J Mol Biol, 2001 Mar 2, 306(4), 631 - 42 Mapping the molecular interface between the sigma(70) subunit of E . coli RNA polymerase and T4 AsiA; Minakhin L et al.; Bacteriophage T4 antisigma protein AsiA (10 kDa) orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of E . coli RNA polymerase . The molecular determinants of sigma(70)-AsiA complex formation are not known . Here, we used combinatorial peptide chemistry, protein-protein crosslinking, and mutational analysis to study the interaction between AsiA and its target, the 33 amino acid residues-long sigma(70) peptide containing conserved region 4.2 . Many region 4.2 amino acid residues contact AsiA, which likely completely occludes the DNA-binding surface of region 4.2 . Though none of region 4.2 amino acid residues is singularly responsible for the very tight interaction with AsiA, sigma(70) Lys593 and Arg596 which lie outside the putative DNA recognition element of region 4.2, contribute the most . In AsiA, the first 20 amino acid residues are both necessary and sufficient for interactions with sigma(70) . Our results clarify details of sigma(70)-AsiA interaction and open the way for engineering AsiA derivatives with altered specificities . Nature, 2001 Mar 8, 410(6825), 235 - 40 A mechanism for initiating RNA-dependent RNA polymerization; Butcher SJ et al.; In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase . Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA . Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail . Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses . By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route . The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction . Our results provide a working model for the initiation of replication and transcription. Microbiology, 2001 Mar, 147(Pt 3), 535 - 47 Regulation of the switch from early to late bacteriophage lambda DNA replication; Baranska S et al.; There are two modes of bacteriophage lambda DNA replication following infection of its host, Escherichia coli . Early after infection, replication occurs according to the theta (theta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode . It is not known how this switch, occurring at a specific time in the infection cycle, is regulated . Here it is demonstrated that in wild-type cells the replication starting from orilambda proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from orilambda is predominantly unidirectional . The regulation of directionality of replication from orilambda is mediated by positive control of lambda p(R) promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the p(tet) promoter instead of p(R) was found to be independent of DnaA function . These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional theta mechanism and is switched early after infection to the sigma mode . It is proposed that in wild-type E . coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional theta mechanism early after infection due to efficient transcriptional activation of orilambda, stimulated by the host DnaA protein . After a few rounds of this type of replication, the resulting increased copy number of lambda genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA . It is proposed that this may lead to inefficient transcriptional activation of orilambda resulting in unidirectional theta replication followed by sigma type replication. Genetics, 2001 Mar, 157(3), 1077 - 87 Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not; Doan PL et al.; Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes . The phage replication origin ori(34) is located in a region that has a hotspot in both assays . To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located) . The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates . As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes . However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes . The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage . The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms. Genetics, 2001 Mar, 157(3), 979 - 90 The Neurospora crassa genome: cosmid libraries sorted by chromosome; Kelkar HS et al.; A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi . The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N . crassa genome was resolved using two translocation strains . Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N . crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes . Assignments of cosmids to linkage groups on the basis of the genetic map vs . the electrophoretic karyotype are 93 +/- 3% concordant . The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome . Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N . crassa genome . By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N . crassa gene sequence as a starting point for gene identification. Clin Immunol, 2001 Mar, 98(3), 313 - 8 Immunologic reconstitution following bone marrow transplantation for X-linked hyper IgM syndrome; Duplantier JE et al.; X-linked hyper IgM syndrome (XHIM), caused by mutations of the CD40 ligand (CD40L) gene, is characterized by recurrent bacterial and opportunistic infections, an increased incidence of autoimmunity and malignancies, and immunodeficiency due to abnormal T/B cell interaction . Because of poor long-term prognosis, bone marrow transplantation (BMT) has been proposed as an alternative treatment . An 8-month-old boy with XHIM and a splice site mutation of CD40L underwent BMT using a fully matched sibling donor . Markers of engraftment and immunologic reconstitution were measured serially . After BMT, activated T cells expressed functional CD40L, and genomic DNA obtained from circulating white cells contained predominantly wild-type CD40L sequences . Serum immunoglobulin levels including IgE and antibody responses to recall antigens normalized, and immunization with the T-cell-dependent neoantigen, bacteriophage &phi;X174, demonstrated amplification of the response and isotope switching . BMT provides a permanent cure for XHIM if a fully matched sibling donor is available and the procedure is performed before complications have occurred . Biotechniques, 2001 Feb, 30(2), 404 - 8, 410, 412-3 Selection of scFv phages on intact cells under low pH conditions leads to a significant loss of insert-free phages; Tur MK et al.; Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens . However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking antibody fragments . Moderate adhesive binding activities and production advantages of these "empty" phages results in their subsequent enrichment during selection on target cells . To date, very limited effort has been made to develop strategies removing nonspecific binding phages during the selection processes . To efficiently reduce insert-free phages when panning on intact cells, we increased the washing stringency by lowering the pH of the buffer with citric acid . Under standard washing procedures (pH 7.4), only approximately 73% of recovered phages were insert-free after three rounds of selection . Using stringent washing procedures (pH 5.0), approximately 12% of recovered phages contained no scFv . Using this protocol, we have cloned an antibody fragment from a mouse/human hybridoma cell line directed against the disialoganglioside GD2 . This study confirms that selection of phage antibodies on cells is efficiently enhanced by assays augmenting the stringency to remove nonspecific binding phages. Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2301 - 5 Epub 2001 Feb 13. Contributions of distinct quaternary contacts to cooperative operator binding by Mnt repressor; Berggrun A et al.; Mnt, a tetrameric repressor encoded by bacteriophage P22, uses N-domain dimers to contact each half of its operator site . Experiments with a double mutant and structural homology with the P22 Arc repressor suggest that contacts made by Arg-28 and stabilized by Glu-33 are largely responsible for dimer-dimer cooperativity in Mnt . These dimer-dimer contacts are energetically more important for operator binding than solution tetramerization, which is mediated by an independent C-terminal coiled-coil domain . Indeed, once one dimer of the Mnt tetramer contacts an operator half site, binding of the second dimer occurs with an effective concentration much lower than that expected if both dimers were flexibly tethered . These results suggest that binding of the second dimer introduces some strain into the protein-DNA complex, a mechanism that could serve to limit the affinity of operator binding and to prevent strong binding of the Mnt tetramer to nonoperator sites. J Immunol Methods, 2001 Feb 1, 248(1-2), 31 - 45 Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapeutics; Irving RA et al.; Protein affinity maturation using molecular evolution techniques to produce high-affinity binding proteins is an important step in the generation of reagents for cancer diagnosis and treatment . Currently, the most commonly used molecular evolution processes involve mutation of a single gene into complex gene repertoires followed by selection from a display library . Fd-bacteriophage are the most popular display vectors, but are limited in their capacity for library presentation, speed of processing and mutation frequency . Recently, the potential of ribosome display for directed molecular evolution was recognised and developed into a rapid and simple affinity selection strategy using ribosome complexes to display antibody fragments (scFv) . Ribosome display and selection has the potential to generate and display large libraries more representative of the theoretical optima for naive repertoires (10(14)) . Even more important is the application of ribosome display for the affinity maturation of individual proteins by rapid mutation and selection cycles . These display strategies can apply to other members of the immunoglobulin superfamily; for example single V-domains which have an important application in providing specific targeting to either novel or refractory cancer markers . We discuss the application of ribosome display and selection in conjunction with variable domain (CTLA-4) libraries as the first step towards this objective and review affinity maturation strategies for in vitro ribosome display systems. Nucleic Acids Res, 2001 Mar 1, 29(5), 1175 - 84 RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities; Sengupta TK et al.; The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs . RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein . To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays . These assays revealed that RB69 RegA protein protects nucleotides -9 to -3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU . On RB69 gene 45, the protected site (nucleotides -8 to -3) contains a similar purine-rich sequence: GAAAUA . Interestingly, T4 RegA protein protected the same nucleotides on these RNAs . To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs . Comparative gel shift assays demonstrated that RB69 RegA protein has an approximately 7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein . RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein . On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA . With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA . Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA . The helix-loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein . However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix-loop groove, which may also play a role in RNA binding. J Virol, 2001 Mar, 75(6), 2509 - 15 Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli; Scholl D et al.; A virulent double-stranded DNA bacteriophage, Phi K1-5, has been isolated and found to be capable of infecting Escherichia coli strains that possess either the K1 or the K5 polysaccharide capsule . Electron micrographs show that the virion consists of a small icosohedral head with short tail spikes, similar to members of the Podoviridae family . DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins . The first is the K5 lyase protein gene of Phi K5, which allows this phage to specifically infect K5 E . coli strains . A second open reading frame encodes a protein almost identical in amino acid sequence to the N-acetylneuraminidase (endosialidase) protein of Phi K1E, which allows this phage to specifically infect K1 strains of E . coli . We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated . A comparison of the tail gene regions of Phi K5, Phi K1E, and Phi K1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer. J Bacteriol, 2001 Mar, 183(6), 1862 - 9 T7 single strand DNA binding protein but not T7 helicase is required for DNA double strand break repair; Yu M et al.; An in vitro system based on Escherichia coli infected with bacteriophage T7 was used to test for involvement of host and phage recombination proteins in the repair of double strand breaks in the T7 genome . Double strand breaks were placed in a unique XhoI site located approximately 17% from the left end of the T7 genome . In one assay, repair of these breaks was followed by packaging DNA recovered from repair reactions and determining the yield of infective phage . In a second assay, the product of the reactions was visualized after electrophoresis to estimate the extent to which the double strand breaks had been closed . Earlier work demonstrated that in this system double strand break repair takes place via incorporation of a patch of DNA into a gap formed at the break site . In the present study, it was found that extracts prepared from uninfected E . coli were unable to repair broken T7 genomes in this in vitro system, thus implying that phage rather than host enzymes are the primary participants in the predominant repair mechanism . Extracts prepared from an E . coli recA mutant were as capable of double strand break repair as extracts from a wild-type host, arguing that the E . coli recombinase is not essential to the recombinational events required for double strand break repair . In T7 strand exchange during recombination is mediated by the combined action of the helicase encoded by gene 4 and the annealing function of the gene 2.5 single strand binding protein . Although a deficiency in the gene 2.5 protein blocked double strand break repair, a gene 4 deficiency had no effect . This argues that a strand transfer step is not required during recombinational repair of double strand breaks in T7 but that the ability of the gene 2.5 protein to facilitate annealing of complementary single strands of DNA is critical to repair of double strand breaks in T7. Cell Mol Life Sci, 1999 Nov 15, 56(7-8), 580 - 603 Scaffolding proteins and their role in viral assembly; Dokland T; Scaffolding proteins are proteins that are required to catalyse, regulate or modulate some step in the assembly of a macromolecular complex . They associate specifically with the nascent protein complex during assembly, but are subsequently removed, and are absent from the mature structure . Scaffolding proteins have been described primarily from viral systems, in particular from the double-stranded DNA bacteriophages, but most likely play a more general role in macromolecular assembly, a fundamental process in all biological systems . Scaffolding proteins may act in a specific fashion, by actively encouraging the formation of correct protein-protein interactions, or more generally by nucleating and promoting assembly . They may also work to ensure the fidelity of the assembly process by preventing the formation of improper interactions, in many ways similar to the role of molecular chaperones in protein folding . In viruses, scaffolding proteins are found both in the form of internal cores and external bracing, and may form elaborate and complex structures . This review will focus on the viral scaffolding proteins, for which an increasing amount of structural and functional information has recently become available. Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 3 - 16 Genomic approaches to typing, taxonomy and evolution of bacterial isolates; Gurtler V et al.; The current literature on bacterial taxonomy, typing and evolution will be critically examined from the perspective of whole-genome structure, function and organization . The following three categories of DNA band pattern studies will be reviewed: (i) random whole-genome analysis; (ii) specific gene variation and (iii) mobile genetic elements . (i) The use of RAPD, PFGE and AFLP to analyse the whole genome will provide a skeleton of polymorphic sites with exact genomic positions as whole-genome sequence data become available . (ii) Different genes provide different levels of evolutionary information for determining isolate relatedness depending on whether they are highly variable (prone to recombination events and horizontal transfer), housekeeping genes with only a small number of single nucleotide differences between isolates or part of the rrn multigene family that is prone to intragenomic recombination and concerted evolution . Comparative analyses of these different gene classes can provide enhanced information about isolate relatedness . (iii) Mobile genetic elements such as insertion sequences, transposons, plasmids and bacteriophages integrate into the bacterial genome at specific (e.g . tRNA genes) or non-specific sites to alter band patterns produced by PFGE, RAPD or AFLP . From the literature it is not clear what level of genetic element duplication constitutes non-relatedness of isolates . A model is presented that incorporates all of the above genomic characteristics for the determination of isolate relatedness in taxonomic, typing and evolutionary studies. Virus Genes, 2001 Jan, 22(1), 35 - 45 Identification of a strong promoter of bacteriophage MB78 that interacts with a host coded factor and regulates the expression of a structural protein; Zargar MA et al.; A strong promoter of bacteriophage MB78 which controls the expression of a small structural protein of the phage has been identified and characterized . Analysis of its nucloetide sequence upstream of the translational start site revealed the presence of conserved -10 (TAATAT) and -35 (TTCTCCT) regions . It was observed that transcription initiates with thymidine residue, 86 nt upstream of the translational start site . Transcription seems to undergo alternate up and down regulation . This promoter is efficiently recognized by sigma 70 RNA polymerase and a host factor also binds to it . Binding of RNA polymerase is independent of binding of the host factor. Curr Genet, 2000 Dec, 38(5), 283 - 90 The terminal protein of a linear mitochondrial plasmid is encoded in the N-terminus of the DNA polymerase gene in white-rot fungus Pleurotus ostreatus; Kim EK et al.; The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed . Cleavage by proteinase K and exonucleases indicated that the 5' ends of pMLP1 and pMLP2 DNAs were associated with terminal proteins . Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp with terminal inverted repeat (TIR) sequences of 381 bp . The end sequence of TIR in pMLP1 is 3'-CCCCC-5', similar to those of Escherichia coli phage PRDI . The pMLP1 plasmid harbors two long open reading frames (ORFI and ORF2) and at least one minor ORF (mORF1) . The deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of ORF2 is homologous to the protein-primed DNA polymerases of family B type . The mORF1 encodes a highly basic protein, most likely a TIR-binding protein, with no apparent sequence homology in the database . Expression of the predicted gene products from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of pMLP1 ORFs . A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products . Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively . Western blot analysis indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase. Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 429 - 33 {The high expression of human beta-nerve growth factor in E . coli}; Zhang J et al.; The Hu beta-NGF gene encoding the beta subunit of mature human nerve growth factor was cloned into the pET11c vector under the control of T7 bacteriophage promoter . The SDS-PAGE electrophoresis results of nonreduced recombinant products showed a clear band corresponding to homodimeric (27 kD) form of the molecule . But the SDS-PAGE results of reduced expression protein showed a single band corresponding to monomeric form of Hu beta-NGF (13.5 kD) . It represented approximately 14.5% of the total cellular protein . Both of them were immunopositive on Western-Blots with rabbit anti-m beta-NGF polyclonic antibodies . And the recombinant product was biologically active on cultured chicken dorsal root ganglion neurons . So it demonstrated the feasibility of synthesizing the biologically active forms of Hu beta-NGF in E . coli. J Mol Biol, 2000 Dec 15, 304(5), 731 - 9 Efficient inhibition of Escherichia coli RNA polymerase by the bacteriophage T4 AsiA protein requires that AsiA binds first to free sigma70; Hinton DM et al.; The bacteriophage T4 AsiA protein inhibits transcription from host and phage early promoters and is required, along with the T4 MotA protein, for activation of phage middle promoters . During infection, AsiA is found in a tight association with the sigma70 subunit of RNA polymerase . We show that AsiA binds rapidly to free sigma70 at either 4 degrees C or 30 degrees C to form an AsiA-sigma70 complex that with core efficiently reconstitutes the AsiA-inhibited RNA polymerase . In contrast, AsiA does not inhibit transcription after a 15 minute incubation with RNA polymerase holoenzyme at 4 degrees C, and at 30 degrees C an incubation of several minutes is required to inhibit most of the polymerase . We show that the heat step needed for AsiA is not the formation of an active AsiA protein . However, it is consistent with the momentary dissociation of holoenzyme to give free sigma70 and core . Our results indicate that AsiA is either unable to access holoenzyme directly or does so very slowly . Efficient generation of the AsiA-inhibited RNA polymerase requires that AsiA first binds to free sigma70 and then the AsiA-sigma70 complex binds to core to form the Asi-A-inhibited polymerase. Structure Fold Des, 2000 Dec 15, 8(12), R227 - 35 Crawling and wiggling on DNA: structural insights to the mechanism of DNA unwinding by helicases; Marians KJ; Crystal structures have recently been solved of the monomeric DNA helicase PcrA bound to forked DNA, and of the hexameric helicase domain of the bacteriophage T7 gene 4 protein, a replication fork DNA helicase/primase . These structures have led to the elaboration of the first molecular models to describe DNA helicase action. J