Bacteriophage

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 φX174, 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<c<15 mg/ml . The onset of a narrow and slanted plateaulike region of G'(f ) is located at around 2 Hz . The variation of the plateau modulus with concentration obeys a power law G(')(N) approximately c(1.4+/-0.3), similar to that found for entangled solutions of F-actin . In the low-frequency regime, the frequency dependence of the viscoelastic moduli can be described by power laws G'(f ) approximately f(0.9-1.2) and G"(f ) approximately f(0.7-0.9), which deviate significantly from the simple theoretical predictions of G'(f ) approximately f(2) and G"(f ) approximately f(1) . The latter behavior cannot yet be understood within the framework of current theories of semiflexible filament networks . For the dynamic viscosity at the low shear rate limit, a concentration dependence of eta(0) approximately c(2.6) was found . Finally, a linear scaling of the terminal relaxation time with concentration, tau(d) approximately c, was observed.

Mol Biochem Parasitol, 2000 Nov, 111(1), 67 - 76
Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters; LaCount DJ et al.; The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite {Ngo H, Tschudi C, Gull K, Ullu E . Double-stranded RNA induces mRNA degradation in Trypanosoma brucei . Proc Natl Acad Sci USA 1998;95:14687-14692} . The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded . To simplify the generation of knockout phenotypes in T . brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid . This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA . We have used plasmids encoding either two T . brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T . brucei . We show here that FLA1 is required for flagellar attachment in T . brucei and that H2B is required for parasite growth . Thus, the two-promoter approach efficiently generates dsRNAi in T . brucei and can be used to produce both specific and random knockout phenotypes in T . brucei . This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13702 - 7
Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids; Schmidt EE et al.; The bacteriophage P1 Cre/loxP system has become a powerful tool for in vivo manipulation of the genomes of transgenic mice . Although in vitro studies have shown that Cre can catalyze recombination between cryptic "pseudo-loxP" sites in mammalian genomes, to date there have been no reports of loxP-site infidelity in transgenic animals . We produced lines of transgenic mice that use the mouse Protamine 1 (Prm1) gene promoter to express Cre recombinase in postmeiotic spermatids . All male founders and all Cre-bearing male descendents of female founders were sterile; females were unaffected . Sperm counts, sperm motility, and sperm morphology were normal, as was the mating behavior of the transgenic males and the production of two-celled embryos after mating . Mice that expressed similar levels of a derivative transgene that carries an inactive Cre exhibited normal male fertility . Analyses of embryos from matings between sterile Cre-expressing males and wild-type females indicated that Cre-catalyzed chromosome rearrangements in the spermatids that lead to abortive pregnancies with 100% penetrance . Similar Cre-mediated, but loxP-independent, genomic alterations may also occur in somatic tissues that express Cre, but, because of the greater difficulty of assessing deleterious effects of somatic mutations, these may go undetected . This study indicates that, following the use of the Cre/loxP site-specific recombination systems in vivo, it is prudent to eliminate or inactivate the Cre recombinase gene as rapidly as possible.

Parasitology, 2000 Aug, 121 ( Pt 2), 185 - 91
The major tegumental antigen of Fasciola hepatica contains repeated elements; Trudgett A et al.; In order to provide a better understanding of the interaction between the liver fluke (Fasciola hepatica) and the immune system of its mammalian host immunoreactive lambda bacteriophage clones containing F . hepatica cDNA have been isolated . Plasmids from these clones were sequenced and found to encode a family of proteins containing certain common elementst . All the clones contained a coding repeating sequence (RRRXCA) which is conserved at the nucleic acid level followed by a non-repeating element coding for the C terminal used by the proteins which shows conservation of amino acids at certain positions . Antisera raised against a beta-galactosidase fusion protein with one of these sequences as a terminal extension was used to localize the immunoreactive antigens . Binding was predominantly in the tegument of the juvenile fluke but was reduced in the adult tegument . The wall of the uterus showed strong reactivity in the adult . Rats immunized with the beta-galactosidase fusion protein showed enhanced resistance to challenge infections . The role of these antigens in the host response to infection by F . hepatica is discussed.

J Mol Evol, 2000 Nov, 51(5), 491 - 7
Distribution and evolution of bacteriophage WO in Wolbachia, the endosymbiont causing sexual alterations in arthropods; Masui S et al.; Wolbachia are obligatory intracellular and maternally inherited bacteria, known to infect many species of arthropod . In this study, we discovered a bacteriophage-like genetic element in Wolbachia, which was tentatively named bacteriophage WO . The phylogenetic tree based on phage WO genes of several Wolbachia strains was not congruent with that based on chromosomal genes of the same strains, suggesting that phage WO was active and horizontally transmitted among various Wolbachia strains . All the strains of Wolbachia used in this study were infected with phage WO . Although the phage genome contained genes of diverse origins, the average G+C content and codon usage of these genes were quite similar to those of a chromosomal gene of Wolbachia . These results raised the possibility that phage WO has been associated with Wolbachia for a very long time, conferring some benefit to its hosts . The evolution and possible roles of phage WO in various reproductive alterations of insects caused by Wolbachia are discussed.

EMBO J, 2000 Nov 15, 19(22), 6275 - 84
The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism; Makeyev EV et al.; Bacteriophage φ6 has a three-segmented double-stranded (ds) RNA genome, which resides inside a polymerase complex particle throughout the entire life cycle of the virus . The polymerase subunit P2, a minor constituent of the polymerase complex, has previously been reported to replicate both φ6-specific and heterologous single-stranded (ss) RNAs, giving rise to dsRNA products . In this study, we show that the enzyme is also able to use dsRNA templates to perform semi-conservative RNA transcription in vitro without the assistance of other proteins . The polymerase synthesizes predominantly plus-sense copies of φ6 dsRNA, medium and small segments being more efficient templates than the large one . This distribution of the test-tube reaction products faithfully mimics viral transcription in vivo . Experiments with chimeric ssRNAs and dsRNAs show that short terminal nucleotide sequences can account for the difference in efficiency of RNA synthesis . Taken together, these results suggest a model explaining important aspects of viral RNA metabolism regulation in terms of enzymatic properties of the polymerase subunit.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 443 - 5
Development of shuttle vectors for spirochetes; Girons IS et al.; Constructions of Escherichia coli-spirochete shuttle vectors are based on naturally occurring plasmids, broad host range plasmids or bacteriophages . This review primarily focuses on genetic tools for Treponema denticola which is associated with periodontal diseases . The T . pallidum FlaA protein, E . coli beta-galactosidase, and the green fluorescent protein were successfully expressed in T . denticola from a shuttle vector system.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 365 - 73
Bacteriophages of spirochetes; Eggers CH et al.; Historically, a number of bacteriophage-like particles have been observed in association with members of the bacterial order Spirochetales, the spirochetes . In the last decade, several spirochete bacteriophages have been isolated and characterized at the molecular level . We have recently characterized a bacteriophage of the Lyme disease agent, Borrelia burgdorferi, which we have designated phiBB-1 . Here we review the history of the association between the spirochetes and their bacteriophages, with a particular emphasis on phiBB-1 and its prophage, the 32-kb circular plasmid family of B . burgdorferi.

Trends Genet, 2000 Nov, 16(11), 512 - 7
Virus mutators and antimutators: roles in evolution, pathogenesis and emergence; Mansky LM et al.; Virus mutators (mutant alleles that confer a higher mutant-frequency phenotype than that of the wild type) and antimutators (mutant alleles that confer a lower mutant-frequency phenotype) were discovered in bacteriophage T4 over three decades ago, but there is only limited detailed knowledge about such genetic variants in viruses that infect humans and threaten public health . The creation of mutators and antimutators during the course of viral infection (particularly in the case of RNA viruses) could play a pivotal role in virus evolution, pathogenesis and emergence, and could also frustrate antiviral therapy.

Mol Cell Biol, 2000 Dec, 20(23), 8866 - 78
Effects of histone tail domains on the rate of transcriptional elongation through a nucleosome; Protacio RU et al.; The N-terminal tail domains of the core histones play important roles in gene regulation, but the exact mechanisms through which they act are not known . Recent studies suggest that the tail domains may influence the ability of RNA polymerase to elongate through the nucleosomal DNA and, thus, that posttranslational modification of the tail domains may provide a control point for gene regulation through effects on the elongation rate . We take advantage of an experimental system that uses bacteriophage T7 RNA polymerase as a probe for aspects of nucleosome transcription that are dominated by the properties of nucleosomes themselves . With this system, experiments can analyze the synchronous, real-time, single-passage transcription on the nucleosomal template . Here, we use this system to directly test the hypothesis that the tail domains may influence the "elongatability" of nucleosomal DNA and to identify which of the tail domains may contribute to this . The results show that the tail domains strongly influence the rate of elongation and suggest that the effect is dominated by the N-terminal domains of the (H3-H4)(2) tetramer . They further imply that tail-mediated octamer transfer is not essential for elongation through the nucleosome . Acetylation of the tail domains leads to effects on elongation that are similar to those arising from complete removal of the tail domains.

J Bacteriol, 2000 Dec, 182(23), 6791 - 7
Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system; Damman CJ et al.; The conserved cp32 plasmid family of Borrelia burgdorferi was recently shown to be packaged into a bacteriophage particle (C . H . Eggers and D . S . Samuels, J . Bacteriol . 181:7308-7313, 1999) . This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system . Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage . An Escherichia coli mutant containing the blyAB locus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction of sheA by blyAB expression was responsible for the hemolytic activity observed previously . Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins . Consistent with holin characteristics, subcellular localization studies with E . coli and B . burgdorferi indicated that BlyA is solely membrane associated and that BlyB is a soluble protein . Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene . Finally, induction of the cp32 prophage in B . burgdorferi dramatically stimulated blyAB expression . Our results provide the first evidence of a prophage-encoded holin within Borrelia.

J Bacteriol, 2000 Dec, 182(23), 6714 - 23
The multifunctional bacteriophage P2 cox protein requires oligomerization for biological activity; Eriksson JM et al.; The Cox protein of bacteriophage P2 is a multifunctional protein of 91 amino acids . It is directly involved in the site-specific recombination event leading to excision of P2 DNA out of the host chromosome . In this context, it functions as an architectural protein in the formation of the excisome . Cox is also a transcriptional repressor of the P2 Pc promoter, thereby ensuring lytic growth . Finally it promotes derepression of prophage P4, a nonrelated defective satellite phage, by activating the P4 P(LL) promoter that controls P4 DNA replication . In this case it binds upstream of the P(LL) promoter, which normally is activated by the P4 Delta protein . In this work we have analyzed the native form of the Cox protein in vivo, using a bacteriophage lambda cI-based oligomerization assay system, and in vitro, using gel filtration, cross-linking agents, and gel retardation assays . We found that P2 Cox has a strong oligomerization function in vivo as well as in vitro . The in vitro analysis indicates that its native form is a tetramer that can self-associate to octamers . Furthermore we show that oligomerization is necessary for the biological activity by characterizing different cox mutants and that oligomerization is mediated by the C-terminal region.

Genetics, 2000 Nov, 156(3), 1437 - 48
Models of experimental evolution: the role of genetic chance and selective necessity; Wahl LM et al.; We present a theoretical framework within which to analyze the results of experimental evolution . Rapidly evolving organisms such as viruses, bacteria, and protozoa can be induced to adapt to laboratory conditions on very short human time scales . Artificial adaptive radiation is characterized by a list of common observations; we offer a framework in which many of these repeated questions and patterns can be characterized analytically . We allow for stochasticity by including rare mutations and bottleneck effects, demonstrating how these increase variability in the evolutionary trajectory . When the product Np, the population size times the per locus error rate, is small, the rate of evolution is limited by the chance occurrence of beneficial mutations; when Np is large and selective pressure is strong, the rate-limiting step is the waiting time while existing beneficial mutations sweep through the population . We derive the rate of divergence (substitution rate) and rate of fitness increase for the case when Np is large and illustrate our approach with an application to an experimental data set . A minimal assumption of independent additive fitness contributions provides a good fit to the experimental evolution of the bacteriophage phiX174.

Virology, 2000 Nov 10, 277(1), 204 - 14
Endonuclease and helicase activities of bacteriophage lambda terminase: changing nearby residue 515 restores activity to the gpA K497D mutant enzyme; Hwang Y et al.; Terminase, the DNA packaging enzyme of bacteriophage lambda, is a heteromultimer of gpNu1 and gpA subunits . In an earlier investigation, a lethal mutation changing gpA residue 497 from lysine to aspartic acid (K497D) was found to cause a mild change in the high-affinity ATPase that resides in gpA and a severe defect in the endonuclease activity of terminase . The K497D terminase efficiently sponsored packaging of mature lambda DNA into proheads . In the present work, K497D terminase was found to have a severe defect in the cohesive end separation, or helicase, activity . Plaque-forming pseudorevertants of lambda A K497D were found to carry mutations in A that suppressed the lethality of the A K497D mutation . The two suppressor mutations identified, A E515G and A E515K, affected residue 515, which is located near the putative P-loop of gpA . A codon substitution study of codon 515 showed that hydrophobic and basic residues suppress the K497D defect, but hydrophilic and acidic residues do not . The E515G change was demonstrated to reverse the endonuclease and helicase defects caused by the K497D change . Moreover, the gpA K497D E515G enzyme was found to have kinetic constants for the high-affinity ATPase center similar to those of the wild type enzyme, and the endonuclease activity of the K497D E515G enzyme was stimulated by ATP to an extent similar to the ATP stimulation of the endonuclease activity of the wild type enzyme .

EMBO J, 2000 Nov 1, 19(21), 5625 - 34
The solution structure of the C-terminal domain of the Mu B transposition protein; Hung LH et al.; Mu B is one of four proteins required for the strand transfer step of bacteriophage Mu DNA transposition and the only one where no high resolution structural data is available . Structural work on Mu B has been hampered primarily by solubility problems and its tendency to aggregate . We have overcome this problem by determination of the three-dimensional structure of the C-terminal domain of Mu B (B(223-312)) in 1.5 M NaCl using NMR spectroscopic methods . The structure of Mu B(223-312) comprises four helices (backbone r.m.s.d . 0.46 A) arranged in a loosely packed bundle and resembles that of the N-terminal region of the replication helicase, DnaB . This structural motif is likely to be involved in the inter-domainal regulation of ATPase activity for both Mu A and DnaB . The approach described here for structural determination in high salt may be generally applicable for proteins that do not crystallize and that are plagued by solubility problems at low ionic strength.

Nucleic Acids Res . 2000 Nov 1;28(21):E93.
Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic SELEX; Shtatland T et al.; Genomic SELEX is a method for studying the network of nucleic acid-protein interactions within any organism . Here we report the discovery of several interesting and potentially biologically important interactions using genomic SELEX . We have found that bacteriophage MS2 coat protein binds several Escherichia coli mRNA fragments more tightly than it binds the natural, well-studied, phage mRNA site . MS2 coat protein binds mRNA fragments from rffG (involved in formation of lipopolysaccharide in the bacterial outer membrane), ebgR (lactose utilization repressor), as well as from several other genes . Genomic SELEX may yield experimentally induced artifacts, such as molecules in which the fixed sequences participate in binding . We describe several methods (annealing of oligonucleotides complementary to fixed sequences or switching fixed sequences) to eliminate some, or almost all, of these artifacts . Such methods may be useful tools for both randomized sequence SELEX and genomic SELEX.

Nucleic Acids Res, 2000 Nov 1, 28(21), 4207 - 11
Pre-steady state kinetics of bacteriophage T4 dam DNA-{N(6)-adenine} methyltransferase: interaction with native (GATC) or modified sites; Malygin EG et al.; The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S:-adenosyl-L-methionine (AdoMet) to the N(6)-position of adenine {generating N(6)-methyladenine and S:-adenosyl-L-homocysteine (AdoHcy)} . Pre-steady state kinetic analysis revealed that the methylation rate constant k(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s(-1), respectively) was at least 20-fold larger than the overall reaction rate constant k(cat) (0.023 s(-1)) . This indicates that the release of products is the rate-limiting step in the reaction . Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit k(meth) . Preformed T4 Dam MTase-DNA complexes are less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis . Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-{C5-cytosine}-MTases.

Epidemiol Infect, 2000 Aug, 125(1), 221 - 4
A comparison of Escherichia coli O157 isolates from cattle in Japan and the USA by molecular biological methods; Akiba M et al.; Escherichia coli O157 isolates from cattle in Japan (n = 91) and in the USA (n = 415) were compared by pulsed-field gel electrophoresis of endonuclease-cleaved genomic DNA, location of the stx genes and bacteriophage typing . Three isolates from cattle in Japan with high similarity to isolates from cattle in the USA were found . Isolates from cattle farms in Japan and the USA may share a common source.

