Bacteriophage

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 additional hydrogen bond to the protein compared to adenine that is presumably the principal origin of the increased affinity . There are also slight changes in phosphate backbone positions compared to unmodified F5 that probably also contribute to affinity . Such phosphate movements are common in structures of RNAs bound to the MS2 T = 3 protein shell and highlight problems for de novo design of RNA binding ligands.

Emerg Infect Dis, 2004 Aug, 10(8), 1482 - 5
Human Escherichia coli O157:H7 genetic marker in isolates of bovine origin; Lejeune JT et al.; The antiterminator Q gene of bacteriophage 933W (Q933) was identified upstream of the stx2 gene in 90% of human disease-origin Escherichia coli O157:H7 isolates and in 44.5% of bovine isolates . Shiga toxin production was higher in Q933-positive isolates than Q933-negative isolates . This genetic marker may provide a useful molecular tool for epidemiologic studies.

J Bacteriol, 2004 Nov, 186(21), 7262 - 72
ModA and ModB, two ADP-ribosyltransferases encoded by bacteriophage T4: catalytic properties and mutation analysis; Tiemann B et al.; Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB . These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins . In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes . Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes . In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced . The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control . In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.

J Bacteriol, 2004 Nov, 186(21), 7032 - 68
Genome of bacteriophage P1; Lobocka MB et al.; P1 is a bacteriophage of Escherichia coli and other enteric bacteria . It lysogenizes its hosts as a circular, low-copy-number plasmid . We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100 . The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms . Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny . Four others ensure plasmid maintenance . The majority of the remaining 37 operons are involved in lytic development . Seventeen operons are transcribed from sigma(70) promoters directly controlled by the master phage repressor C1 . Late operons are transcribed from promoters recognized by the E . coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene . Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging . The genome is particularly rich in Chi recombinogenic sites . The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.

Genome Res, 2004 Oct, 14(10B), 2029 - 33
From ORFeomes to protein interaction maps in viruses; Uetz P et al.; Although cloned viral ORFeomes are particularly well suited for genome-wide interaction mapping due to the limited size of viral genomes, only a few such studies have been published . Here, we summarize virus interaction mapping projects involving vaccinia virus, hepatitis C virus (HCV), potato virus A (PVA), pea seed-borne mosaic virus (PSbMV), and bacteriophage T7, as well as some projects in progress . The studies reported suggest that virus-specific coding and replication strategies must be taken into account to yield accurate numbers of protein interactions . In particular, the number of false negatives can be significant for RNA viruses expressing precursor polyproteins (because interactions between full-length mature proteins are often not detected due to incorrect processing) and for viruses replicating in the cytoplasm whose transcripts have not been selected for splicing signals . In conclusion, the studies on viral protein interaction maps suggest that cloned pathogen ORFeomes will contribute to a holistic picture of the pathogenesis of infectious diseases and are ideal starting points for new approaches in systems biology . Both viral ORFeome and interaction mapping projects are being documented on our Web site .

Biophys J, 2005 Jan, 88(1), 751 - 6 Epub 2004 Oct 15.
Measurements of DNA lengths remaining in a viral capsid after osmotically suppressed partial ejection; Evilevitch A et al.; The effect of external osmotic pressure on the extent of DNA ejection from bacteriophage-lambda was recently investigated (Evilevitch et al., 2003) . The total length of DNA ejected was measured via the 260-nm absorption by free nucleotides, after opening of the capsids in the presence of varying amounts of polyethylene glycol 8000 and DNase I . As a function of osmolyte concentration, this absorption was shown to decrease progressively, ultimately vanishing completely for a sufficiently high external osmotic pressure . In this work we report the results of both sedimentation and gel analysis of the length of DNA remaining inside the capsids, as a function of osmolyte concentration . It is confirmed in this way that the progressive inhibition of DNA ejection corresponds to partial ejection from all of the capsids.

J Theor Biol, 2004 Dec 21, 231(4), 525 - 33
Nonspecific binding of the OR repressors CI and Cro of bacteriophage lambda; Bakk A et al.; We estimate the Gibbs free energy for nonspecific binding (DeltaGNSB) to the Escherichia coli DNA for two regulatory proteins of the lambda phage, CI and Cro . By means of a statistical-mechanical approach, we calculate the cI and cro activities associated with the operator OR of an introduced lambda phage genome (prophage) . In this statistical model we apply in vitro-measured binding free energies to fit in vivo experimental data for cI and cro activities, respectively, where DeltaGNSB is introduced as a free (fitting) parameter . Without nonspecific binding included in the model, the quality of the description is fairly poor, whereas data are nicely correlating with our model with nonspecific binding included over the entire data range . The obtained values of DeltaGNSB are -4.1+/-0.9 kcal/mol, for CI, and -4.2+/-0.8 kcal/mol, for Cro . In particular, in a lysogen (approximately 250 CI monomers per cell) we conclude that 86% of the total CI in the cell is nonspecifically bound, leaving on average around 10 CI dimers freely available in the E . coli cytoplasma . These findings corroborate the view that due to low free cellular particle numbers a dynamical analysis of genetic regulation at OR and comparable systems should include a stochastic component . In addition, we perform a stability analysis of the OR system in the presence of nonspecific binding.

Nucleic Acids Res, 2004 Oct 14, 32(18), 5582 - 95 Print 2004.
The sequences and activities of RegB endoribonucleases of T4-related bacteriophages; Piesiniene L et al.; The RegB endoribonuclease encoded by bacteriophage T4 is a unique sequence-specific nuclease that cleaves in the middle of GGAG or, in a few cases, GGAU tetranucleotides, preferentially those found in the Shine-Dalgarno regions of early phage mRNAs . In this study, we examined the primary structures and functional properties of RegB ribonucleases encoded by T4-related bacteriophages . We show that all but one of 36 phages tested harbor the regB gene homologues and the similar signals for transcriptional and post-transcriptional autogenous regulation of regB expression . Phage RB49 in addition to gpRegB utilizes Escherichia coli endoribonuclease E for the degradation of its transcripts for gene regB . The deduced primary structure of RegB proteins of 32 phages studied is almost identical to that of T4, while the sequences of RegB encoded by phages RB69, TuIa and RB49 show substantial divergence from their T4 counterpart . Functional studies using plasmid-phage systems indicate that RegB nucleases of phages T4, RB69, TuIa and RB49 exhibit different activity towards GGAG and GGAU motifs in the specific locations . We expect that the availability of the different phylogenetic variants of RegB may help to localize the amino acid determinants that contribute to the specificity and cleavage efficiency of this processing enzyme.

Nature, 2004 Oct 14, 431(7010), 841 - 4
Adaptation varies through space and time in a coevolving host-parasitoid interaction; Forde SE et al.; One of the central challenges of evolutionary biology is to understand how coevolution organizes biodiversity over complex geographic landscapes . Most species are collections of genetically differentiated populations, and these populations have the potential to become adapted to their local environments in different ways . The geographic mosaic theory of coevolution incorporates this idea by proposing that spatial variation in natural selection and gene flow across a landscape can shape local coevolutionary dynamics . These effects may be particularly strong when populations differ across productivity gradients, where gene flow will often be asymmetric among populations . Conclusive empirical tests of this theory have been particularly difficult to perform because they require knowledge of patterns of gene flow, historical population relationships and local selection pressures . We have tested these predictions empirically using a model community of bacteria and bacteriophage (viral parasitoids of bacteria) . We show that gene flow across a spatially structured landscape alters coevolution of parasitoids and their hosts and that the resulting patterns of adaptation can fluctuate in both space and time.

J Gen Virol, 2004 Nov, 85(Pt 11), 3219 - 27
Development of a reverse-genetics system for Avian pneumovirus demonstrates that the small hydrophobic (SH) and attachment (G) genes are not essential for virus viability; Naylor CJ et al.; Avian pneumovirus (APV) is a member of the genus Metapneumovirus of the subfamily Pneumovirinae . This study describes the development of a reverse-genetics system for APV . A minigenome system was used to optimize the expression of the nucleoprotein, phosphoprotein, M2 and large polymerase proteins when transfected into Vero cells under the control of the bacteriophage T7 promoter . Subsequently, cDNA was transcribed from the virion RNA to make a full-length antigenome, which was also cloned under the control of the T7 promoter . Transfection of the full-length genome plasmid, together with the plasmids expressing the functional proteins in the transcription and replication complex, generated APV in the transfected cells . The recombinant virus was passaged and was identified by cytopathic effect (CPE) that was typical of APV, the presence of a unique restriction-endonuclease site in the cDNA copy of the genome and immunofluorescence staining with anti-APV antibodies . Replacement of the full-length wild-type antigenome with one lacking the small hydrophobic (SH) protein and the attachment (G) genes generated a virus that grew more slowly and produced atypical CPE with syncytia much larger than those seen with wild-type virus.

Plant Cell Rep . 2004 Oct 9; {Epub ahead of print}
Utility of the FLP- FRT recombination system for genetic manipulation of rice; Radhakrishnan P et al.; To develop an FLP- FRT recombination system- (derived from 2 mu plasmid of Saccharomyces cerevisiae) based marker gene removal application for rice, we introduced the gene for FLP recombinase, under the control of the maize ubiquitin-1 promoter, into the rice genome . FLP activity was monitored in callus and regenerated plants by an assay based on the deletion of the FRT-flanked DNA fragment, leading to the activation of the beta-glucuronidase gene . FLP activity was detected both in the callus and leaves of some of the transgenic lines . Based on our comparison of the recombination efficiency of the FLP- FRT system expressed in the transgenic lines with that of the widely used Cre- lox system (derived from bacteriophage P1), we suggest that the FLP- FRT system is a useful tool for the genetic manipulation of rice.

Biotechniques, 2004 Sep, 37(3), 399 - 405
Multiplex ligation-dependent probe amplification using a completely synthetic probe set; Stern RF et al.; The recent development of multiplex ligation-dependent probe amplification (MLPA) has provided an efficient and reliable assay for dosage screening of multiple loci in a single reaction . However, a drawback to this method is the time-consuming process of generating a probe set by cloning in single-stranded bacteriophage vectors . We have developed a synthetic probe set to screen for deletions in a region spanning 18.5 Mb within chromosome 3q . In a pilot study, we tested 15 synthetic probes on 4 control samples and on 2 patients previously found to possess a heterozygous deletion in the region 3q26-q28 . These synthetic probes detected deletions at all previously known deleted loci . Furthermore, using synthetic probes, the variability of results within samples was similar to that reported for commercially available M13-derived probes . Our results demonstrate that this novel approach to MLPA provides a generic solution to the difficulties of probe development by cloning; such synthetically generated probes may be used to screen a large number of loci in a single reaction . We conclude that the use of synthetic probes for MLPA is a rapid, robust, and efficient alternative for research (and potentially diagnostic) deletion and duplication screening of multiple genomic loci.

EMBO J, 2004 Oct 27, 23(21), 4264 - 74 Epub 2004 Oct 07.
Regulated communication between the upstream face of RNA polymerase and the beta' subunit jaw domain; Wigneshweraraj SR et al.; We used bacteriophage T7-encoded transcription inhibitor gene protein 2 (gp2) as a probe to study the contribution of the Escherichia coli RNA polymerase (RNAP) beta' subunit jaw domain--the site of gp2 binding--to activator and ATP hydrolysis-dependent open complex formation by the sigma(54)-RNAP . We show that, unlike sigma(70)-dependent transcription, activated transcription by sigma(54)-RNAP is resistant to gp2 . In contrast, activator and ATP hydrolysis-independent transcription by sigma(54)-RNAP is highly sensitive to gp2 . We provide evidence that an activator- and ATP hydrolysis-dependent conformational change involving the beta' jaw domain and promoter DNA is the basis for gp2-resistant transcription by sigma(54)-RNAP . Our results establish that accessory factors bound to the upstream face of the RNAP, communicate with the beta' jaw domain, and that such communication is subjected to regulation.

Nucleic Acids Res, 2004 Oct 05, 32(17), 5260 - 79 Print 2004.
Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging; Iyer LM et al.; Recently, it has been shown that a predicted P-loop ATPase (the HerA or MlaA protein), which is highly conserved in archaea and also present in many bacteria but absent in eukaryotes, has a bidirectional helicase activity and forms hexameric rings similar to those described for the TrwB ATPase . In this study, the FtsK-HerA superfamily of P-loop ATPases, in which the HerA clade comprises one of the major branches, is analyzed in detail . We show that, in addition to the FtsK and HerA clades, this superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores . The DNA-packaging ATPases of various bacteriophages and eukaryotic double-stranded DNA viruses also belong to the FtsK-HerA superfamily . The FtsK protein is the essential bacterial ATPase that is responsible for the correct segregation of daughter chromosomes during cell division . The structural and evolutionary relationship between HerA and FtsK and the nearly perfect complementarity of their phyletic distributions suggest that HerA similarly mediates DNA pumping into the progeny cells during archaeal cell division . It appears likely that the HerA and FtsK families diverged concomitantly with the archaeal-bacterial division and that the last universal common ancestor of modern life forms had an ancestral DNA-pumping ATPase that gave rise to these families . Furthermore, the relationship of these cellular proteins with the packaging ATPases of diverse DNA viruses suggests that a common DNA pumping mechanism might be operational in both cellular and viral genome segregation . The herA gene forms a highly conserved operon with the gene for the NurA nuclease and, in many archaea, also with the orthologs of eukaryotic double-strand break repair proteins MRE11 and Rad50 . HerA is predicted to function in a complex with these proteins in DNA pumping and repair of double-stranded breaks introduced during this process and, possibly, also during DNA replication . Extensive comparative analysis of the 'genomic context' combined with in-depth sequence analysis led to the prediction of numerous previously unnoticed nucleases of the NurA superfamily, including a specific version that is likely to be the endonuclease component of a novel restriction-modification system . This analysis also led to the identification of previously uncharacterized nucleases, such as a novel predicted nuclease of the Sir2-type Rossmann fold, and phosphatases of the HAD superfamily that are likely to function as partners of the FtsK-HerA superfamily ATPases.

J Biotechnol, 2004 Oct 19, 114(1-2), 55 - 8
Efficient isolation of cDNA clones encoding rheumatoid arthritis autoantigens by lambda phage surface display; Niwa M et al.; Bacteriophage lambda surface display was used to isolate cDNA clones encoding autoantigens recognized by synovial fluid (SF) or sera from patients with rheumatoid arthritis (RA) . We constructed cDNA libraries from human synovial sarcoma cells and synovial tissue, using the surface display vector lambdafoo . The cDNA libraries were screened by affinity selection using 40 SF and 44 sera as probes separately immobilized in microtiter wells . Phage clones isolated encode 13 different autoantigens; an unknown protein, two proteins previously unanalyzed as autoimmune antigens, three proteins previously unknown to be recognized by RA sera, and seven known RA antigens . When analyzed their sensitivity and specificity for RA by phage enzyme-linked immunosorbent assay, frequencies of sera that recognize the newly-isolated autoantigens ranged from 20.5 to 6.8% of a panel of RA sera, and 13.6-0% of other autoimmune disease sera . These results indicate that the lambda phage surface display may be powerful for the isolation of cDNA clones encoding autoantigens recognized by SF or sera from patients with not only RA but also other autoimmune diseases.

Genes Cells, 2004 Oct, 9(10), 877 - 89
A cryptic lysis gene near the start of the Qbeta replicase gene in the +1 frame; Nishihara T et al.; The maturation/lysis (A2) protein encoded by the group B single-stranded RNA bacteriophage Qbeta mediates lysis of host Escherichia coli cells . We found a frameshift mutation in the replicase (beta-subunit) gene of Qbeta cDNA causes cell lysis . The mutant has a single base deletion 73 nucleotides (nt) 3' from the start of the replicase gene with consequent translation termination at a stop codon 129-131 nt further 3' . The 43-amino acid C-terminal part of the 67-amino acid product encoded by what in WT (wild-type) is the +1 frame, is rich in basic amino acids This 67-aa protein can mediate cell lysis whose characteristics indicate that the protein may cause lysis by a different mechanism and via a different target, than that caused by the A2 maturation/lysis protein . Synthesis of a counterpart of the newly discovered lysis product in wild-type phage infection would require a hypothetical ribosomal frameshifting event . The lysis gene of group A RNA phages is also short, 75 codons in MS2, and partially overlaps the first part of their equivalently located replicase gene, raising significant evolutionary implications for the present finding.

Biochemistry, 2004 Oct 12, 43(40), 12723 - 7
On the solution structure of the T4 sliding clamp (gp45); Millar D et al.; Examination by time-resolved fluorescence spectroscopy of the trimeric bacteriophage T4 clamp protein labeled across its three subunit interfaces with a fluorescence resonance energy transfer (FRET) pair indicates that the clamp exists in just one state in solution, with one open and two closed interfaces . This is in contrast to what is observed in the X-ray crystal structure . The population distribution of the trFRET distance is bimodal, giving 67% as 17 A and 33% as 42 A . This leads to the conclusion that gp45 exists in an asymmetric open state in solution . The further increase in the separation of the FRET pair in the presence of the clamp loader and ATP may be ascribed to either further opening of the open interface or the opening of a closed interface . The ramifications for replisome remodeling by this pathway are discussed.

J Microbiol, 2004 Sep, 42(3), 228 - 32
Purification of filamentous bacteriophage M13 by expanded bed anion exchange chromatography; Ling TC et al.; In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock . M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography . In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed . In the single step expanded bed anion exchange adsorption, UpFront FastLine 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (Ho = 15 cm) of STREAMLINE DEAE (rho = 1.2 g/cm3) from Amersham Pharmacia Biotechnology was used as the anion exchanger . The performance of the two methods were evaluated, analysed, and compared . It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86% . The conventional multiple step method yielded the lower recovery percentage, 36.07% . The generic application of this integrated technique has also been assessed .

Mol Biol (Mosk), 2004 Jul-Aug, 38(4), 632 - 41
{Novel site-specific endonucleases F-TflI, F-TflII and F-TflIV encoded by the bacteriophage T5}
{Application of peptide phage libraries for obtaining peptides specific to mouse lung adenocarcinoma}
Nekrasov BG, Kuvshinov VN, Kaledin VI, Nikolin VP, Ushakova TA, Tumanova OIu, Il'ichev AA.

Peptides binding, in vivo, with mouse lung adenocarcinoma, were selected from a peptide phage library containing above 100 million of different permutations . The selected phages carrying specific peptides accumulated in the tumor node, after intravenous injections made in A/Sn mice with induced adenocarcinoma, and persisted there even in 24 h after injections; whereas, they were detected in small quantities or not detected at all in other tissues (e.g . lungs and muscles) . The selected bacteriophages were shown to accumulate not only in the primary tumor node but also in the lung with multiple metastases . Finally, amino acid sequences of exposed peptides were defined.

Vestn Ross Akad Med Nauk, 2004, (8), 22 - 7
{Human mini-antibodies to orthopoxviruses}; Morozova VV et al.; A library of human scFv antibodies displayed on the surface of bacteriophages (MRC, Cambridge, England) was panned against the Elstree strain of vaccinia virus (VACV), which resulted in the phage repertoire enriched with clones positive to the strain . Individual clones from the repertoire were screened for binding, independently, to the vaccinia and ectromelia viruses; phage antibodies to the orthopoxviruses were selected . Ten unique antibodies were identified after their Vh- and Vl-genes were sequenced . All selected antibodies were assayed by ELISA for binding to the vaccinia, cowpox and ectromelia viruses . Furthermore, all selected antibodies were assayed for binding with major alastrim strains of the live variola virus . According to the results, the above phage antibodies recognized genus-specific epitopes, some of which differed in their conformation.

Genetics, 2004 Sep, 168(1), 9 - 19
Co-infection weakens selection against epistatic mutations in RNA viruses; Froissart R et al.; Co-infection may be beneficial in large populations of viruses because it permits sexual exchange between viruses that is useful in combating the mutational load . This advantage of sex should be especially substantial when mutations interact through negative epistasis . In contrast, co-infection may be detrimental because it allows virus complementation, where inferior genotypes profit from superior virus products available within the cell . The RNA bacteriophage phi6 features a genome divided into three segments . Co-infection by multiple phi6 genotypes produces hybrids containing reassorted mixtures of the parental segments . We imposed a mutational load on phi6 populations by mixing the wild-type virus with three single mutants, each harboring a deleterious mutation on a different one of the three virus segments . We then contrasted the speed at which these epistatic mutations were removed from virus populations in the presence and absence of co-infection . If sex is a stronger force, we predicted that the load should be purged faster in the presence of co-infection . In contrast, if complementation is more important we hypothesized that mutations would be eliminated faster in the absence of co-infection . We found that the load was purged faster in the absence of co-infection, which suggests that the disadvantages of complementation can outweigh the benefits of sex, even in the presence of negative epistasis . We discuss our results in light of virus disease management and the evolutionary advantage of haploidy in biological populations.

Anal Biochem, 2004 Oct 15, 333(2), 351 - 7
Accentuation of differentially expressed proteins using phage technology; Suber RL et al.; Protein profiling is frequently used to elucidate disease-specific or differentially expressed proteins . While recent developments have resulted in improved differential profiling, alternative expression platforms that complement existing techniques are continually being explored . We developed a novel method utilizing the amplification and selection capabilities of random peptide-expressing M13 bacteriophage to accentuate differentially expressed proteins in biologic specimens . While the current study used this method to demonstrate differentially expressed proteins in lung cancer tissue in comparison to normal lung tissue, this approach is applicable to a wide range of sample types.