Bacteriol, 2001 Feb, 183(3), 921 - 7 Lowering S-adenosylmethionine levels in Escherichia coli modulates C-to-T transition mutations; Macintyre G et al.; Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro either directly by enzyme-induced deamination of cytosine to uracil in the absence of the methyl donor, S-adenosylmethionine (SAM), or indirectly through spontaneous deamination of {5-methyl}cytosine to thymine . Using a Lac reversion assay, we investigated the contribution of the first mechanism to Dcm mutagenesis in vivo by lowering the levels of SAM . Escherichia coli SAM levels were lowered by reducing SAM synthetase activity via the introduction of a metK84 allele or by hydrolyzing SAM using the bacteriophage T3 SAM hydrolase . The metK84 strains exhibited increased C-to-T mutagenesis . Expression of the T3 SAM hydrolase gene, under the control of the arabinose-inducible P(BAD) promoter, effectively reduced Dcm-mediated genomic DNA methylation . However, increased mutagenesis was not observed until extremely high arabinose concentrations were used, and genome methylation at Dcm sites was negligible. Biochem Biophys Res Commun, 2001 Feb 23, 281(2), 520 - 8 The regulatory region of the human desmocollin 3 promoter forms a DNA four-way junction; Gadhavi PL et al.; Adhesion between desmosomal junctions is mediated by structural proteins of the cadherin family, viz . three desmocollins (DSC) and three desmogleins (DSG) . Promoter and primer extension analysis of human DSC3 showed a TATA-less sequence initiating transcription via a cluster of sites upstream of the coding region . Deletion analysis of 1 kb of the promoter showed that expression is regulated between --303 and --203 bp upstream of the start-site of translation . Tertiary structure analysis of this cis-active region (cis 1) revealed a potential DNA 4-way junction which is notably G/C-rich in sequence . PAGE analysis of this region identified four differently migrating forms of the DNA . Structure-specific cleavage of the DNA with bacteriophage T7 endonuclease I showed the slowest migrating form to be either an extended/cruciform or stacked-X 4-way junction . DNA-binding, gel retardation assays of the cis 1 region showed distinct DNA-protein complexes and by competition experiments and using purified junction DNA we show that one of these complexes bound with both sequence and structure specificity to the 4-way junction DNA . Methods, 2001 Feb, 23(2), 169 - 78 Construction of high-complexity combinatorial phage display peptide libraries; Noren KA et al.; Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors . Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification . Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity . A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented . The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli . Library design strategies and a protocol for rapid sequence characterization are also presented . Gene, 2001 Jan 10, 262(1-2), 231 - 8 High-level autoenhanced expression of a single-copy gene in Escherichia coli: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements; Marchand I et al.; Bacteriophage T7 early gene 0.7 assists phage growth under suboptimal conditions ('helper' function) . Whereas the C-terminal one-third of the encoded protein participates in host transcription shutoff, the N-terminal two-thirds harbours a protein kinase ('PK') activity with broad specificity . However, how this activity relates to helper function is unclear . Here, a truncated gene 0.7 encoding PK was fused to an IPTG-inducible T7 late promoter and to a translation initiation region from a T7 late gene, and inserted into the chromosome of an E . coli strain expressing T7 RNA polymerase . After induction, total protein synthesis remains unchanged but with over 40% devoted to PK synthesis, an amazing figure for the expression of a single-copy gene . Mutations abolishing PK activity reduce this expression by 3-fold . Thus, PK activity stimulates PK expression when the latter is controlled by T7 late genetic elements . Further experiments show that stimulation occurs at both transcriptional and post-transcriptional levels . The helper function may therefore correspond to a PK-mediated stimulation of late expression, the mechanism of which is discussed . The possibility of exploiting the PK activity for improving E . coli expression systems is also considered. Infect Immun, 2001 Mar, 69(3), 1934 - 7 Human neutrophils and their products induce Shiga toxin production by enterohemorrhagic Escherichia coli; Wagner PL et al.; The Shiga toxins (Stx) are critical virulence factors for Escherichia coli O157:H7 and other serotypes of enterohemorrhagic E . coli (EHEC) . These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages . We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain . Since several factors produced by human cells, including hydrogen peroxide (H2O2), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC . Here, we studied whether H2O2 and also human neutrophils, an important endogenous source of H2O2, induced Stx2 expression by an EHEC clinical isolate . Both H2O2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis. J Mol Biol, 2001 Feb 23, 306(3), 479 - 87 Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010; Niedenzu T et al.; Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis . The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 A resolution . The hexamer shows an annular structure with 6-fold rotational symmetry and a approximately 17 A wide central hole, suggesting that single-stranded DNA may be threaded during unwinding . Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein . In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the "arginine finger" Arg207 contributing to the active site in the adjacent monomer . This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA . The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain. J Mol Biol, 2001 Feb 23, 306(3), 469 - 77 Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1; Hashimoto H et al.; The crystal structure of family B DNA polymerase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) was determined . KOD DNA polymerase exhibits the highest known extension rate, processivity and fidelity . We carried out the structural analysis of KOD DNA polymerase in order to clarify the mechanisms of those enzymatic features . Structural comparison of DNA polymerases from hyperthermophilic archaea highlighted the conformational difference in Thumb domains . The Thumb domain of KOD DNA polymerase shows an "opened" conformation . The fingers subdomain possessed many basic residues at the side of the polymerase active site . The residues are considered to be accessible to the incoming dNTP by electrostatic interaction . A beta-hairpin motif (residues 242-249) extends from the Exonuclease (Exo) domain as seen in the editing complex of the RB69 DNA polymerase from bacteriophage RB69 . Many arginine residues are located at the forked-point (the junction of the template-binding and editing clefts) of KOD DNA polymerase, suggesting that the basic environment is suitable for partitioning of the primer and template DNA duplex and for stabilizing the partially melted DNA structure in the high-temperature environments . The stabilization of the melted DNA structure at the forked-point may be correlated with the high PCR performance of KOD DNA polymerase, which is due to low error rate, high elongation rate and processivity. J Mol Biol, 2001 Feb 23, 306(3), 389 - 96 Hydrogen-deuterium exchange as a probe of folding and assembly in viral capsids; Tuma R et al.; The dynamics of proteins within large cellular assemblies are important in the molecular transformations that are required for macromolecular synthesis, transport, and metabolism . The capsid expansion (maturation) accompanying DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation . A novel method, based on hydrogen-deuterium exchange was devised to investigate the dynamics of capsid expansion . Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dynamics irrespective of the size of the assembly . Partial digestion of the exchanged protein with pepsin allows for region-specific assignment of the exchange . Procapsids and mature capsids were probed under native and slightly denaturing conditions . These experiments revealed regions that exhibit different degrees of flexibility in the procapsid and in the mature capsid . In addition, exchange and deuterium trapping during the process of expansion itself was observed and allowed for the identification of segments of the protein subunit that become buried or stabilized as a result of expansion . This approach may help to identify residues participating in macromolecular transformations and uncover novel patterns and hierarchies of interactions that determine functional movements within molecular machines. Biochemistry (Mosc), 2000 Dec, 65(12), 1346 - 51 Transformation of a fragment of beta-structural bacteriophage T4 adhesin to stable alpha-helical trimer; Miroshnikov KA et al.; Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells . Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a beta-helix, is supposed to be a major structural feature of this protein . We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively . When expressed in E . coli cells, these gp12 fragments formed labile beta-structural trimers . Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant alpha-helical structure . This structure is probably similar to that of fibritin, which has a triple alpha-helical coiled-coil structure . Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization. Mol Cell, 2001 Jan, 7(1), 31 - 41 Bacteriophage T4 proteins replicate plasmids with a preformed R loop at the T4 ori(uvsY) replication origin in vitro; Nossal NG et al.; Bacteriophage T4 DNA replication proteins catalyze complete unidirectional replication of plasmids containing the T4 ori(uvsY) replication origin in vitro, beginning with a preformed R loop at the position of the origin R loop previously identified in vivo . T4 DNA polymerase, clamp, clamp loader, and 32 protein are needed for initial elongation of the RNA, which serves as the leading-strand primer . Normal replication is dependent on T4 41 helicase and 61 primase and is strongly stimulated by the 59 helicase loading protein . 59 protein slows replication without the helicase . As expected, leading-strand synthesis stalls prematurely in the absence of T4 DNA topoisomerase . A DNA unwinding element (DUE) is essential for replication, but the ori(uvsY) DUE can be replaced by other DUE sequences. Biochemistry, 2001 Jan 23, 40(3), 665 - 74 Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid; Tuma R et al.; P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses . The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure . Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups . Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed {Tuma, R., Prevelige, P . E., Jr., and Thomas, G . J., Jr . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 9885-9890} . Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations . Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering . Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies . We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone . We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice . However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core . The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit . The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid. Cytometry, 2001 Mar 1, 43(3), 217 - 22 Novel rapid method for visualization of extent and location of aerosol contamination during high-speed sorting of potentially biohazardous samples; Oberyszyn AS et al.; BACKGROUND: Containment of potentially biohazardous aerosols that result from high-speed sorting of human cells has been an increasingly important problem in analytical cytometry . The current method for assessing the efficiency of aerosol containment involves detection of aerosols containing sorted T4 bacteriophage on lawns of T4-susceptible Escherichia coli on plates that are placed in and around the sort area . Although this method is sensitive, it is time consuming and involves maintenance and handling of bacteria and sorting of bacteriophage that may themselves serve as sources of contamination for sorted viable human cells . METHODS: Glo Germ (5-microm melamine copolymer resin beads), which are fluorescent under black light illumination, were sorted on a Beckman-Coulter Elite ESP sorter in order to visualize deposition of aerosols under normal and mock failure modes . RESULTS: Glo Germ was successfully used under both normal sorting conditions, as well as mock failure mode, to visualize aerosol formation . CONCLUSIONS: We have developed a method to examine aerosol containment using modified Glo Germ, a product used for teaching aseptic technique in hospitals, industry, restaurants, and schools . Use of this technique represents a rapid, inexpensive, qualitative analysis of the extent and location of aerosol contamination from cell sorters . Mol Microbiol, 2001 Feb, 39(3), 731 - 46 Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA replication; Meijer WJ et al.; An early expressed operon, located at the right end of the linear bacteriophage phi29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in phi29-related phages . Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection . p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization . The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized . Purified p16.7A was shown to form dimers in solution . To study the in vivo role of p16.7, a phi29 mutant containing a suppressible mutation in gene 16.7 was constructed . In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times . Based on the results, the putative role of p16.7 in in vivo phi29 DNA replication is discussed. Gene, 2000 Dec 23, 259(1-2), 223 - 33 Structural/functional assignment of unknown bacteriophage T4 proteins by iterative database searches; Kawabata T et al.; Among the total of 274 orfs within bacteriophage T4, only half have been reasonably well characterized, and the functions of the rest have remained obscure . In order to predict the molecular functions of the orfs, a position-specific iterated (PSI)-BLAST search of bacteriophage T4 against the sequence database of known 3D structures was carried out . PSI-BLAST is one of the most powerful iterative sequence search methods using multiple sequence alignment, with the ability to detect many more proteins with distant homology than standard pairwise methods . The 3D structures of proteins are considered to be better preserved than the sequences, and the detected distantly homologous proteins are likely to possess highly similar 3D structures . Thirteen orfs of phage T4, whose homologues were not detected by standard pairwise methods, were found to have significantly homologous counterparts by this method . The plausibility of the results was confirmed by checking whether important residues at substrate/ligand-binding sites were conserved . Among them, two orfs, vs.1 and e.1, which are similar to Escherichia coli lytic enzyme and MutT protein, respectively, had not been studied previously . Also, gp rIIA, a rapid lysis protein, whose gene structure had been intensively studied during the development of molecular biology in the 1950s and yet whose molecular function remains unknown, has an N-terminal domain that is significantly similar to the N-terminal region of the heat shock protein Hsp90. Virology, 2001 Jan 20, 279(2), 385 - 91 The structure of isometric capsids of bacteriophage T4; Olson NH et al.; The three-dimensional structure of DNA-filled, bacteriophage T4 isometric capsids has been determined by means of cryoelectron microscopy and image reconstruction techniques . The packing geometry of protein subunits on the capsid surface was confirmed to be that of the triangulation class T = 13 . The reconstruction clearly shows pentamers, attributed to capsid protein gp24*, surrounded by hexamers of the major capsid protein, gp23* . Positions of the accessory proteins, Hoc and Soc, are also clearly delineated in the surface lattice . The Hoc protein is the most prominent surface feature and appears as an extended molecule with a rounded base from which a thin neck and a globular head protrude . One Hoc molecule associates with each hexamer . Nearly continuous "ridges" are formed at the periphery of the gp23* hexamers by an association of 12 Soc molecules; however, Soc is absent along the boundaries between the hexamers and the pentamers . The duplex DNA genome forms a highly condensed series of concentric layers, spaced about 2.36 nm apart, that follow the general contour of the inner wall of the protein capsid. Biochem Biophys Res Commun, 2001 Jan 26, 280(3), 813 - 7 Functional expression on bacteriophage of the mustard trypsin inhibitor MTI-2; Volpicella M et al.; The mustard trypsin inhibitor MTI-2 is a potential tool in the study of interactions between pest insects and plants . It can be applied to study the adaptations of digestive proteases in pest insects . Phage display allows a rapid and exhaustive system for the selection of heterologous protein variants with novel specificities . Here we describe a bacteriophage expression system which permits functional expression of MTI-2 variants . Active and inactive mutants of MTI-2 are constructed and displayed on phage . These are used to demonstrate that an active variant can be selected from a background of 10,000 inactive mutants in four rounds of selection and amplification . Biochem Biophys Res Commun, 2001 Jan 19, 280(2), 548 - 52 An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library; Shadidi M et al.; The display of human antibody repertoire on the cell surface of the filamentous bacteriophage has offered a novel strategy for selecting antibodies to a diverse range of purified targets . However, the selection of antibodies with biological functions has not yet been fully investigated . To select phage antibodies with therapeutic potential, a synthetic human single chain Fv (scFv) phage antibody library was panned on whole premyelocytic leukemia cell line (HL60) . Phages binding to common receptors and undesirable phages were subtracted by incubating the library with human glioma cells . High affinity binding phages to HL60 cells were enriched by fluorescence-activated cell sorting . After the 6th round of selection, 50% of the selected phage antibodies showed significant binding to HL60 cells, whereas none of the analyzed phage antibodies bound to human pre-B cells (Nalm-6) . In addition to binding, one scFv antibody inhibited HL60 cell proliferation by 90% compared to irrelevant scFv antibodies . Taken together the data demonstrate that specific scFv antibodies with biological functions can be isolated by using whole cells as affinity matrix . Protein Expr Purif, 2001 Feb, 21(1), 183 - 91 Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus; Nunez E et al.; The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies . A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6x His tag at the carboxy-terminal end of the polypeptides . Codons for the 6x His were added in reverse primers used to amplify the corresponding cDNAs . The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter . Upon induction with isopropyl-beta-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni-nitrilotriacetic acid agarose column . This method yielded 20-40 mg of highly pure and very stable proteins per liter of cell culture . Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6x His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions . J Mol Biol, 2001 Feb 2, 305(5), 1131 - 44 Probing the kinetics of formation of the bacteriophage MS2 translational operator complex: identification of a protein conformer unable to bind RNA; Lago H et al.; We have investigated the kinetics of complex formation between bacteriophage MS2 coat protein subunits and synthetic RNA fragments encompassing the natural translational operator site, or the consensus sequences of three distinct RNA aptamer families, which are known to bind to the same site on the protein . Reactions were assayed using stopped-flow fluorescence spectroscopy and either the intrinsic tryptophan fluorescence of the protein or the signals from RNA fragments site-specifically substituted with the fluorescent adenosine analogue 2'-deoxy, 2-aminopurine . The kinetics observed were independent of the fluorophore being monitored or its position within the complex, indicating that the data report global events occurring during complex formation . Competition assays show that the complex being formed consists of a single coat protein dimer and one RNA molecule . The binding reaction is at least biphasic . The faster phase, constituting 80-85 % of the amplitude, is a largely diffusion driven RNA-protein interaction (k1 approximately 2x10(9) M(-1) s(-1)) . The salt dependence of the forward reaction and the similarities of the on-rates of lower-affinity RNA fragments are consistent with a diffusion-controlled step dominated by electrostatic steering . The slower phase is independent of reactant concentration, and appears to correspond to isomerisation of the coat protein subunit(s) prior to RNA binding (k(iso) approximately 0.23 s(-1)) . Measurements with a coat protein mutant (Pro78Asn) show that this phase is not due to cis-trans isomerisation at this residue . The conformational changes in the protein ligand during formation of an RNA-protein complex might play a role in the triggering of capsid self-assembly and a model for this is discussed. J Theor Biol, 2001 Jan 7, 208(1), 37 - 48 Understanding bacteriophage therapy as a density-dependent kinetic process; Payne RJ et al.; Studies of bacteriophage as therapeutic agents have had mixed and unpredictable outcomes . We argue that interpretation of these apparently paradoxical results requires appreciation of various density-dependent threshold effects . We use a mathematical model to delineate different categories of outcome, including therapy by simple inundation, by active biocontrol, and by delayed active biocontrol . Counter-intuitively, there are situations in which earlier inoculation can be less efficacious, and simultaneous inoculation with antibiotics can be detrimental . Predictions of therapeutic responses are made using formulae dependent on biologically meaningful parameters; experimental measurement of the parameters will be a prerequisite of application of the model to particular study systems . Such modelling can point to which aspects of phage biology might most fruitfully be engineered so as to enhance the viability of bacteriophage therapy . Nucleic Acids Res . 