Bioessays, 2000 Nov, 22(11), 997 - 1006
The puzzle of PCNA's many partners; Warbrick E; The identification of proteins that interact with proliferating cell nuclear antigen (PCNA) has recently been a rapidly expanding field of discovery . PCNA is involved in many aspects of DNA replication and processing, forming a sliding platform that can mediate the interaction of proteins with DNA . It is striking that many proteins bind to PCNA through a small region containing a conserved motif; these include proteins involved in cell cycle regulation as well as those involved in DNA processing . Sequential and regulated binding of motif-containing proteins to PCNA may contribute to the ordering of events during DNA replication and repair . Results from bacteriophages and archaea show that the structural basis for the interaction of this motif with PCNA is extremely ancient . The analysis of how such functional motifs have been recruited to proteins in present day organisms helps us to understand how these complex systems arose from ancestral organisms.

Metab Eng, 2000 Jul, 2(3), 210 - 7
Patterns of regulation from mRNA and protein time series; You L et al.; The rapid advance of genome sequencing projects challenges biologists to assign physiological roles to thousands of unknown gene products . We suggest here that regulatory functions and protein-protein interactions involving specific products may be inferred from the trajectories over time of their mRNA and free protein levels within the cell . The level of a protein in the cytoplasm is governed not only by the level of its mRNA and the rate of translation, but also by the protein's folding efficiency, its biochemical modification, its complexation with other components, its degradation, and its transport from the cytoplasmic space . All these co- and post translational events cause the concentration of the protein to deviate from the level that would result if we only accounted for translation of its mRNA . The dynamics of such deviations can create patterns that reflect regulatory functions . Moreover, correlations among deviations highlight protein pairs involved in potential protein-protein interactions . We explore and illustrate these ideas here using a genetically structured simulation for the intracellular growth of bacteriophage T7 .

Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1993 - 7
Recognition of receptor lipopolysaccharides by spike G protein of bacteriophage phiX174; Kawaura T et al.; The spike G protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged G protein (HisG) . In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the phiX174-sensitive strains, and did not bind to LPSs of the phiX174-insensitive strains . The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.

Gene, 2000 Oct 3, 256(1-2), 229 - 36
Affinity selection of cDNA libraries by lambda phage surface display; Niwa M et al.; Bacteriophage lambda surface display was used to isolate cDNA clones encoding autoantigens recognized by sera from patients with Sjogren's syndrome (SS) . We made cDNA libraries from human HeLa and HepG2 cells, using the expression vector lambdafoo . By repeating affinity selection of the libraries with the sera immobilized in microtiter wells, we isolated three clones that encode previously unknown antigens as well as four clones previously known as SS autoantigens . The newly identified autoantigens include TRK-fused gene product (TFG), survival motor neuron gene product (SMN) and pM5, which has a similarity to the metal-binding domain of human fibroblast collagenase . Thus, the bacteriophage lambda surface display is powerful for isolating cDNA clones by affinity screening.

Tumour Biol, 2001 Jan-Feb, 22(1), 36 - 44
Selection of human single chain Fv antibody fragments binding and inhibiting Helicobacter pylori urease; Houimel M et al.; Single chain Fv antibody fragments (scFv) binding to purified Helicobacter pylori urease were selected from a nonimmune human antibody repertoire displayed on filamentous phage . After three rounds of screening on solid phase urease, 44 clones were found to bind the enzyme and four distinct scFv were identified by sequencing their heavy and light chain variable region genes (V(H) and V(L)) . Two of the selected human scFv (scFv B4 and scFv D9) inhibited the activity of H . pylori urease with inhibitory constants (K(i)) of 7 and 2 microM, respectively . Their affinity (K(d)) for H . pylori urease as determined by surface plasmon resonance ranged from 17 to 42 nM . Both scFv were able to bind to urease present on the surface of living H . pylori organisms as demonstrated by flow cytometry analysis . The binding sites of scFv B4 and D9 were mapped by the use of two random hexapeptide libraries (X6 and CX6C) displayed on filamentous bacteriophage . The selected peptide sequences were shown to inhibit scFv binding to H . pylori urease and thus could be used in a vaccination strategy as epitopes mimicking (mimotopes) the region of urease recognized by these human scFv antibody fragments .

Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1473 - 5
Crystallization and preliminary X-ray crystallographic studies on the bacteriophage phi6 RNA-dependent RNA polymerase; Butcher SJ et al.; The RNA-dependent RNA polymerase (P2) from bacteriophage Phi6 has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme . A fully substituted selenomethionyl version of the protein has also been produced . Crystals of both proteins have been grown; most belong to the monoclinic space group P2(1), with unit-cell parameters a = 105.9, b = 94.0, c = 140.9 A, beta = 101.4 degrees, but some are trigonal (space group P3(1) or P3(2)), with unit-cell parameters a = b = 110.1, c = 159.4 A, gamma = 120 degrees . Both crystal forms occur in the same crystallization drop and are morphologically indistinguishable . Native data sets have been collected from both types of crystals to better than 3 A resolution.

Vestn Otorinolaringol, 2000, (5), 64 - 5
{New method of treatment of oropharyngeal leptotrichosis}; Chelidze ND et al.; Thirty six patients with oropharyngeal leptotrichosis were examined and treated . Antistaphylococcal bacteriophage solution was used for their treatment . Tonsillar lacunae were washed with bacteriophage 2-3 times a week, the course of treatment included 6-8 washings . Thirty four patients showed clinical recovery shown as full clearance of the affected sites and elimination of subjective manifestations of leptotrichosis.

J Biol Chem, 2001 Jan 26, 276(4), 2509 - 16 Epub 2000 Oct 25.
Domain effects on the DNA-interactive properties of bacteriophage T4 gene 32 protein; Waidner LA et al.; Bacteriophage T4 gene 32 protein, a model for single-strand specific nucleic acid-binding proteins, consists of three structurally and functionally distinct domains . We have studied the effects of the N and C domains on the protein structure and its nucleic acid-interactive properties . Although the presence of the C domain decreases the proteolytic susceptibility of the core (central) domain, quenching of the core tryptophan fluorescence by iodide is unaltered by the presence of the terminal domains . These results suggest that the overall conformation of the core domain remains largely independent of the flanking domains . Removal of the N or the C terminus does not abolish the DNA renaturation activity of the protein . However, intact protein and its three truncated forms differ in DNA helix-destabilizing activity . The C domain alone is responsible for the kinetic barrier to natural DNA helix destabilization seen with intact protein . Intact protein and core domain potentiate the DNA helix-destabilizing activity of truncated protein lacking only the C domain (*I), enhancing the observed hyperchromicity while increasing the melting temperature . Proteolysis experiments suggest that the affinity of core domain for single-stranded DNA is increased in the presence of *I . We propose that *I can "mingle" with intact protein or core domain while bound to single-stranded DNA.

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12469 - 74
A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization; Chowdhury K et al.; The three-dimensional structure of bacteriophage T7 DNA polymerase reveals the presence of a loop of 4 aa (residues 401-404) within the DNA-binding groove; this loop is not present in other members of the DNA polymerase I family . A genetically altered T7 DNA polymerase, T7 polDelta401-404, lacking these residues, has been characterized biochemically . The polymerase activity of T7 polDelta401-404 on primed M13 single-stranded DNA template is one-third of the wild-type enzyme and has a 3'-to-5' exonuclease activity indistinguishable from that of wild-type T7 DNA polymerase . T7 polDelta401-404 polymerizes nucleotides processively on a primed M13 single-stranded DNA template . T7 DNA polymerase cannot initiate de novo DNA synthesis; it requires tetraribonucleotides synthesized by the primase activity of the T7 gene 4 protein to serve as primers . T7 primase-dependent DNA synthesis on single-stranded DNA is 3- to 6-fold less with T7 polDelta401-404 compared with the wild-type enzyme . Furthermore, the altered polymerase is defective (10-fold) in its ability to use preformed tetraribonucleotides to initiate DNA synthesis in the presence of gene 4 protein . The location of the loop places it in precisely the position to interact with the tetraribonucleotide primer and, presumably, with the T7 gene 4 primase . Gene 4 protein also provides helicase activity for the replication of duplex DNA . T7 polDelta401-404 and T7 gene 4 protein catalyze strand-displacement DNA synthesis at nearly the same rate as does wild-type polymerase and T7 gene 4 protein, suggesting that the coupling of helicase and polymerase activities is unaffected.

J Biol Chem, 2001 Feb 16, 276(7), 4981 - 7 Epub 2000 Oct 24.
The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction: humans versus Escherichia coli; Richardson A et al.; Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins . While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31 . Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a beta-hairpin conformation upon binding to the chaperonin . A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin . Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence . Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity . Two of these substitutions are predicted to preorganize the beta-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site . Together, they result in a GroES that binds chaperonins with higher affinity . It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.

Photochem Photobiol, 2000 Oct, 72(4), 472 - 6
Photochemical and photobiological properties of new bispsoralen derivatives (Bis{PsCn}PIP, n = 4, 6, 8); Kim SK et al.; Bispsoralen derivatives possessing two psoralens and one piperazine molecule, 1,4-bis{n'-(8-psoralenoxy) alkyl} piperazine (Bis{PsCn}PIP, n = 4, 6, 8), show high water solubility, efficient intercalation into DNA and good photocrosslinking efficiency of DNA . Bis(PsC4)PIP shows high lethality on bacteriophage T7 and can effectively inhibit the amplification of DNA by stopping the polymerase chain reactions in a short period of irradiation time.

Prikl Biokhim Mikrobiol, 2000 Sep-Oct, 36(5), 569 - 74
{Antioxidant features of fungal melanin pigments}; Shcherba VV et al.; Fungal melanin pigments were shown to display a high antioxidant activity . An increase in the number of methyl substituents in benzidine molecules of melanins obtained from micromycetes and macromycetes was accompanied by a decrease in the efficiency of inhibition of peroxidase-catalyzed oxidation . Melanins were found to have considerable gene-protecting properties . Pigments isolated from macromycetes and applied at a much lower concentration than those obtained from micromycetes prevented damage to bacteriophage-lambda DNA induced by products of peroxidase-catalyzed degradation of aminobiphenyls.

Biochemistry (Mosc), 2000 Sep, 65(9), 1068 - 74
Structure and folding of bacteriophage T4 gene product 9 triggering infection . II . Study Of conformational changes of gene product 9 mutants using monoclonal antibodies; Zhemaeva LV et al.; Gene product 9 (gp9) of bacteriophage T4, whose spatial structure we have recently solved to 2.3 A resolution, is a convenient model for studying the folding and oligomerization mechanisms of complex proteins . The gp9 polypeptide chain consists of 288 amino acids forming three domains . Three monomers, packed in parallel, assemble to a functionally active protein . The main aim of this work was to study conformational changes and trimerization of gp9 deletion mutants using monoclonal antibodies (mAbs) . We selected a set of mAbs interacting with the amino, middle, and carboxyl regions of the protein, respectively . Eighteen mAbs bind to native as well as to denatured protein, and two mAbs bind to denatured protein only . Using mAbs, we found that deletions of the gp9 N-terminal region result in conformational changes in the middle and C-terminal domains . The study of mAb binding to the CDelta . truncated mutant by competitive ELISA and immunoblotting shows that the C-terminus of the gp9 sequence is essential for protein trimerization and stability . A single point substitution of the Gln282 residue causes formation of a labile trimer that has significant conformational changes in the protein domains . The results of our study show that folding and trimerization of gp9 is a cooperative process that involves all domains of the protein.

J Struct Biol, 2000 Aug, 131(2), 159 - 63
Crystallization and preliminary X-ray analysis of receptor-binding protein P2 of bacteriophage PRD1; Xu L et al.; Bacteriophage PRD1 has remarkable structural similarities to adenovirus, but is unusual in containing a membrane beneath its icosahedral capsid . Its monomeric receptor-binding protein, P2, is part of a complex at each capsid vertex and so is the functional equivalent of adenovirus fiber . P2 has been crystallized by the "hanging-drop" method of vapor diffusion and two different crystal forms were obtained . Macroseeding, used to increase the size of the initial small needles, gave rod-shaped crystals . These grew to a size of 0.08 x 0.08 x 0.50 mm(3) and diffracted to 2.6 A resolution . They have the orthorhombic space group P222(1), with unit cell dimensions a = 137.8 A, b = 46.5 A, c = 136.4 A . A few single crystals of a second form were grown without seeding under slightly different conditions . A parallelepiped crystal (0.10 x 0.10 x 0.35 mm(3)), with space group C222(1) and unit cell dimensions a = 182.3 A, b = 204.8 A, c = 133.3 A, diffracted to 3.5 A resolution . A rotation function for the second form revealed that four monomers of P2 are related by a noncrystallographic twofold axis . The structure of P2 will reveal how this arrangement relates to the trimeric adenovirus fiber .

J Struct Biol, 2000 Aug, 131(2), 146 - 55
Structural analysis of the bacteriophage T3 head-to-tail connector; Valpuesta JM et al.; The connector protein of bacteriophage T3, p8, has been overexpressed in Escherichia coli . Purification of the oligomers built by several copies of p8 reveals a mixed population of dodecamers and tridecamers . The percentages of these two types of oligomers differ in every culture growth, indicating that assembly of this protein depends upon the conditions of the expression system . Those cultures that generated a majority of dodecamers allowed, after purification of the connectors, the two-dimensional crystallization of the dodecamers in a tetragonal arrangement, while the tridecamers did not form crystals . The processing and averaging of several images of frozen-hydrated crystals and their internal phase comparison shows that the crystals are arranged in a P42(1)2 space group, with cell unit dimensions of 165 x 165 A . The three-dimensional reconstruction generated with images of crystals ranging from 0 degrees to 60 degrees tilt reveals a wide domain surrounded by 12 protrusions and a narrow domain that serves to interact with the tail of the bacteriophage . A channel runs along the connector wide enough to allow the translocation of a double-stranded DNA molecule into the prohead . The general structure of the T3 connector is very similar to those obtained for other nonrelated bacteriophages and strongly suggests that the shape of this important viral structure is intimately related to its function .

Hum Reprod, 2000 Jul, 15 Suppl 2, 11 - 7
Transcription and replication of mitochondrial DNA; Clayton DA; The physical isolation of mammalian mitochondrial DNA (mtDNA) over 30 years ago marked the beginning of studies of its structure, replication and the expression of its genetic content . Such analyses have revealed a number of surprises: novel DNA structural features of the circular genome such as the displacement loop (D-loop); multiple sized and deleted forms of the circular genome; a minimal set of mitochondrially encoded rRNAs and tRNAs needed for translation; a bacteriophage-like, nuclear-encoded mitochondrial RNA polymerase for transcription; and a direct linkage between transcription and the commitment to replication of the leading mtDNA strand that centres on the nuclear encoded mitochondrial transcription factor A . One of the more recent revelations is the existence, near the D-loop, of an atypical, stable RNA-DNA hybrid (or R-loop) at the origin of mammalian leading-strand DNA replication, composed of the parent DNA strands and an RNA transcript . In mammalian mitochondrial systems, all of the proteins known to be involved in DNA replication are encoded in the nucleus . Thus alterations and deficiencies in mtDNA replication must arise from mutations in mtDNA regulatory sequences and nuclear gene defects . Further studies of the relationships between nuclear-encoded proteins and their mtDNA target sequences could result in strategies to manipulate genotypes within cellular mtDNA populations.

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12655 - 60
Detection of mutations in transgenic fish carrying a bacteriophage lambda cII transgene target; Winn RN et al.; To address the dual needs for improved methods to assess potential health risks associated with chemical exposure in aquatic environments and for new models for in vivo mutagenesis studies, we developed transgenic fish that carry multiple copies of a bacteriophage lambda vector that harbors the cII gene as a mutational target . We adapted a forward mutation assay, originally developed for lambda transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by lambda in vitro packaging . After infecting and plating phage on a hfl- bacterial host, cII mutants were detected under selective conditions . We demonstrated that many fundamental features of mutation analyses based on lambda transgenic rodents are shared by transgenic fish . Spontaneous mutant frequencies, ranging from 4.3 x 10(-5) in liver, 2.9 x 10(-5) in whole fish, to 1.8 x 10(-5) in testes, were comparable to ranges in lambda transgenic rodents . Treatment with ethylnitrosourea resulted in concentration-dependent, tissue-specific, and time-dependent mutation inductions consistent with known mechanisms of action . Frequencies of mutants in liver increased insignificantly 5 days after ethylnitrosourea exposure, but increased 3.5-, 5.7- and 6 . 7-fold above background at 15, 20, and 30 days, respectively . Mutants were induced 5-fold in testes at 5 days, attaining a peak 10-fold induction 15 days after treatment . Spontaneous and induced mutational spectra in the fish were also consistent with those of lambda transgenic rodent models . Our results demonstrate the feasibility of in vivo mutation analyses using transgenic fish and illustrate the potential value of fish as important comparative animal models.