J Struct Biol, 2004 Sep, 147(3), 291 - 301
Molecular dynamics of protein complexes from four-dimensional cryo-electron microscopy; Heymann JB et al.; Cryo-electron microscopy of single particles offers a unique opportunity to detect and quantify conformational variation of protein complexes . Different conformers may, in principle, be distinguished by classification of individual projections in which image differences arising from viewing geometry are disentangled from variability in the underlying structures by "multiple particle analysis"--MPA . If the various conformers represent dynamically related states of the same complex, MPA has the potential to visualize transition states, and eventually to yield movies of the dynamic process . Ordering the various conformers into a time series is facilitated if cryo-EM data are taken at successive times from a system that is known to be developing in time . Virus maturation represents a relatively tractable dynamic process because the changes are large and irreversible and the rate of the natural process may be conveniently slowed in vitro by adjusting the environmental conditions . We describe the strategy employed in a recent analysis of herpes simplex virus procapsid maturation (Nat . Struct . Biol . 10 (2003) 334-341), compare it with previous work on the maturation of bacteriophage HK97 procapsid, and discuss various factors that impinge on the feasibility of performing similar experimental analyses of molecular dynamics in the general case .

J Pak Med Assoc, 2004 Jul, 54(7), 379 - 82
Rapid detection of rifampicin susceptibility of Mycobacterium tuberculosis in sputum specimens by mycobacteriophage assay; Butt T et al.; OBJECTIVE: To evaluate the performance of FASTPlaqueTB-RIF, a newly introduced bacteriophage assay for rapid detection of rifampicin susceptibility of Mycobacterium tuberculosis in sputum specimens . METHODS: A comparative study of 40 sputum specimens from patients of pulmonary tuberculosis, using FASTPlaqueTB-RIF and Bactec 460 TB system carried out at the Armed Forces Institute of Pathology, Rawalpindi between September and November 2001 . RESULTS: Of the 40 clinical isolates of Mycobacterium tuberculosis tested for rifampicin (RIF) susceptibility using the Bactec 460 TB system, 28 isolates were resistant to RIF and 12 isolates were susceptible . FASTPlaqueTB-RIF identified 24 specimens as resistant to RIF . Three specimens that revealed susceptible isolates on Bactec 460, were resistant by FASTPlaqueTB-RIF while four specimens which revealed resistant isolates on Bactec 460, demonstrated susceptibility to RIF by FASTPlaqueTB-RIF . The sensitivity and specificity of FASTPlaqueTB-RIF were 86% and 73% respectively . The predictive values of positive and negative tests were 0.89 and 0.67 respectively . The overall accuracy of the technique was 82% . The phage assay took 48 hours to perform . CONCLUSION: Early detection of rifampicin resistance by the mycobacteriophage technique direct from sputum specimens is a potentially useful new test which would allow decision regarding appropriate therapy to be made early thus having a positive impact on patient care and on prevention of spread of MDR TB.

Biotechnol Bioeng, 2004 Oct 20, 88(2), 148 - 56
Energy-efficient growth of phage Q Beta in Escherichia coli; Kim H et al.; The role of natural selection in the optimal design of organisms is controversial . Optimal forms, functions, or behaviors of organisms have long been claimed without knowledge of how genotype contributes to phenotype, delineation of design constraints, or reference to alternative designs . Moreover, arguments for optimal designs have been often based on models that were difficult, if not impossible, to test . Here, we begin to address these issues by developing and probing a kinetic model for the intracellular growth of bacteriophage Q beta in Escherichia coli . The model accounts for the energetic costs of all template-dependent polymerization reactions, in ATP equivalents, including RNA-dependent RNA elongation by the phage replicase and synthesis of all phage proteins by the translation machinery of the E . coli host cell . We found that translation dominated phage growth, requiring 85% of the total energy expenditure . Only 10% of the total energy was applied to activities other than the direct synthesis of progeny phage components, reflecting primarily the cost of making the negative-strand RNA template that is needed for replication of phage genomic RNA . Further, we defined an energy efficiency of phage growth and showed its direct relationship to the yield of phage progeny . Finally, we performed a sensitivity analysis and found that the growth of wild-type phage was optimized for progeny yield or energy efficiency, suggesting that phage Q beta has evolved to optimally utilize the finite resources of its host cells.

Phys Rev E Stat Nonlin Soft Matter Phys . 2004 Aug;70(2 Pt 1):021503 . Epub 2004 Aug 11.
Microrheology of solutions of semiflexible biopolymer filaments using laser tweezers interferometry; Addas KM et al.; Semiflexible polymers are of great biological importance in determining the mechanical properties of cells . Techniques collectively known as microrheology have recently been developed to measure the viscoelastic properties of solutions of submicroliter volumes . We employ one such technique, which uses a focused laser beam to trap a micron-sized silica bead and interferometric photodiode detection to measure passively the position fluctuations of the trapped bead with nanometer resolution and high bandwidth . The frequency-dependent complex shear modulus G*(f) can be extracted from the position fluctuations via the fluctuation-dissipation theorem and the generalized Stokes-Einstein relation . Using particle tracking microrheology, we report measurements of shear moduli of solutions of fd viruses, which are filamentous, semiflexible, and monodisperse bacteriophages, each 0.9 microm long, 7 nm in diameter, and having a persistence length of 2.2 microm . Recent theoretical treatments of semiflexible polymer dynamics provide quantitative predictions of the rheological properties of such a model system . The fd samples measured span the dilute, semidilute, and concentrated regimes . In the dilute regime G*(f) is dominated by (rigid rod) rotational relaxation, whereas the high-frequency regime reflects single-semiflexible filament dynamics consistent with the theoretical prediction . Due to the short length of fd viruses used in this study, the intermediate regime does not exhibit a well-developed plateau . A dynamic scaling analysis gives rise to a concentration scaling of c(1.36) (r=0.99) in the transition regime and a frequency scaling of f(0.63) (r=0.98) at high frequencies.

RNA, 2004 Nov, 10(11), 1820 - 30 Epub 2004 Sep 23.
Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase; Gopalakrishna S et al.; UV-induced photochemical crosslinking is a powerful approach that can be used for the identification of specific interactions involving nucleic acid-protein and nucleic acid-nucleic acid complexes . 8-AzidoATP (8-N(3)ATP) is a photoaffinity-labeling agent which has been widely used to elucidate the ATP binding site of a variety of proteins . However, its true potential as a photoactivatable nucleotide analog could not be exploited due to the lack of 8-azidoadenosine phosphoramidite, a monomer used in the synthesis of RNA, and the inability of 8-N(3)ATP to serve as an efficient substrate for bacteriophage RNA polymerase . In this study, we explored the ability of SP6, T3, and T7 RNA polymerases and metal ion cofactors to catalyze the incorporation of 8-N(3)AMP into RNA . Whereas transcription buffer containing 2.0-2.5 mM Mn(2+) supports T7 RNA polymerase-mediated insertion of 8-N(3)AMP into RNA, a mixture of 2.5 mM Mn(2+) and 2.5 mM Mg(2+) further improves the yield of 8-N(3)AMP-containing transcript . In addition, both RNA transcription and reverse transcription proceed with high fidelity for the incorporation of 8-N(3)AMP and complementary residue, respectively . Finally, we show that a high-affinity MS2 coat protein binding sequence, in which adenosine residues were replaced by 8-azidoadenosine, crosslinks to the coat protein of the Escherichia coli phage MS2.

Nucleic Acids Res, 2004 Sep 23, 32(17), 4992 - 5002 Print 2004.
DNA-mediated assembly of weakly interacting DNA-binding protein subunits: in vitro recruitment of phage 434 repressor and yeast GCN4 DNA-binding domains; Guarnaccia C et al.; The specificity of DNA-mediated protein assembly was studied in two in vitro systems, based on (i) the DNA-binding domain of bacteriophage 434 repressor cI (amino acid residues 1-69), or (ii) the DNA-binding domain of the yeast transcription factor GCN4, (amino acids 1-34) and their respective oligonucleotide cognates . In vivo, both of these peptides are part of larger protein molecules that also contain dimerization domains, and the resulting dimers recognize cognate palindromic DNA sequences that contain two half-sites of 4 bp each . The dimerization domains were not included in the peptides tested, so in solution-in the presence or absence of non-cognate DNA oligonucleotides-these molecules did not show appreciable dimerization, as determined by pyrene excimer fluorescence spectroscopy and oxidative cross-linking monitored by mass spectrometry . Oligonucleotides with only one 4 bp cognate half-site were able to initiate measurable dimerization, and two half-sites were able to select specific dimers even from a heterogeneous pool of molecules of closely related specificity (such as DNA-binding domains of the 434 repressor and their engineered mutants that mimic the binding helix of the related P22 phage repressor) . The fluorescent technique allowed us to separately monitor the unspecific, ionic interaction of the peptides with DNA which produced a roughly similar signal in the case of both cognate and non-cognate oligonucleotides . But in the former case, a concomitant excimer fluorescence signal showed the formation of correctly positioned dimers . The results suggest that DNA acts as a highly specific template for the recruitment of weakly interacting protein molecules that can thus build up highly specific complexes.

Nature, 2004 Sep 23, 431(7007), 476 - 81
Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements; Doulatov S et al.; Bordetella bacteriophages generate diversity in a gene that specifies host tropism . This microevolutionary adaptation is produced by a genetic element that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, adenine-specific mutagenesis . Central to this process is a reverse transcriptase-mediated exchange between two repeats; one serving as a donor template (TR) and the other as a recipient of variable sequence information (VR) . Here we describe the genetic basis for diversity generation . The directionality of information transfer is determined by a 21-base-pair sequence present at the 3' end of VR . On the basis of patterns of marker transfer in response to variant selective pressures, we propose that a TR reverse transcript is mutagenized, integrated into VR as a single non-coding strand, and then partially converted to the parental VR sequence . This allows the diversity-generating system to minimize variability to the subset of bases under selection . Using the Bordetella phage cassette as a signature, we have identified numerous related elements in diverse bacteria . These elements constitute a new family of retroelements with the potential to confer selective advantages to their host genomes.

Mol Divers, 2004, 8(3), 231 - 45
Multiplexed sorting of libraries on libraries: a novel method for empirical protein design by affinity-driven phage enrichment on synthetic peptide arrays; Hultschig C et al.; Chemically synthesized peptide arrays on planar cellulose carriers are proposed as libraries of ligands suitable for the multiplexed simultaneous capture of peptide-specific acceptor proteins from a large randomly mutagenized library of acceptor proteins presented on bacteriophage M13 particles . This experimental set-up can be exploited to rapidly screen for individual new, distinct binding partners from two complementary libraries (two-dimensional screening) . The technical feasibility of this empirical protein design approach was demonstrated with calmodulin as an aceptor protein using an array of mastoparan variants for multiplexed phage affinity enrichment.

Mol Cell, 2004 Sep 24, 15(6), 991 - 7
Conformational switching by the scaffolding protein D directs the assembly of bacteriophage phiX174; Morais MC et al.; The three-dimensional structure of bacteriophage phiX174 external scaffolding protein D, prior to its interaction with other structural proteins, has been determined to 3.3 angstroms by X-ray crystallography . The crystals belong to space group P4(1)2(1)2 with a dimer in the asymmetric unit that closely resembles asymmetric dimers observed in the phiX174 procapsid structure . Furthermore, application of the crystallographic 4(1) symmetry operation to one of these dimers generates a tetramer similar to the tetramer in the icosahedral asymmetric unit of the procapsid . These data suggest that both dimers and tetramers of the D protein are true morphogenetic intermediates and can form independently of other proteins involved in procapsid morphogenesis . The crystal structure of the D scaffolding protein thus represents the state of the polypeptide prior to procapsid assembly . Hence, comparison with the procapsid structure provides a rare opportunity to follow the conformational switching events necessary for the construction of complex macromolecular assemblies .

Biochem Biophys Res Commun, 2004 Oct 22, 323(3), 809 - 15
Bacteriophage T4 alpha-glucosyltransferase: a novel interaction with gp45 and aspects of the catalytic mechanism; Sommer N et al.; The bacteriophage T4 alpha- and beta-glucosyltransferases (AGT and BGT) catalyse the transfer of glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine of T4 DNA in an alpha- and beta-conformation, respectively . Following the 3D structure of BGT and a secondary structure alignment of AGT and BGT, we performed a site-directed mutagenesis of AGT . A two-domain structure was deduced, with an open substrate-free and a closed substrate-bound conformation . We also identified specific amino acids involved in DNA binding . The identification of a protein-protein interaction of AGT and gp45 which is a part of the T4 replication complex supports the idea that T4 DNA is alpha-glucosylated immediately after synthesis . BGT then glucosylates those hydroxymethyl cytosines not previously served by AGT.

Water Res, 2004 Nov, 38(18), 3821 - 32
Removal of biological and non-biological viral surrogates by spiral-wound reverse osmosis membrane elements with intact and compromised integrity; Mi B et al.; The removal of bacteriophage MS2 and fluorescent-dyed polystyrene microspheres with intact and purposely compromised spiral-wound RO membrane elements was investigated . MS2 rejection with intact membrane elements was >99.9995% . A model developed for data evaluation revealed that the advective passage of MS2 through imperfections of intact membrane elements was <2 x 10(-5)% of the overall product water flow produced . The advective passage of MS2 and microspheres through a pinhole induced in one of the elements was 0.05-0.1% of the overall product water flow . Prolonged testing of both intact and compromised elements resulted in increased MS2 rejection corresponding to advective MS2 passage through membrane imperfections of <3 x 10(-7)% of the overall product water flow . The permeate flow rate obtained with an element with a larger pinhole was 5-13% greater than that of the intact element, and the corresponding rejection of MS2 and microspheres was similar to that observed for sodium chloride . The use of a cracked o-ring in the connection of the permeate tube to the element vessel end-cup resulted in advective passage of MS2 through the crack of <0.0001% of the overall permeate flow.

Nat Biotechnol, 2004 Oct, 22(10), 1297 - 301 Epub 2004 Sep 19.
The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination; Xie J et al.; A recently developed method makes it possible to genetically encode unnatural amino acids with diverse physical, chemical or biological properties in Escherichia coli and yeast . We now show that this technology can be used to efficiently and site-specifically incorporate p-iodo-L-phenylalanine (iodoPhe) into proteins in response to an amber TAG codon . The selective introduction of the anomalously scattering iodine atom into proteins should facilitate single-wavelength anomalous dispersion experiments on in-house X-ray sources . To illustrate this, we generated a Phe153 --> iodoPhe mutant of bacteriophage T4 lysozyme and determined its crystal structure using considerably less data than are needed for the equivalent experiment with cysteine and methionine . The iodoPhe residue, although present in the hydrophobic core of the protein, did not perturb the protein structure in any meaningful way . The ability to selectively introduce this and other heavy atom-containing amino acids into proteins should facilitate the structural study of proteins.

J Biol Chem, 2004 Nov 26, 279(48), 50609 - 18 Epub 2004 Nov 26.
The highly processive DNA polymerase of bacteriophage T5 . Role of the unique N and C termini; Andraos N et al.; The DNA polymerase encoded by bacteriophage T5 has been reported previously to be processive and to catalyze extensive strand displacement synthesis . The enzyme, purified from phage-infected cells, did not require accessory proteins for these activities . Although T5 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I and T7 DNA polymerase, it contains unique regions of 130 and 71 residues at its N and C termini, respectively . We cloned the gene encoding wild-type T5 DNA polymerase and characterized the overproduced protein . We also examined the effect of N- and C-terminal deletions on processivity and strand displacement synthesis . T5 DNA polymerase lacking its N-terminal 30 residues resembled the wild-type enzyme albeit with a 2-fold reduction in polymerase activity . Deletion of 24 residues at the C terminus resulted in a 30-fold reduction in polymerase activity on primed circular DNA, had dramatically reduced processivity, and was unable to carry out strand displacement synthesis . Deletion of 63 residues at the C terminus resulted in a 20,000-fold reduction in polymerase activity . The 3' to 5' double-stranded DNA exonuclease activity associated with T5 DNA polymerase was reduced by a factor of 5 in the polymerase truncated at the N terminus but was stimulated by a factor of 7 in the polymerase truncated at the C terminus . We propose a model in which the C terminus increases the affinity of the DNA for the polymerase active site, thus increasing processivity and decreasing the accessibility of the DNA to the exonuclease active site.

J Biol Chem, 2004 Nov 26, 279(48), 50012 - 8 Epub 2004 Nov 26.
Bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase . Comparison of pre-steady state and single turnover methylation of 40-mer duplexes containing two (un)modified target sites; Malygin EG et al.; We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5'-GATC/5'-GATC) or some modified variant(s) . The results extend a model from studies with single-site 20-mer duplexes . Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites . During processive movement of T4Dam to the next site, the rate-limiting step was the exchange of the product S-adenosyl-l-homocysteine (AdoHcy) for AdoMet without T4Dam dissociating from the duplex . Consequently, instead of a single exponential rate dependence, complex methylation curves were obtained with at least two pre-steady state steps . With 40-mer duplexes containing a single target site, the kinetics were simpler, fitting a single exponential followed by a linear steady state phase . Single turnover methylation of 40-mer duplexes also proceeded in two stages . First, two dimeric T4Dam-AdoMet molecules bound, and each catalyzed a two-step methylation . Instead of processive movement of T4Dam, a conformational adaptation occurred . We propose that following methyl transfer to one strand, dimeric (T4Dam-AdoMet)-(T4Dam-AdoHcy) was capable of rapidly reorienting itself and catalyzing methyl transfer to the target adenine on the complementary, unmethylated strand . This second stage methyl transfer occurred at a rate about 25-fold slower than in the first step; it was rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand . Under single turnover conditions, there was complete methylation of all target adenine residues with each of the two-site 40-mer duplexes.

Cell, 2004 Sep 17, 118(6), 743 - 55
Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation; Mancini EJ et al.; Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP . The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor . We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound . Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations . These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes . Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding . This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes.

J Biol Chem, 2004 Nov 26, 279(48), 50031 - 41 Epub 2004 Nov 26.
Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation; Kim SJ et al.; The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome . We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates . Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity . Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2 . In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187) . Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling . Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody . Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication . In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication . Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2.

Photochem Photobiol, 2004 Sep-Oct, 80(2), 294 - 300
Photoinactivation of hepatitis A virus by synthetic porphyrins; Casteel MJ et al.; Porphyrins are photosensitizers and may be applicable in situations where viral inactivation is required, as for in vitro inactivation of nonenveloped viruses in blood components or in other aqueous media . No study has examined the efficacy of porphyrin inactivation on human pathogens such as hepatitis A virus (HAV) in plasma or other liquids . Experiments were conducted to evaluate the effect of synthetic porphyrins on HAV in porphyrin-containing human plasma and phosphate-buffered saline exposed to long-wavelength (365 nm) UV light . Inactivation of bacteriophage MS2 (MS2) also was determined in some trials . Solutions containing cationic, anionic or amphiphilic porphyrins irradiated with an average light dose of 4.3 J/cm(2) for 90 min resulted in >3 log(10) (>99.9%) to >4 log(10) (>99.99%) inactivation of both HAV and MS2 . Viral inactivation may have been greater than observed because the limits of detection of the assay had been reached . Under ambient lighting conditions, none of the porphyrins was mutagenic in the Ames assay and only the congener with the longest chain-length, tetrakis (N-{n-hexadecyl}-4-pyridiniumyl) porphyrin, was appreciably toxic to mammalian cells . Disinfection by photoactivated synthetic porphyrins therefore can offer an effective and relatively safe approach to removal of nonenveloped viruses from aqueous media.

J Mol Recognit, 2004 Sep-Oct, 17(5), 390 - 6
Bacteriophage Ø29 protein p6: an architectural protein involved in genome organization, replication and control of transcription; Gonzalez-Huici V et al.; Protein p6 of B . subtilis bacteriophage O29 binds to DNA forming a nucleoprotein complex in which the DNA wraps a protein core forming a right-handed superhelix, therefore restraining positive supercoiling and compacting the DNA . The protein does not specifically recognize a nucleotide sequence but rather a structural feature and it binds as a dimer through the minor groove . Protein p6 is in a monomer-dimer equilibrium that shifts to higher-order structures at a concentration of about 1 mM . These structures are probably present in vivo as the intracellular concentration of p6 is estimated to be in this range, and in fact the effective concentration should be still higher due to the macromolecular crowding . The p6 oligomers show an elongated shape compatible with a helical structure reminiscent of the superhelical DNA of the nucleoprotein complex, therefore it was proposed that protein p6 forms a scaffold on which the DNA folds . Since protein p6 is very abundant in infected cells, enough to bind the entire viral progeny, it was proposed to have an architectural role organizing and compacting the viral genome . It has been demonstrated that protein p6 binds in vivo to most, if not all, the O29 genome, although with different affinity, the highest one corresponding to the genome ends . Binding to plasmidic DNA was much lower, although it increased dramatically when the negative superhelicity was decreased . Hence, protein p6 binding specificity for O29 DNA is based on supercoiling, providing that the O29 genome, although topologically constrained, has a negative superhelicity lower than that of plasmid DNA . The formation of the nucleoprotein complex has functional implications in DNA replication and the control of transcription . It activates the initiation of replication that occurs at the genome ends for which the binding affinity is highest . It represses early transcription from promoter C2, and, together with protein p4, it represses transcription from promoters A2b and A2c and activates late transcription from promoter A3; therefore, protein p6 is involved in the early to late transcription switch.

