the PRM promoter and a repressor at the PR promoter . In contrast to other lambdoid bacteriophages, 933W left operator (OL) contains only two repressor binding sites, but the OL-bound repressor still efficiently represses PL transcription . Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity . In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a "linker " region that joins the two domains of these proteins . However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence . We show here that the autocleavage occurs at a Leu-Gly dipeptide . Thus, the specificity of the repressor autocleavage site is more variable than thought previously. J Bacteriol, 2004 Nov, 186(22), 7571 - 4 Chlamydiaphage Chp2, a skeleton in the phiX174 closet: scaffolding protein and procapsid identification; Clarke IN et al.; Chlamydiaphage Chp2 is a member of the family Microviridae, of which bacteriophage phiX174 is the type species . Although grouped in the same family, the relationship between the Microviridae coliphages and the Chp2-like viruses, which infect obligate intracellular parasitic bacteria, is quite distant, with major differences in structural protein content and scaffolding protein dependence . To investigate the morphogenesis of Chp2, large particles were isolated from infected Chlamydophila abortus by equilibrium and rate zonal sedimentation . A monoclonal antibody that recognizes only assembled viral coat proteins was used in these detection assays . Thus, the detected particles represent virions and/or postcapsid formation assembly intermediates . Two distinct particle types were detected, differing in both protein and DNA content . Filled particles lacked VP3, the putative internal scaffolding protein, whereas empty particles contained this protein . These results indicate that VP3 is a scaffolding protein and that the isolated VP3-containing particles most likely represent Chp2 procapsids. J Virol, 2004 Nov, 78(22), 12668 - 71 Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1; Trus BL et al.; By electron microscopy and image analysis, we find that baculovirus-expressed UL6 is polymorphic, consisting of rings of 11-, 12-, 13-, and 14-fold symmetry . The 12-mer is likely to be the oligomer incorporated into procapsids: at a resolution of 16 A, it has an axial channel, peripheral flanges, and fits snugly into a vacant vertex site . Its architecture resembles those of bacteriophage portal/connector proteins. Virol J . 2004 Sep 17;1(1):4. Divergence of the mRNA targets for the Ssb proteins of bacteriophages T4 and RB69; Borjac-Natour JM et al.; The single-strand binding (Ssb) protein of phage T4 (T4 gp32, product of gene 32) is a mRNA-specific autogenous translational repressor, in addition to being a sequence-independent ssDNA-binding protein that participates in phage DNA replication, repair and recombination . It is not clear how this physiologically essential protein distinguishes between specific RNA and nonspecific nucleic acid targets . Here, we present phylogenetic evidence suggesting that ssDNA and specific RNA bind the same gp32 domain and that plasticity of this domain underlies its ability to configure certain RNA structures for specific binding . We have cloned and characterized gene 32 of phage RB69, a relative of T4 We observed that RB69 gp32 and T4 gp32 have nearly identical ssDNA binding domains, but diverge in their C-terminal domains . In T4 gp32, it is known that the C-terminal domain interacts with the ssDNA-binding domain and with other phage-induced proteins . In translation assays, we show that RB69 gp32 is, like T4 gp32, an autogenous translational repressor . We also show that the natural mRNA targets (translational operators) for the 2 proteins are diverged in sequence from each other and yet can be repressed by either gp32 . Results of chemical and RNase sensitivity assays indicate that the gp32 mRNA targets from the 2 related phages have similar structures, but differ in their patterns of contact with the 2 repressors . These and other observations suggest that a range of gp32-RNA binding specificities may evolve in nature due to plasticity of the protein-nucleic acid interaction and its response to modulation by the C-terminal domain of this translational repressor. Biochemistry, 2004 Nov 2, 43(43), 13715 - 23 Three-dimensional structure of the R115E mutant of T4-bacteriophage 2'-deoxycytidylate deaminase; Almog R et al.; 2'-Deoxycytidylate deaminase (dCD) converts deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate and is a major supplier of the substrate for thymidylate synthase, an important enzyme in DNA synthesis and a major target for cancer chemotherapy . Wild-type dCD is allosterically regulated by the end products of its metabolic pathway, deoxycytidine 5'-triphosphate and deoxythymidine 5'-triphosphate, which act as an activator and an inhibitor, respectively . The first crystal structure of a dCD, in the form of the R115E mutant of the T4-bacteriophage enzyme complexed with the active site inhibitor pyrimidin-2-one deoxyribotide, has been determined at 2.2 A resolution . This mutant of dCD is active, even in the absence of the allosteric regulators . The molecular topology of dCD is related to that of cytidine deaminase (CDA) but with modifications for formation of the binding site for the phosphate group of dCMP . The enzyme has a zinc ion-based mechanism that is similar to that of CDA . A second zinc ion that is present in bacteriophage dCD, but absent in mammalian dCD and CDA, is important for the structural integrity of the enzyme and for the binding of the phosphate group of the substrate or inhibitor . Although the R115E mutant of dCD is a dimer in solution, it