Comb Chem High Throughput Screen, 2000 Oct, 3(5), 373 - 92
Targeting of phage display vectors to mammalian cells; Uppala A et al.; Phage display libraries offer a strategy to isolate peptide ligands to target proteins and to define potential interaction sites between proteins . Recent studies have indicated a novel utility for phage display in that bacteriophage engineered to express peptide ligands to specific cell surface receptors are internalized by mammalian cells . Thus, reporter genes such as green fluorescent protein and lacZ harbored in the phage genome can be delivered to mammalian cells using targeting peptides displayed on the surface of phage . There is also the possibility to generate novel types of peptide libraries expressed intracellularly using a phage capable of inducing expression of its coding genes in human cells.

Parasite, 2000 Sep, 7(3), 233 - 6
Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis; Siddiqui AA et al.; A full length cDNA (1463 bp) encoding isocitrate lyase (EC 4.1.3.1) of Strongyloides stercoralis is described . The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase . The conceptually translated amino acid sequence of the open reading frame for S . stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39 . The sequence is 69% A/T, reflecting a characteristic A/T codon bias of S . stercoralis . The amino acid sequence of S . stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus . The full length cDNA of S . stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system . S . stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels . The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S . stercoralis is also discussed.

J Bacteriol, 2000 Nov, 182(21), 6130 - 6
Discovery, purification, and characterization of a temperate transducing bacteriophage for Bordetella avium; Shelton CB et al.; We discovered and characterized a temperate transducing bacteriophage (Ba1) for the avian respiratory pathogen Bordetella avium . Ba1 was initially identified along with one other phage (Ba2) following screening of four strains of B . avium for lysogeny . Of the two phage, only Ba1 showed the ability to transduce via an allelic replacement mechanism and was studied further . With regard to host range, Ba1 grew on six of nine clinical isolates of B . avium but failed to grow on any tested strains of Bordetella bronchiseptica, Bordetella hinzii, Bordetella pertussis, or Bordetella parapertussis . Ba1 was purified by CsCl gradient centrifugation and was found to have an icosahedral head that contained a linear genome of approximately 46.5 kb (contour length) of double-stranded DNA and a contractile, sheathed tail . Ba1 readily lysogenized our laboratory B . avium strain (197N), and the prophage state was stable for at least 25 generations in the absence of external infection . DNA hybridization studies indicated the prophage was integrated at a preferred site on both the host and phage replicons . Ba1 transduced five distinctly different insertion mutations, suggesting that transduction was generalized . Transduction frequencies ranged from approximately 2 x 10(-7) to 1 x 10(-8) transductants/PFU depending upon the marker being transduced . UV irradiation of transducing lysates markedly improved transduction frequency and reduced the number of transductants that were lysogenized during the transduction process . Ba1 may prove to be a useful genetic tool for studying B . avium virulence factors.

J Bacteriol, 2000 Nov, 182(21), 6082 - 90
Genetic and biochemical analysis of dimer and oligomer interactions of the lambda S holin; Grundling A et al.; Bacteriophage lambda uses a holin-endolysin system for host cell lysis . R, the endolysin, has muralytic activity . S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis . lambda S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function . A collection of 27 single-cysteine products of alleles created from lambda S(C51S) were tested for holin function . Most of the single-cysteine variants retained the ability to support lysis . Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains . Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane . Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless S(C51S) (i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein . In contrast, the lysis-defective S(A52V) mutant dimerizes but does not form cross-linkable oligomers . Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective S(A52V/C51S) is found in dimers after treatment of the membrane with a cross-linking agent . Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for S(A52V) . Both S(A52V) (dominant negative) and S(A48V) (antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation . Thus, productive and unproductive heterodimer formation between the parental protein and the mutants S(A52V) and S(A48V), respectively, may account for the dominant and antidominant lysis phenotypes . Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface . These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.

Biochem Biophys Res Commun, 2000 Sep 24, 276(2), 600 - 6
The PCNA from Thermococcus fumicolans functionally interacts with DNA polymerase delta; Henneke G et al.; We have cloned the gene encoding proliferating cell nuclear antigen (PCNA) from the hyperthermophilic euryarchaeote Thermococcus fumicolans (Tfu) . Tfu PCNA contains 250 amino acids with a calculated M(r) of 28,000 and is 26% identical to human PCNA . Next, Tfu PCNA was overexpressed in Escherichia coli and it showed an apparent molecular mass of 33.5 kDa . The purified Tfu PCNA was tested first with recombinant Tfu DNA polymerase I (Tfu pol) and second with calf thymus DNA polymerase delta (pol delta) . When tested with the homologous Tfu pol on bacteriophage lambda DNA, large amounts of Tfu PCNA were required to obtain two- to threefold stimulation . Surprisingly, however, Tfu PCNA was much more efficient than human PCNA in stimulating calf thymus pol delta . Our data suggest that PCNA has been functionally conserved not only within eukaryotes but also from hyperthermophilic euryarchaeotes to mammals .

J Hosp Infect, 2000 Sep, 46(1), 61 - 6
Transmission of viruses via contact in ahousehold setting: experiments using bacteriophage straight phiX174 as a model virus; Rheinbaben F et al.; Contamination of the environment with pathogens is the prerequisite for contact infections . The aim of this study was to elucidate how viruses can be transmitted from a primary contact person to further individuals . Bacteriophage straight phiX174 was chosen as a model virus . In its stability straight phiX174 is comparable with the most resistant human pathogenic viruses, e.g . polio- or parvoviruses . About 10(7)pfu were applied to exposed contact points such as door handles or the hands of volunteers . After touching of these handles and common social contacts like hand shaking, re-isolation rates were determined from the hands of our test persons . Contaminated door handles and skin surfaces were found to be efficient sources for potential infection . At least 14 persons could be contaminated by horizontal spread, one after the other by touching the same door handle . Successive transmission from one person to another could be followed up to the sixth contact person . These results were confirmed under everyday life conditions in a flat shared by four students . The transmission could not be prevented by the usual standards of hand hygiene, practised in this household . straight phiX174 could be reisolated after 24h from the hands of all persons tested even after normal use and cleaning of their hands . This might be improved by the use of liquid soap dispensers.

Annu Rev Microbiol, 2000, 54, 799 - 825
Holins: the protein clocks of bacteriophage infections; Wang IN et al.; Two proteins, an endolysin and a holin, are essential for host lysis by bacteriophage . Endolysin is the term for muralytic enzymes that degrade the cell wall; endolysins accumulate in the cytosol fully folded during the vegetative cycle . Holins are small membrane proteins that accumulate in the membrane until, at a specific time that is "programmed" into the holin gene, the membrane suddenly becomes permeabilized to the fully folded endolysin . Destruction of the murein and bursting of the cell are immediate sequelae . Holins control the length of the infective cycle for lytic phages and so are subject to intense evolutionary pressure to achieve lysis at an optimal time . Holins are regulated by protein inhibitors of several different kinds . Holins constitute one of the most diverse functional groups, with >100 known or putative holin sequences, which form >30 ortholog groups.

Clin Chem, 2000 Oct, 46(10), 1562 - 73
Enzymatic mutation detection in the P53 gene; Inganas M et al.; BACKGROUND: The enzymatic mutation detection (EMD) assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves preformed heteroduplex molecules at mismatch sites, forming two shorter fragments that can be resolved by gel electrophoresis . The method can be used to detect single and multiple base changes, as well as insertions and deletions . METHODS: The sensitivity, specificity, and positional accuracy of mutation detection by EMD with the PASSPORT(TM) Mutation Scanning Kit were assessed in a blind fashion for three analytical platforms (radioactive detection and automated laser sequencers ALFexpress and ABI PRISM 377) . PCR products of 703 bp covering codons 188-393 of the P53 gene were prepared from colorectal tumor samples and analyzed by EMD; the results were compared to data from cDNA sequencing . A 1362-bp PCR product prepared from IL4r gene was used to test detection of multiple base changes in long PCR products . RESULTS: The sensitivity for detection of mutations using EMD exceeded 90%, and the specificity exceeded 80% on all analysis platforms . The method localized 90% of mutations to within two codons and four codons for automated laser sequencers and detection by radioactivity, respectively . The method detected at least five mismatches in heteroduplexes >1 kb . CONCLUSIONS: The EMD system facilitates efficient detection of genetic variation in fragments exceeding 1 kb irrespective of location and type . The technology is particularly well suited to the detection of mutations in genes frequently mutated at unpredictable locations.

Virology, 2000 Sep 15, 275(1), 218 - 24
Characterization of phi 13, a bacteriophage related to phi 6 and containing three dsRNA genomic segments; Qiao X et al.; The three dsRNA genomic segments of bacteriophage Phi 13 were copied as cDNA and the nucleotide sequences were determined . The organization of the genome is similar to that of Phi 6, and there is significant similarity in the amino acid sequences of the proteins of the polymerase complex and one of the membrane proteins, P6 . There is little or no similarity in the nucleotide sequences . Several features of the viral proteins differ markedly from those of Phi 6 . Although both phages are covered by a lipid-containing membrane, the protein compositions are different . The host attachment protein consists of two peptides rather than one and the phage attaches directly to the LPS of the host rather than to a Type IV pilus . Despite the differences in the structure of the membranes, the two viruses can successfully exchange the genes for host attachment proteins and thereby change their host specificities .

Virology, 2000 Sep 15, 275(1), 133 - 44
In vitro assembly of bacteriophage P4 procapsids from purified capsid and scaffolding proteins; Wang S et al.; Bacteriophage P4 is a satellite virus of bacteriophage P2, which has acquired the ability to utilize the structural gene products of P2 to assemble its own capsid . The normal P2 capsid has a T = 7 icosahedral structure comprised of the gpN-derived capsid protein, whereas the capsid produced under the control of P4 has a smaller, T = 4 structure . The protein responsible for this size determination is the P4-coded gene product Sid, which forms an external scaffold on the P4 procapsid . Using an in vitro assembly system, we show that gpN and Sid can coassemble into procapsid-like particles, indistinguishable from those produced in vivo, in the absence of any other gene products . The fidelity of the assembly reaction is enhanced by the inclusion of PEG and has a pH optimum between 8.0 and 8.5 . Analysis of the assembly properties of truncated versions of Sid and gpN suggests that the amino-terminal part of Sid is involved in gpN binding, while the carboxyl-terminal part forms trimeric Sid-Sid interactions, and that the first 31 amino acids of gpN are required for binding to Sid as well as for size determination .

Clin Pharmacol Ther, 2000 Sep, 68(3), 225 - 30
Phage therapy: the peculiar kinetics of self-replicating pharmaceuticals; Payne RJ et al.; The specter of antibiotic-resistant bacteria has provoked renewed interest in the possible use of bacteriophages to control bacterial infections . We argue that clinical application of phage therapy has been held back by a failure to appreciate the extent to which the pharmacokinetics of self-replicating agents differ from those of normal drugs . For self-replicating pharmaceutical agents, treatment outcome depends critically on various density-dependent thresholds, often with apparently paradoxical consequences . An ability to predict these thresholds and associated critical time points is a necessity if phage therapy is to become clinically practicable.

Eur J Biochem, 2000 Oct, 267(20), 6231 - 8
The assembly factor P17 from bacteriophage PRD1 interacts with positively charged lipid membranes; Holopainen JM et al.; The interactions of the assembly factor P17 of bacteriophage PRD1 with liposomes were investigated by static light scattering, fluorescence spectroscopy, and differential scanning calorimetry . Our data show that P17 binds to positively charged large unilamellar vesicles composed of the zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine and sphingosine, whereas only a weak interaction is evident for 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles . P17 does not bind to negatively charged membranes composed of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and 1-palmitoyl-2-oleoyl-phosphatidylcholine . Our differential scanning calorimetry results reveal that P17 slightly perturbs the phase behaviour of neutral phosphatidylcholine and negatively charged multilamellar vesicles . In contrast, the phase transition temperature of positively charged dimyristoylphosphatidylcholine/sphingosine multilamellar vesicles (molar ratio 9 : 1, respectively) is increased by approximately 2.4 degrees C and the half width of the enthalpy peak broadened from 1.9 to 5.6 degrees C in the presence of P17 (protein : lipid molar ratio 1 : 47) . Moreover, the enthalpy peak is asymmetrical, suggesting that lipid phase separation is induced by P17 . Based on the far-UV CD spectra, the alpha-helicity of P17 increases upon binding to positively charged micelles composed of Triton X-100 and sphingosine . We propose that P17 can interact with positively charged lipid membranes and that this binding induces a structural change on P17 to a more tightly packed and ordered structure.

Can J Microbiol, 2000 Sep, 46(9), 841 - 7
High-frequency interconversion of turbid and clear plaque strains of bacteriophage f1 and associated host cell death; Kuo MY et al.; Under normal cultivation conditions, a mixture of turbid and clear plaques is often apparent in cultures of bacterial cells infected with filamentous bacteriophages . Beginning with a culture of wild-type filamentous phage f1, which itself produces turbid plaques, a clear plaque strain (c1) was isolated . From c1, the turbid plaque strain t1 was isolated; from t1, the clear plaque strain c2 was isolated; and from c2, the turbid plaque strain t2 was isolated . Each of these strains was generated with a frequency of approximately 1 x 10(-4) . Although filamentous phages have been thought not to induce host cell death, both turbid and clear plaque strains of f1 killed host bacteria . Plating of bacterial cells 1 h after infection revealed that colonies produced by cells infected with either wild-type f1 or strain c2 were smaller than those derived from uninfected cells, and that colony formation by infected cells was reduced by 15% and 38%, respectively . The time course of bacterial growth revealed that, at 4 h after infection, the number of CFU per milliliter of culture of cells infected with wild-type f1 or with strain c2 was reduced by 27% and 95%, respectively, compared with that for uninfected cells . Microculture analysis also revealed that the percentages of nondividing cells in f1 or c2 infected were 19% and 52%, respectively, 4 h after infection with wild-type f1 or with strain c2; no such cells were detected in cultures of uninfected cells . Negative staining and electron microscopy showed that 20% and 61% of cells infected with wild-type f1 or with strain c2 were dead 4 h postinfection . Finally, although the rates of DNA synthesis were similar for infected and uninfected cells, the rates of RNA and protein synthesis were markedly reduced in infected cells.

Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 286 - 92
Stoichiometry and inter-subunit interaction of the wedge initiation complex, gp10-gp11, of bacteriophage T4; Zhao L et al.; Association of gp10 and gp11 (gp=gene product) is the first step in the assembly pathway of the wedge part of the baseplate of bacteriophage T4 . The gp10-gp11 complex constitutes the six tail pins at the corners of the baseplate hexagon on the distal side . The stoichiometry of the subunits, gp10 and gp11, of this complex was determined in combination with sedimentation equilibrium, Edman degradation of the complex and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . From the results of Edman degradation and SDS-PAGE, the molar ratio of gp10 and gp11 was approximately 1 . On the other hand, the molecular weight of the purified gp10-gp11 complex was determined by sedimentation equilibrium to be 284000+/-7000, which is in good agreement with the expected value of 269840 if the stoichiometry is 3:3 . Furthermore, comparison of the results in the presence and in the absence of reducing reagent, 2-mercaptoethanol (2-ME), in SDS-PAGE revealed that two molecules of gp10 in the complex formed a disulfide bond, while the third gp10 molecule does not participate in the disulfide bond formation.

J Bacteriol, 2000 Oct, 182(20), 5807 - 12
Characterization of bacteriophage lambda excisionase mutants defective in DNA binding; Cho EH et al.; The bacteriophage lambda excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination . Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA . Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase . The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28 . Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T . E . Numrych, R . I . Gumport, and J . F . Gardner, EMBO J . 11:3797-3806, 1992) . We substituted alanines for the polar amino acids in this region . Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding . In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding . This indicates that the hydrophilic face of the alpha-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.