J Contam Hydrol, 2004 Oct, 74(1-4), 231 - 52
Bacteriophage transport through a fining-upwards sedimentary sequence: laboratory experiments and simulation; Flynn R et al.; A column containing four concentric layers of progressively finer-grained glass beads (graded column) was used to study the transport of the bacteriophage T7 in water flowing parallel to layering through a fining-upwards (FU) sedimentary structure . By passing a pulse of T7, and a conservative solute tracer upwards through a column packed with a single bead size (uniform column), the capacity of each bead type to attenuate the bacteriophage was determined . Solute and bacteriophage responses were modelled using an analytical solution to the advection-dispersion equation, with first-order kinetic deposition simulating bacteriophage attenuation . Resulting deposition constants for different flow velocities indicated that filtration theory-determined values differed from experimentally determined values by less than 10% . In contrast, the responses of solute and bacteriophage tracers passing upwards through graded columns could not be reproduced with a single analytical solution . However, a flux-weighted summation of four one-dimensional advective-dispersive analytical terms approximated solute breakthrough curves . The prolonged tailing observed in the resulting curve resembled that typically generated from field-based tracer test data, reflecting the potential importance of textural heterogeneity in the transport of dissolved substances in groundwater . Moreover, bacteriophage deposition terms, determined from filtration theory, reproduced the T7 breakthrough curve once desorption and inactivation on grain surfaces were incorporated . To evaluate the effect of FU sequences on mass transport processes in more detail, bacteriophage passage through sequences resembling those sampled from a FU bed in a fluvioglacial gravel pit were carried out using an analogous approach to that employed in the laboratory . Both solute and bacteriophage breakthrough responses resembled those generated from field-based test data and in the graded column experiments . Comparisons with the results of simulations using averaged hydraulic conductivities show that simulations employing averaged parameters overestimate bacteriophage travel times and underestimate masses recovered and peak concentrations.

J Microbiol, 2004 Jun, 42(2), 99 - 102
Biochemical quantitation of PM2 phage DNA as a substrate for endonuclease assay; Joo YJ et al.; Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome . This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range . Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA . The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol . The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation . A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214 . Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range . This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.

Microbiology, 2004 Sep, 150(Pt 9), 2959 - 71
Diversity of stx2 converting bacteriophages induced from Shiga-toxin-producing Escherichia coli strains isolated from cattle; Muniesa M et al.; The presence of bacteriophages encoding Shiga toxin 2 (stx(2) phages) was analysed in 168 strains of Shiga-toxin-producing Escherichia coli (STEC) isolated from cattle . Following mitomycin C induction, strains carrying stx(2) phages were screened by plaque blot and hybridization with an stx(2)A-probe . In the stx(2)-phage-carrying strains, the amounts of phage production, phage DNA extracted and Stx(2) produced after induction were assessed . The induced stx(2) phages were characterized morphologically and genetically . Assays to obtain lysogens from different strains were also carried out and phages induced from the lysogens were compared with those induced from the STEC isolates . Results indicated that 18 % of the strains carried an inducible stx(2) phage . Most of them showed a direct relationship between phage induction and toxin production . Each strain carried only one inducible stx(2) phage, although a few strains had two copies of the stx(2) in the chromosome . The stx(2) phages showed diverse morphology and a wide variability in their genome . Assays to obtain lysogens showed that not all the phages were transduced with the same frequency and only six lysogens were obtained . Phages in the lysogens were the same as those induced from their respective initial STEC host strains, although the induction and relative toxin production of the lysogens varied . Most phages carried the stx(2) gene, while a few carried stx(2) variants . Infectivity of the phages depended on the different hosts, although O157 : H7 was preferentially infected by phages induced from O157 strains . The results show that inducible stx(2) phages are common among STEC of animal origin and that they may enhance the spread of stx(2).

Appl Environ Microbiol, 2004 Sep, 70(9), 5089 - 93
Calicivirus inactivation by nonionizing (253.7-nanometer-wavelength {UV}) and ionizing (gamma) radiation; De Roda Husman AM et al.; Noroviruses (previously Norwalk-like viruses) are the most common viral agents associated with food- and waterborne outbreaks of gastroenteritis . In the absence of culture methods for noroviruses, animal caliciviruses were used as model viruses to study inactivation by nonionizing (253.7-nm-wavelength {UV}) and ionizing (gamma) radiation . Here, we studied the respiratory feline calicivirus (FeCV) and the presumed enteric canine calicivirus (CaCV) and compared them with the well-studied bacteriophage MS2 . When UV irradiation was used, a 3-log(10) reduction was observed at a fluence of 120 J/m(2) in the FeCV suspension and at a fluence of 200 J/m(2) for CaCV; for the more resistant phage MS2 there was a 3-log(10) reduction at a fluence of 650 J/m(2) . Few or no differences were observed between levels of UV inactivation in high- and low-protein-content virus stocks . In contrast, ionizing radiation could readily inactivate MS2 in water, and there was a 3-log(10) reduction at a dose of 100 Gy, although this did not occur when the phage was diluted in high-protein-content stocks of CaCV or FeCV . The low-protein-content stocks showed 3-log(10) reductions at a dose of 500 Gy for FeCV and at a dose of 300 for CaCV . The inactivation rates for both caliciviruses with ionizing and nonionizing radiation were comparable but different from the inactivation rates for MS2 . Although most FeCV and CaCV characteristics, such as overall particle and genome size and structure, are similar, the capsid sequences differ significantly, making it difficult to predict human norovirus inactivation . Adequate management of UV and gamma radiation processes for virus inactivation should limit public health risks.

J Bacteriol, 2004 Sep, 186(18), 6335 - 9
Processing of the tail lysozyme (gp5) of bacteriophage T4; Ye N et al.; The processing site of gp5 has been determined to be between residues Val-390 and His-391, instead of Ser-351 and Ala-352 as previously reported (H . Kanamaru, N . C . Gassner, N . Ye, S . Takeda, and F . Arisaka, J . Bacteriol . 181:2739-2744) . Moreover, the maturation of gp5 is abolished by null mutations in other hub genes, indicating that cleavage requires the interactions of several baseplate proteins.

Genetics, 2004 Aug, 167(4), 2015 - 26
Evolution of specialists in an experimental microcosm; Dykhuizen DE et al.; The impact of adaptation on the persistence of a balanced polymorphism was explored using the lactose operon of Escherichia coli as a model system . Competition in chemostats for two substitutable resources, methylgalactoside and lactulose, generates stabilizing frequency-dependent selection when two different naturally isolated lac operons (TD2 and TD10) are used . The fate of this balanced polymorphism was tracked over evolutionary time by monitoring the frequency of fhuA-, a linked neutral genetic marker that confers resistance to the bacteriophage T5 . In four out of nine chemostats the lac polymorphism persisted for 400-600 generations when the experiments were terminated . In the other five chemostats the fhuA polymorphism, and consequently the lac operon polymorphism, was lost between 86 and 219 generations . Four of 13 chemostat cultures monomorphic for the lac operon retained the neutral fhuA polymorphism for 450-550 generations until they were terminated; the remainder became monomorphic at fhuA between 63 and 303 generations . Specialists on each galactoside were isolated from chemostats that maintained the fhuA polymorphism, whether polymorphic or monomorphic at the lac operon . Strains isolated from three of four chemostats in which the lac polymorphism was preserved had switched their galactoside preference . Most of the chemostats where the fhuA polymorphism was lost also contained specialists . These results demonstrate that the initial polymorphism at lac was of little consequence to the outcome of long-term adaptive evolution . Instead, the fitnesses of evolved strains were dominated by mutations arising elsewhere in the genome, a fact confirmed by showing that operons isolated from their evolved backgrounds were alone unable to explain the presence of both specialists . Our results suggest that, once stabilized, ecological specialization prevented selective sweeps through the entire population, thereby promoting the maintenance of linked neutral polymorphisms .

Biometrics, 2004 Sep, 60(3), 573 - 81; discussion 581-8
A Bayesian approach to DNA sequence segmentation; Boys RJ et al.; Many deoxyribonucleic acid (DNA) sequences display compositional heterogeneity in the form of segments of similar structure . This article describes a Bayesian method that identifies such segments by using a Markov chain governed by a hidden Markov model . Markov chain Monte Carlo (MCMC) techniques are employed to compute all posterior quantities of interest and, in particular, allow inferences to be made regarding the number of segment types and the order of Markov dependence in the DNA sequence . The method is applied to the segmentation of the bacteriophage lambda genome, a common benchmark sequence used for the comparison of statistical segmentation algorithms.

Trends Microbiol, 2004 Sep, 12(9), 401 - 4
Antibiotic-induced lateral transfer of antibiotic resistance; Hastings PJ et al.; As do many temperate bacteriophages, integrating conjugative elements (ICEs) recruit the SOS DNA damage response to mobilize themselves from the bacterial chromosome and infect other cells . This transfers resistance to multiple antibiotics . Several commonly used antibiotics induce the SOS response, potentially hastening genetic change and the evolution to resistance of pathogenic populations . The use of such antibiotics should be reconsidered.

Curr Biol, 1992 Mar, 2(3), 150 - 1
Phage introns on the hop: an alternative view; Bickle TA; The nuclease encoded by a rare bacteriophage intron is very similar to one class of bacterial restriction enzyme, and may provide clues as to the co-evolution of host and phage.

Curr Biol, 1993 Mar, 3(3), 141 - 8
Structural similarity of DNA-binding domains of bacteriophage repressors and the globin core; Subbiah S et al.; Background: In recent years, the determination of large numbers of protein structures has created a need for automatic and objective methods for the comparison of structures or conformations . Many protein structures show similarities of conformation that are undetectable by comparing their sequences . Comparison of structures can reveal similarities between proteins thought to be unrelated, providing new insight into the interrelationships of sequence, structure and function . Results: Using a new tool that we have developed to perform rapid structural alignment, we present the highlights of an exhaustive comparison of all pairs of protein structures in the Brookhaven protein database . Notably, we find that the DNA-binding domain of the bacteriophage repressor family is almost completely embedded in the larger eight-helix fold of the globin family of proteins . The significant match of specific residues is correlated with functional, structural and evolutionary information . Conclusion: Our method can help to identify structurally similar folds rapidly and with high-sensitivity, providing a powerful tool for analyzing the ever-increasing number of protein structures being elucidated.

J Virol, 2004 Sep, 78(18), 9790 - 7
Integral membrane protein P16 of bacteriophage PRD1 stabilizes the adsorption vertex structure; Jaatinen ST et al.; The icosahedral membrane-containing double-stranded DNA bacteriophage PRD1 has a labile receptor binding spike complex at the vertices . This complex, which is analogous to that of adenovirus, is formed of the penton protein P31, the spike protein P5, and the receptor binding protein P2 . Upon infection, the internal phage membrane transforms into a tubular structure that protrudes through a vertex and penetrates the cell envelope for DNA injection . We describe here a new class of PRD1 mutants lacking virion-associated integral membrane protein P16 . P16 links the spike complex to the viral membrane and is necessary for spike stability . We also show that the unique vertex used for DNA packaging is intact in the P16-deficient particle, indicating that the 11 adsorption vertices and the 1 portal vertex are functionally and structurally distinct .

Chem Biol, 2004 Aug, 11(8), 1081 - 91
Design and validation of a bifunctional ligand display system for receptor targeting; Chen L et al.; Here we developed a bacteriophage display particle designed to serve as a bifunctional entity that can target tumors while delivering an agent . We engineered a chimera phage vector containing a pIII-displayed alphav integrins-targeting moiety and a pVIII-displayed streptavidin binding adaptor moiety . By using the chimeric phage particle, targeting of alphav integrins on cells in culture and tumor-related blood vessels was shown through different applications, including luminescent quantum dots localization, surface plasmon resonance-based binding detection, and an in vivo tumor model . The strategy validated here will accelerate the discovery and characterization of receptor-ligand binding events in high throughput, and cell-specific delivery of diagnostics or therapeutics to organs of choice without the need for chemical conjugation.

Mol Hum Reprod, 2004 Oct, 10(10), 767 - 72 Epub 2004 Aug 20.
Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease; Handyside AH et al.; Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA . Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers . Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting . As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells . With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA . The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.

Water Sci Technol, 2004, 50(1), 155 - 8
Removal of F-specific RNA bacteriophages in artificial recharge of groundwater--a field study; Niemi RM et al.; Artificial recharge of groundwater offers a semi-natural means to produce raw water for drinking-water plants . Surface water works are increasingly being replaced by artificial groundwater works in Finland . Two municipalities, one serving 30,000 and the other 170,000 inhabitants, have considered filtering river water through eskers for the production of potable water . In this study the removal of bacteriophages during infiltration of river water was estimated, for the evaluation of treatment adequacy in a field study . A 5-m-deep column of sand was constructed and used to mimic the percolating phase in infiltration . An artificial esker was constructed on the riverbank by isolating a 2-m-wide, 2-m-deep and 18-m-long bed of coarse sand with plastic . The sand bed represented the saturated zone . River water was pumped at a rate of 40 L/h to the sand column . The river water was spiked with F+ specific RNA phage MS2 by adding phage suspension during one week at an average concentration of 4.3 x 10(9) PFU/mL . Samples for phage assays were taken during one month, from four sampling sites, on the basis of detention time as estimated by a tracer experiment with sodium chloride . The median count of MS2 for percolated water was 2.4 x 10(5) PFU/mL, representing a 96.7% reduction . During the passage of 6 m in the saturated zone, a further reduction of 98.5% occurred . During the passage from 6 m to 12 m the additional reduction was 99.97% . The overall reduction was between 6 and 7 log10 units . The removal of MS2 phages was rather efficient, although the esker material was coarse, mainly sandy, gravel.

Methods Mol Biol, 2004, 278, 79 - 88
Media for studies of partially aligned states; Fleming K et al.; Measurement of residual dipolar couplings for proteins in nuclear magnetic resonance (NMR) requires a degree of molecular alignment . This may be achieved through the use of liquid crystals or compressed hydrated gels . Several media have been described in the literature, and this chapter describes five of the most commonly used systems . For two of these systems, bicelles and filamentous bacteriophage, the media can be purchased in a form ready for experiments . The remainder must be made by the investigator, yet they are generally straightforward to synthesize in the laboratory . Poly(ethylene glycol)/alcohol mixtures and Helfrich phases are made by simply mixing the ingredients in the correct manner, and strained gels use techniques familiar to all molecular biologists.

J Bacteriol, 2004 Sep, 186(17), 5715 - 20
The vir gene of bacteriophage MAV1 confers resistance to phage infection on Mycoplasma arthritidis; Clapper B et al.; Lysogenization of Mycoplasma arthritidis with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats . The MAV1 vir gene is one of only two constitutively transcribed phage genes in the lysogen . We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane . To investigate whether Vir is a virulence factor, the vir gene was cloned into the transposon vector Tn4001T and inserted in the genome of the nonlysogen strain 158 . The virulence of the resulting transformants was no different from that of the parent strain . Interestingly, all vir-containing transformants were resistant to infection by MAV1 . Vir had no effect on MAV1 adsorption . We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm.

J Bacteriol, 2004 Sep, 186(17), 5699 - 707
Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli McrA endonuclease; Anton BP et al.; McrA is one of three functions that restrict modified foreign DNA in Escherichia coli K-12, affecting both methylated and hydroxymethylated substrates . We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system . We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein . Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified lambda and T4gt, respectively) and for induction of the E . coli SOS response in the presence of M.HpaII methylation, indicative of DNA damage . Our findings suggest the presence of an N-terminal DNA-binding domain and a C-terminal catalytic nuclease domain connected by a linker region largely tolerant of amino acid insertions . DNA damage inflicted by a functional C-terminal domain is required for restriction of phage T4gt . Disruption of the N-terminal domain abolishes restriction of both substrates . Surprisingly, truncation mutations that spare the N-terminal domain do not mediate DNA damage, as measured by SOS induction, but nevertheless partially restrict M.HpaII-modified lambda in vivo . We suggest a common explanation for this "restriction without damage" and a similar observation seen in vivo with McrB, a component of another of the modified-DNA restriction functions . Briefly, we propose that unproductive site-specific binding of the protein to a vulnerable position in the lambda genome disrupts the phage development program at an early stage . We also identified a single mutant, carrying an insertion in the N-terminal domain, which could fully restrict lambda but did not restrict T4gt at all . This mutant may have a selective impairment in substrate recognition, distinguishing methylated from hydroxymethylated substrates . The study shows that the technically easy insertion scanning method can provide a rich source of functional information when coupled with effective phenotype tests.

Mol Genet Genomics, 2004 Sep, 272(2), 227 - 34 Epub 2004 Aug 14.
Escherichia coli mazEF-mediated cell death as a defense mechanism that inhibits the spread of phage P1; Hazan R et al.; The Escherichia coli gene pair mazEF is a regulatable chromosomal toxin-antitoxin module: mazF encodes a stable toxin and mazE encodes for a labile antitoxin that overcomes the lethal effect of MazF . Because MazE is labile, inhibition of mazE expression results in cell death . We studied the effect of mazEF on the development of bacteriophage P1 upon thermoinduction of the prophage P1CM c1ts and upon infection with virulent phage particles (P1( vir)) . In several E . coli strains, we showed that the Delta mazEF derivative strains produced significantly more phages than did the parent strain . In addition, upon induction of K38(P1CM c1ts), nearly all of the Delta mazEF mutant cells lysed; in contrast, very few of the parental mazEF + K38 cells underwent lysis . However, most of these cells did not remain viable . Thus, while the Delta mazEF cells die as a result of the lytic action of the phage, most of the mazEF(+) cells are killed by a different mechanism, apparently through the action of the chromosomal mazEF system itself . Furthermore, the introduction of lysogens into a growing non-lysogenic culture is lethal to Delta mazEF but not for mazEF(+) cultures . Thus, although mazEF action causes individual cells to die, upon phage growth this is generally beneficial to the bacterial culture because it causes P1 phage exclusion from the bacterial population . These results provide additional support for the view that bacterial cultures may share some of the characteristics of multicellular organisms.

Cell, 2004 Aug 20, 118(4), 419 - 29
Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host; Leiman PG et al.; The contractile tail of bacteriophage T4 undergoes major structural transitions when the virus attaches to the host cell surface . The baseplate at the distal end of the tail changes from a hexagonal to a star shape . This causes the sheath around the tail tube to contract and the tail tube to protrude from the baseplate and pierce the outer cell membrane and the cell wall before reaching the inner cell membrane for subsequent viral DNA injection . Analogously, the T4 tail can be contracted by treatment with 3 M urea . The structure of the T4 contracted tail, including the head-tail joining region, has been determined by cryo-electron microscopy to 17 A resolution . This 1200 A-long, 20 MDa structure has been interpreted in terms of multiple copies of its approximately 20 component proteins . A comparison with the metastable hexagonal baseplate of the mature virus shows that the baseplate proteins move as rigid bodies relative to each other during the structural change.

Cell, 2004 Aug 20, 118(4), 403 - 4
Signal transduction at a protein synapse; Steven AC; Contraction of the bacteriophage T4 tail in the act of host cell penetration represents a massive structural change powered by conformational free energy . A paper in this issue of Cell by compares cryo-electron microscopic reconstructions of the initial and final states and reveals that the basic underlying mechanism is concerted rigid-body movements of the constituent protein subunits, akin to the tumbling of gears in a lock.

J Mol Biol, 2004 Sep 3, 342(1), 219 - 27
Adenovirus fibre shaft sequences fold into the native triple beta-spiral fold when N-terminally fused to the bacteriophage T4 fibritin foldon trimerisation motif; Papanikolopoulou K et al.; Adenovirus fibres are trimeric proteins that consist of a globular C-terminal domain, a central fibrous shaft and an N-terminal part that attaches to the viral capsid . In the presence of the globular C-terminal domain, which is necessary for correct trimerisation, the shaft segment adopts a triple beta-spiral conformation . We have replaced the head of the fibre by the trimerisation domain of the bacteriophage T4 fibritin, the foldon . Two different fusion constructs were made and crystallised, one with an eight amino acid residue linker and one with a linker of only two residues . X-ray crystallographic studies of both fusion proteins shows that residues 319-391 of the adenovirus type 2 fibre shaft fold into a triple beta-spiral fold indistinguishable from the native structure, although this is now resolved at a higher resolution of 1.9 A . The foldon residues 458-483 also adopt their natural structure . The intervening linkers are not well ordered in the crystal structures . This work shows that the shaft sequences retain their capacity to fold into their native beta-spiral fibrous fold when fused to a foreign C-terminal trimerisation motif . It provides a structural basis to artificially trimerise longer adenovirus shaft segments and segments from other trimeric beta-structured fibre proteins . Such artificial fibrous constructs, amenable to crystallisation and solution studies, can offer tractable model systems for the study of beta-fibrous structure . They can also prove useful for gene therapy and fibre engineering applications.

Philos Transact A Math Phys Eng Sci, 2004 Jul 15, 362(1820), 1497 - 517
Statics and dynamics of condensed DNA within phages and globules; Odijk T; Several controversial issues concerning the packing of linear DNA in bacteriophages and globules are discussed . Exact relations for the osmotic pressure, capsid pressure and loading force are derived in terms of the hole size inside phages under the assumption that the DNA globule has a uniform density . A new electrostatic model is introduced for computing the osmotic pressure of rod-like polyelectrolytes at very high concentrations . At intermediate packing, a reptation model is considered for DNA diffusing within a toroidal globule . Under tight-packing conditions a model of Coulomb sliding friction is proposed . A general discussion is given of our current understanding of the statics and dynamics of confined DNA in the context of the following experiments: characterization of the liquid crystalline phases, X-ray scattering by phages, osmotic-stress measurements, cyclization within globules and single-molecule determination of the loading forces.