crystallizes as a hexamer, mimicking the natural state of the wild-type enzyme . Residues 112 and 115, which are known to be important for the binding of the allosteric regulators, are found in a pocket that is at the intersubunit interfaces in the hexamer but distant from the substrate-binding site . The substrate-binding site is composed of residues from a single protein molecule and is sequestered in a deep groove . This groove is located at the outer surface of the hexamer but ends at the subunit interface that also includes residue 115 . It is proposed that the absence of subunit interactions at this interface in the dimeric R115E mutant renders the substrate-binding site accessible . In contrast, for the wild-type enzyme, binding of dCTP induces an allosteric effect that affects the subunit interactions and results in an increase in the accessibility of the binding site. Methods Mol Biol, 2004, 291, 145 - 54 Quantifying in vivo somatic mutations using transgenic mouse model systems; Swiger RR; This chapter describes the use of the bacteriophage cII positive selection assay with the Mutatrade markMouse transgenic model system . The assay is similar to others involving a transgenic target, including the cII and lacI assays in the Big Blue(R) Mouse, lacZ in the MutaMouse, and the gpt delta assay . Briefly, high-molecular-weight DNA is purified from the tissue of interest and used as substrate during in vitro packaging reactions, in which the lambda transgenes are excised from the genome and assembled into viable phage . Phage containing the mutational targets are then adsorbed into an appropriate bacterial host, and mutations sustained in vivo are evidenced by either standard recombinant screening or selection assays . Mutant frequencies are reported as the ratio of mutant phage to total phage units analyzed . The lambda-based transgenic mouse assays are used to study and characterize in vivo mutagenesis, as well as for mutagenicity assessment . The models permit the enumeration of mutations sustained in virtually any tissue of the mouse and are sensitive and robust . Application of the assays is simple, not requiring resources beyond those commonly found in most academic laboratories. Science, 2004 Oct 22, 306(5696), 644 - 7 Gene order and dynamic domains; Kosak ST et al.; When considering the daunting complexity of eukaryotic genomes, some comfort can be found in the fact that the human genome may contain only 30,000 to 40,000 genes . Moreover, growing evidence suggests that genomes may be organized in such a way as to take advantage of space . A gene's location in the linear DNA sequence and its position in the three-dimensional nucleus can both be important in its regulation . Contrary to prevailing notions in this postgenomic era, the bacteriophage lambda, a paragon of simplicity, may still have a few things to teach us with respect to these facets of nonrandom genomes. J Water Health, 2004 Sep, 2(3), 201 - 14 Assessment of drinking water quality using indicator bacteria and bacteriophages; Mendez J et al.; Bacterial indicators and bacteriophages suggested as potential indicators of water quality were determined by public laboratories in water from springs, household water wells, and rural and metropolitan water supplies in north-eastern Spain . Indicator bacteria were detected more frequently than bacteriophages in springs, household water wells and rural water supplies . In contrast, positive bacteriophage detections were more numerous than those of bacteria in metropolitan water supplies . Most of the metropolitan water supply samples containing indicators had concentrations of chlorine below 0.1 mg l(-1), their indicator loads resembling more closely those of rural water supplies than any other samples taken from metropolitan water supplies . The number of samples from metropolitan water supplies containing more than 0.1 mg l(-1) of chlorine that contained phages clearly outnumbered those containing indicator bacteria . Some association was observed between rainfall and the presence of indicators . Sediments from service reservoirs and water from dead ends in the distribution network of one of the metropolitan water supplies were also tested . Bacterial indicators and phages were detected in a higher percentage than in samples of tap water from the same network . Additionally, indicator bacteria were detected more frequently than bacteriophages in sediments of service reservoirs and water from dead end samples . We conclude that naturally occurring indicator bacteria and bacteriophages respond differently to chlorination and behave differently in drinking water distribution networks . Moreover, this study has shown that testing for the three groups of phages in routine laboratories is easy to implement and feasible without the requirement for additional material resources for the laboratories. RNA, 2004 Nov, 10(11), 1776 - 82 The crystal structure of a high affinity RNA stem-loop complexed with the bacteriophage MS2 capsid: further challenges in the modeling of ligand-RNA interactions; Horn WT et al.; We have determined the structure to 2.8 A of an RNA aptamer (F5), containing 2'-deoxy-2-aminopurine (2AP) at the -10 position, complexed with MS2 coat protein by soaking the RNA into precrystallised MS2 capsids . The -10 position of the RNA is an important determinant of binding affinity for coat protein . Adenine at this position in other RNA stem-loops makes three hydrogen bonds to protein functional groups . Substituting 2AP for the -10 adenine in the F5 aptamer yields an RNA with the highest yet reported affinity for coat protein . The refined X-ray structure shows that the 2AP base makes an additio