J Biotechnol, 2000 Sep 29, 83(1-2), 161 - 72
Improved protection against lung colonization by Actinobacillus pleuropneumoniae ghosts: characterization of a genetically inactivated vaccine; Huter V et al.; Pigs immunized with Actinobacillus pleuropneumoniae ghosts or a formalin-inactivated bacterin were found to be protected against clinical disease in both vaccination groups, whereas colonization of the lungs with A . pleuropneumoniae was only prevented in ghost-vaccinated pigs . Bacterial ghosts are empty cell envelopes created by the expression of a cloned bacteriophage lysis gene and, unlike formalin-inactivated bacteria, suffer no denaturing steps during their production . This quality may lead to a superior presentation of surface antigens to the immune system . Analysis by SDS-PAGE and immunoblotting of the two vaccine preparations revealed different contents of antigenic proteins . In order to better understand the immunogenic properties of A . pleuropneumoniae ghosts and formalin-inactivated bacteria, we compared the serum antibody response induced in both treatment groups . Immune sera were tested on whole cell antigen or purified virulence factors including outer membrane protein preparations (OMPs), outer membrane lipoprotein OmlA1, transferrin binding proteins (TfbA1, TfbA7 and TfbB) and Apx toxins (ApxI, II and III) . SDS-PAGE and immunoblots revealed no specific antibody response against the single virulence factors tested in any vaccinated animal . The two vaccination groups showed different recognition patterns of whole cell antigen and OMP-enriched preparations . A 100 kDa protein was recognized significantly stronger by ghost-vaccinated pigs than convalescent pigs . This unique antibody population induced by ghosts could play a determining role in the prevention of lung colonization . The same 100 kDa antigen was recognized by ghost-sera in homologous as well as heterologous serotype A . pleuropneumoniae protein preparations . Indications for a crossprotective potential in the ghost vaccine were supported by studies on rabbit hyperimmune sera.

Science, 2000 Sep 22, 289(5487), 2129 - 33
Topologically linked protein rings in the bacteriophage HK97 capsid; Wikoff WR et al.; The crystal structure of the double-stranded DNA bacteriophage HK97 mature empty capsid was determined at 3.6 angstrom resolution . The 660 angstrom diameter icosahedral particle contains 420 subunits with a new fold . The final capsid maturation step is an autocatalytic reaction that creates 420 isopeptide bonds between proteins . Each subunit is joined to two of its neighbors by ligation of the side-chain lysine 169 to asparagine 356 . This generates 12 pentameric and 60 hexameric rings of covalently joined subunits that loop through each other, creating protein chainmail: topologically linked protein catenanes arranged with icosahedral symmetry . Catenanes have not been previously observed in proteins and provide a stabilization mechanism for the very thin HK97 capsid.

Genetika, 2000 Jul, 36(7), 920 - 4
{Gene rIIB from bacteriophage T4 in genetic recombination}; Shcherbakov VP et al.; The effect of the rIIB gene on genetic recombination in bacteriophage T4 was studied . Relationships between recombination frequency and the physical distance were determined in three series of isomarker two-factor crosses between rII mutants . In the first series of intergenic crosses (rIIa x rIIb), the rII gene function was restored owing to complementation . In the second series of crosses, identical to the first one, the rIIB gene function was suppressed, because the rIIa parent carried an additional amberlike mutation in the rIIB gene . The recombinants were scored by plating lysates on the amber-suppressor Escherichia coli strain, on which an amberlike mutation was not expressed phenotypically . In the third series, all crosses were intragenic (rIIb x rIIb) . In two series of crosses in the absence of the rIIB function, the relationships between recombination frequency and the physical distance were identical, whereas enhanced recombination frequencies were observed in the rIIB+ background . The magnitude of the rIIB-related effect depended on distance, reaching the maximum in the region located 100 to 200 bp from the beginning of the rIIB gene . The possible role of the rIIB protein in genetic recombination is discussed.

J Mol Biol, 2000 Sep 29, 302(4), 777 - 95
ATPase center of bacteriophage lambda terminase involved in post-cleavage stages of DNA packaging: identification of ATP-interactive amino acids; Hang JQ et al.; Terminase is the enzyme that mediates lambda DNA packaging into the viral prohead . The large subunit of terminase, gpA (641 amino acid residues), has a high-affinity ATPase activity (K(m)=5 microM) . To directly identify gpA's ATP-interacting amino acids, holoterminase bearing a His(6)-tag at the C terminus of gpA was UV-crosslinked with 8-N(3)-{alpha-(32)P}ATP . Tryptic peptides from the photolabeled terminase were purified by affinity chromatography and reverse-phase HPLC . Two labeled peptides of gpA were identified . Amino acid sequencing failed to show the tyrosine residue of the first peptide, E(43)SAY(46)QEGR(50), or the lysine of the second peptide, V(80)GYSK(84)MLL(87), indicating that Y(46) and K(84) were the 8-N(3)-ATP-modified amino acids . To investigate their roles in lambda DNA packaging, Y(46) was changed to E, A, and F, and K(84) was changed to E and A . Purified His(6)-tagged terminases with changes at residues 46 and 84 lacked the gpA high-affinity ATPase activity, though the cos cleavage and cohesive end separation activities were near to those of the wild-type enzyme . In virion assembly reactions using virion DNA as a packaging substrate, the mutant terminases showed severe defects . In summary, the results indicate that Y(46) and K(84) are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post-cos cleavage stages of lambda DNA packaging .

Int J Radiat Biol, 2000 Sep, 76(9), 1289 - 94
Methylene blue inhibits polymerase 1 enzyme and sensitizes Escherichia coli bacteria to X-rays; Menezes S et al.; PURPOSE: To investigate the mechanism of methylene blue-induced radiosensitization in Escherichia coli cells . MATERIALS AND METHODS: Bacteriophage lambda15 was irradiated with X-rays in the presence or absence of methylene blue (MB) and infected into bacteria with different repair capabilities preincubated with or without MB . The survival of the bacteriophage in each bacterial strain was used to quantify MB-induced radiosensitization . DNA repair in bacteria irradiated with X-rays and incubated with or without MB was examined by alkaline and neutral sucrose gradients . An in vitro repair system of pBR322 plasmid DNA irradiated with X-rays was designed to determine the repair enzyme targeted by MB . CONCLUSIONS: MB impairs the repair activity of the polymerase 1 enzyme in E . coli cells, sensitizing these bacteria to the lethal effects of ionizing radiation . Since MB accumulates preferentially in some malignant tumours, it will be of interest to investigate its effects on the repair of irradiated human cells.

Photochem Photobiol, 2000 Sep, 72(3), 365 - 73
Partial complementation of the DNA repair defects in cells from xeroderma pigmentosum groups A, C, D and F but not G by the denV gene from bacteriophage T4; Francis MA et al.; Endonuclease V (denV) from bacteriophage T4 was examined for its ability to complement the DNA repair defect in xeroderma pigmentosum (XP) cells from complementation groups A, C, D, F and G . The denV gene was introduced into SV40-transformed normal and XP cells using a retroviral vector . Expression of denV resulted in partial correction of UV sensitivity and increased host cell reactivation (HCR) of a UV-damaged reporter gene for XP cells from groups A, C and D, but not those from group G . Expression of denV in XP-F cells resulted in enhanced HCR of a UV-damaged reporter but did not affect UV sensitivity . The observed partial complementation is thought to reflect denV-mediated repair of cyclobutane-pyrimidine dimers (CPD), and is incomplete as denV does not recognize other UV-induced lesions, and may not even efficiently remove all CPD . As XP-F cells are believed to retain near-normal levels of CPD repair in the bulk of the genome, we believe that the disparity in the ability of denV to complement the repair deficiency in these cells results from an increased rate, but not level, of CPD repair . Furthermore, we suggest that the lack of correction in the XP-G cells examined results from an inability to process denV-incised CPD by the base excision repair pathway, as has been suggested for cells from the related genetic disorder, Cockayne syndrome . Expression of denV in repair proficient normal cells also resulted in increased HCR of the UV-damaged reporter construct, possibly arising from an increased rate of CPD repair in these cells.

J Immunol Methods, 2000 Aug 28, 242(1-2), 33 - 42
The cellular immune recognition of proteins expressed by an African swine fever virus random genomic library; Jenson JS et al.; The cellular immune recognition of peptides expressed by an African swine fever virus (ASFV) random genomic library has been studied . DNA from the Malawi (LIL20/1) ASFV isolate was randomly sheared by sonication, cloned into a plasmid vector downstream of a bacteriophage T7 promoter, and 72 recombinant plasmids were arbitrarily selected . These plasmids were transiently expressed following transfection into major histocompatibility complex (MHC) class I(+) class II(-) matched pig skin cells, which had been co-infected with vTF7-3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase . Such cells served as antigen presenting cells and each recombinant plasmid was screened in a proliferation assay for recognition by CD8(+) lymphocytes from inbred pigs previously exposed to ASFV . This assay was demonstrated to measure CD8(+) T cell proliferation, as predicted by the phenotype of the antigen presenting cell . Of the 72 randomly selected clones, 14 were reproducibly recognised by immune pig lymphocytes and 10 corresponded to non-overlapping and distinct nucleic acid sequences . This high frequency of ASFV encoded antigenic epitopes supports the concept that cellular immunity to the virus may play an important role in resistance to ASF.

J Mol Biol, 2000 Sep 22, 302(3), 625 - 38
Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers; Darling PJ et al.; The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch . While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites . Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined . The dimerization constant for cro has been measured as a prelude to these binding studies . Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques . Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites . Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2 . Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol . Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation . This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity .

Biochemistry, 2000 Sep 19, 39(37), 11500 - 7
Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding I: analysis of cro dimerization from nanomolar to micromolar concentrations; Darling PJ et al.; The cro repressor from bacteriophage lambda is an important and classical transcription regulatory protein that binds DNA operator sites as a dimer . Therefore, a complete understanding of gene regulation by cro requires knowledge of the coupled energetics of its protein dimerization and site-specific DNA binding . A method is described by which cro repressor can be labeled in vivo with {(35)S}methionine to a specific activity of 2 x 10(15) cpm/mol . As a prelude to binding studies, the association equilibrium of cro was determined over the range 10(-)(9)-10(-)(3) M using large-zone analytical gel chromatography with radiolabeled repressor . The data are best described by a monomer-dimer stoichiometry with an equilibrium constant of 3.07 (+/-1.08) x 10(6) M(-)(1) total cro monomer . Stokes radii for monomers and dimers were evaluated from the resolved gel partition coefficients . Under the conditions employed in this study (10 mM Bis-Tris, 200 mM KCl, 2.5 mM MgCl(2), 1 mM CaCl(2), 100 microg/mL BSA, pH 7.0, 20 degrees C), self-association of cro to species with assembly states greater than dimers is not observed.

Arch Microbiol, 2000 Jul-Aug, 174(1-2), 89 - 96
ClpP/ClpX-mediated degradation of the bacteriophage lambda O protein and regulation of lambda phage and lambda plasmid replication; Wegrzyn A et al.; The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex . This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease . Nevertheless, the physiological role of this rapid degradation remains unclear . Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria . However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium . Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants . The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate . However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates . These results indicate that the O protein (whose level in E . coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates . However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells . Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.

Mutat Res, 2000 Sep 15, 461(1), 41 - 58
Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNA glycosylase; Radany EH et al.; The Ugi protein inhibitor of uracil-DNA glycosylase encoded by bacteriophage PBS2 inactivates human uracil-DNA glycosylases (UDG) by forming a tight enzyme:inhibitor complex . To create human cells that are impaired for UDG activity, the human glioma U251 cell line was engineered to produce active Ugi protein . In vitro assays of crude cell extracts from several Ugi-expressing clonal lines showed UDG inactivation under standard assay conditions as compared to control cells, and four of these UDG defective cell lines were characterized for their ability to conduct in vivo uracil-DNA repair . Whereas transfected plasmid DNA containing either a U:G mispair or U:A base pairs was efficiently repaired in the control lines, uracil-DNA repair was not evident in the lines producing Ugi . Experiments using a shuttle vector to detect mutations in a target gene showed that Ugi-expressing cells exhibited a 3-fold higher overall spontaneous mutation frequency compared to control cells, due to increased C:G to T:A base pair substitutions . The growth rate and cell cycle distribution of Ugi-expressing cells did not differ appreciably from their parental cell counterpart . Further in vitro examination revealed that a thymine DNA glycosylase (TDG) previously shown to mediate Ugi-insensitive excision of uracil bases from DNA was not detected in the parental U251 cells . However, a Ugi-insensitive UDG activity of unknown origin that recognizes U:G mispairs and to a lesser extent U:A base pairs in duplex DNA, but which was inactive toward uracil residues in single-stranded DNA, was detected under assay conditions previously shown to be efficient for detecting TDG.

Mol Gen Mikrobiol Virusol, 2000, (3), 33 - 6
{Expression of a fragment of the env gene, coding for the GP46 surface glycoprotein of the human T-cell leukemia virus, in Escherichia coli cells}; Bondarenko VO et al.; Designing of recombinant plasmids pSB2 and pSB3 with the 932 bp HTLV-II env gene inserts encoding the full-length surface membrane glycoprotein gp46 is described . Vectors pGOmpF and pET32a expressing genes cloned under control of the late bacteriophage T7 promoter were used . Western blot analysis of cellular proteins derived from E . coli B834/pSB2 and E . coli B834/pSB3 revealed that 34 kD and 31 kD polypeptides corresponding to the full-length gp46 and its processed form without signal peptide were synthesized under control of these recombinant plasmids . Cytotoxic activity of the recombinant proteins towards bacterial cells was demonstrated . Both polypeptides specifically reacted to sera from humans infected with HTLV-II . High antigenic specificity of P34-HTLV-II proteins makes a promising candidate for diagnostic confirmation tests.

J Biol Chem, 2000 Dec 1, 275(48), 37951 - 6
In vivo and in vitro function of GroEL mutants with impaired allosteric properties; Fridmann Y et al.; Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction . Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo . Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 degrees C . This strain supports the growth of phages T4 and T5 but not phage lambda and produces light at 28 degrees C when transformed with a second plasmid containing the lux operon . In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 degrees C . In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested . Our results show that allostery in GroEL is important in vivo in a manner that depends on the physiological conditions and is protein substrate specific.

Mol Microbiol, 2000 Aug, 37(4), 800 - 10
Genetic analysis of the strong gyrase site (SGS) of bacteriophage Mu: localization of determinants required for promoting Mu replication; Pato ML et al.; The Mu strong gyrase site (SGS), located in the centre of the Mu genome, is required for efficient Mu replication, as it promotes synapsis of the prophage termini . Other gyrase sites tested, even very strong ones, were unable to substitute for the SGS in Mu replication . To determine the features required for its unique properties, a deletion analysis was performed on the SGS . For this analysis, we defined the 20 bp centred on the midpoint of the 4 bp staggered cleavage made by gyrase to be the 'core' and the flanking sequences to be the 'arms' . The deletion analysis showed that (i) approximately 40 bp of the right arm is required, in addition to core sequences, for both efficient Mu replication and gyrase cleavage; and (ii) the left arm was not required for efficient Mu replication, although it was required for efficient gyrase cleavage . These observations implicated the right arm as the unique feature of the SGS . The second observation showed that strong gyrase cleavage and Mu replication could be dissociated and suggested that even weak gyrase sites, if supplied with the right arm of the SGS, could promote Mu replication . Hybrid sites were constructed with gyrase sites that could not support efficient Mu replication . The SGS right arm was used to replace one arm of the strong pSC101 gyrase site or the weaker pBR322 site . The pSC101 hybrid site allowed efficient Mu replication, whereas the pBR322 hybrid site allowed substantial, but reduced, replication . Hence, it appears that optimal Mu replication requires a central strong gyrase site with the properties imparted by the right arm sequences . Possible roles for the SGS right arm in Mu replication are addressed.

Chromosoma, 2000 Jul, 109(4), 226 - 34
Double-strand breaks on artificial chromosomes in yeast; Ross LO et al.; Yeast artificial chromosomes composed primarily of bacteriophage gamma DNA exhibit very low levels of meiotic crossing over compared with similarly sized intervals of natural yeast DNA . When these recombinationally quiet chromosomes were augmented with a 12.5 kb insert of sequences from yeast chromosome VIII, genetic studies demonstrated that the artificial chromosomes had acquired recombination properties characteristic of this region of chromosome VIII . On authentic yeast chromosomes, most meiotic recombination events are initiated at sites where the DNA is cleaved to create a double-strand break (DSB) . This report describes physical analyses that were carried out to examine the relationship between DSB sites and the recombination behavior of the artificial chromosomes . The results show that DSBs are rare on these artificial chromosomes, except for the 12.5 kb insert . Mapping of the DSB sites shows that their positions correlate with the previously determined positions of DSB sites on chromosome VIII . Deletion of two characterized chromosome VIII DSB sites from the 12.5 kb insert on the artificial chromosome resulted in the loss of the predicted DSB fragments and a reduction in crossing over between artificial chromosomes.