Mol Microbiol, 2004 Aug, 53(4), 1251 - 65
Bacteriophage T7 DNA ejection into cells is initiated by an enzyme-like mechanism; Kemp P et al.; In a normal infection about 850 bp of the bacteriophage T7 genome is ejected into the cell, the remainder of the genome is internalized through transcription by Escherichia coli and then T7 RNA polymerase . Rates of T7 DNA internalization by the E . coli enzyme in vivo are constant across the whole genome . As expected for an enzyme-catalysed reaction, rates vary with temperature and can be fitted to Arrhenius kinetics . Phage virions containing a mutant gp16, a protein known to be ejected from the phage capsid into the cell at the initiation of infection, allow complete entry of the T7 genome in the absence of transcription . The kinetics of DNA ejection from such a mutant virion into the bacterial cytoplasm have also been measured at different temperatures in vivo . Between 15 and 43 degrees C the entire 40 kb T7 genome is translocated into the cell at a constant rate that is characteristic for each temperature, and the temperature-dependence of DNA translocation rates can be fitted to Arrhenius kinetics . The data are consistent with the idea that transcription-independent DNA translocation from the T7 virion is also enzyme-catalysed . The proton motive force is necessary for this mode of DNA translocation, because collapsing the membrane potential while the T7 genome is entering the cell abruptly halts further DNA transfer.

Virology, 2004 Sep 1, 326(2), 340 - 52
F exclusion of bacteriophage T7 occurs at the cell membrane; Cheng X et al.; The F plasmid PifA protein, known to be the cause of F exclusion of bacteriophage T7, is shown to be a membrane-associated protein . No transmembrane domains of PifA were located . In contrast, T7 gp1.2 and gp10, the two phage proteins that trigger phage exclusion, are both soluble cytoplasmic proteins . The Escherichia coli FxsA protein, which, at higher concentrations than found in wild-type cells, protects T7 from exclusion, is shown to interact with PifA . FxsA is a polytopic membrane protein with four transmembrane segments and a long cytoplasmic C-terminal tail . This tail is not important in alleviating F exclusion and can be deleted; in contrast, the fourth transmembrane segment of FxsA is critical in allowing wild-type T7 to grow in the presence of F PifA . These data suggest that the primary event that triggers the exclusion process occurs at the cytoplasmic membrane and that FxsA sequesters PifA so that membrane damage is minimized.

Nat Biotechnol, 2004 Sep, 22(9), 1161 - 5 Epub 2004 Aug 08.
Aggregation-resistant domain antibodies selected on phage by heat denaturation; Jespers L et al.; We describe a method for selecting aggregation-resistant proteins by heat denaturation . This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate . The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs . After cooling, the dAbs were selected for binding to protein A (a ligand common to these folded dAbs) . Phage displaying dAbs that unfold reversibly were thereby enriched with respect to those that do not . From a repertoire of phage dAbs, six dAbs were characterized after selection; they all resisted aggregation, and were soluble, well expressed in bacteria and could be purified in good yields . The method should be useful for making aggregation-resistant proteins and for helping to identify features that promote or prevent protein aggregation, including those responsible for misfolding diseases.

Acta Crystallogr D Biol Crystallogr, 1997 Mar, 53(Pt 2), 217 - 9
Crystallization and preliminary X-ray analysis of bacteriophage lambda lysozyme in which all tryptophans have been replaced by aza-tryptophans; Evrard C; After many unsuccessful attempts to crystallize the bacteriophage lambda lysozyme, a mutant where all the tryptophan residues have been replaced by aza-tryptophans has been crystallized by the vapor-diffusion method . The crystals are orthorhombic and belong to space group P2(1)2(1)2(1) with cell dimensions a = 73.01, b = 78.80, c = 82.31 A . Diffraction data were collected using synchrotron radiation sources . Crystals diffract to a resolution of 2.3 A . Data from two different platinum derivatives were also recorded to 2.8 and 2.5 A, respectively.

Acta Crystallogr D Biol Crystallogr, 1995 Sep, 51(Pt 5), 792 - 804
Pf1 filamentous bacteriophage: refinement of a molecular model by simulated annealing using 3.3 A resolution X-ray fibre diffraction data; Gonzalez A; The filamentous bacteriophage Pf1 is structurally similar to the well known Ff (fd, fl, M13) strains, but it gives much better X-ray diffraction patterns, enabling a more detailed analysis of the molecular structure . The 46-residue protein subunit can be closely approximated by a single gently curved stretch of alpha-helix . The axes of the subunits are at a small angle to the virion axis, and several thousand subunits form an overlapping inter-digitated helical array surrounding a DNA core . We have derived a detailed model of the virion based on X-ray data and stereochemical constraints . We have considered potential sources of error in the diffraction data, and used the improved data to study regions where the protein subunit of Pf1 may deviate from a continuous alpha-helix . We use simulated annealing to escape from local minima, and various kinds of electron-density maps to guide the model building . Refinement of the model shows that the first few residues at the N terminus are non-helical, and there is a slight discontinuity in the alpha-helix near the middle of the sequence . The model is consistent both with general structural principles derived from high-resolution analysis of other proteins, and with specific chemical and spectroscopic data about Pf1 . We apply the same refinement techniques to an alternative model with a non-helical surface loop between residues 13 and 19 . Comparative analysis of models with and without a loop shows that the loop model is not supported by 3.3 A resolution X-ray diffraction data.

Acta Crystallogr D Biol Crystallogr, 1996 Jan, 52(Pt 1), 226 - 8
Crystallization and preliminary X-ray analysis of bacteriophage T4 deoxynucleotide kinase; Sebastiao P; T4 deoxynucleotide kinase catalyzes the phosphorylation of 5-hydroxymethyldeoxycytidylate, dTMP and dGMP while excluding dCMP and dAMP . In order to understand the mechanism of this remarkable specificity, the enzyme was over-expressed in Escherichia coli, purified and crystallized for X-ray diffraction analysis . The crystals belong to the monoclinic space group C2 with cell dimensions a = 155.2, b = 58.5, c = 75.7 A, beta = 108.1 degrees . There are two protein monomers in the asymmetric unit related by a twofold axis . Diffraction data to 2.0 A resolution have been collected.

Acta Crystallogr D Biol Crystallogr, 1994 Jan, 50(Pt 1), 105 - 9
Crystallization and preliminary X-ray diffraction studies of the bacteriophage Qbeta; Valegard K; Crystals of bacteriophage Qbeta have been obtained by the vapor-diffusion technique . The crystals diffract to at least 3.5 A resolution . The crystal space group is C222(1) with the unit-cell parameters a = 478, b = 296, c = 477 A, alpha = beta = gamma = 90 degrees . The unit cell contains four virus particles . A pattern of systematic extinctions has been used to deduce the packing of the particles in the cell . A limited data set to 3.9 A resolution has been collected, and the predicted position has been confirmed by the self-rotation and the Patterson functions.

Acta Crystallogr D Biol Crystallogr, 1995 May, 51(Pt 3), 347 - 53
Reciprocal-space molecular-replacement averaging; Tong L; The molecular-replacement equations, in which electron-density averaging and skew averaging have been unified, were used in reciprocal space to refine and extend the resolution of phased reflections . A procedure has been developed for the treatment of molecular envelopes of general shape . The equations were successfully applied to the reflection data of bacteriophage phiX174 (60-fold redundancy) . Truncation of the G diffraction function beyond the first few nodes did not have a significant effect on the quality of the molecular-replacement equations . Reciprocal-space molecular-replacement averaging should prove to be a useful alternative to real-space averaging . Strategies are discussed that are possible only in reciprocal space.

J Biol Chem, 2004 Oct 15, 279(42), 43568 - 73 Epub 2004 Aug 06.
In vivo bypass efficiencies and mutational signatures of the guanine oxidation products 2-aminoimidazolone and 5-guanidino-4-nitroimidazole; Neeley WL et al.; The in vivo mutagenic properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole, two products of peroxynitrite oxidation of guanine, are reported . Two oligodeoxynucleotides of identical sequence, but containing either 2-aminoimidazolone or 5-guanidino-4-nitroimidazole at a specific site, were ligated into single-stranded M13mp7L2 bacteriophage genomes . Wild-type AB1157 Escherichia coli cells were transformed with the site-specific 2-aminoimidazolone- and 5-guanidino-4-nitroimidazole-containing genomes, and analysis of the resulting progeny phage allowed determination of the in vivo bypass efficiencies and mutational signatures of the DNA lesions . 2-Aminoimidazolone was efficiently bypassed and 91% mutagenic, producing almost exclusively G to C transversion mutations . In contrast, 5-guanidino-4-nitroimidazole was a strong block to replication and 50% mutagenic, generating G to A, G to T, and to a lesser extent, G to C mutations . The G to A mutation elicited by 5-guanidino-4-nitroimidazole implicates this lesion as a novel source of peroxynitrite-induced transition mutations in vivo . For comparison, the error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation . SOS induction caused little change in the efficiency of DNA polymerase bypass of 2-aminoimidazolone; however, bypass of 5-guanidino-4-nitroimidazole increased nearly 10-fold . Importantly, the mutation frequencies of both lesions decreased during replication in SOS-induced cells . These data suggest that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole in DNA are substrates for one or more of the SOS-induced Y-family DNA polymerases and demonstrate that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole are potent sources of mutations in vivo.

Appl Environ Microbiol, 2004 Aug, 70(8), 4864 - 71
Characterization of coliphage PR772 and evaluation of its use for virus filter performance testing; Lute S et al.; Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody, and plasma-derived biopharmaceuticals . Recently, a consensus has developed among filter manufacturers and end users about the desirability of a common nomenclature and a standardized test for classifying and identifying virus-retentive filters . The Parenteral Drug Association virus filter task force has chosen PR772 as the model bacteriophage to standardize nomenclature for large-pore-size virus-retentive filters (filters designed to retain viruses larger than 50 to 60 nm in size) . Previously, the coliphage PR772 (Tectiviridae family) has been used in some filtration studies as a surrogate for mammalian viruses of around 50 to 60 nm . In this report, we describe specific properties of PR772 critical to the support of its use for the standardization of virus filters . The complete genomic sequence of virulent phage PR772 was determined . Its genome contains 14,946 bp with an overall G+C content of 48.3 mol%, and 32 open reading frames of at least 40 codons . Comparison of the PR772 nucleotide sequence with the genome of Tectiviridae family prototype phage PRD1 revealed 97.2% identity at the DNA level . By dynamic light-scattering analysis, its hydrodynamic diameter was measured as 82 +/- 6 nm, consistent with use in testing large-virus-retentive filters . Finally, dynamic light-scattering analysis of PR772 preparations purified on CsCl gradients showed that the phage preparations are largely monodispersed . In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger-pore-size virus-retentive filters.

Protein Expr Purif, 2004 Sep, 37(1), 203 - 6
The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3); Dumon-Seignovert L et al.; Two mutant strains of Escherichia coli BL21(DE3), called C41(DE3) and C43(DE3) and originally described by Miroux and Walker, are frequently used to overcome the toxicity associated with overexpressing recombinant proteins using the bacteriophage T7 RNA polymerase expression system . Even when the toxicity of the plasmids is so high that it prevents transformation in the strain BL21(DE3), the toxic proteins can often be expressed successfully in C41(DE3) and/or C43(DE3) . In this work, using a range of plasmids coding for several types of proteins, we investigated in BL21(DE3), C41(DE3), and C43(DE3) their ability to undergo transformation and to express . While transformation was always possible in C41(DE3) and C43(DE3), we could not obtain transformants in BL21(DE3) for 62% of the expression vectors tested . Moreover, after induction, the expression of heterologous proteins in both mutant strains is generally better than in BL21(DE3) . In this study, we also enhanced the stability of plasmids in culture during the expression of proteins by adding the par locus from the plasmid pSC101 to the vector backbone . The stability of a subset of the plasmids (measured 3 h after induction) was determined in C41(DE3) and C43(DE3) and varies from 62 to 92% for C43(DE3) and from 10 to 90% for C41(DE3) . This study demonstrates the usefulness of these strains C41(DE3) and C43(DE3) in solving the problem of plasmid instability during the expression of toxic recombinant proteins.

Microbiol Res, 2004, 159(2), 97 - 102
Two-triplet CGA repeats impede DNA replication in bacteriophage M13 in Escherichia coli; Pan X; Expansion and contraction instabilities associated with CAG, CGG, GAA and CGA (GAC) repeats propagation cause more than a dozen human genetic diseases and cancers . In this work, the propagation behavior of a bacteriophage M13 carrying a calf prochymosin cDNA fragment with a (CGA)2 repeat in a small hairpin forming region is reported . Such a M13 derivative when propagated in Escherichia coli, produces small plaques by decreasing phage yield and also mitigates the inhibition on host cell growth, compared to those control bacteriophages either containing a "CTGCTA" sequence or wildtype, suggesting that CGA2 repeat impedes DNA replication in vivo . Moreover, an increased internal free energy is found associated with (CGA)2 sequence compared to those "CTGCTA" and wildtype, which ruled out a possibility of CGA2 repeat effects on propagation is through influencing the hairpin structure formation.

J Biol Chem, 2004 Oct 1, 279(40), 42018 - 25 Epub 2004 Jul 29.
A unique region in bacteriophage t7 DNA polymerase important for exonucleolytic hydrolysis of DNA; Kumar JK et al.; A crystal structure of the bacteriophage T7 gene 5 protein/Escherichia coli thioredoxin complex reveals a region in the exonuclease domain (residues 144-157) that is not present in other members of the E . coli DNA polymerase I family . To examine the role of this region, a genetically altered enzyme that lacked residues 144-157 (T7 polymerase (pol) Delta144-157) was purified and characterized biochemically . The polymerase activity and processivity of T7 pol Delta144-157 on primed M13 DNA are similar to that of wild-type T7 DNA polymerase implying that these residues are not important for DNA synthesis . The ability of T7 pol Delta144-157 to catalyze the hydrolysis of a phosphodiester bond, as judged from the rate of hydrolysis of a p-nitrophenyl ester of thymidine monophosphate, also remains unaffected . However, the 3'-5' exonuclease activity on polynucleotide substrates is drastically reduced; exonuclease activity on single-stranded DNA is 10-fold lower and that on double-stranded DNA is 20-fold lower as compared with wild-type T7 DNA polymerase . Taken together, our results suggest that residues 144-157 of gene 5 protein, although not crucial for polymerase activity, are important for DNA binding during hydrolysis of polynucleotides.

J Bacteriol, 2004 Aug, 186(16), 5533 - 7
Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development; Oviedo de Anda NA et al.; Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth) . Single-base-pair mutations (bar(-)) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells) . When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering tRNA(2)(Ile) as pep-tRNA(2)(Ile) . Either tRNA(2)(Ile) or Pth may reverse these effects . In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester tRNA(2)(Ile) as pep-tRNA(2)(Ile) . In addition, overexpression of the barI minigene impairs the development even of bar(-) phages in rap cells . Interestingly, tRNA or Pth may reestablish lambda phage development . These results suggest that lambda bar minigenes are expressed and tRNA(2)(Ile) is sequestered as pep-tRNA(2)(Ile) during lambda phage development.

J Bacteriol, 2004 Aug, 186(16), 5523 - 8
Complete genomic nucleotide sequence of the temperate bacteriophage Aa Phi 23 of Actinobacillus actinomycetemcomitans; Resch G et al.; The entire double-stranded DNA genome of the Actinobacillus actinomycetemcomitans bacteriophage Aa Phi 23 was sequenced . Linear DNA contained in the phage particles is circularly permuted and terminally redundant . Therefore, the physical map of the phage genome is circular . Its size is 43,033 bp with an overall molar G+C content of 42.5 mol% . Sixty-six potential open reading frames (ORFs) were identified, including an ORF resulting from a translational frameshift . A putative function could be assigned to 23 of them . Twenty-three other ORFs share homologies only with hypothetical proteins present in several bacteria or bacteriophages, and 20 ORFs seem to be specific for phage Aa Phi 23 . The organization of the phage genome and several genetic functions share extensive similarities to that of the lambdoid phages . However, Aa Phi 23 encodes a DNA adenine methylase, and the DNA packaging strategy is more closely related to the P22 system . The attachment sites of Aa Phi 23 (attP) and several A . actinomycetemcomitans hosts (attB) are 49 bp long.

J Bacteriol, 2004 Aug, 186(16), 5202 - 9
An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts; Bidlack JE et al.; F(+) strains of Escherichia coli infected with donor-specific bacteriophage such as M13 are sensitive to bile salts . We show here that this sensitivity has two components . The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili . The latter component was manifested as reduced or no growth of an F(+) strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F(-) strain or as a reduced plating efficiency of the F(+) strain on solid media; at 2% bile salts, plating efficiency was reduced 10(4)-fold . Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain . Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate) . F(+) cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F(-) cells, suggesting a selectivity for amphipathic anions . A mutation in any but one F tra gene required for the assembly of F pili, including the traA gene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion . The exception was traW . A traW mutant was 100-fold more sensitive to cholate than the tra(+) strain but only marginally more sensitive to taurocholate or glycocholate . Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F(+) cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic beta-lactamase into the medium.

Cytometry, 2004 Aug, 60A(2), 125 - 34
An analytical system based on a compact flow cytometer for DNA fragment sizing and single-molecule detection; Habbersett RC et al.; BACKGROUND: Previous reports have demonstrated accurate DNA fragment sizing of linear DNA fragments, from 564 to approximately 4 x 10(5) bp, in a flow system . B-phycoerythrin (B-PE), commonly used in conventional cytometric applications that require high-sensitivity, was the first fluorophore detected in flow at the single-molecule level . METHODS: Dilute solutions of stained DNA fragments or B-PE were analyzed in a simplified, compact flow system, with enhanced performance and lower cost, utilizing a solid-state laser and a single-photon sensing avalanche photodiode detector (SSAPD) . Extensive data processing and display software, developed specifically for the photon-counting data stream, extracts correlated height, width, and area features from bursts of photons due to discrete molecules passing through the sensing region in the flow channel . RESULTS: DNA fragment sizing in flow has now been demonstrated for SYTOX-orange-stained fragments ranging in size over 3.4 orders of magnitude, from 125 to 5 x 10(5) bp . For Lambda bacteriophage DNA (lambda DNA; 48.5 kbp) a CV of 1.2 % has been achieved . Analysis of a femtomolar B-PE solution demonstrates that the bursts of photons from individual molecules can be baseline-resolved with 0.5 mW of laser power at a signal to noise ratio (SNR) of approximately 30, with approximately 100 photons detected from each molecule . CONCLUSIONS: A compact, low-power, high-sensitivity system detects DNA fragments as small as 125 bp or individual B-PE molecules in a flowing liquid stream . Demonstrated linearity, sensitivity, and resolution indicate that <1.0 mW of laser power is optimal, permitting further miniaturization of the system and additional cost reduction . Comprehensive analytical software exploits the standard cytometric paradigm of multiple 2D graphs and gating to extract features from classes of individually analyzed biomolecules . This complete system is thus poised to engage high-sensitivity applications not amenable to conventional flow cytometric instrumentation.

J Mol Biol, 2004 Aug 13, 341(3), 869 - 79
Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy; Thiriot DS et al.; The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein . These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process . The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle . The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively . The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A . The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.

Nat Struct Mol Biol, 2004 Sep, 11(9), 850 - 6 Epub 2004 Aug 01.
The PM2 virion has a novel organization with an internal membrane and pentameric receptor binding spikes; Huiskonen JT et al.; Biological membranes are notoriously resistant to structural analysis . Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid . Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid . The viral genome closely interacts with the inner leaflet . The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization . Pentameric receptor-binding spikes protrude from the surface . It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle . First, it acts as a scaffold to nucleate capsid assembly . Second, after host recognition, it fuses with the host outer membrane to promote genome entry . The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.

Parasitol Today, 1996 Sep, 12(9), 357 - 62
Organelle DNAs: The bit players in malaria parasite DNA replication; Williamson DH et al.; The replication mechanics of the extrachromosomal DNAs of the malaria parasite are beginning to be anravelled . At 6 kb, the mitochondrial genome is the smallest known and, unlike higher eukaryotes, its multiple copies per cell occur as polydisperse linear concatemers . Here, Don Williamson, Peter Preiser and Iain Wilson discuss recent evidence that this DNA replicates by a process akin to those of certain bacteriophages, which make use of extensive recombination coupled with rolling circles . The parasite's second extrachromosomal DNA, a 35 kb circular molecule thought to be a plastid remnant inherited from a remote photoautotroph, probably replicates in a more familiar fashion from conventional origins or D loops . Improved understanding of both organelle's replicative mechanisms could give new leads to malaria chemotherapy.

Protein Sci, 2004 Aug, 13(8), 2260 - 9
Double-stranded DNA bacteriophage prohead protease is homologous to herpesvirus protease; Cheng H et al.; Double-stranded DNA bacteriophages and herpesviruses assemble their heads in a similar fashion; a pre-formed precursor called a prohead or procapsid undergoes a conformational transition to give rise to a mature head or capsid . A virus-encoded prohead or procapsid protease is often required in this maturation process . Through computational analysis, we infer homology between bacteriophage prohead proteases (MEROPS families U9 and U35) and herpesvirus protease (MEROPS family S21), and unify them into a procapsid protease superfamily . We also extend this superfamily to include an uncharacterized cluster of orthologs (COG3566) and many other phage or bacteria-encoded hypothetical proteins . On the basis of this homology and the herpesvirus protease structure and catalytic mechanism, we predict that bacteriophage prohead proteases adopt the herpesvirus protease fold and exploit a conserved Ser and His residue pair in catalysis . Our study provides further support for the proposed evolutionary link between dsDNA bacteriophages and herpesviruses.