2001 Feb 15;29(4):E22. Directional cDNA library construction assisted by the in vitro recombination reaction; Ohara O et al.; We report here a new directional cDNA library construction method using an in vitro site-specific recombination reaction, based on the integrase-excisionase system of bacteriophage lambda . Preliminary experiments revealed that in vitro recombinational cloning (RC) provided important advantages over conventional ligation-assisted cloning: it eliminated restriction digestion for directional cloning, generated low levels of chimeric clones, reduced size bias and, in our hands, gave a higher cloning efficiency than conventional ligation reactions . In a cDNA cloning experiment using an in vitro synthesized long poly(A)(+) RNA (7.8 kb), the RC gave a higher full-length cDNA clone content and about 10 times more transformants than conventional ligation-assisted cloning . Furthermore, characterization of rat brain cDNA clones yielded by the RC method showed that the frequency of cDNA clones >2 kb having internal NotI sites was approximately 6%, whereas these cDNAs could not be cloned at all or could be isolated only in a truncated form by conventional methods . Taken together, these results indicate that the RC method makes it possible to prepare cDNA libraries better representing the entire population of cDNAs, without sacrificing the simplicity of current conventional ligation-assisted methods. Nucleic Acids Res . 2001 Feb 1;29(3):E11. DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry; Kwon Y et al.; Sets of RNA ladders can be synthesized by transcription of a bacteriophage-encoded RNA polymerase using 3'-deoxynucleotides as chain terminators . These ladders can be used for sequencing of DNA . Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders . Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of chain-terminated RNA ladders allowed DNA sequence determination of up to 56 nt . It is also demonstrated that A-->G and C-->T variations in heterozygous and homozygous samples can be unambiguously identified by the mass spectrometric analysis . As a step towards single-tube sequencing reactions, alpha-thiotriphosphate nucleotide analogs were used to overcome problems caused by chain terminator-independent, premature termination and by the small mass difference between natural pyrimidine nucleotides. J Bacteriol, 2001 Feb, 183(4), 1277 - 83 The initiation codon affects ribosome binding and translational efficiency in Escherichia coli of cI mRNA with or without the 5' untranslated leader; O'Donnell SM et al.; Translational efficiency of an AUG, CUG, GUG, or UUG initiation codon was measured for the naturally leaderless cI mRNA from bacteriophage lambda . In a cI-lacZ translational fusion, only AUG supported a high level of expression; GUG supported a low level of expression, while UUG and CUG expression was barely above background levels . Addition of an untranslated lac leader and Shine-Dalgarno sequence to cI increased expression but still showed a dependence on an AUG for maximum expression . cI-lacZ mRNA with an AUG initiation codon showed a greater in vitro ribosome binding strength and a higher level of full-length in vivo mRNA, suggesting that the initiation codon is an important determinant of ribosome binding strength and translational efficiency for mRNA with or without the 5' untranslated leader. Appl Environ Microbiol, 2001 Feb, 67(2), 539 - 45 Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold; Chen F et al.; A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples . Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry . The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses . Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software . Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy . Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method . Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures . Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample . Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts . The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed. J Virol, 2000 May, 74(9), 4229 - 35 The phage infection process: a functional role for the distal linker region of bacteriophage protein 3; Nilsson N et al.; The filamentous bacteriophage infects Escherichia coli by interaction with the F pilus and the TolQRA complex . The virus-encoded protein initiating this process is the gene 3 protein (g3p) . The g3p molecule can be divided into three different domains separated by two glycine-rich linker regions . Though there has been extensive evaluation of the importance of the diverse domains of g3p, no proper function has so far been assigned to these linker regions . Through the design of mutated variants of g3p that were displayed on the surface of bacteriophage, we were able to elucidate a possible role for the distal glycine-rich linker region . A phage that displayed a g3p comprised of only the N1 domain, the first linker region, and the C-terminal domain was able to infect cells at almost the same frequency as the wild-type phage . This infection was proven to be dependent on the motif between amino acid residues 68 and 86 (i.e., the first glycine-rich linker region of g3p) and on F-pilus expression. J Virol, 2000 May, 74(9), 4057 - 63 The interaction of bacteriophage P2 B protein with Escherichia coli DnaB helicase; Odegrip R et al.; Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC . Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B . However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene . Here we demonstrate that P2 B associates with DnaB . This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where (35)S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein . Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB . In this respect, P2 B was comparable to lambda P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader . Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication. PDA J Pharm Sci Technol, 1999 Mar-Apr, 53(2), 75 - 82 Application of bacteriophages as surrogates for mammalian viruses: a case for use in filter validation based on precedents and current practices in medical and environmental virology; Aranha-Creado H et al.; Infectivity-based assays are the assays of choice for the detection of pathogenic mammalian viruses . While it is intuitively appropriate to conduct testing and validation studies with the known viral burden or a closely related mammalian species, logistic considerations often dictate otherwise . Consequently, bacteriophages have served as suitable surrogates for mammalian viruses in both medical and environmental virology applications . The wide range of bacteriophages available offers a powerful analytical tool amenable to several different applications: filter validation studies (where removal is based on size exclusion), investigations into virus contamination control issues, evaluation of barrier materials, etc . There is a considerable body of evidence to suggest and support the use of bacteriophages as surrogates for mammalian viruses . Use of appropriately sized bacteriophages provides an innocuous, efficacious and expeditious method for economical testing and validation of viral clearance capabilities of virus removal filters, thus facilitating performance of filter validation studies in biopharmaceuticals under product- and process-specific conditions in an overall effort towards ensuring the virological safety of biologicals . This paper discusses the limitations associated with mammalian virus assays and provides a rationale for the use of bacteriophages as surrogates for mammalian viruses . Data from published literature documenting applicability of bacteriophages in filter validation studies, especially when removal is based on size exclusion, is reviewed along with examples of studies from the fields of medical and environmental virology. Arch Virol, 2000, 145(2), 397 - 405 Infectious cDNA clones of two grapevine viruses; Saldarelli P et al.; Full-length cDNA copies of the genomes of Grapevine virus A (GVA) and Grapevine virus B (GVB) under the control of bacteriophage T7 promoter have been synthesized, which were refractory to cloning in Escherichia coli . However, both transcribed cDNAs were infectious when mechanically inoculated to Nicotiana plants . A full-length cDNA copy of GVB was engineered in pCass2, a plasmid containing a partially duplicated copy of the Ca35S promoter, but was rather unstable in Escherichia coli . No infection of Nicotiana plants was obtained following mechanical inoculation but detached Nicotiana leaves, inoculated by particle bombardment, supported the multiplication of a GVB isolate seemingly identical to the wild-type used for cloning . Nicotiana seedling inoculated with sap expressed from these leaves became infected showing typical GVB symptoms . Transient transcription of Ca35S driven cDNA clones was also detected by RT-PCR in leaves of the grapevine hybrid LN33 following inoculation by particle bombardment . The availability of infectious cDNA clones of GVA and GVB constitutes a tool for the study of genome expression and pathogenesis, and for the ultimate establishment of the aetiological role of these viruses. J Biol Chem, 2001 Mar 30, 276(13), 10387 - 97 Epub 2000 Dec 22. Interacting fidelity defects in the replicative DNA polymerase of bacteriophage RB69; Bebenek A et al.; The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase alpha class) enzymes that determine the fidelity of phage DNA replication . A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli . We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme . RB69 gp43s lacking proofreading function (Exo(-) enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr(567) (Pol(Y567(A/S/T)) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene . The results reveal that Tyr(567) is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme . In vitro assays show that the Pol(Y567A) Exo(-) enzyme generates mispairs more frequently but extends them less efficiently than does a Pol(+) Exo(-) enzyme . Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43. J Mol Biol, 2001 Jan 19, 305(3), 559 - 66 Pre-steady-state kinetics of initiation of transcription by T7 RNA polymerase: a new kinetic model; Kuzmine I et al.; In order to begin to understand the mechanism of the initiation of transcription in the model bacteriophage T7 RNA polymerase system, the simplest possible reaction, the synthesis of a dinucleotide, has been followed by quench-flow kinetics and numerical integration of mechanism-specific rate equations has been used to test specific kinetic models . In order to fit the observed time dependence in the pre-steady-state kinetics, a model for dinucleotide synthesis is proposed in which rebinding of the dinucleotide to the enzyme-DNA complex must be included . Separate reactions using dinucleotide as a substrate confirm this mechanism and the determined rate constants . The dinucleotide rebinding observed as inhibition under these conditions forms a productive intermediate in the synthesis of longer transcripts, and must be included in future kinetic mechanisms . The rate-limiting step leading to product formation shows a substrate dependence consistent with the binding of two substrate GTP molecules, and at saturating levels of GTP, is comparable in magnitude to the product release rate . The rate of product release shows a positive correlation with the concentration of GTP, suggesting that the reaction shows base-specific substrate activation . The binding of another substrate molecule, presumably via interaction with the triphosphate binding site, likely facilitates displacement of the dinucleotide product from the complex . Biochemistry, 2001 Jan 16, 40(2), 543 - 8 Unwinding of unnatural substrates by a DNA helicase; Tackett AJ et al.; Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination . These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice . The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined . Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding . The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand . We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA) . PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates . The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts . Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates . The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates. Mol Microbiol, 2001 Jan, 39(2), 361 - 9 Increased bar minigene mRNA stability during cell growth inhibition; Valadez JG et al.; Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl-tRNA hydrolase (Pth) activity . Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome . Expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth-defective cells . Two of these wild-type bar regions, barI+ and barII+, contain minigenes with similar AUG-AUA-stop codon sequences preceded by different Shine-Dalgarno (SD) and spacer regions . The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest . Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two-codon 'ORF' (open reading frame) . To test for this possibility, we assayed the effect of point mutations within the barI minigene . The results showed that a base pair substitution within the SD and the two-codon 'ORF' sequences affected protein synthesis and cell growth inhibition . In addition, mRNA stability was altered in each mutant . Higher mRNA stability correlated with the more toxic minigenes . We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA. J Virol, 2001 Jan, 75(2), 687 - 98 Herpes simplex virus DNA cleavage and packaging proteins associate with the procapsid prior to its maturation; Sheaffer AK et al.; Packaging of DNA into preformed capsids is a fundamental early event in the assembly of herpes simplex virus type 1 (HSV-1) virions . Replicated viral DNA genomes, in the form of complex branched concatemers, and unstable spherical precursor capsids termed procapsids are thought to be the substrates for the DNA-packaging reaction . In addition, seven viral proteins are required for packaging, although their individual functions are undefined . By analogy to well-characterized bacteriophage systems, the association of these proteins with various forms of capsids, including procapsids, might be expected to clarify their roles in the packaging process . While the HSV-1 UL6, UL15, UL25, and UL28 packaging proteins are known to associate with different forms of stable capsids, their association with procapsids has not been tested . Therefore, we isolated HSV-1 procapsids from infected cells and used Western blotting to identify the packaging proteins present . Procapsids contained UL15 and UL28 proteins; the levels of both proteins are diminished in more mature DNA-containing C-capsids . In contrast, UL6 protein levels were approximately the same in procapsids, B-capsids, and C-capsids . The amount of UL25 protein was reduced in procapsids relative to that in more mature B-capsids . Moreover, C-capsids contained the highest level of UL25 protein, 15-fold higher than that in procapsids . Our results support current hypotheses on HSV DNA packaging: (i) transient association of UL15 and UL28 proteins with maturing capsids is consistent with their proposed involvement in site-specific cleavage of the viral DNA (terminase activity); (ii) the UL6 protein may be an integral component of the capsid shell; and (iii) the UL25 protein may associate with capsids after scaffold loss and DNA packaging, sealing the DNA within capsids. J Biol Chem, 2001 Mar 30, 276(13), 10306 - 13 Epub 2000 Dec 21. Scanning mutagenesis reveals roles for helix n of the bacteriophage T7 RNA polymerase thumb subdomain in transcription complex stability, pausing, and termination; Brieba LG et al.; Deletions within the thumb subdomain (residues 335-408) of T7 RNA polymerase decrease elongation complex stability and processivity, but the structure of a T7RNAP initial transcription complex containing a 3-nucleotide RNA reveals no interactions between the thumb and the RNA or DNA . Modeling of a longer RNA in this structure, using a T7DNAP-primer-template structure as a guide, suggests that the phosphate ribose backbone of the RNA contacts a stretch of mostly positively charged side chains between residues 385 and 395 of helix N of the thumb . Scanning mutagenesis of this region reveals that alanine substitutions of Arg(391), Ser(393), and Arg(394) destabilize elongation complexes and that substitutions at 393 and 394 increase termination of transcripts 5 or more bases in length . The alpha-carbons of all 3 of these residues lie on the side of helix N, which faces into the template-binding cleft of the RNA polymerase, and modeling suggests that they can contact the RNA 4-5 bases away from the 3'-end . Alanine substitutions of other residues within 385-395 do not have marked effects on transcription complex stability, but alanine substitutions of Asp(388) and Tyr(385) reduce pausing and termination at the T7 concatemer junction . Both of these side chains lie on the outer side of helix N, pointing away from the template binding cleft . The thumb subdomain of T7RNAP therefore has roles both in transcription complex stabilization and in pausing and termination at the T7 concatemer junction. Philos Trans R Soc Lond B Biol Sci, 2000 Nov 29, 355(1403), 1677 - 84 Experimental evolution recapitulates natural evolution; Wichman HA et al.; Genomes of the closely related bacteriophages phiX174 and S13 are 5386 bases long and differ at 114 nucleotides, affecting 28 amino acids . Both parental phages were adapted to laboratory culture conditions in replicate lineages and analysed for nucleotide changes that accumulated experimentally Of the 126 experimental substitutions, 90% encoded amino-acid changes, and 62% of the substitutions occurred in parallel in more than one experimental line . Furthermore, missense changes at 12 of the experimental sites were at residues differing between the parental phages; in ten cases the phiX174 experimental lineages were convergent with the S13 parent, or vice versa, at both the nucleotide and amino-acid levels . Convergence at a site was even obtained in both directions in three cases . These results point to a limited number of pathways taken during evolution in these viruses, and also raise the possibility that much of the amino-acid variation in the natural evolution of these viruses has been selected. Oncogene, 2000 Nov 23, 19(50), 5781 - 7 Reduced ligation during DNA base excision repair supported by BRCA2 mutant cells; Bogliolo M et al.; The breast cancer predisposing genes BRCA1 and BRCA2 appear to be involved in DNA repair . In particular, the sensitivity of BRCA2-deficient mouse embryonic fibroblasts to ionizing radiation and the demonstrated interaction of the BRCA2 protein with Rad51, a major factor in recombinational repair, indicate that BRCA2 is important for double strand break repair . The human BRCA2-deficient human cell line Capan-1, whilst being sensitive to ionizing radiation, is also sensitive to the alkylating agent methymethanesulfonate . The major lesions induced by this agent are methylated bases which are removed primarily by the base excision repair (BER) pathway . We have investigated the efficiency of BER in Capan-1 cells by an in vitro assay in which plasmid substrates containing a single lesion are repaired by mammalian cell extracts . In comparison to the control cell lines BxPC-3, T24 and MCF7, Capan-1 cells exhibited a reduced rate of DNA ligation during both the single-nucleotide insertion and PCNA-dependent pathways of BER . The reduced rate of DNA ligation exhibited by Capan-1 cell extracts was complemented by addition of bacteriophage T4 DNA ligase or human DNA ligase III . BRCA2-mutant Capan-1 cells may possess reduced DNA ligase activity during BER. J Gen Virol, 2001 Jan, 82(Pt 1), 9 - 15 Rapid identification of a tobacco mosaic virus epitope by using a coat protein gene-fragment-pVIII fusion library; Holzem A et al.; This study describes the identification of the epitope recognized by the tobacco mosaic virus (TMV) coat protein (CP)-specific monoclonal antibody 29 (MAb29) by displaying a CP gene-fragment library on pVIII of filamentous phage M13 . More than 80% of the clones isolated after one round of panning bound specifically to MAb29 . DNA sequencing of ten randomly chosen MAb29-specific clones and subsequent sequence comparison revealed a common seven amino acid epitope (ELIRGTG) representing amino acids 131-137 of the TMV CP . The reactivity of MAb29 in competition ELISA towards glutathione S:-transferase fused to this epitope was stronger than that towards full-length wild-type TMV CP, confirming the epitope sequence determined by gene-fragment phage display . This demonstrated that gene-fragment libraries displayed on the phage surface as fusion proteins with the filamentous bacteriophage gene VIII are useful tools for rapid identification of linear epitopes recognized by MAbs. Drug Metab Dispos, 2001 Jan, 29(1), 4 - 7 Identification of a new human CYP2A gene fragment with close linkage to CYP2GP1; Sheng J et al.; Human genomic libraries were screened to identify CYP2G-related cytochrome P450 genes . A genomic fragment comprising exons 7 through 9 of CYP2GP1 and exons 6 through 9 of a previously unidentified CYP2A gene, designated CYP2A7P2, was isolated from an EMBL3 library; the two genes were arranged in outward opposite directions with about 8 kbp of intervening sequence . The same structure was also detected in a bacteriophage P1 clone, which contained a full-length CYP2GP1 gene, exons 6 through 9 of CYP2A7P2, and the CYP2B7 gene . However, additional CYP2A-related exons as well as other CYP2A genes, CYP2A7P1, CYP2B6, CYP2F1, and CYP2GP2 were not detected . These results indicate that CYP2A7P2 is located near CYP2B7 in the middle of the CYP2A-2B-2F gene cluster on chromosome 19 . Furthermore, an analysis of CYP2A sequence alignment suggests that CYP2A7P2 may be derived from the same ancestral gene that gave rise to CYP2A7P1, which was corrupted by a large insertion at intron 5. J Mol Biol, 2000 Dec 15, 304(5), 883 - 96 From minichaperone to GroEL 2: importance of avidity of the multisite ring structure; Chatellier J et al.; Structural studies on minichaperones and GroEL imply a continuous ring of binding sites around the neck of GroEL . To investigate the importance of this ring, we constructed an artificial heptameric assembly of minichaperones to mimic their arrangement in GroEL . The