J Biol Chem, 2000 Nov 24, 275(47), 37127 - 36
Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4; Leffers G et al.; Double-stranded DNA-packaging in icosahedral bacteriophages is believed to be driven by a packaging "machine" constituted by the portal protein and the two packaging/terminase proteins assembled at the unique portal vertex of the empty prohead shell . Although ATP hydrolysis is evidently the principal driving force, which component of the packaging machinery functions as the translocating ATPase has not been elucidated . Evidence suggests that the large packaging subunit is a strong candidate for the translocating ATPase . We have constructed new phage T4 terminase recombinants under the control of phage T7 promoter and overexpressed the packaging/terminase proteins gp16 and gp17 in various configurations . The hexahistidine-tagged-packaging proteins were purified to near homogeneity by Ni(2+)-agarose chromatography and were shown to be highly active for packaging DNA in vitro . The large packaging subunit gp17 but not the small subunit gp16 exhibited an ATPase activity . Although gp16 lacked ATPase activity, it enhanced the gp17-associated ATPase activity by >50-fold . The gp16 enhancement was specific and was due to an increased catalytic rate for ATP hydrolysis . A phosphorylated gp17 was demonstrated under conditions of low catalytic rates but not under high catalytic rates in the presence of gp16 . The data are consistent with the hypothesis that a weak ATPase is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.

J Mol Biol, 2000 Aug 25, 301(4), 975 - 85
Structure of bacteriophage T4 gene product 11, the interface between the baseplate and short tail fibers; Leiman PG et al.; Bacteriophage T4, like all other viruses, is required to be stable while being transmitted from host to host, but also is poised to eject efficiently and rapidly its double-stranded DNA genome to initiate infection . The latter is coordinated by the recognition of receptors on Escherichia coli cells by the long tail fibers and subsequent irreversible attachment by the short tail fibers . These fibers are attached to the baseplate, a multi-subunit assembly at the distal end of the tail . Recognition and attachment induce a conformational transition of the baseplate from a hexagonal to a star-shaped structure.The crystal structure of gene product 11 (gp11), a protein that connects the short tail fibers to the baseplate, has been determined to 2.0 A resolution using multiple wavelength anomalous dispersion with Se . This structure is compared to the trimeric structure of gp9, which connects the baseplate with the long tail fibers . The structure of gp11 is a trimer with each monomer consisting of 218 residues folded into three domains . The N-terminal domains form a central, trimeric, parallel coiled coil surrounded by the middle "finger" domains . The fingers emanate from the carboxy-terminal beta-annulus domain, which, by comparison with the T4 whisker "fibritin" protein, is probably responsible for trimerization . The events leading from recognition of the host to the ejection of viral DNA must be communicated along the assembled trimeric (gp9)(3) attached to the long tail fibers via the trimeric baseplate protein (gp10)(3) to the trimeric (gp11)(3) and the trimeric short tail fibers .

J Mol Biol, 2000 Aug 18, 301(3), 575 - 84
Preferential binding of fd gene 5 protein to tetraplex nucleic acid structures; Oliver AW et al.; The gene 5 protein of filamentous bacteriophage fd is a single-stranded DNA-binding protein that binds non-specifically to all single-stranded nucleic acid sequences, but in addition is capable of specific binding to the sequence d(GT(5)G(4)CT(4)C) and the RNA equivalent r(GU(5)G(4)CU(4)C), the latter interaction being important for translational repression . We show that this sequence preference arises from the formation of a tetraplex structure held together by a central block of G-quartets, the structure of which persists in the complex with gene 5 protein . Binding of gene 5 protein to the tetraplex leads to formation of a approximately 170 kDa nucleoprotein complex consisting of four oligonucleotide strands and eight gene 5 protein dimers, with a radius of gyration of 45 A and an overall maximum dimension of 120-130 A . A model of the complex is presented that is consistent with the data obtained . It is proposed that the G-quartet may act as a nucleation site for binding gene 5 protein to adjacent single-stranded regions, suggesting a novel mechanism for translational repression .

Annu Rev Biochem, 2000, 69, 651 - 97
Structure and function of hexameric helicases; Patel SS et al.; Helicases are motor proteins that couple the hydrolysis of nucleoside triphosphate (NTPase) to nucleic acid unwinding . The hexameric helicases have a characteristic ring-shaped structure, and all, except the eukaryotic minichromosomal maintenance (MCM) helicase, are homohexamers . Most of the 12 known hexameric helicases play a role in DNA replication, recombination, and transcription . A human genetic disorder, Bloom's syndrome, is associated with a defect in one member of the class of hexameric helicases . Significant progress has been made in understanding the biochemical properties, structures, and interactions of these helicases with DNA and nucleotides . Cooperativity in nucleotide binding was observed in many, and sequential NTPase catalysis has been observed in two proteins, gp4 of bacteriophage T7 and rho of Escherichia coli . The crystal structures of the oligomeric T7 gp4 helicase and the hexamer of RepA helicase show structural features that substantiate the observed cooperativity, and both are consistent with nucleotide binding at the subunit interface . Models are presented that show how sequential NTP hydrolysis can lead to unidirectional and processive translocation . Possible unwinding mechanisms based on the DNA exclusion model are proposed here, termed the wedge, torsional, and helix-destabilizing models.

Appl Environ Microbiol, 2000 Sep, 66(9), 3868 - 77
Mathematical analysis of growth and interaction dynamics of streptomycetes and a bacteriophage in soil; Burroughs NJ et al.; We observed the infection cycle of the temperate actinophage KC301 in relation to the growth of its host Streptomyces lividans TK24 in sterile soil microcosms . Despite a large increase in phage population following germination of host spores, there was no observable impact on host population numbers as measured by direct plate counts . The only change in the host population following infection was the establishment of a small subpopulation of KC301 lysogens . The interaction of S . lividans and KC301 in soil was analyzed with a population-dynamic mathematical model to determine the underlying mechanisms of this low susceptibility to phage attack relative to aquatic environments . This analysis suggests that the soil environment is a highly significant component of the phage-host interaction, an idea consistent with earlier observations on the importance of the environment in determining host growth and phage-host dynamics . Our results demonstrate that the accepted phage-host interaction and host life cycle, as determined from agar plate studies and liquid culture, is sufficient for quantitative agreement with observations in soil, using soil-determined rates . There are four significant effects of the soil environment: (i) newly germinated spores are more susceptible to phage lysis than are hyphae of developed mycelia, (ii) substrate mycelia in mature colonies adsorb about 98% of the total phage protecting susceptible young hyphae from infection, (iii) the burst size of KC301 is large in soil (>150, 90% confidence) relative to that observed in liquid culture (120, standard error of the mean {SEM}, 6), and (iv) there is no measurable impact on the host in terms of reduced growth by the phage . We hypothesize that spatial heterogeneity is the principal cause of these effects and is the primary determinant in bacterial escape of phage lysis in soil.

J Neurosci, 2000 Sep 1, 20(17), 6385 - 93
CaMKIIalpha 3' untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage; Rook MS et al.; The CaMKIIalpha mRNA extends into distal hippocampal dendrites, and the 3' untranslated region (3'UTR) is sufficient to mediate this localization . We labeled the 3'UTR of the CaMKIIalpha mRNA in hippocampal cultures by using a green fluorescent protein (GFP)/MS2 bacteriophage tagging system . The CaMKIIalpha 3'UTR formed discrete granules throughout the dendrites of transfected cells . The identity of the fluorescent granules was verified by in situ hybridization . Over 30 min time periods these granules redistributed without a net increase in granule number; with depolarization there is a tendency toward increased numbers of granules in the dendrites . These observations suggest that finer time resolution of granule motility might reveal changes in the motility characteristics of granules after depolarization . So that motile granules could be tracked, shorter periods of observation were required . The movements of motile granules can be categorized as oscillatory, unidirectional anterograde, or unidirectional retrograde . Colocalization of CaMKIIalpha 3'UTR granules and synapses suggested that oscillatory movements allowed the granules to sample several local synapses . Neuronal depolarization increased the number of granules in the anterograde motile pool . Based on the time frame over which the granule number increased, the translocation of granules may serve to prepare the dendrite for mounting an adequate local translation response to future stimuli . Although the resident pool of granules can respond to signals that induce local translation, the number of granules in a dendrite might reflect its activation history.

Virology, 2000 Sep 1, 274(2), 429 - 37
Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay; Vassiliou W et al.; We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp) . ATP and GTP with a p-nitrophenyl moiety attached to the gamma-phosphate were synthesized (PNP-NTPs) . These PNP-NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6 . When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophenylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed . These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity . Furthermore, the PNP-NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma . This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis .

Plasmid, 2000 Sep, 44(2), 111 - 26
Replication of oriJ-based plasmid DNA during the stringent and relaxed responses of Escherichia coli; Potrykus K et al.; The oriJ-based plasmids contain the origin of DNA replication from the cryptic Rac prophage, present in the chromosomes of most Escherichia coli K-12 strains . The organization of the oriJ replication region resembles that of the bacteriophage lambda, although sequence similarity is small . Here we investigated the regulation of replication of the oriJ-based plasmid in E . coli relA(+) and relA(-) hosts during amino acid starvation and limitation, i.e., during the stringent and relaxed responses . We found that, contrary to plasmids derived from phage lambda, replication of the oriJ-based plasmid proceeds efficiently during both stringent and relaxed responses . On the other hand, density shift experiments and measurement of the stability of a putative replication initiator protein (the lambda O protein homologue) suggest that this replication may be carried out by the heritable replication complex, as previously demonstrated for lambda plasmids . We demonstrate that contrary to bacteriophage lambda p(R) promoter, an analogous promoter from the oriJ region is activated rather than inhibited at increased ppGpp levels . We propose that various responses of these promoters (p(R) and p(R-Rac), which are necessary for transcriptional activation of orilambda and perhaps oriJ, respectively) to ppGpp are responsible for differences in the replication regulation between orilambda- and oriJ-based plasmids during the stringent response .

Int J Med Microbiol, 2000 Jul, 290(3), 269 - 78
Clonal diversity of Shiga toxin-producing Escherichia coli O157:H7/H- in Germany--a ten-year study; Liesegang A et al.; Two hundred and ten E . coli O157:H7/H- strains isolated from single cases and outbreaks of diarrhoea and haemolytic uraemic syndrome (HUS) in Germany between 1988 and 1998 were characterised by a range of molecular subtyping methods and phage typing in order to analyse their clonal nature . A high clonal heterogeneity, together with a considerable clonal stability, has been identified among the bacterial isolates and no single clonal type appeared to be geographically dominant . It is recommended to apply pulsed-field gel electrophoresis (PFGE) together with P gene profile determination (number and genomic positions of lambdoid bacteriophages) as laboratory tools for an extended epidemiological surveillance of E . coli OOFF phage typing will remain helpful as a first line of analysis, particularly in outbreak situations.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1187 - 90
Purification, crystallization and initial X-ray analysis of the head-tail connector of bacteriophage phi29; Badasso MO et al.; The head-tail connector of bacteriophage phi29, an oligomer of gene product 10 (gp10), was crystallized into various forms . The most useful of these were an orthorhombic P22(1)2(1) form (unit-cell parameters a = 143.0, b = 157.0, c = 245.2 A), a monoclinic C2 form (a = 160.7, b = 143.6, c = 221.0 A, beta = 97.8 degrees ) and another monoclinic C2 form (a = 177.0, b = 169.1, c = 185.2 A, beta = 114.1 degrees ) . Frozen crystals diffracted to about 3.2 A resolution . There is one connector per crystallographic asymmetric unit in each case . Rotation functions show the connector to be a dodecamer . Translation functions readily determined the position of the 12-fold axis in each unit cell . The structure is being determined by 12-fold electron-density averaging within each crystal and by averaging between the various crystal forms.

Biochemistry, 2000 Aug 29, 39(34), 10566 - 73
Assembly of bacteriophage PRD1 spike complex: role of the multidomain protein P5; Caldentey J et al.; The spike structure of bacteriophage PRD1 is comprised of proteins P2, P5, and P31 . It resembles the corresponding receptor-binding structure of adenoviruses . We show that purified recombinant protein P5 is an elongated (30 x 2.7 nm; R(h) = 5.5 nm), multidomain trimer which can slowly associate into nonamers . Cleavage of the 340 amino acid long P5 with collagenase yields 2 fragments . The larger, 205 amino acid long C-terminal fragment appears to contain the residues responsible for the trimerization of the protein, whereas the smaller N-terminal part mediates the interaction of P5 with the pentameric vertex protein P31 (24 x 2.5 nm, R(h) = 4.2 nm) . In addition, the presence of the N-terminal sequence is required for the formation of the P5 nonamer . The results presented here suggest that P5 and P31 form an elongated adaptor complex at the 5-fold vertexes of the virion which anchors the adsorption protein P2 (21 x 2.5 nm; R(h) = 4.1 nm) . Our results also suggest that the P5 trimer forms a substantial part of the viral spike shaft that was previously thought to be composed exclusively of protein P2.

Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 10068 - 73
Novel folded protein domains generated by combinatorial shuffling of polypeptide segments; Riechmann L et al.; It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments . To investigate this proposal, we fused DNA encoding the N-terminal half of a beta-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage . Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames . Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold . Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding . Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself . These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.

Eur J Biochem, 2000 Sep, 267(17), 5438 - 49
Structure and expression of the mouse gene encoding the endozepine-like peptide from haploid male germ cells; Valentin M et al.; The endozepine-like peptide (ELP) represents a testis-specific isoform of the ubiquitous acyl-CoA binding protein (ACBP) and is highly expressed in late haploid stages of male germ cell development . The genomic sequence of the functional ELP gene as well as that of a pseudogene were analysed from independent bacteriophage clones of a 129sv mouse genomic library . Unlike the ACBP gene, which comprises four exons, the ELP gene has only a single intron within the region of the 5' untranslated region, suggesting that, like some other haploid expressed genes, the ELP gene might have evolved by retroposon-mediated gene duplication . Primer extension analysis was used to define the start site for transcription and hence the 5' promoter region . Electrophoretic mobility shift analysis was carried out on this region comparing nuclear extracts from adult mouse testis with those from mouse liver . Several testis-specific DNA-protein complexes were observed throughout 700 bp upstream of the transcription start site . One of these could be identified as corresponding to a steroidogenic factor-1 (SF-1) binding element . Further analysis using pure transcription factors showed that this element at position -340 was able to bind specifically to both SF-1 and to the germ cell nuclear factor (GCNF) . Immunohistochemical analysis using an ELP-specific antibody showed that expression was very restricted within the testis to the postmeiotic germ cells, and in the ovary to interstitial/luteal cells, cell-types known to express GCNF and SF-1, respectively . Testes of CREM-tau knockout mice, lacking all spermatogenic stages later than round spermatids, were devoid of ELP immunoreactivity, whereas in RAD6 knockout mice the few remaining elongated spermatids were clearly defined by this excellent late haploid marker product . The ELP gene and its product thus offer an ideal system with which to investigate the differentiation of late haploid stages of spermatogenesis.

Cancer Chemother Pharmacol, 2000, 46 Suppl, S8 - 12
Recombinant anti-carcinoembryonic antigen antibodies for targeting cancer; Chester KA et al.; Antibodies can be used to target cancer therapies to malignant tissue; the approach is attractive because conventional treatments such as chemo- and radiotherapy are dose limited due to toxicity in normal tissues . Effective targeting relies on appropriate pharmacokinetics of antibody-based therapeutics, ideally showing maximum uptake and retention in tumor and rapid clearance from normal tissue . We have studied the factors influencing these dynamics for antibodies against carcinoembryonic antigen (CEA) . Protein engineering of anti-CEA antibodies, in vivo biodistribution models, and mathematical models have been employed to improve understanding of targeting parameters, define optimal characteristics for the antibody-based molecules employed, and develop new therapies for the clinic . Engineering antibodies to obtain the desired therapeutic characteristics is most readily achieved using recombinant antibody technology, and we have taken the approach of immunizing mice to provide high-affinity anti-CEA single-chain Fv antibodies (sFvs) from filamentous bacteriophage libraries . MFE-23, the most characterized of these sFvs, has been expressed in bacteria and purified in our laboratory for two clinical trials: a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumor deposits are detected by a hand-held probe during surgery . Both these trials showed that MFE-23 is safe and effective in localizing tumor deposits in patients with cancer . We are now developing fusion proteins that use the MFE-23 antibody to deliver a therapeutic moiety; MFE-23:: carboxypeptidase G2 (CPG2) targets the enzyme CPG2 for use in the antibody-directed enzyme prodrug therapy system and MFE::tumor necrosis factor alpha (TNFalpha) aims to reduce sequestration and increase tumor concentrations of systemically administered TNFalpha.