Insect Mol Biol, 2004 Aug, 13(4), 365 - 9
Molecular discrimination of Wolbachia in the Culex pipiens complex: evidence for variable bacteriophage hyperparasitism; Sanogo YO et al.; The medically important members of the Culex pipiens species complex provide an enigma for systematists, evolutionary biologists, and vector biologists . The species complex is composed of forms that differ in their ecology, behaviour, physiology and vector competence . Cytoplasmic incompatibility (CI) caused by endosymbiotic Wolbachia bacteria is thought to play an important role in restricting gene flow and the evolution of the Culex complex . Here we describe the first molecular marker useful for discriminating between Wolbachia infections in Culex . A putative bacteriophage locus (orf7) varies between Culex forms in copy number and sequence . We provide evidence that the orf7 loci are strictly associated with Wolbachia and are maternally inherited.

Biotechnol Lett, 2004 Jun, 26(11), 897 - 900
Rapid detection of 'rare' actinomycetes in environmental samples; Gathogo EW et al.; Natural products continue to be a useful source of new leads for the pharmaceutical industry . Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections . We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples . This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.

J Biol Chem, 2004 Sep 24, 279(39), 40795 - 801 Epub 2004 Jul 19.
The functional domains of bacteriophage t4 terminase; Kanamaru S et al.; The packaging of double-stranded genomic DNA into some viral and all bacteriophage capsids is driven by powerful molecular motors . In bacteriophage T4, the motor consists of the portal protein assembly composed of twelve copies of gene product 20 (gp20, 61 kDa) and an oligomeric terminase complex composed of gp16 (18 kDa) and gp17 (70 kDa) . The packaging motor drives the 171-kbp T4 DNA into the capsid utilizing the free energy of ATP hydrolysis . Evidence suggests that gp17 is the key component of the motor; it exhibits ATPase, nuclease, and in vitro DNA-packaging activities . The N- and C-terminal halves of gp17 were expressed and purified to homogeneity and found to have ATPase and nuclease activities, respectively . The N-terminal domain exhibited 2-3-fold higher Kcat values for gp16-stimulated ATPase than the full-length gp17 . Neither of the domains, individually or together, exhibited in vitro DNA-packaging activity, suggesting that communication between the domains is essential for DNA packaging . The domains, in particular the C-terminal domain or a mixture of both the N- and C-terminal domains, inhibited in vitro DNA packaging that is catalyzed by full-length gp17 . In conjunction with genetic evidence, these data suggest that the domains compete with the full-length gp17 for binding sites on the portal protein . A model for the assembly of the T4 DNA-packaging machine is presented .

Proc Natl Acad Sci U S A, 2004 Jul 27, 101(30), 11007 - 12 Epub 2004 Jul 19.
Genetic organization of the psbAD region in phages infecting marine Synechococcus strains; Millard A et al.; The discovery of the genes psbA and psbD, encoding the D1 and D2 core components of the photosynthetic reaction center PSII (photosystem II), in the genome of the bacteriophage S-PM2 (a cyanomyovirus) that infects marine cyanobacteria begs the question as to how these genes were acquired . In an attempt to answer this question, it was established that the occurrence of the genes is widespread among marine cyanomyovirus isolates and may even extend to podoviruses . The phage psbA genes fall into a clade that includes the psbA genes from their potential Synechococcus and Prochlorococcus hosts, and thus, this phylogenetic analysis provides evidence to support the idea of the acquisition of these genes by horizontal gene transfer from their cyanobacterial hosts . However, the phage psbA genes form distinct subclades within this lineage, which suggests that their acquisition was not very recent . The psbA genes of two phages contain identical 212-bp insertions that exhibit all of the canonical structural features of a group I self-splicing intron . The different patterns of genetic organization of the psbAD region are consistent with the idea that the psbA and psbD genes were acquired more than once by cyanomyoviruses and that their horizontal transfer between phages via a common phage gene pool, as part of mobile genetic modules, may be a continuing process . In addition, genes were discovered encoding a high-light inducible protein and a putative key enzyme of dark metabolism, transaldolase, extending the areas of host-cell metabolism that may be affected by phage infection.

Virology, 2004 Aug 15, 326(1), 41 - 6
A fragile lattice: replacing bacteriophage lambda's head stability gene D with the shp gene of phage 21 generates the Mg2+-dependent virus, lambda shp; Wendt JL et al.; Phage lambda DNA packaging is accompanied by prohead expansion, due to structural changes in gpE, the major capsid protein . Rearrangement of the gpE lattice creates binding sites for trimers of gpD, the head stabilization protein . lambda-Like phage 21's shp gene is homologous to lambda's D gene . gpD and gpShp share 49% amino acid identity . To ask whether gpShp could stabilize the lambda head shell, we replaced lambda's D gene with shp, creating lambda shp . Unlike lambda or 21, lambda shp was strictly dependent on the presence of 10(-2) M Mg2+, and lambda shp virions were very sensitive to chelating agents . Density gradient studies indicated that the lambda gpE lattice was underpopulated with gpShp . gpD's N-terminus has been proposed to contact gpE, and we found that lambda D/shp, which produces a chimeric protein with the N-terminus of gpD and the C-terminus of gpShp, was Mg2+-independent and more stable than lambda shp.

Int J Tuberc Lung Dis, 2004 Jul, 8(7), 899 - 902
Rapid diagnosis of pulmonary tuberculosis by mycobacteriophage assay; Butt T et al.; We evaluated FASTPlaqueTB, a recently introduced bacteriophage assay for rapid detection of Mycobacterium tuberculosis complex in sputum specimens, using 169 non-duplicate sputum specimens from patients suspected of pulmonary tuberculosis . The results of 160 specimens were analysed . FASTPlaqueTB assay detected tuberculosis in 77% (46/60) of culture-positive cases . Among the AFB smear-positive cases (n = 47) it had a sensitivity of 76% and specificity of 60% while among AFB smear-negative cases (n = 113) its sensitivity and specificity were 78% and 98%, respectively . The overall sensitivity and specificity of the technique were 77% and 96%, respectively, and the positive and negative predictive values were respectively 92% and 87% . The overall efficiency of the test was 89% . Test results were available in 48 h.

EMBO J, 2004 Aug 4, 23(15), 2952 - 62 Epub 2004 Jul 15.
T4 AsiA blocks DNA recognition by remodeling sigma(70) region 4; Lambert LJ et al.; Bacteriophage T4 AsiA is a versatile transcription factor capable of inhibiting host gene expression as an 'anti-sigma' factor while simultaneously promoting gene-specific expression of T4 middle genes in conjunction with T4 MotA . To accomplish this task, AsiA engages conserved region 4 of Eschericia coli sigma(70), blocking recognition of most host promoters by sequestering the DNA-binding surface at the AsiA/sigma(70) interface . The three-dimensional structure of an AsiA/region 4 complex reveals that the C-terminal alpha helix of region 4 is unstructured, while four other helices adopt a completely different conformation relative to the canonical structure of unbound region 4 . That AsiA induces, rather than merely stabilizes, this rearrangement can be realized by comparison to the homologous structures of region 4 solved in a variety of contexts, including the structure of Thermotoga maritima sigma(A) region 4 described herein . AsiA simultaneously occupies the surface of region 4 that ordinarily contacts core RNA polymerase (RNAP), suggesting that an AsiA-bound sigma(70) may also undergo conformational changes in the context of the RNAP holoenzyme.

Mol Microbiol, 2004 Aug, 53(3), 821 - 8
Translation repression by an RNA polymerase elongation complex; Wilson HR et al.; Bacteriophage lambda N and bacterial Nus proteins together with a unique site NUT in the leader of the early viral N gene transcript bind RNA polymerase (RNAP) and form a highly processive antitermination complex; N bound at NUT also represses N translation . In this study, we investigate whether N and NUT cause N translation repression as part of the antitermination complex by testing conditions that inhibit the formation of the N-modified transcription complex for their effect on N-mediated translation repression . We show that nus and nut mutations that in combination destabilize multiple interactions in the antitermination complex prevent N-mediated translation repression . Likewise, transcription of the nut-N region by T7 RNAP, which does not lead to the assembly of an effective antitermination complex when N is supplied, eliminates translation repression . We also demonstrate that a unique mutant beta subunit of RNAP reduces N-mediated translation repression, and that overexpression of transcription factor NusA suppresses this defect . We conclude that the N-modified RNAP transcription complex is necessary to repress N translation .

Methods Mol Biol, 2004, 259, 47 - 65
Generation, use, and validation of receptor-selective antibodies; Mackrill JJ; Antibodies have proved invaluable in the study of G-protein-coupled receptors (GPCRs) . The utility of these immunoglobulin probes for investigation of protein structures and functions arises from their selectivity as well as their versatility . Antibodies can be used to analyze GPCR size, abundance, distribution, turnover, modification, interaction with other proteins, and functional properties . In this chapter, techniques for the generation and characterization of receptor-selective antibodies are described . Two protocols are given for the generation of antibodies: (1) development of polyclonal antibodies (PAbs) against synthetic peptides corresponding to a specific site within a GPCR and (2) selection of synthetic single-chain fragment variable (scFv) monoclonal antibodies (MAbs) from libraries expressed on the surface of bacteriophage . Immunoblot and enzyme-linked immunosorbent assays for characterization of the selectivity and affinity of such antibodies are described . Finally, methods are given for improvement of the titer and specificity of PAbs.

Biochemistry, 2004 Jul 20, 43(28), 9151 - 9
Binding of cationic porphyrin to isolated and encapsidated viral DNA analyzed by comprehensive spectroscopic methods; Zupan K et al.; The complexation of tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with free and encapsidated DNA of T7 bacteriophage was investigated . To identify binding modes and relative concentrations of bound TMPyP forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed . Spectral decomposition, fluorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of TMPyP, e.g., external binding and intercalation both in free and in encapsidated DNA . Optical melting studies revealed that TMPyP increases the strand separation temperature of both free and native phage DNA and does not change the phase transition temperature of phage capsid proteins . From these findings we concluded that TMPyP binding does not influence the protein structure and/or the protein-DNA interaction . A combined analysis of absorption spectra and fluorescence decay curves made possible the determination of concentrations of free, externally bound, and intercalated porphyrin . As a perspective, our results facilitate a qualitative analysis of the TMPyP binding process at various experimental conditions.

Nucleic Acids Res, 2004 Jul 06, 32(12), 3515 - 21 Print 2004.
Packaging motor from double-stranded RNA bacteriophage phi12 acts as an obligatory passive conduit during transcription; Kainov DE et al.; Double-stranded RNA viruses sequester their genomes within a protein shell, called the polymerase complex . Translocation of ssRNA into (packaging) and out (transcription) of the polymerase complex are essential steps in the life cycle of the dsRNA bacteriophages of the Cystoviridae family (phi6-phi14) . Both processes require a viral molecular motor P4, an NTPase, which bears structural and functional similarities to hexameric helicases . In effect, switching between the packaging and the transcription mode requires the translocation direction of the P4 motor to reverse . However, the mechanism of the reversal remains elusive . Here we characterize the P4 protein from bacteriophage phi12 and exploit its purine nucleotide specificity to delineate P4 role in transcription . The results indicate that while P4 actively translocates RNA during packaging it acts as a passive conduit for RNA export . The directionality switching is accomplished via the regulation of P4 NTPase activity within the polymerase core.

Nucleic Acids Res . 2004 Jun 28;32(11):e91 . Print 2004 Jun.
A novel 4-base-recognizing RNA cutter that can remove the single 3' terminal nucleotides from RNA molecules; Takaku H et al.; Mammalian tRNase ZL shows versatility in substrate recognition . This enzyme can not only process pre-tRNAs by cleaving off their 3' trailer sequences, but also recognize and cleave pre-tRNA-like complexes and micro-pre-tRNAs . Here we demonstrate that 24-27 nt hairpin RNAs (hook RNAs) can guide cleavages of separate target RNAs by tRNase ZL through the micro-pre-tRNA-like complexes between the targets and the hook RNAs and that tRNase ZL together with hook RNA works as 4-7-base-recognizing RNA cutters . The cleavage sites were located only after the nucleotide corresponding to the discriminator nucleotide . Cleavage assays for various substrate/hooker complexes showed that the cleavage efficiency changes depending on the maximum number of substrate/hooker recognition base pairings and the stem length of hook RNA and that a 5 nt recognition sequence and a hook RNA containing a 6 or 7 bp stem are the best combination for the optimal target cleavage . We also show that a 4-base RNA cutter can remove the single 3' terminal nucleotides from RNA molecules . These results indicate that this new type of RNA cutter can be utilized to homogenize at their 3' termini RNA transcripts synthesized in vitro with a bacteriophage RNA polymerase.

Anal Biochem, 2004 Aug 1, 331(1), 60 - 7
High-throughput fluorescence spectroscopic analysis of affinity of peptides displayed on bacteriophage; Landon LA et al.; Fluorescence spectroscopy titrations, although widely used to analyze binding affinity, are not an efficient screening method for detecting high-affinity binding among a large number of available ligands, such as during bacteriophage display selections . We hypothesize that a miniaturized, high-throughput fluorescence spectroscopy assay can be used to efficiently analyze selection results by applying the Langmuir equation to the binding data to estimate affinity constants for a large number of ligands, either as synthesized molecules or as displayed on bacteriophage . Here, bacteriophage-display-derived peptides specific for the Thomsen-Friedenreich disaccharide are used to develop a high-throughput fluorescence spectroscopy screening method, which uses one binding partner labeled with a fluorescent dye and different concentrations of a second partner to analyze binding affinity in bacteriophage display selections . The affinity constants derived from binding isotherms prepared using the new system accurately replicate those derived from standard spectroscopy titrations . Furthermore, the technique correctly defined the affinity constant describing binding of a cognate epitope peptide by a monoclonal antibody . Finally, we have applied the technique to analysis of binding affinity by ligands displayed on bacteriophage, which suggests that this technique could be used to monitor bacteriophage enrichment during selections.

Nucleic Acids Res . 2004 Jul 07;32(12):e97.
Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification; Spottke B et al.; Several DNA/RNA sequencing strategies have been developed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) . In the reverse Sanger sequencing approach alpha-thiophosphate-containing NTPs are employed . Sequencing ladders are produced by the subsequent exonuclease cleavage, which is inhibited by the alpha-S-NTP at the 3' terminus . Here the reverse Sanger sequencing of RNA is described . The stability of RNA during the UV-MALDI process is higher relative to DNA, and RNA can be easily synthesized by transcription using bacteriophage RNA polymerase . alpha-S-rNTP was added to the reaction in a ratio of 1:3 to the native rNTPs and was incorporated statistically by the RNA polymerase . Four separate sequence ladders were produced, to avoid the problem of the only 1u mass difference between uridine and cytidine . However, it was shown that RNA transcription does not produce homogeneous transcripts . Therefore isolation of the full-length transcript is required to attain a non-ambiguous interpretation of cleavage spectra . This is achieved by the exclusive immobilization of the full-length transcript on a solid phase . The full-length transcripts were hybridized to magnetic beads, coated with short universal sequences, complementary to the in vitro RNA . After purification and isolation the RNA full-length transcript is cleaved by snake venom phosphodiesterase (SVP) and the obtained sequence ladder is analyzed by MALDI-MS.

Genetics, 2004 Jun, 167(2), 559 - 67
Epistasis and its relationship to canalization in the RNA virus phi 6; Burch CL et al.; Although deleterious mutations are believed to play a critical role in evolution, assessing their realized effect has been difficult . A key parameter governing the effect of deleterious mutations is the nature of epistasis, the interaction between the mutations . RNA viruses should provide one of the best systems for investigating the nature of epistasis because the high mutation rate allows a thorough investigation of mutational effects and interactions . Nonetheless, previous investigations of RNA viruses by S . Crotty and co-workers and by S . F . Elena have been unable to detect a significant effect of epistasis . Here we provide evidence that positive epistasis is characteristic of deleterious mutations in the RNA bacteriophage phi 6 . We estimated the effects of deleterious mutations by performing mutation-accumulation experiments on five viral genotypes of decreasing fitness . We inferred positive epistasis because viral genotypes with low fitness were found to be less sensitive to deleterious mutations . We further examined environmental sensitivity in these genotypes and found that low-fitness genotypes were also less sensitive to environmental perturbations . Our results suggest that even random mutations impact the degree of canalization, the buffering of a phenotype against genetic and environmental perturbations . In addition, our results suggest that genetic and environmental canalization have the same developmental basis and finally that an understanding of the nature of epistasis may first require an understanding of the nature of canalization.

Nat Struct Mol Biol, 2004 Aug, 11(8), 784 - 90 Epub 2004 Jul 04.
Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7; Li Y et al.; Ultraviolet-induced DNA damage poses a lethal block to replication . To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD) . When the 3' thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation . When the 5' thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3' base of the primer, while the fingers are in a closed conformation . These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.

J Chromatogr A, 2004 Jun 4, 1038(1-2), 121 - 30
Isolation of a peptide ligand for affinity purification of factor VIII using phage display; Kelley BD et al.; Polypeptides for use in affinity chromatography of factor VIII were identified using phage display technology . Phage libraries were designed to express polypeptide fusions containing five to seven residues flanked by two cysteines that form a disulfide bond . Individual bacteriophage were selected for the ability of these polypeptides to bind factor VIII, and then release the protein under mild elution conditions . Strong consensus sequences were observed that appear to be necessary for this reversible interaction . Chemically synthesized ligands identified by this screening were immobilized onto a chromatographic support and used for affinity purification of factor VIII from a complex feedstream . A chromatographic step was developed that provided a 10000-fold reduction in host cell proteins and DNA, while providing exceptional product recovery.

J Bacteriol, 2004 Jul, 186(14), 4628 - 37
P2 growth restriction on an rpoC mutant is suppressed by alleles of the Rz1 homolog lysC; Markov D et al.; Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase beta' subunit and is nonpermissive for plating of bacteriophage P2 . P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB . The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage lambda . Indeed, coexpression of lysB and lysC complemented the growth defect of lambda Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function . Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC . We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation .

Anal Chem, 2004 Jul 1, 76(13), 3770 - 6
Quantitative microfluidic separation of DNA in self-assembled magnetic matrixes; Minc N et al.; We present an experimental study of the microfluidic electrophoresis of long DNA in self-assembling matrixes of magnetic bead columns . Results are presented for the rapid separation of lambda-phage, 2lambda-DNA, and bacteriophage T4 DNA, where separation resolutions greater than 2 between lambda and T4 are achieved in times as short as 150 s . The use of a computer-piloted flow control system and injection results in high reproducibility between separations . We compare the experimentally measured mobility and dispersion with an exactly solvable lattice Monte Carlo model . The theory predicts that the mean velocity scales linearly with the field, the band broadening scales with the inverse of the field, and the resolution is independent of the field for intermediate fields-all of which are in accord with the experimental results . Moreover, reasonable quantitative agreement is achieved for band broadening for longer DNA (2lambda and T4) when the average postengagement time is measured experimentally . This work demonstrates the possibility of achieving fast microfluidic separation of large DNA on a routine basis.

Proc Natl Acad Sci U S A, 2004 Jul 13, 101(28), 10416 - 21 Epub 2004 Jun 28.
Treating cocaine addiction with viruses; Carrera MR et al.; Cocaine addiction continues to be a major health and social problem in the United States and other countries . Currently used pharmacological agents for treating cocaine abuse have proved inadequate, leaving few treatment options . An alternative is to use protein-based therapeutics that can eliminate the load of cocaine, thereby attenuating its effects . This approach is especially attractive because the therapeutic agents exert no pharmacodynamic action of their own and therefore have little potential for side effects . The effectiveness of these agents, however, is limited by their inability to act directly within the CNS . Bacteriophage have the capacity to penetrate the CNS when administered intranasally . Here, a method is presented for engineering filamentous bacteriophage to display cocaine-binding proteins on its surface that sequester cocaine in the brain . These antibody-displaying constructs were examined by using a locomotor activity rodent model to assess the ability of the phage-displayed proteins to block the psychoactive effects of cocaine . Results presented demonstrate a strategy in the continuing efforts to find effective treatments for cocaine addiction and suggest the application of this protein-based treatment for other drug abuse syndromes.

J Mol Biol, 2004 Jul 16, 340(4), 707 - 30
Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection; Zhang X et al.; T7 RNA polymerase selectively transcribes T7 genes during infection but is also involved in DNA replication, maturation and packaging . T7 lysozyme is an amidase that cuts a bond in the peptidoglycan layer of the cell wall, but it also binds T7 RNA polymerase and inhibits transcription, and it stimulates replication and packaging of T7 DNA . To better understand the roles of these two proteins during T7 infection, mutants of each were constructed or selected and their biochemical and physiological behavior analyzed . The amidase activity of lysozyme is needed for abrupt lysis and release of phage particles but appears to have no role in replication and packaging . The interaction between polymerase and lysozyme stimulates both replication and packaging . Polymerase mutants that gain the ability to grow normally in the absence of an interaction with lysozyme still fail to shut down late transcription and, remarkably, have become hypersensitive to inhibition when lysozyme is able to bind . These lysozyme-hypersensitive polymerases behave without lysozyme similarly to wild-type polymerase with lysozyme: both remain longer at the promoter before establishing a lysozyme-resistant elongation complex and both increase the length of pausing when elongation complexes encounter an eight-base recognition sequence involved in DNA packaging . Replication origins contain T7 promoters, but the role of T7 RNA polymerase in initiating replication is not understood well enough to more than speculate how the lysozyme-polymerase interaction stimulates replication . Maturation and packaging is apparently initiated through interaction between prohead-terminase complexes and transcription elongation complexes paused at the sequence TATCTGT(T/A), well conserved at the right-end of the concatemer junction of T7-like phages . A model that is consistent with the structure of an elongation complex and a large body of mutational and biochemical data is proposed to explain sequence-specific pausing and potential termination at the consensus recognition sequence (C/T)ATCTGT(T/A).