heptameric Gp31 co-chaperonin from bacteriophage T4, an analogue of GroES, was used as a scaffold to display the GroEL minichaperones . A fusion protein, MC(7), was generated by replacing a part of the highly mobile loop of Gp31 (residues 23-44) with the sequence of the minichaperone (residues 191-376 of GroEL) . The purified recombinant protein assembled into a heptameric ring composed of seven 30.6 kDa subunits . Although single minichaperones (residues 193-335 to 191-376 of GroEL) have certain chaperone activities in vitro and in vivo, they cannot refold heat and dithiothreitol-denatured mitochondrial malate dehydrogenase (mtMDH), a reaction that normally requires GroEL, its co-chaperonin GroES and ATP . But, MC(7) refolded MDH in vitro . The expression of MC(7) complements in vivo two temperature-sensitive Escherichia coli alleles, groEL44 and groEL673, at 43 degrees C . Although MC(7) could not compensate for the complete absence of GroEL in vivo, it enhanced the colony-forming ability of cells containing limiting amounts of wild-type GroEL at 37 degrees C . MC(7 )also reduces aggregate formation and cell death in mammalian cell models of Huntington's disease . The assembly of seven minichaperone subunits on a heptameric ring significantly improves their activity, demonstrating the importance of avidity in GroEL function . J Mol Biol, 2000 Dec 15, 304(5), 779 - 91 The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex; Wentzell LM et al.; Quinolone drugs can inhibit bacterial DNA replication, via interaction with the type II topoisomerase DNA gyrase . Using a DNA template containing a preferred site for quinolone-induced gyrase cleavage, we have demonstrated that the passage of the bacteriophage T7 replication complex is blocked in vitro by the formation of a gyrase-drug-DNA complex . The majority of the polymerase is arrested approximately 10 bp upstream of this preferred site, although other minor sites of blocking have been observed . The ability of mutant gyrase proteins to arrest DNA replication in vitro has been investigated . Gyrase containing mutations in the A subunit at either the active-site tyrosine (Tyr122) or Ser83 (a residue known to be involved in quinolone interaction) failed to halt the progress of the polymerase . A low-level, quinolone-resistant mutation in the B subunit of gyrase showed reduced blocking compared to wild-type . We have demonstrated that DNA cleavage and replication blocking occur on similar time-scales and we conclude that formation of the cleavable complex is a prerequisite for polymerase blocking . Additionally, we have shown that collision of the replication proteins with the gyrase-drug-DNA complex is not sufficient to render this complex irreversible and that further factors must be involved in processing this stalled complex . Biochemistry, 2000 Dec 26, 39(51), 16155 - 62 Role of aromatic residues at the lipid-water interface in micelle-bound bacteriophage M13 major coat protein; Yuen CT et al.; Analyses of transmembrane domains of proteins have revealed that aromatic residues tend to cluster at or near the lipid-water interface of the membrane . To assess protein-membrane interactions of such residues, a viable mutant library was generated of the major coat protein of bacteriophage M13 (a model single membrane-spanning protein) in which one or the other of its interfacial tyrosine residues (Tyr-21 and Tyr-24) is mutated . Using the interfacial tryptophan (Trp-26) as an intrinsic probe, blue shifts in fluorescence emission spectra and quenching constants indicated that mutants with a polar amino acid substitution (such as Y24D or Y24N) are less buried in a deoxycholate micelle environment than in the wild type protein . These polar mutants also exhibited alpha-helix to beta-structure transition temperatures in incremental-heating circular dichroism studies relatively lower than those of wild type and nonpolar mutants (such as Y21V, Y21I, and Y24A), indicating that specific side chains in the lipid-water interface influence local protein-micelle interactions . Mutant Y21F exhibited the highest transition temperature, suggesting that phenylalanine is ostensibly the most effective interfacial anchoring residue . Using phage viability as the assay in a combination of site-directed and saturation mutagenesis experiments, it was further observed that both Tyr residues could not simultaneously be "knocked out" . The overall results support the notion that an interfacial Tyr is a primary recognition element for precise strand positioning in vivo, a function that apparently cannot be performed optimally by residues with simple aliphatic character. Parasite Immunol, 2000 Dec, 22(12), 639 - 50 Antibodies raised against the flagellar pocket fraction of Trypanosoma brucei preferentially recognize HSP60 in cDNA expression library; Radwanska M et al.; A purified flagellar pocket fraction of the Trypanosoma brucei AnTat 1.1E clone was used for the generation of polyclonal antiserum in rats . Anti-flagellar pocket antibodies present in this serum recognized several proteins distinct from the major variant surface glycoprotein (VSG) . In Balb/c mice, flagellar pocket immunization resulted in partial resistance towards the challenge with a low dose of parasites . This was accompanied by the induction of specific IgG2a antibodies . In an attempt to discover protective parasite antigens, antiflagellar pocket serum was used for the screening of a T . brucei bloodstream form cDNA library constructed in the lambdagt11 bacteriophage expression system . Through antibody panning and VSG elimination, 15 specific cDNA inserts were selected . Most intriguing was the observation that in addition to two clones encoding the invariant surface glycoprotein 75 (ISG75), 10 out of 15 independently selected cDNA inserts encoded the trypanosome heat shock protein 60 (tHSP60). Trends Microbiol, 2000 Nov, 8(11), 504 - 8 The origins and ongoing evolution of viruses; Hendrix RW et al.; Genome analyses of double strand DNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes and by the acquisition of novel genes as simple genetic elements termed morons . These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution. Biochim Biophys Acta, 2000 Dec 20, 1509(1-2), 311 - 23 Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13; Spruijt RB et al.; During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli . This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state . For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part . All coat protein mutants used are successfully produced in mg quantities by overexpression in E . coli . Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids . 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 . Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid . By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded . Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19 . The hinge region (residues 17-24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane . Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation . The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio . The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo . From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane. J Biol Chem, 2001 Mar 23, 276(12), 8720 - 6 Epub 2000 Dec 04. Pseudo-T-even bacteriophage RB49 encodes CocO, a cochaperonin for GroEL, which can substitute for Escherichia coli's GroES and Bacteriophage T4's Gp31; Ang D et al.; Bacteriophage T4-encoded Gp31 is a functional ortholog of the Escherichia coli GroES cochaperonin protein . Both of these proteins form transient, productive complexes with the GroEL chaperonin, required for protein folding and other related functions in the cell . However, Gp31 is specifically required, in conjunction with GroEL, for the correct folding of Gp23, the major capsid protein of T4 . To better understand the interaction between GroEL and its cochaperonin cognates, we determined whether the so-called "pseudo-T-even bacteriophages" are dependent on host GroEL function and whether they also encode their own cochaperonin . Here, we report the isolation of an allele-specific mutation of bacteriophage RB49, called epsilon22, which permits growth on the E . coli groEL44 mutant but not on the isogenic wild type host . RB49 epsilon22 was used in marker rescue experiments to identify the corresponding wild type gene, which we have named cocO (cochaperonin cognate) . CocO has extremely limited identity to GroES but is 34% identical and 55% similar at the protein sequence level to T4 Gp31, sharing all of the structural features of Gp31 that distinguish it from GroES . CocO can substitute for Gp31 in T4 growth and also suppresses the temperature-sensitive phenotype of the E . coli groES42 mutant . CocO's predicted mobile loop is one residue longer than that of Gp31, with the epsilon22 mutation resulting in a Q36R substitution in this extra residue . Both the CocO wild type and epsilon22 proteins have been purified and shown in vitro to assist GroEL in the refolding of denatured citrate synthase. Biochemistry, 2000 Dec 19, 39(50), 15365 - 74 Structure of the bacteriophage lambda Ser/Thr protein phosphatase with sulfate ion bound in two coordination modes; Voegtli WC et al.; The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin) . These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center . The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled . To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution . The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif . Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center . The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes . In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule . Two additional solvent molecules are coordinated to Mn1 . In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2 . The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands . In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162 . The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues. Genomics, 2000 Dec 1, 70(2), 165 - 70 The assembly of large BACs by in vivo recombination; Mejia JE et al.; We have developed a method for recombining bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) containing large genomic DNA fragments into a single vector using the Cre-lox recombination system from bacteriophage P1 in vivo . This overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of DNA into Escherichia coli cells . We used the method to construct a human artificial chromosome vector of 404 kb encompassing long tracts of alpha satellite DNA, telomeric sequences, and the human hypoxanthine phosphoribosyltransferase gene . The specificity of Cre recombinase for loxP sites minimizes the possibility of intramolecular rearrangements, unlike previous techniques using general homologous recombination in E . coli, and makes our method compatible with the presence of large arrays of repeated sequences in cloned DNA . This methodology may also be applied to retrofitting PACs or BACs with markers and functional sequences. Gene, 2000 Nov 27, 258(1-2), 127 - 39 Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak; Yokoyama K et al.; Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains . Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected . We have determined the complete nucleotide sequence of the prophage VT1-Sakai . The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome . The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously . The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21 . The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported . These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai . Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages. Int Microbiol, 1999 Dec, 2(4), 227 - 32 Enumeration and isolation of viral particles from oligotrophic marine environments by tangential flow filtration; Alonso MC et al.; A method for concentrating, enumerating and isolating viral particles from marine water samples was developed and evaluated . The method consists of a concentration step by a tangential flow filtration (TFF) system, ultrafiltration by centrifugal concentrator, and visualization by transmission electron microscopy (TEM) . This procedure allows to reduce volumes of ca . 21 of seawater to 10-20 microliters, which can be dispensed on electron microscopy grids to count total viral particles . This method allows the recovery of small numbers of viral particles from oligotrophic seawater samples, in which viral numbers ranged from 10(5) to 10(6) viral particles/ml . The tangential flow filtration system was evaluated as quantitative technique using suspensions of two different bacteriophages (T6 and phi X174) in autoclaved seawater . Recovery rates varied depending on both the viral morphology and flow rate; recovery percentages reached 117.4% for T6 and 60.6% for phi X174 using low flow rate. Int Microbiol, 1998 Sep, 1(3), 191 - 6 Human enteric viruses in the water environment: a minireview; Bosch A; Water virology started around half a century ago, with scientists attempting to detect poliovirus in water samples . Since that time, other enteric viruses responsible for gastroenteritis and hepatitis, among a great variety of virus strains, have replaced enteroviruses as the main target for detection in the water environment . Technical molecular developments, polymerase-chain reaction (PCR) amplification being the method of choice, enable the detection of fastidious health-significant viruses . However, shortcomings of molecular procedures include their potential incompatibility with concentration methods, indispensable to reduce the water sample volume to assay for viruses, the inability to discern between infectious and non infectious material . On the other hand, these procedures are restrained to sophisticated laboratories and detection of alternative indicator organisms has been proposed . Bacterial indicators fail to give a reliable clue of the virological quality of water . Selected bacteriophage groups appear as a better choice for their use as virus indicators. Mutat Res, 2001 Jan 5, 461(4), 289 - 99 tert-Butoxyl radicals generate mainly 7,8-dihydro-8-oxoguanine in DNA; Mahler HC et al.; Like hydroxyl radicals, alkoxyl radicals have been implicated in the generation of cellular oxidative DNA damage under physiological conditions; however, their genotoxic potential has not yet been established . We have analyzed the DNA damage induced by a photochemical source of tert-butoxyl radicals, the water soluble peroxy ester {4-(tert-butyldioxycarbonyl)benzyl}triethylammonium chloride (BCBT), using various repair endonucleases as probes . The irradiation (UV(360)) of BCBT in the presence of bacteriophage PM2 DNA was found to generate a DNA damage profile that consisted mostly of base modifications sensitive to the repair endonuclease Fpg protein . Approximately 90% of the modifications were identified as 7,8-dihydro-8-oxoguanine (8-oxoGua) residues by HPLC/ECD analysis . Oxidative pyrimidine modifications (sensitive to endonuclease III), sites of base loss (AP sites) and single-strand breaks were only minor modifications . Experiments with various scavengers and quenchers indicated that the DNA damage by BCBT+UV(360) was caused by tert-butoxyl radicals as the ultimate reactive species . The mutagenicity associated with the induced damage was analyzed in the gpt gene of plasmid pSV2gpt, which was exposed to BCBT+UV(360) and subsequently transfected into Escherichia coli . The results were in agreement with the specific generation of 8-oxoGua . Nearly all point mutations (20 out of 21) were found to be GC-->TA transversions known to be characteristic for 8-oxoGua . In conclusion, alkoxyl radicals generated from BCBT+UV(360) induce 8-oxoGua in DNA with a higher selectivity than any other reactive oxygen species analyzed so far. Int J Med Microbiol, 2000 Oct, 290(6), 519 - 27 Pathogenicity islands and phage conversion: evolutionary aspects of bacterial pathogenesis; Dobrindt U et al.; Horizontal gene transfer plays a key role in the generation of novel bacterial pathogens . Besides plasmids and bacteriophages, large genomic regions termed pathogenicity islands (PAIs) can be transferred horizontally . All three mechanisms for DNA exchange or transfer may be important for the evolution of bacterial pathogens. J Mol Biol, 2000 Dec 8, 304(4), 621 - 32 The TolA-recognition site of colicin N . ITC, SPR and stopped-flow fluorescence define a crucial 27-residue segment; Gokce I et al.; Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors . After first binding to the outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal region . Colicin N residues critical to TolA binding have been discovered, but the full extent of any colicin TolA site is unknown . We present, for the first time, a fully mapped TolA binding site for a colicin . It was determined through the use of alanine-scanning mutants, glutathione S-transferase fusion peptides and Biacore/fluorescence binding studies . The minimal TolA binding region is 27 residues and of similar size to the TolA binding region of bacteriophage g3p-D1 protein . Stopped-flow kinetic studies show that the binding to TolA follows slow association kinetics . The role of other E . coli Tol proteins in colicin translocation was also investigated . Isothermal titration microcalorimetry (ITC) and in vivo studies conclusively show that colicin N translocation does not require the presence of TolB . ITC also demonstrated colicin A interaction with TolB, and that colicin A in its native state does not interact with TolAII-III . Colicin N does not bind TolR-II . The TolA protein is shown to be unsuitable for direct immobilisation in Biacore analysis . Mol Biotechnol, 2000 Sep, 16(1), 77 - 86 New insights into protein-DNA interactions obtained by electron microscopy; Schnos M et al.; The electron microscopic study of DNA-protein complexes can yield valuable information that is often not easily available by other methods . In this article we give a number of examples that were chosen to illustrate the utility of electron microscopy . Along with the strategy used are protocols that allow such experiments to be carried out . The first example employs the following strategy . Points of close proximity between nucleic acid and protein within a bacteriophage or virus are made permanent by crosslinking . Bacteriophage or virus are then partially disrupted so that individual components can be visualized . With bacteriophages, such experiments show which DNA end first enters the host on infection and therefore can in principle indicate which phage genes would be first available for transcription . This type of experiment can also show which DNA end is first to be encapsulated during formation of the bacteriophage . Information on direction of encapsulation and indirectly, direction of replication of the rolling circles that lead to concatermeric DNA to be encapsulated, can also be derived . Such experiments can additionally accurately define the degree of DNA permutation, if present, within a bacteriophage population . Finally, examples are shown for in vitro reactions involving DNA, RecA, RecO, RecF, RecR, and SSB that lead to a further understanding of recombinational repair . Additionally antibody-gold labeling is used to locate various proteins in such complexes. Annu Rev Genet, 2000, 34, 439 - 456 Genetic analysis of bacteriophage-encoded cochaperonins; Ang D et al.; Early genetic studies identified the Escherichia coli groES and groEL genes because mutations in them blocked the growth of bacteriophages lambda and T4 . Subsequent genetic and biochemical analyses have shown that GroES and GroEL constitute a chaperonin machine, absolutely essential for E . coli growth, because it is needed for the correct folding of many of its proteins . In spite of very little sequence identity to GroES, the bacteriophage T4-encoded Gp31 protein and the bacteriophage RB49-encoded CocO protein are bona fide GroEL cochaperonins, even capable of substituting for GroES in E . coli growth . A major functional distinction is that only Gp31 and CocO can assist GroEL in the correct folding of Gp23, the major bacteriophage capsid protein . Conserved structural features between CocO and Gp31, which are absent from GroES, highlight their potential importance in specific cochaperonin function. Mol Cell, 2000 Oct, 6(4), 803 - 14 Analysis of the Okazaki fragment distributions along single long DNAs replicated by the bacteriophage T4 proteins; Chastain PD 2nd et al.; Rolling circle replication from M13 DNA circles was previously reconstituted in vitro using purified factors encoded by bacteriophage T4 . The products are duplex circles with linear tails >100 kb . When T4 DNA polymerase deficient in 3' to 5' exonuclease activity was employed, electron microscopy revealed short single-stranded DNA "flaps" along the replicated tails . This marked the beginning of each Okazaki fragment, allowing an analysis of the lengths of sequential Okazaki fragments on individual replicating molecules . DNAs containing runs of Okazaki fragments of similar length were found, but most showed large length variations over runs of six or more fragments reflecting the broad population distribution. J Virol, 2000 Dec, 74(24), 11681 - 9 Identification of conserved residues contributing to the activities of adenovirus DNA polymerase; Liu H et al.; Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase alpha family and uses a protein primer for initiation of DNA synthesis . It contains motifs characteristic of a proofreading 3'-5'-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region . To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system . Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication . The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein . Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J . Wang et al., Cell 89:1087-1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics, 2000 Oct, 62(4 Pt B), 5509 - 17 Viscoelastic properties of semiflexible filamentous bacteriophage fd; Schmidt FG et al.; The cytoskeletal protein filament F-actin has been treated in a number of recent studies as a model physical system for semiflexible filaments . In this work, we studied the viscoelastic properties of entangled solutions of the filamentous bacteriophage fd as an alternative to F-actin with similar physical parameters . We present both microrheometric and macrorheometric measurements of the viscoelastic storage and loss moduli, G'(f ) and G"(f ), respectively, in a frequency range 0.01<f<4 Hz, for fd solutions in the concentration range 5