Cell Mol Life Sci, 2000 Jan 20, 57(1), 128 - 48
The terminase enzyme from bacteriophage lambda: a DNA-packaging machine; Catalano CE; This review focuses on the biochemical, biophysical, and catalytic properties of terminase, an enzyme involved in bacteriophage lambda genome packaging . The holoenzyme possesses ATPase, DNA strand-separation, and site-specific nuclease activities that work in concert to insert a viral genome into the confines of a performed capsid . Moreover, the terminase subunits are part of a series of nucleoprotein complexes involved in genome packaging, including remarkably stable intermediates that transition to a highly mobile DNA packaging 'machine.' Models for the assembly and interconversion of these complexes are presented . Interactions between the catalytic sites in the enzyme complex, and modulation of these catalytic activities as it relates to the assembly and relative stability of the packaging intermediates are discussed . This ordered progression of nucleoprotein intermediates is a common theme in biology as demonstrated by mechanistic similarities between viral DNA packaging, the initiation of chromosomal replication, and the initiation of transcription . Terminase is thus part of a growing number of examples of biological 'machines' or molecular 'motors.'

Nature, 2000 Aug 10, 406(6796), 625 - 8
Evolvability of an RNA virus is determined by its mutational neighbourhood; Burch CL et al.; The ubiquity of mechanisms that generate genetic variation has spurred arguments that evolvability, the ability to generate adaptive variation, has itself evolved in response to natural selection . The high mutation rate of RNA viruses is postulated to be an adaptation for evolvability, but the paradox is that whereas some RNA viruses evolve at high rates, others are highly stable . Here we show that evolvability in the RNA bacteriophage phi6 is also determined by the accessibility of advantageous genotypes within the mutational neighbourhood (the set of mutants one or a few mutational steps away) . We found that two phi6 populations that were derived from a single ancestral phage repeatedly evolved at different rates and toward different fitness maxima . Fitness measurements of individual phages showed that the fitness distribution of mutants differed between the two populations . Whereas population A, which evolved toward a higher maximum, had a distribution that contained many advantageous mutants, population B, which evolved toward a lower maximum, had a distribution that contained only deleterious mutants . We interpret these distributions to measure the fitness effects of genotypes that are mutationally available to the two populations . Thus, the evolvability of phi6 is constrained by the distribution of its mutational neighbours, despite the fact that this phage has the characteristic high mutation rate of RNA viruses.

Mutat Res, 2000 Aug 21, 469(1), 115 - 26
The mutagenic spectrum of acridine-linked aniline nitrogen mustards in AS52 cells: implications of DNA targeting with high selectivity for adenine or guanine bases; Ferguson LR et al.; The mutational spectra generated in AS52 cells at the gpt gene locus by aniline mustards were studied by the isolation of resistant clones and sequencing of the altered gene . A set of four aniline mustards (both mono- and bifunctional) linked to a DNA-affinic 9-aminoacridine (9-AA) carrier was used, together with the untargeted mustards chlorambucil (CHL) and its half-mustard, and the DNA binding carrier, 9-AA . Both 9-AA and CHL were weak cytotoxins, with the DNA-targeted mustards being markedly (10-40-fold) more dose potent, and the bifunctional ones somewhat more toxic than the monofunctional ones . 9-AA produced a different spectrum of mutations to the spontaneous background, with more minor addition events and less base pair substitutions, and showing for the first time that frameshift events so characteristic of 9-AA in bacteria or bacteriophage also occur in mammalian cells . The mutational spectra of the DNA-targeted mustards were quite different both from this and from the lesions caused by the untargeted mustards, which cause largely transition mutations at AT sites (despite a clear preference for formation of N(7)-guanine adducts) . There were very few transition mutations, suggesting that the initial O(6)-alkylguanine/O(4)-alkylthymine lesions considered to give rise to these are relatively rare . There was also a lower incidence of complete deletions, usually attributed to DNA cross-links . For the short chain length targeted mustards, which form initial stable adducts largely (95%) at guanine N(7) sites, base pair substitution mutations, predominantly transversions, involved AT and GC base pairs equally . In contrast, the longer chain length targeted mustards, which form >90% of initial adducts at adenine N(1) sites, generated also formed transversion mutations, but these overwhelmingly (24/27) involved AT base pairs.

Proteins, 2000 Oct 1, 41(1), 123 - 32
Measured and calculated effects of mutations in bacteriophage T4 lysozyme on interactions in solution; Chang RC et al.; Understanding the molecular determinants of protein interactions in solution has fundamental implications for understanding protein solution thermodynamics and, hence, processes as diverse as separations performance and cellular self-organization . Our earlier theoretical calculations indicate that the protein-protein interactions are dominated by a small number of configurations in which highly complementary surface regions are apposed, rather than by the overall colloidal interactions . To examine this paradigm more explicitly, we investigated the effects of protein structural modifications on protein-protein interactions . Experimental measurements are presented of B(22)(') values of a set of mutants of Ser44 in bacteriophage T4 lysozyme . Effects are seen with both charged and uncharged substitutions . The results with the charged substitutions follow the expected trends, whereas those with the uncharged substitutions may be explained by the impact of the mutations on the local protein geometry, which directly affects the complementarity of protein interactions . These effects are also captured well by molecular calculations that account for the mutations . The interaction energetics between protein pairs could provide information on the propensity for adventitious interactions, which can have important implications for separations and for normal and pathological self-assembly . Thus, protein structural data implicit in genomic information, coupled with appropriate calculational and experimental tools, can ultimately provide insights into protein interactions in vivo and in bioprocessing.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 959 - 64
Effects of crystal twinning on the ability to solve a macromolecular structure using multiwavelength anomalous diffraction; Yang F et al.; The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved by multiwavelength anomalous diffraction (MAD) for a selenomethionine (SeMet) derivative of the protein at 1.8 A resolution, using crystals in space group P2(1) {Yang et al . (2000), Nature Struct . Biol . 7, 230-237} . Subsequent analysis showed that the crystals of both the original protein and the SeMet derivative were pseudo-merohedrally twinned with a twinning fraction approximately 0.36, owing to the near-identity of the a and c axes . An analysis of the crystal structure solution is presented and the utility of twinned crystals for solving the structure using MAD and of different phasing strategies is discussed; the results obtained with several software packages are compared.

J Biol Chem, 2000 Nov 24, 275(47), 36949 - 56
Characterization of a mutation of bacteriophage lambda integrase . Putative role in core binding and strand exchange for a conserved residue; Bankhead T et al.; Site-specific recombination is involved in processes ranging from resolution of bacterial chromosome dimers to adeno-associated viral integration and is a versatile tool for mammalian genetics . The bacteriophage lambda-encoded site-specific recombinase integrase (Int) is one of the best studied site-specific recombinases and mediates recombination via four distinct pathways . We have characterized a mutant version of lambda Int, IntT236I; this mutant can perform the bent-L pathway only, whereas the corresponding IntT236A mutant can perform bent-L, excision and integration pathways . Experiments with both IntT236I and IntT236A show that the hydroxyl group of threonine is necessary for wild-type recombination . Substitution of the threonine by serine leads to nearly complete rescue of the mutant phenotypes . In addition, our data show that the IntT236I mutant is defective partially due to obstructive steric interactions . Comparisons of crystal structures reveal that the threonine at residue 236 may play an important role in stabilizing recombination intermediates through solvent-mediated protein-DNA interactions at the core-binding sites and that the hydroxyl group is important for effective cleavage and Holliday junction formation . Our data also indicate that Int contacts the core sites differently in intermediates assembled in excisive versus bent-L recombination.

Mol Cell Biol, 2000 Sep, 20(17), 6287 - 99
The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site; Del Gatto-Konczak F et al.; Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5' splice site . We show that IAS1 can activate the use of several heterologous 5' splice sites in vitro . Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5' splice sites linked to IAS1 . TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5' splice sites, only one of which is adjacent to IAS1 . Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts . This binding is stronger if IAS1 is adjacent to a 5' splice site and is U1 snRNP dependent . Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner . If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1 . Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5' splice site . Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5' splice site in a U1 snRNP-dependent fashion . TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.

Evolution Int J Org Evolution, 2000 Apr, 54(2), 686 - 91
Effect of deleterious mutation-accumulation on the fitness of RNA bacteriophage MS2; de la Pena M et al.; RNA viruses show the highest mutation rate in nature . It has been extensively demonstrated that, in the absence of purifying selection, RNA viruses accumulate deleterious mutations at a high rate . However, the parameters describing this accumulation are, in general, poorly understood . The present study reports evidences for fitness declines by the accumulation of deleterious mutations in the bacteriophage MS2 . We estimated the rate of fitness decline to be as high as 16% per bottleneck transfer . In addition, our results agree with an additive model of fitness effects.

Evolution Int J Org Evolution, 2000 Apr, 54(2), 397 - 405
Independent contrasts succeed where ancestor reconstruction fails in a known bacteriophage phylogeny; Oakley TH et al.; Methods of ancestor reconstruction are important tools for evolutionary inference that are difficult to test empirically because ancestral states are rarely known with certainty . We evaluated reconstruction methods for continuous phenotypic characters using taxa from an experimentally generated bacteriophage phylogeny . Except for one slowly evolving character, the estimated ancestral states of continuous phenotypic characters were highly inaccurate and biased, even when including a known ancestor at the root . This error was caused by a directional trend in character evolution and by rapid rates of character evolution . Computer simulations confirmed that such factors affect reconstruction of continuous characters in general . We also used phenotypic viral characters to evaluate two methods that attempt to estimate the correlation between characters during evolution . Whereas a nonphylogenetic regression was relatively inaccurate and biased, independent contrasts accurately estimated the correlation between characters with little bias.

Nat Biotechnol, 2000 Aug, 18(8), 873 - 6
Phage display of peptide epitopes from HIV-1 elicits strong cytolytic responses; De Berardinis P et al.; Although much effort has been expended on evaluating recombinant proteins and synthetic peptides as immunogens, they have generally proved incapable of inducing an efficient cytotoxic T-cell (CTL) response . Filamentous bacteriophage fd can display multiple copies of foreign peptides in the N-terminal region of its major coat protein pVIII, 2,700 copies of which make up the virus capsid . Here we show that fd virions displaying peptide RT2 (ILKEPVHGV), corresponding to residues 309-317 of the reverse transcriptase (RTase) of HIV-1, are able to prime a CTL response specific for this HIV-1 epitope in human cell lines . Successful priming also requires a T-helper epitope, pep23 (KDSWTVNDIQKLVGK), corresponding to residues 249-263 of HIV-1 RTase . Supplying this by displaying it on either the same or a separate bacteriophage virion led to activation of antigen-specific CD4+ T cells . Likewise, HLA-A2 transgenic mice immunized with bacteriophage virions displaying peptide RT2 were shown to mount an effective, specific anti-HIV-RT2 CTL response . This unexpected ability to elicit a designated cytolytic T-cell response, in addition to a B-cell response, has important implications for access to the class I major histocompatibility complex (MHC) loading compartment and the development of recombinant vaccines.

J Mol Recognit, 2000 Jul-Aug, 13(4), 167 - 87
Engineered protein scaffolds for molecular recognition; Skerra A; The use of so-called protein scaffolds has recently attracted considerable attention in biochemistry in the context of generating novel types of ligand receptors for various applications in research and medicine . This development started with the notion that immunoglobulins owe their function to the composition of a conserved framework region and a spatially well-defined antigen-binding site made of peptide segments that are hypervariable both in sequence and in conformation . After the application of antibody engineering methods along with library techniques had resulted in first successes in the selection of functional antibody fragments, several laboratories began to exploit other types of protein architectures for the construction of practically useful binding proteins . Properties like small size of the receptor protein, stability and ease of production were the focus of this work . Hence, among others, single domains of antibodies or of the immunoglobulin superfamily, protease inhibitors, helix-bundle proteins, disulphide-knotted peptides and lipocalins were investigated . Recently, the scaffold concept has even been adopted for the construction of enzymes . However, it appears that not all kinds of polypeptide fold which may appear attractive for the engineering of loop regions at a first glance will indeed permit the construction of independent ligand-binding sites with high affinities and specificities . This review will therefore concentrate on the critical description of the structural properties of experimentally tested protein scaffolds and of the novel functions that have been achieved on their basis, rather than on the methodology of how to best select a particular mutant with a certain activity . An overview will be provided about the current approaches, and some emerging trends will be identified . (c) 2000 John Wiley & Sons, Ltd . Abbreviations used: ABD albumin-binding domain of protein G APPI Alzheimer's amyloid beta-protein precursor inhibitor BBP bilin-binding protein BPTI bovine (or basic) pancreatic trypsin inhibitor BSA bovine serum albumin CBD cellulose-binding domain of cellobiohydrolase I CD circular dichroism Cdk2 human cyclin-dependent kinase 2 CDR complementarity-determining region CTLA-4 human cytotoxic T-lymphocyte associated protein-4 FN3 fibronectin type III domain GSH glutathione GST glutathione S-transferase hIL-6 human interleukin-6 HSA human serum albumin IC(50) half-maximal inhibitory concentration Ig immunoglobulin IMAC immobilized metal affinity chromatography K(D) equilibrium constant of dissociation K(i) equilibrium dissociation constant of enzyme inhibitor LACI-D1 human lipoprotein-associated coagulation inhibitor pIII gene III minor coat protein from filamentous bacteriophage f1 PCR polymerase-chain reaction PDB Protein Data Bank PSTI human pancreatic secretory trypsin inhibitor RBP retinol-binding protein SPR surface plasmon resonance TrxA E . coli thioredoxin

Mol Microbiol, 2000 Jul, 37(2), 356 - 63
Bacteriophage PRD1 DNA entry uses a viral membrane-associated transglycosylase activity; Rydman PS et al.; Amino acid sequence analyses have indicated that the amino-terminal part of bacteriophage PRD1 structural protein P7 carries a conserved transglycosylase domain . We analysed wild-type PRD1 and different mutant particles in zymograms and found a glycolytic activity that was associated with protein P7 . This is the first time a putative bacteriophage or plasmid lytic transglycosylase has been shown to have an enzymatic activity . In the absence of protein P7, the phage DNA replication and host cell lysis were delayed . Gene VII of PRD1 is known to encode proteins P7 and P14 . In this investigation, the open reading frame coding for P14 was mapped to the 3' end of gene VII . Proteins P7 and P14 probably form a heteromultimeric complex, which is located at the particle vertices and is involved in the early steps of the PRD1 life cycle

Mol Microbiol, 2000 Jul, 37(2), 345 - 55
Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection; Moak M et al.; The predicted catalytic glutamate residue for transglycosylase activity of bacteriophage T7 gp16 is not essential for phage growth, but is shown to be beneficial during infection of Escherichia coli cells grown to high cell density, conditions in which murein is more highly cross-linked . In the absence of the putative transglycosylase, internalization of the phage genome is significantly delayed during infection . The lytic transglycosylase motif of gp16 is essential for phage growth at temperatures below 20 degrees C, indicating that these growth conditions also lead to increased cross-linking of peptidoglycan . Overexpression of sltY, E . coli soluble lytic transglycosylase, partially complements the defect in infection of mutant phage particles, allowing them to infect at higher efficiencies . Conversely, an sltY deletion increases the latent period of wild-type phage.

Mol Microbiol, 2000 Jun, 36(6), 1425 - 35
A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro; Spears PA et al.; We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization . The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis . The wlb genetic locus of B . pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis . Polyacrylamide gel analysis of LPS from B . avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile . Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B . avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B . avium . Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid . Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B . pertussis, B . bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B . avium . The mutants also acquired resistance to a newly identified B . avium-specific bacteriophage, Ba1 . Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B . avium, but not the heterologous B . pertussis locus, restored sensitivity to Ba1 . Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B . parapertussis restored partial sensitivity to Ba1 . Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B . avium suggested that phage sensitivity required the presence of O-antigen . At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro . In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B . avium restored serum resistance to wild-type levels . However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant) . This is the first characterization of LPS mutants of B . avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.