J Biol Chem, 2004 Aug 27, 279(35), 37040 - 8 Epub 2004 Jun 24.
Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis; Petyuk V et al.; Cre is a site-specific recombinase from bacteriophage P1 . It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP . To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein . The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A . The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism . In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity . Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity . We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants . The role of those regions in the recombination process is discussed.

Plasmid, 2004 Jul, 52(1), 31 - 47
Mycoplasma arthritidis bacteriophage MAV1 prophage integration, deletions, and strain-related polymorphisms; Washburn LR et al.; Temperate bacteriophage MAV1 is found in certain highly virulent strains of Mycoplasma arthritidis . Integration sites, portions of the right and left prophage ends, and flanking DNA from eight prophages in seven M . arthritidis strains were characterized in this study . attb and attp sites conformed for the most part to the consensus sequence TATTTTT, although minor polymorphisms were noted . Prophages were integrated into similar sites in four strains, suggesting that these strains may have had a common ancestor . Two strains had three prophage copies each, and integration sites were identical . Two strains had two copies each . One of these shared two of the integration sites occupied in the three-copy strains, while the other shared one of these sites and harbored a second prophage in a unique site . Integration sites in the two strains with one prophage each were unique . Four MAV1 copies contained extensive substitutions within a region encoding a putative structural protein and the putative repressor protein . A 3-kb fragment was deleted from the right side of two of these copies . It is proposed that polymorphisms within MAV1 prophage integration sites and within the prophages themselves may help to identify phylogenetic relationships among virulent M . arthritidis strains.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 91 - 5
Different restriction and modification phenotypes in ruminal lactate-utilizing bacteria; Piknova M et al.; Analysis of restriction and modification activities in lactate-utilizing bacteria belonging to the Megasphaera elsdenii and Mitsuokella multiacida species revealed the presence of GATC-specific, MboI isospecific, restriction-modification (R-M) systems in all strains tested . While restriction endonucleases isolated from M . elsdenii strains were found to be sensitive to Dam methylation, enzymes from M . multiacida cleaved DNA irrespective of Dam methylation . The comparison of type II R-M systems specificities in three closely related lactate-utilizing ruminal bacterial species indicated complete lack of restriction and/or modification enzymes previously characterized from Selenomonas ruminantium in tested M . elsdenii and M . multiacida strains . R-M systems are believed to represent the main defense tool against phage infection . Based on the results of our experiments it could be assumed that M . elsdenii and M . multiacida use the different strategy for bacteriophage protection compared to S . ruminantium.

J Mol Biol, 2004 Jul 9, 340(3), 587 - 97
Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display; Held HA et al.; A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency . Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat . The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation . Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix . In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes . Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display . Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display . The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display . The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions . These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology .

J Mol Biol, 2004 Jul 9, 340(3), 445 - 57
Monovalent cations regulate DNA sequence recognition by 434 repressor; Mauro SA et al.; The bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout . In indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein's DNA-binding site modulate the affinity of DNA for protein . The conformation and flexibility of a DNA sequence can be influenced by the interaction of the DNA bases or backbone with solution components . We examined the effect of changing the cation-type present in solution on the stability and structure of 434 repressor complexes with wild-type and mutant OR1 and OR3, binding sites that differ in their contacted and non-contacted base sequences . We find that the affinity of repressor for OR1, but not for OR3, depends remarkably on the type and concentration of monovalent cation . Moreover, the formation of a stable, specific repressor-OR1 complex requires the presence of monovalent cations; however, repressor-OR3 complex formation has no such requirement . Changing monovalent cation type alters the ability of repressor to protect OR1, but not OR3, from *OH radical cleavage . Altering the relative length of the poly(dA) x poly(dT) tract in the non-contacted regions of the OR1 and OR3 can reverse the cation sensitivity of repressor's affinities for these two sites . Taken together these findings show that cation-dependent alterations in DNA structure underlies indirect readout of DNA sequence by 434 repressor and perhaps other proteins .

J Mol Biol, 2004 Jul 9, 340(3), 419 - 33
Evidence that a local refolding event triggers maturation of HK97 bacteriophage capsid; Lee KK et al.; Bacteriophage capsids are a striking example of a robust yet dynamic genome delivery vehicle . Like most phages, HK97 undergoes a conformational maturation that converts a metastable Prohead into the mature Head state . In the case of HK97, maturation involves a significant expansion of the capsid and concomitant cross-linking of capsid subunits . The final state, termed Head-II, is a 600 angstroms diameter icosahedral structure with catenated subunit rings . Cryo-EM, small angle X-ray scattering (SAXS), and biochemical assays were used previously to characterize the initial (Prohead-II) and final states (Head-II) as well as four maturation intermediates . Here we extend the characterization of the acid-induced expansion of HK97 in vitro by monitoring changes in intrinsic fluorescence, circular dichroism (CD), and SAXS . We find that the greatest changes in all observables occur at an early stage of maturation . Upon acidification, fluorescence emissions from HK97 exhibit a blueshift and decrease in intensity . These spectral changes reveal two kinetic phases of the expansion reaction . The early phase exhibits sensitivity to pH, increasing in rate nearly 200-fold when acidification pH is lowered from 4.5 to 3.9 . The second, slower phase reported by fluorescence is relatively insensitive to pH . Time-resolved SAXS experiments report an increase in overall particle dimension that parallels the fluorescence changes for the early phase . Native agarose gel assays corroborated this finding . By contrast, probes of CD at far-UV indicate that secondary structural changes precede the early expansion phase reported by SAXS and fluorescence . Based on the crystallographic structure of Head-II and the pseudo-atomic model of Prohead-II, we interpret these changes as reflecting the conversion of subunit N-terminal arms (N-arm) from unstructured polypeptide to the mixture of beta-strand and beta-turn observed in the Head-II crystal structure . Refolding of the N-arm may thus represent the conformational trigger that initiates the irreversible expansion of the phage capsid .

Prog Nucleic Acid Res Mol Biol, 2004, 78, 227 - 60
The PCNA-RFC families of DNA clamps and clamp loaders; Majka J et al.; The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways . Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories . When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling . Its modification by ubiquitin directs the cellular response to DNA damage . The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it . Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits . Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E . coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62) . Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA . This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes . Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified . A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit . Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1 . The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

Emerg Infect Dis, 2004 Jun, 10(6), 1134 - 7
Bacteriophages and diffusion of beta-lactamase genes; Muniesa M et al.; We evaluated the presence of various Beta-lactamase genes within the bacteriophages in sewage . Results showed the occurrence of phage particles carrying sequences of bla(OXA-2), bla(PSE-1) or bla(PSE-4) and bla(PSE)-type genes . Phages may contribute to the spread of some Beta-lactamase genes.

J Bacteriol, 2004 Jul, 186(13), 4369 - 75
Displacements of prohead protease genes in the late operons of double-stranded-DNA bacteriophages; Liu J et al.; Most of the known prohead maturation proteases in double-stranded-DNA bacteriophages are shown, by computational methods, to fall into two evolutionarily independent clans of serine proteases, herpesvirus assemblin-like and ClpP-like . Phylogenetic analysis suggests that these two types of phage prohead protease genes displaced each other multiple times while preserving their exact location within the late operons of the phage genomes.

J Mol Biol, 2004 Jul 2, 340(2), 253 - 61
Computational and experimental probes of symmetry mismatches in the Arc repressor-DNA complex; Spector S et al.; Arc repressor from bacteriophage P22 is a homodimeric member of the ribbon-helix-helix family of transcription factors . Two dimers bind cooperatively to adjacent sites on operator DNA with the antiparallel beta-sheet of each Arc dimer contacting the major groove . Despite the 2-fold symmetry of the dimer, the sequence of the DNA half-site is not symmetric, and thus there is an inherent symmetry mismatch in the complex . Here, using a combination of computational and experimental methods, we further characterize this symmetry mismatch . Continuum electrostatic calculations show that the DNA-contacting side-chains of Gln9, Asn11 and Arg13 each make better electrostatic interactions in one subunit of the Arc dimer than in the other . Based on the computational results, one or both Gln9 side-chains were substituted with Arg or His and Asn11 was similarly substituted with Ile in the context of a single-chain variant of Arc repressor . Although the double mutants that maintain the symmetry of the protein bind much more weakly than scArc, mutants with single Q9R or N11I substitutions bind operator DNA with affinities close to wild-type . Whereas the effects are nearly additive for the mutations at position 9, the effects of single N11I mutations are non-additive . The calculations thus enabled the identification of functionally tolerated mutations in the DNA-binding surface of Arc repressor, which in turn revealed energetic consequences of asymmetric environments in this protein-DNA interaction.

Nucleic Acids Res . 2004 Jun 15;32(10):e84.
Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography; Cheong HK et al.; Knowledge of the three-dimensional structures of RNA and its complexes is important for understanding the molecular mechanism of RNA recognition by proteins or ligands . Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallography . However, this run-off transcription method results in heterogeneity at the RNA 3-terminus . For structural studies, RNA purification requires a single nucleotide resolution . Usually PAGE purification is used, but it is tedious, time-consuming and cost ineffective . To overcome these problems in high-throughput RNA synthesis, we devised a method of RNA preparation that uses trans-acting DNAzyme and sequence-specific affinity column chromatography . A tag sequence is added at the 3' end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence . The 3' end tag is then removed by sequence-specific cleavage using trans-acting DNAzyme, the arm lengths of which are optimized for turnover number . This purification method is simpler and faster than the conventional method.

Biochemistry, 2004 Jun 22, 43(24), 7948 - 53
Identification of essential residues within Lit, a cell death peptidase of Escherichia coli K-12; Copeland NA et al.; Bacteriophage exclusion is a suicide response to viral infection . In strains of Escherichia coli K-12 infected with T4 phage this process is mediated by the host-encoded Lit peptidase . Lit is activated by a unique sequence in the major head protein of the T4 phage (the Gol sequence) which then cleaves site-specifically the host translation factor EF-Tu, ultimately leading to cell death . Lit has very low sequence identity with other peptidases, with only a putative metallopeptidase motif, H(160)EXXH, giving an indication of its catalytic activity . The aim of the present study was to ascertain if Lit is a metallopeptidase, identify residues essential for Lit activity, and probe the involvement of the Gol sequence in the activation of enzymatic activity . Lit activity was inhibited by the zinc chelator 1,10-phenanthroline, consistent with the suggestion that it is a metallopeptidase . Preliminary covalent modification experiments found that Lit was susceptible to inactivation by diethyl pyrocarbonate, with about three histidines reversibly modified, one of which was found to be essential for proteolytic activity . Subsequently, 13 mutants of the Lit enzyme were constructed that included all 10 histidines as well as other residues within the metallopeptidase motif . This demonstrated that the residues within the HEXXH motif are required for Lit activity and further defined the essential catalytic core as H(160)EXXHX(67)H, with additional residues such as His169 being important but not essential for activity . Kinetic analysis of Lit activation by a synthetic Gol peptide highlighted that elevated concentrations of the peptide (>10-fold above activation K(M)) are inhibitory to Lit, with this effect also seen in partially active Lit mutants . The susceptibility of Lit to inhibition by its own activating peptide suggests that the Gol sequence may be able to bind nonproductively to the enzyme at high concentration . We discuss these data in the context of the currently understood models for Gol-mediated activation of the Lit peptidase and its mechanism of action.

Biochemistry, 2004 Jun 22, 43(24), 7750 - 65
Altering DNA polymerase incorporation fidelity by distorting the dNTP binding pocket with a bulky carcinogen-damaged template; Yan SF et al.; Fidelity of DNA polymerases is predominantly governed by an induced fit mechanism in which the incoming dNTP in the ternary complex fits tightly into a binding pocket whose geometry is determined by the nature of the templating base . However, modification of the template with a bulky carcinogen may alter the dNTP binding pocket and thereby the polymerase incorporation fidelity . High fidelity DNA polymerases, such as bacteriophage T7 DNA polymerase, are predominantly blocked by bulky chemical lesions on the template strand during DNA replication . However, some mutagenic bypass can occur, which may lead to carcinogenesis . Experimental studies have shown that a DNA covalent adduct derived from (+)-anti-BPDE {(+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene}, a carcinogenic metabolite of benzo{a}pyrene (BP), primarily blocks Sequenase 2.0, an exo(-) T7 DNA polymerase; however, a mismatched dATP can be preferentially inserted opposite the damaged adenine templating base within the active site of the polymerase {Chary, P., and Lloyd, R . S . (1995) Nucleic Acids Res . 23, 1398-1405} . The goal of this work is to elucidate structural features that contribute to DNA polymerase incorporation fidelity in the presence of this bulky covalent adduct and to interpret the experimental findings on a molecular level . We have carried out molecular modeling and molecular dynamics simulations with AMBER 6.0, investigating a T7 DNA polymerase primer-template closed ternary complex containing this 10S (+)-trans-anti-{BP}-N(6)-dA adduct in the templating position within the polymerase active site . All four incoming dNTPs were studied . The simulations show that the BP ring system fits well into an open pocket on the major groove side of the modified template adenine with anti glycosidic bond conformation, without disturbing critical polymerase-DNA interactions . However, steric hindrance between the BP ring system and the primer-template DNA causes displacement of the modified template adenine, so that the dNTP base binding pocket is enlarged . This alteration can explain the experimentally observed preference for incorporation of dATP opposite this lesion . These studies also rationalize the observed lower probabilities of incorporation of the other three nucleotides . Our results suggest that the differences in incorporation of dGTP, dCTP, and dTTP are due to the effects of imperfect geometric complementarity . Thus, the simulations suggest that altered DNA polymerase incorporation fidelity can result from adduct-induced changes in the dNTP base binding pocket geometry . Furthermore, plausible structural explanations for the observed effects of {BP}-N(6)-dA adduct stereochemistry on the observed stalling patterns are proposed.

J Biol Chem, 2004 Aug 20, 279(34), 35735 - 40 Epub 2004 Jun 11.
UvsX recombinase and Dda helicase rescue stalled bacteriophage T4 DNA replication forks in vitro; Kadyrov FA et al.; The rescue of stalled replication forks via a series of steps that include fork regression, template switching, and fork restoration often has been proposed as a major mechanism for accurately bypassing non-coding DNA lesions . Bacteriophage T4 encodes almost all of the proteins required for its own DNA replication, recombination, and repair . Both recombination and recombination repair in T4 rely on UvsX, a RecA-like recombinase . We show here that UvsX plus the T4-encoded helicase Dda suffice to rescue stalled T4 replication forks in vitro . This rescue is based on two sequential template-switching reactions that allow DNA replication to bypass a non-coding DNA lesion in a non-mutagenic manner.

Vaccine, 2004 Jun 23, 22(19), 2413 - 9
Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle; Jepson CD et al.; The stability of whole bacteriophage lambda particles, used as a DNA vaccine delivery system has been examined . Phage were found to be highly stable under normal storage conditions . In liquid suspension, no decrease in titre was observed over a 6-month period at 4 and -70 degrees C, and phage stability was unaffected by freeze/thawing . The measured half life of phage in suspension was 36 days at 20 degrees C, 3.4 days at 37 degrees C and 2.3 days at 42 degrees C . Freeze drying of a phage suspension (with or without the stabilizers dry skim milk or trehalose) resulted in 5-20% residual viability . Following desiccation (with or without stabilizers), measured half lives ranged from 20 to 100 days at 20 degrees C, 2.6 to 38 days at 37 degrees C, 2.1 to 26 days at 42 degrees C, 7 to 33 h at 70 degrees C, and 1.3 to 6m at 100 degrees C . In all cases the addition of trehalose significantly increased the stability of the desiccated phage . When stored at -70 degrees C, desiccated phage appeared to be stable in the absence of stabilizers . When phage lambda was diluted into water, a marginal loss in titre was observed over a 2-week period . Over a 24 h period, liquid phage suspensions were stable within the pH range pH 3-11, therefore oral administration of bacteriophage DNA vaccines via drinking water may be possible.

EMBO Rep, 2004 Jul, 5(7), 721 - 7 Epub 2004 Jun 11.
Pharmacological-based translational induction of transgene expression in mammalian cells; Boutonnet C et al.; In the quest for the development of pharmacological switches that control gene expression, no system has been reported that regulates at the translational level . To permit small-molecule control of transgene translation, we have constructed a farnesyl transferase inhibitor-responsive translation initiation factor . This artificial protein is a three-component chimaera consisting of the ribosome recruitment core of the eIF4G1 eukaryotic translation initiation factor, the RNA-binding domain of the R17 bacteriophage coat protein and the plasma membrane localization CAAX motif of farnesylated H-Ras . This membrane-delocalized translation factor is inactive unless liberated in the cytosol . Farnesyl transferase inhibitor FTI-277 prevents the membrane association of the CAAX motif and thus increases the cytoplasmic levels of the eIF4G fusion protein, which is then capable of inducing translation of the second cistron of a bicistronic messenger RNA containing an R17-binding site in its intercistronic space . Such direct translational control by farnesyl transferase inhibitors provides a system for fast, graded and reversible regulation of transgene expression.

Br J Cancer, 2004 Jun 14, 90(12), 2384 - 9
Candidate target genes for loss of heterozygosity on human chromosome 17q21; De Marchis L et al.; Loss of heterozygosity (LOH) on chromosome 17q21 has been detected in 30% of primary human breast tumours . The smallest common region deleted occurred in an interval between the D17S746 and D17S846 polymorphic sequences tagged sites that are located on two recombinant P1-bacteriophage clones of chromosome 17q21: 122F4 and 50H1, respectively . To identify the target gene for LOH, we defined a map of this chromosomal region . We found the following genes: JUP, FK506BP10, SC65, Gastrin (GAS) and HAP1 . Of the genes that have been identified in this study, only JUP is located between D17S746 and D17S846 . This was of interest since earlier studies have shown that JUP expression is altered in breast, lung and thyroid tumours as well as cell lines having LOH in chromosome 17q21 . However, no mutations were detected in JUP using single-strand conformation polymorphism analysis of primary breast tumour DNAs having LOH at 17q21 . We could find no evidence that the transcription promoter for JUP is methylated in tumour DNAs having LOH at 17q21 . We suspect that the target gene for LOH in primary human breast tumours on chromosome 17q21 is either JUP and results in a haploinsufficiency for expression or may be an unidentified gene located in the interval between D17S846 and JUP.

Proc Natl Acad Sci U S A, 2004 Jun 22, 101(25), 9327 - 32 Epub 2004 Jun 08.
Selection-subtraction approach (SSA): a universal genetic screening technique that enables negative selection; Singhi AD et al.; Screening of expression libraries for bioactive clones that modulate the growth of mammalian cells has been limited largely to positive selections incapable of revealing growth suppressive or lethal genetic elements . We have developed a technique, selection-subtraction approach (SSA), that allows growth-modulating clones to be isolated based on alterations in their relative abundance in growing cell populations that have been transduced with an expression library . SSA utilizes tagged retroviral libraries in bacteriophage lambda vectors (retrophages) . Nylon prints from retrophage libraries are used to determine the relative abundance of tags in library-transduced cells to identify biological activity of individual clones . Applications of SSA for gene discovery, target discovery, and generation of mutant proteins have been demonstrated, by using p53 and ataxia telangiectasia mutated (ATM) as models to isolate growth inhibitory proteins, peptides and antisense RNAs, and temperature-sensitive mutant proteins.

Environ Microbiol, 2004 Jul, 6(7), 716 - 25
Free Shiga toxin bacteriophages isolated from sewage showed diversity although the stx genes appeared conserved; Muniesa M et al.; Phages carrying the stx2 gene were detected in a range of sewage samples using a plaque hybridization-based method . After detection, phages were isolated and propagated with a laboratory strain of Escherichia coli as host for characterization purposes . Although it was not possible to conduct propagation or transduction experiments on most of the phages, 11 reached a sufficiently high titre for studies of host infectivity, electron microscopy and sequencing of the stx2 flanking regions to be performed . These phages showed a wide range of host infectivity and morphology . The genetic structure of the 5' stx flanking region appeared conserved whereas the 3' region differed from that of previously described phages . This is the first description of infectious stx-phages isolated as free particles in the environment, and as such constitutes a new contribution to the study of the ecology of these phages.

Environ Microbiol, 2004 Jul, 6(7), 707 - 15
Bacteria and viruses in the water column of tropical freshwater reservoirs; Peduzzi P et al.; In tropical freshwater reservoirs of Sri Lanka, which are linked in an aquatic network, bacterial abundance and production as well as virus abundance, frequency of viral infection and virus production were investigated together with a set of nutrient species (Kjeldahl-N, NO3-N, total P, soluble P, PO4-P) . At two characteristic seasons (wet season, dry season), samples were taken from two types of reservoirs (new upland impoundment and ancient, shallow lowland reservoir), each during 4 days at various depths of the entire water columns . Kjeldahl-N and total P were greatly elevated in the wind-mixed water body of the shallow impoundment during the dry season, whereas the deeper reservoir type exhibited no obvious seasonality . In SYBR green trade mark -stained samples, bacterial abundance showed no seasonal pattern in either reservoir type . Bacterial secondary production, however, was significantly elevated in the entire water column of the shallow impoundment under wind-mixed conditions in the dry season . Highest abundance of virus particles and elevated frequency of bacteria containing mature phages were also observed in the shallow reservoir during the dry season indicating favourable conditions for virus propagation . Data from this aquatic network show that most virus parameters, such as abundance or frequency of visibly infected cells, were positively linked to bacterial abundance and production, but also to organic nitrogen or some phosphorus species . We calculated that between 13.2% and 46.1% of the bacterial standing stocks would be subjected to virus-mediated mortality . Estimates of bacteriophage production revealed that from 10 x 10(9) up to 98 x 10(9) phages were produced per litre and day . Bacteria and viruses in the studied tropical freshwater system appear to be linked to various environmental conditions and may affect processes at the ecosystem scale.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Nov, 19(6), 598 - 600
{Construction and characterization of osteosarcoma 9901 cell cDNA library}; Liao B et al.; AIM: To construct human osteosarcoma 9901 cell cDNA expression library for screening osteosarcoma-specific antigens . METHODS: Total RNA was extracted from human osteosarcoma cell line 9901 and mRNA was purified . cDNA was synthesized by reverse transcription, and cDNA fragments larger than 400 bp were ligated with dephosphorylated arms of lambdagt11 . The recombinants were packaged in-vitro, and a small portion of packaged phage was used to infect E.coli Y1090 for titration . The size of cDNA inserts and the diversity of library were detected by PCR . RESULTS: The osteosarcoma 9901 cell line cDNA library consisting of 1.5x10(6) recombinant bacteriophages was constructed . The average length of exogenous inserts in the recombinants was about 1.4 kb . CONCLUSION: The cDNA library reported herein is suitable for screening osteosarcoma-specific antigens.

Nucleic Acids Res, 2004 Jun 04, 32(10), 3093 - 100 Print 2004.
Sequence-specific Rho-RNA interactions in transcription termination; Graham JE; The bacteriophage lambda tR1 terminator encodes a region of the nascent cro transcript containing RNA residues recognized by termination factor Rho . To identify ribonucleotide-protein interactions contributing to termination, a library of reporter gene plasmids was constructed containing predominantly single-nucleotide substitutions in a 24 nt region previously shown to be critical for efficient termination . Screening 16 822 bacterial transformants identified 110 terminator mutants, most of which contained two or more nucleotide substitutions . Although the vast majority of single base changes did not reduce tR1 function, 11 specific single-nucleotide substitutions at eight positions interspersed in the upstream part of the target region (5'-ATAACCCCGCTCTT ACACATTCCA-3') did reduce termination . About half of these substitutions also reduced Rho-dependent termination on cro gene templates transcribed by purified RNA polymerase, indicating specific residues critical for optimal terminator function . Other termination defects were not reproduced in these in vitro assays, and likely resulted from indirect effects of altering interactions between tR1 and additional cellular factors capable of attenuating Rho function . Our results indicate that while Rho is able to recognize a wide variety of similar rut site sequences by interacting with alternate nucleotides at critical positions, interactions with specific individual ribonucleotides of the tR1 transcript provide highly efficient Rho-dependent termination.

Protein Expr Purif, 2004 Jul, 36(1), 40 - 7
High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells; Chambers SP et al.; We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells . In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator . This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide . This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression . Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E . coli and insect cells . In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E . coli and insect cells . Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production.

Mol Cell, 2004 Jun 4, 14(5), 559 - 69
Control of crosslinking by quaternary structure changes during bacteriophage HK97 maturation; Gan L et al.; Radical structural changes drive the maturation of the capsid of HK97, a lambda-like, dsDNA bacteriophage of Escherichia coli . These include expansion from approximately 560 to approximately 660 A in diameter, metamorphosis from a round to an angular shape, and formation of covalent crosslinks between adjacent capsomers . Analogous transformations also occur in unrelated viruses and protein complexes . We find that expansion and crosslinking happen concurrently during maturation at low pH . Expansion causes residues on three different subunits to move up to 35 A to form 420 active sites that each catalyze the formation of a lysine-asparagine crosslink between adjacent subunits, making crosslink formation an indirect reporter of structural change . Intermediate crosslinking patterns support a previously proposed model of expansion, while hydrophobic properties aid in distinguishing discrete intermediates . A structure derived from cryo-EM images reveals the free intermediate conformation of penton arms, supporting our model for coordinated movement of hexons and pentons on the capsid lattice.

Antiviral Res, 2004 Mar, 61(3), 141 - 51
Methylene blue photoinactivation of RNA viruses; Floyd RA et al.; We present a review of the current status of the use of methylene blue (MB) photoinactivation of viruses starting with the first early observations up to its current use to inactivate HIV-1 in blood products . Basic mechanism of action studies conducted with model bacteriophages indicate that MB-photomediated viral RNA-protein crosslinkage is a primary lesion and that oxygen, specifically singlet oxygen, is very important also . Basic studies on the mechanism of action with HIV are lacking; however, we do show new data illustrating that viral reverse transcriptase inactivation per se cannot account for MB-mediated photoinactivation . We also show data illustrating that MB photomediates the inactivation of West Nile Virus, a flavivirus, which poses a significant new threat to the continental US . MB photoinactivation of viruses show significant promise because the technology not only offers significant potency but the history of safe MB use in human therapy makes it attractive also.

FEBS Lett, 2004 Jun 1, 567(1), 35 - 41
Replicable and recombinogenic RNAs; Chetverin AB; This paper summarizes results of the 40-year studies on replication and recombination of RNA molecules in the cell-free amplification system of bacteriophage Q . Special attention is paid to the molecular colony technique that has provided for the discovery of the nature of "spontaneous" RNA synthesis by Q replicase and of the ability of RNA molecules to spontaneously rearrange their sequences under physiological conditions . Also discussed is the impact of these data on the concept of RNA World and on the development of new in vitro cloning and diagnostic tools.

J Mol Biol, 2004 Jun 11, 339(4), 927 - 35
Design and crystal structure of bacteriophage T4 mini-fibritin NCCF; Boudko SP et al.; Fibritin is a fibrous protein that forms "whiskers" attached to the neck of bacteriophage T4 . Whiskers interact with the long tail fibers regulating the assembly and infectivity of the virus . The fibritin trimer includes the N-terminal domain responsible for attachment to the phage particle and for the collar formation, the central domain forming a 500 A long segmented coiled-coil structure, and the C-terminal "foldon" domain . We have designed a "mini" fibritin with most of the coiled-coil domain deleted, and solved its crystal structure . The non-helical N-terminal part represents a new protein fold that tightly interacts with the coiled-coil segment forming a single domain, as revealed by calorimetry . The analysis of the crystal structure and earlier electron microscopy data on the collar-whisker complex suggests the necessity of other proteins to participate in the collar formation . Crystal structure determination of the N-terminal domain of fibritin is the first step towards elucidating the detailed structure and assembly mechanism of the collar-whisker complex.

Peptides, 2004 Apr, 25(4), 629 - 35
A conformation-constrained peptide library based on insect defensin A; Zhao A et al.; Here, we reported a conformation-constrained peptide library, that was constructed based on the scaffold of a 29 amino acids peptide derived from insect defensin A . The peptide scaffold was designed utilizing the InsightII molecular modeling software and then displayed on M13 filamentous bacteriophage by fusion with coat protein III . The library was constructed by randomization of seven positions located within the two loops of the peptide scaffold generating approximately 8.3 x 10(8) transformants . Sequences from 14 randomly selected phage clones indicated that the distribution of nucleotides and amino acids paralleled with the expected frequency . Screening against the target proteins: tumor necrosis factor alpha, TNF receptor 1, TNF receptor 2 and monoclonal antibody against BMP-2 showed significant enrichment in all cases . The results presented here show that the reconstructed insect defensin A domain will be a promising non-antibody protein scaffold for the presentation of a phage-displayed constrained peptide library .

Mol Microbiol, 2004 Jun, 52(5), 1403 - 11
Bacteriophage T4 endonuclease II: concerted single-strand nicks yield double-strand cleavage; Carlson K et al.; In vivo, endonuclease II (EndoII) of coliphage T4 cleaves sites with conserved sequence elements (CSEs) to both the left and the right of the cleaved bonds, 16 bp altogether with some variability tolerated . In vitro, however, single-strand nicks in the lower strand predominate at sites containing only the left-side CSE that determines the precise position of lower strand nicks . Upper strand nick positions vary both in vivo and in vitro . A 24 bp substrate was nicked with the same precision as in longer substrates, showing that the conserved sequence suffices for precise nicking by EndoII . Using DNA ligase in vitro, we found that EndoII nicked both strands simultaneously at an in vivo-favoured site but not at an in vitro-favoured site . This indicates that the right-side CSE at in vivo-favoured sites is important for simultaneous nicking of both strands, generating double-strand cleavage . Separate analysis of the two strands following in vitro digestion at two in vitro-favoured sites showed that EndoII nicked the lower strand about 1.5-fold faster than the upper strand . In addition, the upper and lower strands were nicked independently of each other, seldom resulting in double-strand cleavage . Thus, cleavage by EndoII is the fortuitous outcome of two separate nicking events.

J Huazhong Univ Sci Technolog Med Sci, 2004, 24(1), 52 - 4, 58
Construction and significance of directional expression cDNA library from human NB4 cells; Chen G et al.; Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen . Total RNA and purified mRNA were extracted from human premyeloid cell line NB4 . First and second strands of cDNA were synthesized by reverse transcription . After blunting, the cDNA fragments were ligated with EcoR I adapters . Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector . The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E . coli XL1-Blue-MRF' for titration . The recombinants were examined by color selection . In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I . The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6% . The average length of the recombinant exogenous inserts was about 1.7 kb . It was concluded that the constructed cDNA library are deserved to screen target clones.

Mol Gen Mikrobiol Virusol, 2004, (2), 28 - 32
{Expression regulation of the protelomerase gene of the bacteriophage N15}; Dorokhov BD et al.; The N15 bacteriophage, when in the lysogenic state, does not integrate into the chromosome; in fact, it exists as a linear plasmid with the covalently closed ends . Upon infection, the phage DNA circularizes via its cohesive ends, after which a specific enzyme, the N15 protelomerase, cuts the circular molecule thus generating a linear plasmid with the covalently closed telomeres . Protelomerase generates, as the replication of plasmid prophage proceeds, the hairpin telomeres in replicated molecules . We identified the promoter of the protelomerase gene and demonstrated that it could be repressed presumably due to its binding with 3 tosL sites overlapping the promoter . We also found the transformation efficiency of E . coli cells of linear DNA with hairpin telomeres to be approximately 100-fold lower versus the circular DNA of the same size . At the same time, presence of the N15 prophage or of the protelomerase-expressing vector enhances, in a strain being transformed, the efficiency of its transformation by linear DNA up to a level ensured in transformation by circular plasmids . We believe that protelomerase, while binding with the hairpin telomeres, protects the latter from degradation by cellular nucleases.

J Biochem Biophys Methods, 2004 May 31, 59(2), 167 - 80
Analysis of solid-phase immobilized antibodies by atomic force microscopy; Johnson JC et al.; Antibody adsorption to solid surfaces creates a number of constraints that may interfere with epitope recognition and ligand-antibody interaction . By optimizing the conditions of adsorption, one may minimize these constraints . We have studied several factors that affect the antibody adsorption using atomic force microscopy (AFM) as a readout mechanism . AFM provides a highly sensitive, label-free method for detecting and analyzing molecular interactions . In this report, AFM was used to study antibody properties, the efficiency of particle capture and ligand-antibody interaction using anti-bacteriophage fd antibodies in a solid phase assay format . The capture efficiencies of anti-fd preparations adsorbed onto gold surfaces under various conditions including pH and antibody concentration were determined and compared . The relative sensitivities of each antibody for the capture of phage fd as a function of applied phage concentrations was evaluated . The collective data indicates that AFM is effective as an analytical instrument for studying the functionality of surface adsorbed antibodies in particle capture assays . This method of analysis can be extended to rapidly screen and select antibodies or other ligands with a specific set of characteristics . As the number and complexity of chip-based analytical platforms in proteomics increases, rapid selection/screening processes such as that described here will become invaluable.

J Biotechnol, 2004 Jun 10, 110(3), 227 - 33
In vivo excision and amplification of large human genomic segments using the Cre/loxP-and large T antigen/SV40 ori-mediated machinery; Min Kim J et al.; In vivo excision and amplification of pre-determined large genomic segments, directly from the genome of a natural host, can be a powerful tool for obtaining the genomic sequences with minimum rearrangements . In this study, an in vivo excision and amplification system in human BJAB cells was devised by combining the Cre/loxP system of bacteriophage P1 and the large T antigen/SV40 ori system of Simian virus 40 . Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into 5'- and 3'-untranslated regions (UTRs) of the human iNOS . An SV40 ori sequence, which serves as a conditional replication system, was inserted between the loxP sites . Trans-acting genes cre and large T antigen, which were under the control of a tetracycline responsive promoter, were also inserted into the 5'- and 3'-UTRs of the iNOS, respectively, by homologous recombination . Upon induction by doxycycline, the 45-kb iNOS genomic fragment of human chromosome 17 flanked by two loxP sites was excised and amplified up to about 45 copies per cell . Our method is very useful for obtaining large genomic fragments in quantities directly from human cells without using foreign hosts . Therefore, our approach can be used effectively for gap sequencing of a genome, gene therapy, and functional analysis of unknown genes in human cells .

Nucleic Acids Res, 2004 May 24, 32(9), 2937 - 46 Print 2004.
2'-deoxyribonolactone lesion produces G->A transitions in Escherichia coli; Faure V et al.; 2'-deoxyribonolactone (dL) is a C1'-oxidized abasic site damage generated by a radical attack on DNA . Numerous genotoxic agents have been shown to produce dL including UV and gamma-irradiation, ene-dye antibiotics etc . At present the biological consequences of dL present in DNA have been poorly documented, mainly due to the lack of method for introducing the lesion in oligonucleotides . We have recently designed a synthesis of dL which allowed investigation of the mutagenicity of dL in Escherichia coli by using a genetic reversion assay . The lesion was site-specifically incorporated in a double-stranded bacteriophage vector M13G*1, which detects single-base-pair substitutions at position 141 of the lacZalpha gene by a change in plaque color . In E.coli JM105 the dL-induced reversion frequency was 4.7 x 10(-5), similar to that of the classic abasic site 2'-deoxyribose (dR) . Here we report that a dL residue in a duplex DNA codes mainly for thymidine . The processing of dL in vivo was investigated by measuring lesion-induced mutation frequencies in DNA repair deficient E.coli strains . We showed a 32-fold increase in dL-induced reversion rate in AP endonuclease deficient (xth nfo) mutant compared with wild-type strain, indicating that the Xth and Nfo AP endonucleases participate in dL repair in vivo.

FEMS Microbiol Lett, 2004 Jun 1, 235(1), 157 - 62
Enzymes used for the determination of HbA1C; Hirokawa K et al.; To develop an enzymatic measurement of HbA(1C), two key enzymes, i.e., fructosyl peptide oxidase and Aspergillus protease were characterized . Fructosyl peptide oxidase from Eupenicillium terrenum was a flavoenzyme that could catalyze the oxidation of N-(1-deoxyfructosyl)-Val-His . The enzyme showed high specificity toward alpha-glycated molecules, therefore it seemed suitable for the HbA(1C) assay . Since high levels of FPOX expression seemed toxic to host cells, we applied a gene expression system using a bacteriophage vector and achieved high levels of expression in Escherichia coli . Next, we found that Aspergillus protease was able to digest N-(1-deoxyfructosyl)-hexapeptide, a glycated peptide that was released from the beta-chain of HbA(1C) by Glu-C endoproteinase . We showed that the N-(1-deoxyfructosyl)-Val-His released from N-(1-deoxyfructosyl)-hexapeptide by Aspergillus protease could be assayed enzymatically using fructosyl peptide oxidase, therefore these enzymes could be applied to the enzymatic measurement of HbA(1C).

Evolution Int J Org Evolution, 2004 Apr, 58(4), 692 - 701
Genome properties and the limits of adaptation in bacteriophages; Bull JJ et al.; Eight bacteriophages were adapted for rapid growth under similar conditions to compare their evolved, endpoint fitnesses . Four pairs of related phages were used, including two RNA phages with small genomes (MS2 and Qbeta) two single-stranded DNA phages with small genomes (phiX174 and G4), two T-odd phages with medium-sized, double-stranded DNA genomes (T7 and T3), and two T-even phages with large, double-stranded DNA genomes (T6 and RB69) . Fitness was measured as absolute growth rate per hour under the same conditions used for adaptation . T7 and T3 achieved the highest fitnesses, able to increase by 13 billionfold and three-quarters billionfold per hour, respectively . In contrast, the RNA phages achieved low fitness maxima, with growth rates approximately 400-fold and 4000-fold per hour . The highest fitness limits were not attributable to high mutation rates or small genome size, even though both traits are expected to enhance adaptation for fast growth . We suggest that major differences in fitness limits stem from different "global" constraints, determined by the organization and composition of the phage genome affecting whether and how it overcomes potentially rate-limiting host processes, such as transcription, translation, and replication . Adsorption rates were also measured on the evolved phages . No consistent pattern of adsorption rate and fitness was observed across the four different types of phages, but within each pair of related phages, higher adsorption was associated with higher fitness . Different adsorption rate limits within pairs may stem from "local" constraints-sequence differences leading to different local optima in the sequence space.

J Biol Chem, 2004 Jul 16, 279(29), 30228 - 35 Epub 2004 May 18.
Interaction of nick-directed DNA mismatch repair and loop repair in human cells; Huang YM et al.; In human cells, large DNA loop heterologies are repaired through a nick-directed pathway independent of mismatch repair . However, a 3'-nick generated by bacteriophage fd gene II protein heterology is not capable of stimulating loop repair . To evaluate the possibility that a mismatch near a loop could induce both repair types in human cell extracts, we constructed and tested a set of DNA heteroduplexes, each of which contains a combination of mismatches and loops . We have demonstrated that a strand break generated by restriction endonucleases 3' to a large loop is capable of provoking and directing loop repair . The repair of 3'-heteroduplexes in human cell extracts is very similar to that of 5'-heteroduplex repair, being strand-specific and highly biased to the nicked strand . This observation suggests that the loop repair pathway possesses bidirectional repair capability similar to that of the bacterial loop repair system . We also found that a nick 5' to a coincident mismatch and loop can apparently stimulate the repair of both . In contrast, 3'-nick-directed repair of a G-G mismatch was reduced when in the vicinity of a loop (33 or 46 bp between two sites) . Increasing the distance separating the G-G mismatch and loop by 325 bp restored the efficiency of repair to the level of a single base-base mismatch . This observation suggests interference between 3'-nick-directed large loop repair and conventional mismatch repair systems when a mispair is near a loop . We propose a model in which DNA repair systems avoid simultaneous repair at adjacent sites to avoid the creation of double-stranded DNA breaks.

J Bacteriol, 2004 Jun, 186(11), 3599 - 608
DNA binding regions of Q proteins of phages lambda and phi80; Guo J et al.; Bacteriophage lambda gene Q protein and the related proteins of other lambdoid phages are transcription antiterminators that interact both with DNA in the late gene promoter segment and with RNA polymerase subunits . Using hybrids between Q of lambda and the related Q of phage 80, we characterized elements of both Q and DNA that contribute to the DNA binding function . In particular, we found a C-terminal segment of the protein that is responsible for binding specificity and an approximately 15 residue segment on a predicted alpha helix within this segment at which alanine substitutions decrease DNA binding . We identified a six-nucleotide segment located between the -35 and -10 promoter elements that confers binding specificity and is the site of point mutants that impair binding, and we isolated suppressors in lambda Q that restore binding function by increasing the overall binding affinity . We also identified putative zinc finger structures in both proteins.

Nat Struct Mol Biol, 2004 Jun, 11(6), 531 - 8 Epub 2004 May 16.
Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA; Byrd AK et al.; Helicases are molecular motors that unwind double-stranded DNA or RNA . In addition to unwinding nucleic acids, an important function of these enzymes seems to be the disruption of protein-nucleic acid interactions . Bacteriophage T4 Dda helicase can displace proteins bound to DNA, including streptavidin bound to biotinylated oligonucleotides . We investigated the mechanism of streptavidin displacement by varying the length of the oligonucleotide substrate . We found that a monomeric form of Dda catalyzed streptavidin displacement; however, the activity increased when multiple helicase molecules bound to the biotinylated oligonucleotide . The activity does not result from cooperative binding of Dda to the oligonucleotide . Rather, the increase in activity is a consequence of the directional bias in translocation of individual helicase monomers . Such a bias leads to protein-protein interactions when the lead monomer stalls owing to the presence of the streptavidin block.

Anal Chem, 2004 May 15, 76(10), 2700 - 7
Bead-based electrochemical immunoassay for bacteriophage MS2; Thomas JH et al.; Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox . A paramagnetic bead-based electrochemical immunoassay has been developed for detecting bacteriophage MS2 . The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-beta-galactosidase conjugate to another site on the virus . beta-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP) . PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample . The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode . With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 x 10(10) viral particles/mL with RDE amperometry . A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected . The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 x 10(10) MS2 particles/mL.

J Virol, 2004 Jun, 78(11), 5679 - 85
A bunyamwera virus minireplicon system in mosquito cells; Kohl A et al.; Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses . Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells . To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells . An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established . Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids . Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells . However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs . We also report that promoter strength is different for vertebrate versus invertebrate cells . The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.

Protein Sci, 2004 Jun, 13(6), 1538 - 46 Epub 2004 May 07.
Pressure dissociation studies provide insight into oligomerization competence of temperature-sensitive folding mutants of P22 tailspike; Lefebvre BG et al.; Several temperature-sensitive folding (tsf) mutants of the tailspike protein from bacteriophage P22 have been found to fold with lower efficiency than the wild-type sequence, even at lowered temperatures . Previous refolding studies initiated from the unfolded monomer have indicated that the tsf mutations decrease the rate of structured monomer formation . We demonstrate that pressure treatment of the tailspike aggregates provides a useful tool to explore the effects of tsf mutants on the assembly pathway of the P22 tailspike trimer . The effects of pressure on two different tsf mutants, G244R and E196K, were explored . Pressure treatment of both G244R and E196K aggregates produced a folded trimer . E196K forms almost no native trimer in in vitro refolding experiments, yet it forms a trimer following pressure in a manner similar to the native tailspike protein . In contrast, trimer formation from pressure-treated G244R aggregates was not rapid, despite the presence of a G244R dimer after pressure treatment . The center-of-mass shifts of the fluorescence spectra under pressure are nearly identical for both tsf aggregates, indicating that pressure generates similar intermediates . Taken together, these results suggest that E196K has a primary defect in formation of the beta-helix during monomer collapse, while G244R is primarily an assembly defect.