J Biol Chem, 2000 Oct 27, 275(43), 33759 - 64
Specific recognition of DNA by integration host factor . Glutamic acid 44 of the beta-subunit specifies the discrimination of a T:A from an A:T base pair without directly contacting the DNA; Read EK et al.; Integration host factor (IHF) is a protein that binds to the H' site of bacteriophage lambda with sequence specificity . Genetic experiments implicated amino acid residue Glu(44) of the beta-subunit of IHF in discrimination against substitution of A for T at position 44 of the TTR submotif of the binding site (Lee, E . C., Hales, L . M., Gumport, R . I., Gardner, J . F . (1992) EMBO J., 11, 305-313) . We have extended this observation by generating all possible single-base substitutions at positions 43, 44, and 45 of the H' site . IHF failed to bind these H' site substitution mutants in vivo . The K(d)(app) value for each H' site substitution, except for H'45A mutant, was reduced >2000-fold relative to the wild-type site . Substitution of amino acid beta-Glu(44) with alanine prevented IHF from discriminating against the H'44A variant but not the other H' site substitution mutants . Further analysis with other substitutions at position beta44 demonstrated that both oxygens of the wild-type glutamic acid are necessary for discrimination of AT at position 44 . Because the beta-Glu(44) residue does not contact the DNA, this residue probably enforces binding specificity indirectly through interaction with amino acids that themselves contact the DNA.

J Mol Biol, 2000 Aug 4, 301(1), 101 - 15
Selection of peptidic mimics of digoxin from phage-displayed peptide libraries by anti-digoxin antibodies; Ball WJ Jr et al.; Since the initial report of the development of methodology to generate high-affinity digitalis-specific (digoxin) antibodies, these antibodies have proven extremely useful tools to monitor digoxin levels in digitalized patients and, as Fab fragments, to reverse toxic digoxin effects in life-threatening digoxin overdoses . These antibodies (both digoxin-specific and ouabain-specific) have been used extensively by investigators for the identification and characterization of putative endogenous digitalis-like factors . In this study, we used two well-characterized mouse anti-digoxin monoclonal antibodies (mAbs), designated 26-10 and 45-20, as binding templates with which to select short bacteriophage-displayed (pIII protein inserted) peptides that are capable of binding to these mAbs and mimicking the conformational structure of digoxin . Selective enrichment from two phage-displayed random peptide libraries enabled us to isolate and identify distinct 15 and 26 amino acid residue peptide inserts that bind with high avidity and idiotypic specificity to the selecting mAbs . Among these displayed inserts a subset was identified whose mAb binding is inhibited by digoxin and whose corresponding synthetic peptides inhibit phage binding . They, therefore, appear to bind at the mAbs digoxin-binding sites . These data provide the first clear evidence that short polypeptides can serve as surrogates for the low molecular mass hapten digoxin .

J Mol Biol, 2000 Aug 4, 301(1), 35 - 45
The internal head protein Gp16 controls DNA ejection from the bacteriophage T7 virion; Struthers-Schlinke JS et al.; A wild-type T7 virion ejects about 850 bp of the 40 kb genome into the bacterial cell by a transcription-independent process . Internalization of the remainder of the genome normally requires transcription . Inhibition of transcription-independent DNA translocation beyond the leading 850 bp is not absolute but the time taken by a population of phage genomes in overcoming the block averages about 20 minutes at 30 degrees C . There are additional blocks to transcription-independent translocation and less than 20 % of infecting DNA molecules completely penetrate the cell cytoplasm after four hours of infection . Mutant virions containing an altered gene 16 protein either prevent the blocks to transcription-independent DNA translocation or effect rapid release from blocking sites and allow the entire phage DNA molecule to enter the cell at a constant rate of about 75 bp per second . This rate is likely the same at which the leading 850 bp is ejected into the cell from a wild-type virion . All mutations fall into two clusters contained within 380 bp of the 4 kb gene 16, suggesting that a 127 residue segment of gp16 controls DNA ejection from the phage particle . We suggest that this segment of gp16 acts as a clamp to prevent transcription-independent DNA translocation .

J Biol Chem, 2000 Oct 6, 275(40), 31528 - 35
Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerase in vitro; da Silva EF et al.; DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots . We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion . The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats . DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide . We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction . Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I . The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand . These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.

Genetics, 2000 Aug, 155(4), 1493 - 504
Double-strand break repair in tandem repeats during bacteriophage T4 infection; Tomso DJ et al.; Recombinational repair of double-strand breaks in tandemly repeated sequences often results in the loss of one or more copies of the repeat . The single-strand annealing (SSA) model for repair has been proposed to account for this nonconservative recombination . In this study we present a plasmid-based physical assay that measures SSA during bacteriophage T4 infection and apply this assay to the genetic analysis of break repair . SSA occurs readily in broken plasmid DNA and is independent of the strand exchange protein UvsX and its accessory factor UvsY . We use the unique features of T4 DNA metabolism to examine the link between SSA repair and DNA replication and demonstrate directly that the DNA polymerase and the major replicative helicase of the phage are not required for SSA repair . We also show that the Escherichia coli RecBCD enzyme can mediate the degradation of broken DNA during early, but not late, times of infection . Finally, we consider the status of broken ends during the course of the infection and propose a model for SSA during T4 infections.

J Virol Methods, 2000 Jul, 88(1), 89 - 104
Utilisation of bacteriophage display libraries to identify peptide sequences recognised by equine herpesvirus type 1 specific equine sera; Birch-Machin I et al.; Three filamentous phage random peptide display libraries were used in biopanning experiments with purified IgG from the serum of a gnotobiotic foal infected with equine herpesvirus-1 (EHV-1) to enrich for epitopes binding to anti-EHV-1 antibodies . The sequences of the amino acids displayed were aligned with protein sequences of EHV-1, thereby identifying a number of potential antibody binding regions . Presumptive epitopes were identified within the proteins encoded by genes 7 (DNA helicase/primase complex protein), 11 (tegument protein), 16 (glycoprotein C), 41 (integral membrane protein), 70 (glycoprotein G), 71 (envelope glycoprotein gp300), and 74 (glycoprotein E) . Two groups of sequences, which aligned with either glycoprotein C (gC) or glycoprotein E (gE), identified type-specific epitopes which could be used to distinguish between sera from horses infected with either EHV-1 or EHV-4 in an ELISA using either the phage displaying the peptide or synthetic peptides as antigen . The gC epitope had been previously identified as an immunogenic region by conventional monoclonal antibody screening whereas the gE antibody binding region had not been previously identified . This demonstrates that screening of phage display peptide libraries with post-infection polyclonal sera is a suitable method for identifying diagnostic antigens for viral infections such as EHV-1.

J Virol Methods, 2000 Jul, 88(1), 35 - 40
Green fluorescent protein as a probe of rotational mobility within bacteriophage T4; Mullaney JM et al.; Green fluorescent protein (GFP) was targeted into bacteriophage T4 heads and proheads as a probe of the internal environment . Targeting was accomplished with internal protein III (IPIII) fusion proteins or capsid targeting sequence (CTS)-tagged proteins, where CTS is the 10-amino acid residue CTS of IPIII . Recombinant phage T4{CTS/IPIII/GFP}, T4{CTS/IPIII(T)GFP}, and T4{CTS/GFP} packaged GFP fusion proteins and processed them at cleavage sites designated / . Steady-state and time-resolved fluorescence measurements suggest that packaged GFP is concentrated to a high density, that fusion protein IPIII(T)GFP occurs in a tightly clustered arrangement, and that the internal milieu of the phage head reduces rotational mobility of GFP . Phage, but not proheads, packaged with fusion protein IPIII(T)GFP gave an unexpectedly lower anisotropy than phage and proheads packaged with GFP, which suggests IPIII(T)GFP is bound to DNA in a manner that causes close associations between GFP molecules resulting in homotransfer between fluorophores within packaged phage . Targeting of reporter proteins into active virions is a promising approach for determining the structure of the condensed DNA, and properties of encapsidated viral enzymes.

Gene Ther, 2000 Jul, 7(13), 1121 - 5
A radiation-controlled molecular switch for use in gene therapy of cancer; Scott SD et al.; Ionising radiation induces the expression of a number of radiation-responsive genes and there is current interest in exploiting this to regulate the expression of exogenous therapeutic genes in gene therapy strategies for cancer . However, the radiation-responsive promoters used in these approaches are often associated with low and transient levels of therapeutic gene expression . We describe here a novel radiation-triggered molecular switching device based on promoter elements from the radiation-responsive Egr-1 gene and the cre-LoxP site-specific recombination system of the P1 bacteriophage . Using this system, a single, minimally toxic dose of radiation induced cre-mediated excision of a lox-P flanked stop cassette in a silenced expression vector and this resulted in amplified levels of CMV-promoter-driven expression of the exogenous tumour-sensitising gene, HSV-tk . This strategy could be used in combination with targeted delivery and tumour-specific promoters to elicit the tumour-targeted and prolonged expression of a variety of tumour-sensitising genes and provide an unprecedented level of control and tumour selectivity.

Curr Microbiol, 2000 Sep, 41(3), 157 - 60
Formation and stability of the bacteriophage lambda replication complexes in UV-irradiated Escherichia coli; Wegrzyn A et al.; Bacteriophage lambda replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter lambda DNA copies after a replication round in Escherichia coli . In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage lambda, such a complex may be stable for many cell generations . However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock . Therefore, we asked whether other environmental stresses may cause disassembly of the lambda replication complex . We found that UV irradiation of the host cells prevented formation of the stable lambda replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation . These results indicate that the stable lambda replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of lambda replication complexes is possible . Both stable and unstable lambda replication complexes are functional because replication of lambda DNA under conditions preventing formation of the stable complex proceeds efficiently.

J Infect Dis, 2000 Aug, 182(2), 435 - 41 Epub 2000 Jul 21.
Evaluation of CD4+ T cell function In vivo in HIV-infected patients as measured by bacteriophage phiX174 immunization; Fogelman I et al.; Bacteriophage phiX174 immunization was used to measure CD4(+) T cell function in vivo in human immunodeficiency virus (HIV)-infected patients across all disease stages . Function was evaluated by measuring the ability of T cells to provide help to B cells in antibody production, amplification, and isotype switching . A total of 33 patients and 10 controls received 3 bacteriophage phiX174 immunizations 6 weeks apart . The patients' responses regarding bacteriophage-specific total antibody titers and IgG titers were quantitatively and qualitatively inferior to the controls' responses . Overall, 7 of 33 patients had normal T cell function . Baseline CD4 counts provided the strongest correlation with total antibody and IgG titers . HIV RNA had a weaker association with responses but had some predictive power among patients with a CD4 count >200 cells/microL . Bacteriophage phiX174 immunization seems to be a useful tool for measuring immune function in vivo, which suggests that most HIV-infected patients may have abnormal CD4(+) T cell function despite adequate antiretroviral treatment.

J Leukoc Biol, 2000 Jul, 68(1), 58 - 64
Monitoring of neutrophil priming in whole blood by antibodies isolated from a synthetic phage antibody library; Koenderman L et al.; Neutrophil activation is a multistep process . In vitro activation of neutrophils with semiphysiological activators is optimal only after preactivation or priming with cytokines, chemotaxins, and/or bacterial products . Until now, no antibodies have been developed that can distinguish between resting and (cytokine) primed neutrophils with a sufficient dynamic range necessary for screening clinical samples . We have isolated two human phage antibodies, designated MoPhab A17 and A27, from a synthetic bacteriophage antibody library . These phage antibodies recognize epitopes that are upregulated on neutrophils present in whole blood treated with low priming concentrations of cytokines, such as GM-CSF and TNF-alpha . This induction was time- and concentration-dependent and optimal at concentrations that are sufficient for priming functional responses in neutrophils: GM-CSF (10 pM) and TNF-alpha (100 IU/ml) . PMNs, isolated from the peripheral blood of chronic obstructive pulmonary disease (COPD) patients with a clinical exacerbation, exhibited a partial in vivo primed phenotype . These antibodies promise to be an ideal tool to monitor disease activity in whole blood of patients with inflammatory diseases.

FEBS Lett, 2000 Jul 7, 476(3), 224 - 8
Probing sugar translocation through maltoporin at the single channel level; Bezrukov SM et al.; Sugar permeation through maltoporin of Escherichia coli, a trimer protein that facilitates maltodextrin translocation across outer bacterial membranes, was investigated at the single channel level . For large sugars, such as maltohexaose, elementary events of individual sugar molecule penetration into the channel were readily observed . At small sugar concentrations an elementary event consists of maltoporin channel closure by one third of its initial conductance in sugar-free solution . Statistical analysis of such closures at higher sugar concentrations shows that all three pores of the maltoporin channel transport sugars independently . Interestingly, while channel conductance is only slightly asymmetric showing about 10% higher values at -200 mV than at +200 mV (from the side of protein addition), asymmetry in dependence of the sugar binding constant on the voltage polarity is about 20 times higher . Combining our data with observations made with bacteriophage-lambda we conclude that the sugar residence time is much more sensitive to (and is decreased by) voltages that are negative from the intra-cell side of the bacterial membrane.

J Biol Chem, 2000 Oct 6, 275(40), 31496 - 504
Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems; Villemain JL et al.; The gene 32 protein (gp32) of bacteriophage T4 is the essential single-stranded DNA (ssDNA)-binding protein required for phage DNA replication and recombination . gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by DNA polymerase or recombination enzymes . The precise roles of gp32 affinity and cooperativity in promoting replication and recombination have yet to be defined, however . Previous work established that the N-terminal "B-domain" of gp32 is essential for cooperativity and that point mutations at Arg(4) and Lys(3) positions have varying and dramatic effects on gp32-ssDNA interactions . Therefore, we examined the effects of six different gp32 B-domain mutants on T4 in vitro systems for DNA synthesis and homologous pairing . We find that the B-domain is essential for gp32's stimulation of these reactions . The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e . wild-type gp32 approximately R4K > K3A approximately R4Q > R4T > R4G gp32-B . However, the functional defect of a particular mutant is often greater than can be explained simply by its ability to saturate the ssDNA at equilibrium, suggesting additional defects in the proper assembly and activity of DNA polymerase and recombinase complexes on ssDNA, which may derive from a decreased lifetime of gp32-ssDNA clusters.

J Mol Biol, 2000 Jul 28, 300(5), 1057 - 65
Mutations induced by bacteriophage T7 RNA polymerase and their effects on the composition of the T7 genome; Beletskii A et al.; We show here that transcription by the bacteriophage T7 RNA polymerase increases the deamination of cytosine bases in the non-transcribed strand to uracil, causing C to T mutations in that strand . Under optimal conditions, the mutation frequency increases about fivefold over background, and is similar to that seen with the Escherichia coli RNA polymerase . Further, we found that a mutant T7 RNA polymerase with a slower rate of elongation caused more cytosine deaminations than its wild-type parent . These results suggest that promoting cytosine deamination in the non-transcribed strand is a general property of transcription in E . coli and is dependent on the length of time the transcription bubble stays open during elongation . To see if transcription-induced mutations have influenced the evolution of bacteriophage T7, we analyzed its genome for a bias in base composition . Our analysis showed a significant excess of thymine over cytosine bases in the highly transcribed regions of the genome . Moreover, the average value of this bias correlated well with the levels of transcription of different genomic regions . Our results indicate that transcription-induced mutations have altered the composition of bacteriophage T7 genome and suggest that this may be a significant force in genome evolution .

Chemosphere, 2000 Oct, 41(8), 1279 - 86
Forces dictating colloidal interactions between viruses and soil; Chattopadhyay S et al.; The fate and transport of viruses in soil and aquatic environments were studied with respect to the different forces involved in the process of sorption of these viruses on soil particles . In accordance with the classical DLVO theory, we have calculated the repulsive electrostatic forces and the attractive van der Waals forces . Bacteriophages have been used as model sorbates, while different clays have been used as model sorbents . The equations used for the determination of the change in free energy for the process (deltaG) takes into consideration the roughness of the sorbent surfaces . Results indicate that attractive van der Waals forces predominate the process of sorption of the selected bacteriophages on clays.

Ultramicroscopy, 2000 Jul, 84(1-2), 85 - 99
Mapping and fuzzy classification of macromolecular images using self-organizing neural networks; Pascual A et al.; In this work the effectiveness of the fuzzy kohonen clustering network (FKCN) in the unsupervised classification of electron microscopic images of biological macromolecules is studied . The algorithm combines Kohonen's self-organizing feature maps (SOFM) and Fuzzy c-means (FCM) in order to obtain a powerful clustering technique with the best properties inherited from both . Exploratory data analysis using SOFM is also presented as a step previous to final clustering . Two different data sets obtained from the G40P helicase from B . Subtilis bacteriophage SPP1 have been used for testing the proposed method, one composed of 2458 rotational power spectra of individual images and the other composed by 338 images from the same macromolecule . Results of FKCN are compared with self-organizing feature maps (SOFM) and manual classification . Experimental results prove that this new technique is suitable for working with large, high-dimensional and noisy data sets and, thus, it is proposed to be used as a classification tool in electron microscopy.