J Biol Chem, 2004 Jul 16, 279(29), 30554 - 62 Epub 2004 May 08.
A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis; Kato M et al.; The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site . We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase . These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases . The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template . We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.

J Clin Microbiol, 2004 May, 42(5), 2115 - 20
Development of a bacteriophage phage replication assay for diagnosis of pulmonary tuberculosis; McNerney R et al.; Successful infection and replication of bacteriophages is indicative of the presence of viable bacteria . We describe here the development of a bacteriophage replication assay for the detection of Mycobacterium tuberculosis by using mycobacteriophage D29 . Optimization of phage inoculate and incubation times allowed highly sensitive detection of M . bovis BCG . Fewer than 10 CFU (100 CFU/ml) were detected . No false-positive results were observed in negative samples . Application of the assay to 496 sputum specimens in the National Reference Laboratory of Zambia produced sensitivity, specificity, and positive and negative predictive values of 44.1, 92.6, 82.2, and 67.5%, respectively, compared to culture on Lowenstein-Jensen medium . The equivalent corresponding results for direct fluorescent smear microscopy were 42.3, 96.8, 91.2, and 67.6% . The small increase in sensitivity over that of direct microscopy does not justify the introduction of this technique for routine diagnosis of pulmonary tuberculosis at this time.

Appl Bioinformatics, 2003, 2(1), 47 - 62
Comparison of responses by bacteriophages and bacteria to pressures on the base composition of open reading frames; Mortimer JR et al.; Differences in the base composition of genomes can occur because of GC pressure, purine-loading pressure (AG pressure) and RNY pressure, for which there are possible functional explanations, and because of the more abstract pressures exerted by individual bases . The graphical approach of Muto and Osawa was used to analyse how bacteriophages and bacteria balance potentially conflicting pressures on their genomes . Phages generally respond to AG pressure by increasing A while keeping T constant, and by decreasing C while keeping G constant . In contrast, bacteria generally increase both A and T, the former more so, and decrease both G and C, the latter more so . These differences largely occur at third codon positions, which are more responsive than first and second codon positions to AG pressure and GC pressure . Phages respond to AG pressure more in the third codon position than bacteria, whereas bacteria respond more in the first codon position than phages . Conversely, bacteria respond to GC pressure more in the third codon position than phages, whereas phages respond more in the first codon position than bacteria . As GC pressure increases, A is traded for C and AG pressure decreases; first and second codon positions, having more A than T, are most responsive to this negative effect of increased GC pressure; third positions either do not respond (phages) or respond weakly (bacteria) . In a set of 48 phage-host pairs, degrees of purine loading were less correlated between phage and host than were GC percentages . These results suggest that pressures on conventional and genome phenotypes operate differentially in phages and bacteria, generating both general differences in base composition and specific differences characteristic of particular phage-host pairs . The reciprocal relationship between GC pressure and AG pressure implies that effects attributed to GC pressure may actually be due to AG pressure, and vice versa.

Mol Biol (Mosk), 2004 Mar-Apr, 38(2), 297 - 302
{Structural organization and control of expression of the sop-operon of linear plasmid prophage N15}; Ravin NV et al.; Stable inheritance of bacterial chromosomes and low-copy-number plasmids depends on the active partition of replicated molecules between daughter cells . The partition mechanism is well known for circular plasmids F and P1 . The mechanism of partition of linear replicons was studied with the example of bacteriophage N15, which persists as a linear plasmid with covalently closed ends on lysogeny, rather than integrating into the Escherichia coli chromosome . Since stable inheritance of N15 is due to the sop operon homologous to sop of the F plasmid, the control of expression of the N15 sop genes was analyzed . The sop promoter (Psop) contains a binding site for bacterial IHF and five CTTTGC copies, which overlap the -35 and -10 elements . The Sop proteins were shown to interact with a Psop-containing DNA fragment in vitro . Transcription of the sop operon is regulated by the Sop proteins: SopA represses Psop, and SopB enhances the repression, having no effect on the promoter activity in the absence of SopA . In N15 lysogenic cells, Psop proved to be repressed . This regulatory mechanism was assumed to ensure production of SopA and SopB in amounts required for the segregation stability of N15 and to neutralize occasional fluctuations of their concentration in the cell.

Proc Natl Acad Sci U S A, 2004 May 11, 101(19), 7264 - 9 Epub 2004 May 03.
The DNA-unwinding mechanism of the ring helicase of bacteriophage T7; Jeong YJ et al.; Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation . Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication . Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding . We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters . The comparison of the unwinding properties of T7 helicase with its translocation properties on single-stranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases . T7 helicase unwinds DNA with a rate of 15 bp/s, which is 9-fold slower than the translocation speed along ssDNA . T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA . We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel . The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.

Nat Struct Mol Biol, 2004 Jun, 11(6), 544 - 50 Epub 2004 May 02.
The sigma 70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter; Nickels BE et al.; The sigma(70) subunit of RNA polymerase plays an essential role in transcription initiation . In addition, sigma(70) has a critical regulatory role during transcription elongation at the bacteriophage lambda late promoter, lambda P(R') . At this promoter, sigma(70) mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element . Here we provide evidence that sigma(70) also mediates a pause in early elongation at the lac promoter (plac) . Like that at lambda P(R'), the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element . Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between sigma(70) and the remainder of the transcription elongation complex . Bioinformatic analysis suggests that promoter-proximal sigma(70)-dependent pauses may play a role in the regulation of many bacterial promoters.

Methods Mol Biol, 2004, 269, 41 - 50
Transient and inducible expression of vaccinia/T7 recombinant viruses; Mohamed MR et al.; Recombinant DNA technology has made it possible to develop molecular cloning vectors that allow the expression of heterologous genes in a variety of animal viruses . This chapter discusses the use of vaccinia virus encoding bacteriophage T7 RNA polymerase as an expression vector system . A chosen gene is inserted into a plasmid vector designed to express genes under the control of the T7 promoter . Transient expression can then be achieved either by transfecting this plasmid into cells infected with the recombinant vaccinia virus expressing T7 RNA polymerase, vTF7-3 or by crossing this plasmid into the vaccinia virus genome and coinfecting cells with both viruses . Moreover, placement of lacO downstream of the vaccinia virus P11 late promoter regulating T7 RNA polymerase expression, and integration of lacI under vaccinia promoter control into the viral genome, vT7lacOI, yielded a recombinant virus capable of IPTG-inducible T7 promoter-controlled expression of foreign genes.

J Biochem (Tokyo), 2004 Mar, 135(3), 397 - 403
Characterization of bacteriophage T3 DNA ligase; Cai L et al.; DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase . Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase . The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme . The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50% . Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min . T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments . Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.

Vet Immunol Immunopathol, 2004 May, 99(1-2), 11 - 24
Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis; Manoutcharian K et al.; The aim of this study was to test the capacity of recombinant phages to deliver antigens for vaccination against porcine cysticercosis . Thus, three peptides (KETc1, KETc12, GK1) and a recombinant antigen KETc7, previously proven to induce high levels of protection against pig cysticercosis, were expressed on the surface of the M13 bacteriophage at multiple copies . The pool of these four recombinant phages induced high levels of protection against an experimental murine cysticercosis . The immunogenicity of the phage vaccine preparation was therefore, tested in pigs, the natural host of Taenia solium . Subcutaneous or oral vaccination with these phages induced antigen-specific cellular immune responses in pigs . Preliminary data also points to the protective capacity of this recombinant phage vaccine against pig cysticercosis . The immunogenicity of these recombinant phages, together with the low cost of their production, make them a realistic candidate to be tested in pigs as an anti-cysticercus phage vaccine for field trials . This is the first report describing the application of a filamentous bacteriophage as a vaccine in large animals such as pigs, the only intermediate hosts of T . solium, a parasite of major medical importance in developing countries . The potential application of phages as a modern platform for vaccines for human and animal diseases is discussed .

Virology, 2004 May 1, 322(2), 253 - 63
The portal protein plays essential roles at different steps of the SPP1 DNA packaging process; Isidro A et al.; A large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection . These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices . The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient . The portal protein is an essential component of this DNA packaging machine . Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action . Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated . Functional regions of gp6 necessary at each step are identified within its primary structure . The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses.

Proteins, 2004 May 15, 55(3), 733 - 42
X-ray structural and simulation analysis of a protein mutant: the value of a combined approach; Mattos C et al.; The effect of the mutation Arg 96 to His on the stability of bacteriophage T4 lysozyme has been previously studied by calorimetric experiments, X-ray crystallography, and free energy simulation techniques . The experimental and calculated values for the difference between the free energy of denaturation of the mutant and the wild type are in reasonable agreement . However, the two approaches led to different explanations for the loss in stability . To analyze the differences, a series of refinements based on the crystallographic data were performed, a number of aspects of the simulations were reexamined, and continuum electrostatic calculations were done to complement the latter . The results of those comparisons provide a better understanding of the origin of the free energy difference in this mutant . Furthermore, they show the importance of the combined use of simulations and crystallography for interpreting the effects of mutations on the energetics of the system .

Mol Microbiol, 2004 May, 52(3), 815 - 22
Morphing molecular specificities between Arm-peptide and NUT-RNA in the antitermination complexes of bacteriophages lambda and P22; Franklin NC; Bacteriophage lambda's N-protein includes a 17-amino-acid segment, Arm, rich in arginine and having specific affinity for a 15-nucleotide RNA stem-loop called BOX-B . Parallel but different Arm/BOX-B sequences in lambda's cousin, phage P22, account for some of the type specificity that distinguishes lambda from P22: the N of each works only with its cognate BOX-B in vivo . We find that the specificity of N(lambda) can be shifted gradually to that of N(22) by substituting sets of particular amino acids from Arm(22) into Arm of N(lambda) . The determinative amino acids are generally those shown by nuclear magnetic resonance to contact BOX-B RNA; gain or loss of these contact amino acids is reasonably expected to contribute to the affinity of each amino acid sequence . Intermediate sequences may show no function with either BOX-B, or weak function with both BOX-B(lambda) and BOX-B(22), the latter suggesting possible evolutionary paths for specificity shifts.

J Mol Biol, 2004 Feb 27, 336(4), 851 - 70
Mechanical measurement of single-molecule binding rates: kinetics of DNA helix-destabilization by T4 gene 32 protein; Pant K et al.; Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein, and is essential for DNA replication, recombination and repair . While gp32 binds preferentially and cooperatively to ssDNA, it has not been observed to lower the thermal melting temperature of natural double-stranded DNA (dsDNA) . However, in single-molecule stretching experiments, gp32 significantly destabilizes lambda DNA . In this study, we develop a theory of the effect of the protein on single dsDNA stretching curves, and apply it to the measured dependence of the DNA overstretching force on pulling rate in the presence of the full-length and two truncated forms of the protein . This allows us to calculate the rate of cooperative growth of single clusters of protein along ssDNA that are formed as the dsDNA molecule is stretched, as well as determine the site size of the protein binding to ssDNA . The rate of cooperative binding (ka) of both gp32 and of its proteolytic fragment *I (which lacks 48 residues from the C terminus) varies non-linearly with protein concentration, and appears to exceed the diffusion limit . We develop a model of protein association with the ends of growing clusters of cooperatively bound protein enhanced by 1-D diffusion along dsDNA, under the condition of protein excess . Upon globally fitting ka versus protein concentration, we determine the binding site size and the non-cooperative binding constants to dsDNA for gp32 and I . Our experiment mimics the growth of clusters of gp32 that likely exist at the DNA replication fork in vivo, and explains the origin of the "kinetic block" to dsDNA melting by gene 32 protein observed in thermal melting experiments.

Curr Opin Struct Biol, 2004 Apr, 14(2), 171 - 80
The bacteriophage T4 DNA injection machine; Rossmann MG et al.; The tail of bacteriophage T4 consists of a contractile sheath surrounding a rigid tube and terminating in a multiprotein baseplate, to which the long and short tail fibers of the phage are attached . Upon binding of the fibers to their cell receptors, the baseplate undergoes a large conformational switch, which initiates sheath contraction and culminates in transfer of the phage DNA from the capsid into the host cell through the tail tube . The baseplate has a dome-shaped sixfold-symmetric structure, which is stabilized by a garland of six short tail fibers, running around the periphery of the dome . In the center of the dome, there is a membrane-puncturing device, containing three lysozyme domains, which disrupts the intermembrane peptidoglycan layer during infection.

Proc Natl Acad Sci U S A, 2004 Apr 27, 101(17), 6415 - 20 Epub 2004 Apr 16.
A signal-arrest-release sequence mediates export and control of the phage P1 endolysin; Xu M et al.; The Lyz endolysin of bacteriophage P1 was found to cause lysis of the host without a holin . Induction of a plasmid-cloned lyz resulted in lysis, and the lytic event could be triggered prematurely by treatments that dissipate the proton-motive force . Instead of requiring a holin, export was mediated by an N-terminal transmembrane domain (TMD) and required host sec function . Exported Lyz of identical SDS/PAGE mobility was found in both the membrane and periplasmic compartments, indicating that periplasmic Lyz was not generated by the proteolytic cleavage of the membrane-associated form . In gene fusion experiments, the Lyz TMD directed PhoA to both the membrane and periplasmic compartments, whereas the TMD of the integral membrane protein FtsI restricts Lyz to the membrane . Thus, the N-terminal domain of Lyz is both necessary and sufficient not only for export of this endolysin to the membrane but also for its release into the periplasm . The unusual N-terminal domain, rich in residues that are weakly hydrophobic, thus functions as a signal-arrest-release sequence, which first acts as a normal signal-arrest domain to direct the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm . Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an N-terminal signal-arrest-release sequence is not unique to Lyz . These observations are discussed in relation to the role of holins in the control of host lysis by bacteriophage encoding a secretory endolysin.

J Bacteriol, 2004 May, 186(9), 2699 - 707
Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12; Poteete AR; The orf gene of bacteriophage lambda, fused to a promoter, was placed in the galK locus of Escherichia coli K-12 . Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Delta(recC ptr recB recD)::P(tac) gam bet exo pae cI DeltarecG background, lacking recF, recO, recR, ruvAB, and ruvC functions . It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid . Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant . In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.

Phys Rev E Stat Nonlin Soft Matter Phys . 2004 Mar;69(3 Pt 1):031915 . Epub 2004 Mar 31.
Atomic force microscopy contact, tapping, and jumping modes for imaging biological samples in liquids; Moreno-Herrero F et al.; The capabilities of the atomic force microscope for imaging biomolecules under physiological conditions has been systematically investigated . Contact, dynamic, and jumping modes have been applied to four different biological systems: DNA, purple membrane, Alzheimer paired helical filaments, and the bacteriophage phi29 . These samples have been selected to cover a wide variety of biological systems in terms of sizes and substrate contact area, which make them very appropriate for the type of comparative studies carried out in the present work . Although dynamic mode atomic force microscopy is clearly the best choice for imaging soft samples in air, in liquids there is not a leading technique . In liquids, the most appropriate imaging mode depends on the sample characteristics and preparation methods . Contact or dynamic modes are the best choices for imaging molecular assemblies arranged as crystals such as the purple membrane . In this case, the advantage of image acquisition speed predominates over the disadvantage of high lateral or normal force . For imaging individual macromolecules, which are weakly bonded to the substrate, lateral and normal forces are the relevant factors, and hence the jumping mode, an imaging mode which minimizes lateral and normal forces, is preferable to other imaging modes.

J Biol Chem, 2004 Jul 23, 279(30), 31337 - 47 Epub 2004 Apr 13.
RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2; Nandakumar J et al.; Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid . The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks . RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn) . Neither dATP, GTP, CTP, nor UTP can substitute for ATP . We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme . Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity . Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.

J Biol Chem, 2004 Jun 11, 279(24), 25721 - 8 Epub 2004 Apr 13.
Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein; Jones CE et al.; Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo . 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein . The helicase loader is an almost completely alpha-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins . We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein . Here, we characterize several mutants of 59 protein in an initial effort to test this model . We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA . As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system . 59 protein with a deletion of the two C-terminal residues, Lys(216) and Tyr(217), binds fork DNA normally . In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA . However, it binds 32 protein in the absence of DNA . The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system . We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.

J Virol, 2004 May, 78(9), 4710 - 9
Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d; Bower JF et al.; DNA vaccines expressing the envelope (Env) of human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting immune responses . Oligomeric or trimeric (gp140) forms of Env that more closely mimic the native proteins on the virion are often more effective immunogens than monomeric (gp120) envelopes . In this study, several forms of Env constructed from the HIV-1 isolate YU-2 (HIV-1(YU-2)) were tested for their immunogenic potential: a trimeric form of uncleaved (-) Env stabilized with a synthetic trimer motif isolated from the fibritin (FT) protein of the T4 bacteriophage, sgp140(YU-2)(-/FT), was compared to sgp140(YU-2)(-) without a synthetic trimerization domain, as well as to monomeric gp120(YU-2) . DNA plasmids were constructed to express Env alone or fused to various copies of murine C3d (mC3d) . BALB/c mice were vaccinated (day 1 and week 4) with DNA expressing a codon-optimized envelope gene insert, alone or fused to mC3d . Mice were subsequently boosted (week 8) with the DNA or recombinant Env protein . All mice had high anti-Env antibody titers regardless of the use of mC3d . Sera from mice vaccinated with DNA expressing non-C3d-fused trimers elicited neutralizing antibodies against homologous HIV-1(YU-2) virus infection in vitro . In contrast, sera from mice inoculated with DNA expressing Env-C3d protein trimers elicited antibody that neutralized both homologous HIV-1(YU-2) and heterologous HIV-1(ADA), albeit at low titers . Therefore, DNA vaccines expressing trimeric envelopes coupled to mC3d, expressed in vivo from codon-optimized sequences, elicit low titers of neutralizing antibodies against primary isolates of HIV-1.

J Biol Chem, 2004 Jun 18, 279(25), 26762 - 7 Epub 2004 Apr 12.
DNA stimulates Mec1-mediated phosphorylation of replication protein A; Bartrand AJ et al.; The cellular single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) becomes phosphorylated periodically during the normal cell cycle and also in response to DNA damage . In Saccharomyces cerevisiae, RPA phosphorylation requires the checkpoint protein Mec1, a protein kinase homologous in structure and function to human ATR . We confirm here that immunocomplexes containing a tagged version of Mec1 catalyze phosphorylation of purified RPA, likely reflecting an RPA kinase activity intrinsic to Mec1 . A significant stimulation of this activity is observed upon the addition of covalently closed ssDNA derived from the bacteriophage M13 . This stimulation is not observed with mutant RPA deficient for DNA binding, indicating that DNA-bound RPA is a preferred substrate . Stimulation is also observed upon the addition of linear ssDNA homopolymers or hydrolyzed M13 ssDNA . In contrast to circular ssDNA, these DNA cofactors stimulate both wild type and mutant RPA phosphorylation . This finding suggests that linear ssDNA can also stimulate Mec1-mediated RPA phosphorylation by activating Mec1 or an associated protein . Although the Mec1-interacting protein Ddc2 is required for RPA phosphorylation in vivo, it is required for neither basal nor ssDNA-stimulated RPA phosphorylation in vitro . Therefore, activation of Mec1-mediated RPA phosphorylation by either circular or linear ssDNA does not operate through Ddc2 . Our results provide insight into the mechanisms that function in vivo to specifically induce RPA phosphorylation upon initiation of DNA replication, repair, or recombination.

Curr Protein Pept Sci, 2004 Apr, 5(2), 73 - 9
What is the structure of the RecA-DNA filament?
Yu X, VanLoock MS, Yang S, Reese JT, Egelman EH.
The bacterial RecA protein has been a model system for understanding how a protein can catalyze homologous genetic recombination . RecA-like proteins have now been characterized from many organisms, from bacteriophage to humans . Some of the RecA-like proteins, including human RAD51, appear to function as helical filaments formed on DNA . However, we currently have high resolution structures of inactive forms of the protein, and low resolution structures of the active complexes formed by RecA-like proteins on DNA in the presence of ATP or ATP analogs . Within a crystal of the E . coli RecA protein, a helical polymer exists, and it has been widely assumed that this polymer is quite similar to the active helical filament formed on DNA . Recent developments have suggested that this may not be the case.

Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 6003 - 8 Epub 2004 Apr 07.
Molecular architecture of the prolate head of bacteriophage T4; Fokine A et al.; The head of bacteriophage T4 is a prolate icosahedron with one unique portal vertex to which the phage tail is attached . The three-dimensional structure of mature bacteriophage T4 head has been determined to 22-A resolution by using cryo-electron microscopy . The T4 capsid has a hexagonal surface lattice characterized by the triangulation numbers T(end) = 13 laevo for the icosahedral caps and T(mid) = 20 for the midsection . Hexamers of the major capsid protein gene product (gp)23* and pentamers of the vertex protein gp24*, as well as the outer surface proteins highly antigenic outer capsid protein (hoc) and small outer capsid protein (soc), are clearly evident in the reconstruction . The size and shape of the gp23* hexamers are similar to the major capsid protein organization of bacteriophage HK97 . The binding sites and shape of the hoc and soc proteins have been established by analysis of the soc(-) and hoc(-)soc(-) T4 structures.

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