Biomol Eng, 2000 Jun, 16(6), 191 - 7
Bacteriophage SP6 RNA polymerase mutants with altered termination efficiency and elongation processivity; Yoo J et al.; An Escherichia coli strain containing two plasmids was developed for in vivo isolation of the phage SP6 RNA polymerase mutants . It was developed to isolate mutants with increased proficiency of termination at the SP6 terminator and/or with reduced elongation processivity . Mutations were randomly introduced into an N-terminal third of the polymerase gene that was placed under a lac promoter in one plasmid . In the other plasmid, a promoter-lacking lacZ gene modified for reduced translation efficiency was placed downstream of a tandem pair of the SP6 terminator located downstream of an SP6 promoter-chloramphenicol acetyltransferase gene . Termination-up mutants were selected in vivo as they rendered LacZ activity level lower than the wild-type, without reducing chloramphenicol resistance substantially . Three such mutants (M15L, M15S, and D117G) were purified, and their termination efficiencies were measured in vitro at two different intrinsic termination signals in the E . coli rrnB terminator t1 that are different in requiring RNA hairpin formation . All three mutations enhanced termination efficiencies in vitro at the SP6 terminator and the upstream signal of rrnB t1, but reduced the efficiency at the downstream signal of it . All the mutations reduced elongation processivity, as the mutants produced much less amounts of large transcripts (2.1 kb) than the wild-type but the similar amounts of small transcripts (up to 670 nt) . Thus, the mutations, all reducing elongation processivity of the polymerase, exhibited opposite effects on the two types of intrinsic termination signals, suggesting that the two mechanisms involve different interactions with the phage RNA polymerase.

J Biol Chem, 2000 Sep 15, 275(37), 28593 - 8
Overproduction of acetyl-CoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli; Davis MS et al.; Acetyl-CoA carboxylase (ACC) catalyzes the first committed step of the fatty acid synthetic pathway . Although ACC has often been proposed to be a major rate-controlling enzyme of this pathway, no direct tests of this proposal in vivo have been reported . We have tested this proposal in Escherichia coli . The genes encoding the four subunits of E . coli ACC were cloned in a single plasmid under the control of a bacteriophage T7 promoter . Upon induction of gene expression, the four ACC subunits were overproduced in equimolar amounts . Overproduction of the proteins resulted in greatly increased ACC activity with a concomitant increase in the intracellular level of malonyl-CoA . The effects of ACC overexpression on the rate of fatty acid synthesis were examined in the presence of a thioesterase, which provided a metabolic sink for fatty acid overproduction . Under these conditions ACC overproduction resulted in a 6-fold increase in the rate of fatty acid synthesis.

J Biol Chem, 2000 Sep 22, 275(38), 29749 - 53
Degradation of HIV-1 integrase by the N-end rule pathway; Mulder LC et al.; Human immunodeficiency virus type-1 (HIV-1) integrase catalyzes the irreversible insertion of the viral genome into host chromosomal DNA . We have developed a mammalian expression system for the synthesis of authentic HIV-1 integrase in the absence of other viral proteins . Integrase, which bears a N-terminal phenylalanine, was found to be a short-lived protein in human embryo kidney 293T cells . The degradation of integrase could be suppressed by proteasome inhibitors . N-terminal phenylalanine is recognized as a degradation signal by a ubiquitin-proteasome proteolytic system known as the N-end rule pathway . The replacement of N-terminal phenylalanine with methionine, valine, or glycine, which are stabilizing residues in the N-end rule, resulted in metabolically stabilized integrase proteins (half-life of N-terminal Met-integrase was at least 3 h) . Conversely, the substitution of N-terminal phenylalanine with other destabilizing residues retained the metabolic instability of integrase . These findings indicate that the HIV-1 integrase is a physiological substrate of the N-end rule . We discuss a possible functional similarity to the better understood turnover of the bacteriophage Mu transposase and functions of integrase instability to the maintenance and integrity of the host cell genome.

Cell, 2000 Jun 9, 101(6), 589 - 600
Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides; Singleton MR et al.; We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7 . The structure reveals how subunit contacts stabilize the hexamer . Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway . The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding . The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer . This model suggests a mechanism for DNA translocation.

Cell, 2000 Jun 23, 101(7), 801 - 11
Crystal structure of the lambda repressor C-terminal domain provides a model for cooperative operator binding; Bell CE et al.; Interactions between transcription factors bound to separate operator sites commonly play an important role in gene regulation by mediating cooperative binding to the DNA . However, few detailed structural models for understanding the molecular basis of such cooperativity are available . The c1 repressor of bacteriophage lambda is a classic example of a protein that binds to its operator sites cooperatively . The C-terminal domain of the repressor mediates dimerization as well as a dimer-dimer interaction that results in the cooperative binding of two repressor dimers to adjacent operator sites . Here, we present the x-ray crystal structure of the lambda repressor C-terminal domain determined by multiwavelength anomalous diffraction . Remarkably, the interactions that mediate cooperativity are captured in the crystal, where two dimers associate about a 2-fold axis of symmetry . Based on the structure and previous genetic and biochemical data, we present a model for the cooperative binding of two lambda repressor dimers at adjacent operator sites.

Microbiology, 2000 Jul, 146 ( Pt 7), 1651 - 60
Microvirus of chlamydia psittaci strain guinea pig inclusion conjunctivitis: isolation and molecular characterization; Hsia RC et al.; The authors report the isolation and molecular characterization of a bacteriophage, φCPG1, which infects CHLAMYDIA: psittaci strain Guinea pig Inclusion Conjunctivitis . Purified virion preparations contained isometric particles of 25 nm diameter, superficially similar to spike-less members of the φX174 family of bacteriophages . The single-stranded circular DNA genome of φCPG1 included five large ORFs, which were similar to ORFs in the genome of a previously described CHLAMYDIA: bacteriophage (Chp1) that infects avian C . psittaci . Three of the ORFs encoded polypeptides that were similar to those in a phage infecting the mollicute Spiroplasma melliferum, a pathogen of honeybees . Lesser sequence similarities were seen between two ORF products and the major capsid protein of the φX174 coliphage family and proteins mediating rolling circle replication initiation in phages, phagemids and plasmids . Phage φCPG1 is the second member of the genus CHLAMYDIAMICROVIRUS:, the first to infect a member of a CHLAMYDIA: species infecting mammals . Similarity searches of the nucleotide sequence further revealed a highly conserved (75% identity) 375 base sequence integrated into the genome of the human pathogen Chlamydia pneumoniae . This genomic segment encodes a truncated 113 residue polypeptide, the sequence of which is 72% identical to the amino-terminal end of the putative replication initiation protein of φCPG1 . This finding suggests that C . pneumoniae has been infected by a phage related to φCPG1 and that infection resulted in integration of some of the phage genome into the C . pneumoniae genome.

J Neurogenet, 1996 Dec, 11(1-2), 59 - 79
Drosophila rosA gene, which when mutant causes aberrant photoreceptor oscillation, encodes a novel neurotransmitter transporter homologue; Burg MG et al.; The Drosophila receptor oscillation A (rosA) mutations, which cause electroretinogram (ERG) defects, including oscillations, were localized to the 24F4-25A2 region of chromosome 2L . Genomic fragments from this region, isolated from bacteriophage P1 clones, included those that detect transcriptional defects in rosA mutants in RNA blot experiments . One of these genomic fragments was used to screen a head cDNA library . The largest cDNA clone (3.6 kb) isolated was shown to rescue a rosA mutant in P element-germline transformation experiments . The ROSA protein deduced from the open reading frame in the 3.6 kb rosA cDNA is 943 amino acids long and is 36-41% identical to members of the superfamily of Na+/Cl(-)-dependent neurotransmitter transporters, with no indication of higher sequence identity to any one subgroup within the superfamily . RNA blot experiments revealed multiple transcripts in various developmental stages, the most abundant one being a 3.7 kb transcript, particularly in the adult head . Tissue in situ experiments identified the rosA transcript to be localized to many tissues, with higher levels of hybridization in the nervous system and digestive tract . The results demonstrate that the rosA gene encodes a novel Na+/Cl(-)-dependent transporter important for normal response properties of the photoreceptor.

Biochem Biophys Res Commun, 2000 Jul 5, 273(2), 528 - 31
Epsilon as an initiator of translation of CAT mRNA in Escherichia coli; Golshani A et al.; Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region . It enhances translation in Escherichia coli via base pairing with nucleotides 458-466 located in the helical domain #17 of 16S rRNA . We have recently reported that when the complementarity to 16S rRNA is extended, the epsilon is converted from an enhancer to an independent initiator of translation . Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of epsilon in E . coli cells . Our results show that epsilon displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon . Under these conditions its efficiency is comparable with that of the consensus Shine-Dalgarno sequence .

J Mol Biol, 2000 Jul 7, 300(2), 307 - 20
Homodimeric peptides displayed by the major coat protein of filamentous phage; Zwick MB et al.; Peptide libraries displayed by filamentous bacteriophage have proven a powerful tool for the discovery of novel peptide agonists, antagonists and epitope mimics . Most phage-displayed peptides are fused to the N terminus of either the minor coat protein, pIII, or the major coat protein, pVIII . We report here that peptides containing cysteine residues, displayed as N-terminal fusions to pVIII, can form disulfide-bridged homodimers on the phage coat . Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodimers by SDS-PAGE analysis that involved pretreatment of the phage with reducing or thiol-modifying agents . For all phage whose recombinant peptide contained a single Cys residue, a significant fraction of the peptide-pVIII molecules were displayed as dimers on the phage coat . The dimeric form was in greater abundance than the monomer in almost all cases in which both forms could be reliably observed . Occasionally, peptides containing two Cys residues also formed dimers . These results indicate that, for a given pVIII-displayed peptide bearing a single Cys residue, a significant fraction of the peptide (>40 %) will dimerize regardless of its sequence; however, sequence constraints probably determine whether all of the peptide will dimerize . Similarly, only occasionally do peptides bearing two Cys residues form intermolecular disulfide bridges instead of intramolecular ones; this indicates that sequence constraints may also determine dimerization versus cyclization . Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is present in the cell prior to its assembly onto phage . A model of the peptide-pVIII homodimer is discussed in light of existing models of the structure and assembly of the phage coat . The unique secondary structures created by the covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive peptides .

J Mol Biol, 2000 Jun 23, 299(5), 1203 - 16
Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases; Klemm M et al.; The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway . For the site-specific recombination reactions catalyzed by the bacteriophage lambda tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals . By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates . Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone . The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways . The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int . The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase . They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes . The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme . Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway .

J Mol Biol, 2000 Jun 23, 299(5), 1193 - 202
Dissection of bacteriophage lambda site-specific recombination using synthetic peptide combinatorial libraries; Cassell G et al.; A wide variety of tools have been used to dissect biochemical pathways, inhibitors being chief among them . Combinatorial approaches have made the search for inhibitors much more efficient . We have applied such an approach to identify hexapeptides which inhibit different steps in a site-specific recombination reaction mediated by the bacteriophage lambda integrase protein . Integrase's mechanism is still incompletely understood, in large part because several pathway intermediates remain hard to isolate . Integrase-catalyzed recombination is very efficient, but if blocked, it is highly reversible to substrates; this combination makes some intermediates exceedingly transient . We have used synthetic peptide combinatorial libraries to screen for hexapeptides that affect the recombination pathway at different stages, and have identified two families of peptides: one probably blocks DNA cleavage, the other may stabilize the Holliday junction intermediates . These peptides do not resemble parts of integrase or any of the other helper functions in the pathway . The deconvolution of hexapeptide libraries based both on inhibition of an enzymatic reaction as well as on accumulation of reaction intermediates is a novel approach to finding useful tools for dissecting a biochemical pathway .

EMBO J, 1983, 2(8), 1357 - 65
Evidence for a repeating cross-beta sheet structure in the adenovirus fibre; Green NM et al.; The amino acid sequence of the adenovirus fibre protein reveals an approximately repeating motif of 15 residues . A diagonal comparison matrix established that these repeats extended from residue 43 to residue 400 of the 581 residue sequence . Assignment of secondary structure combined with model building showed that each 15-residue segment contained two short beta-strands and two beta-bends, one of which incorporated an extra residue in a beta-bulge of the Gx type . The 44 strands together gave a long (210 A) narrow, amphipathic beta-sheet, which could be stabilised by dimer formation to give the shaft of the fibre . The knob could arise from a dimer of the C-terminal 180 residue segment, predicted to be an 8-10 stranded beta-sandwich . This model is consistent with the electron micrographs of the fibre and it was supported by measurements of c.d . and of electron diffraction from microcrystals . The latter gave a pair of wide angle arcs, corresponding to a repeat of 4.7 A, oriented appropriately for a cross-beta structure . The relation of this structure to globular structures is discussed and a folding pathway is proposed . In its general features the structure resembles that proposed for the tail fibre of bacteriophage T4.

J Biol Chem, 2000 Sep 15, 275(37), 29113 - 22
Establishment of lysogeny in bacteriophage 186 . DNA binding and transcriptional activation by the CII protein; Shearwin KE et al.; The CII protein of bacteriophage 186 is a transcriptional activator of the helix-turn helix family required for establishment of the lysogenic state . DNA binding by 186 CII is unusual in that the invertedly repeated half sites are separated by 20 base pairs, or two turns of the DNA helix, rather than the one turn usually associated with this class of proteins . Here, we investigate quantitatively the DNA binding properties of CII and its interaction with RNA polymerase at the establishment promoter, p(E) . The stoichiometry of CII binding was determined by sedimentation equilibrium experiments using a fluorescein-labeled oligonucleotide and purified CII . These experiments indicate that the CII species bound to DNA is a dimer, with additional weak binding of a tetrameric species at high concentrations . Examination of the thermodynamic linkages between CII self-association and DNA binding shows that CII binds to the DNA as a preformed dimer (binding free energy, 9.9 kcal/mol at 4 degrees C) rather than by association of monomers on the DNA . CII binding induces in the DNA a bend of 41 (+/- 5) degrees . The spacing between the binding half sites was shown to be important for CII binding, insertion or removal of just 1 base pair significantly reducing the affinity for CII . Removal of 5 or 10 base pairs between binding half sites eliminated binding, as did insertion of an additional 10 base pairs . CII binding at p(E) was improved marginally by the presence of RNA polymerase (DeltaDeltaG = -0.5 (+/- 0.3) kcal/mol) . In contrast, the binding of RNA polymerase at p(E) was undetectable in the absence of CII but was improved markedly by the presence of CII . Thus, CII appears to recruit RNA polymerase to the promoter . The nature of the base pair changes in mutant phage, selected by their inability to establish lysogeny, are consistent with this mechanism of CII action.

J Biol Chem, 2000 Sep 1, 275(35), 27145 - 54
Interaction of the bacteriophage T4 gene 59 helicase loading protein and gene 41 helicase with each other and with fork, flap, and cruciform DNA; Jones CE et al.; Bacteriophage T4 gene 59 helicase loading protein accelerates the loading of T4 gene 41 DNA helicase and is required for recombination-dependent DNA replication late in T4 phage infection . The crystal structure of 59 protein revealed a two-domain alpha-helical protein, whose N-terminal domain has strong structural similarity to the DNA binding domain of high mobility group family proteins (Mueser, T . C., Jones, C . E., Nossal, N . G., and Hyde, C . C . (2000) J . Mol . Biol . 296, 597-612) . We have previously shown that 59 protein binds preferentially to fork DNA . Here we show that 59 protein binds to completely duplex forks but cannot load the helicase unless there is a single-stranded gap of more than 5 nucleotides on the fork arm corresponding to the lagging strand template . Consistent with the roles of these proteins in recombination, we find that 59 protein binds to and stimulates 41 helicase activity on Holliday junction DNA, and on a substrate that resembles a strand invasion structure . 59 protein forms a stable complex with wild type 41 helicase and fork DNA in the presence of adenosine 5'-O-(thiotriphosphate) . The unwinding activity of 41 helicase missing 20 C-terminal amino acids is not stimulated by 59 protein, and it does not form a complex with 59 protein on fork DNA.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7760 - 5
Assembly and activation of site-specific recombination complexes; Pena CE et al.; Site-specific recombination is responsible for a broad range of biological phenomena, including DNA inversion, resolution of transposition intermediates, and the integration and excision of bacteriophage genomes . Integration of mycobacteriophage L5 is catalyzed by a phage-encoded integrase with recombination occurring between specific attachment sites on the phage and mycobacterial chromosomes (attP and attB, respectively) . Although some site-specific recombination systems simply involve binding of the recombinase to the sites of strand exchange, synapsis, and recombination, phage systems typically require the assembly of higher-order structures within which the recombinational potential of integrase is activated . The requirement for these structures derives from the necessity to regulate the directionality of recombination-either integration or excision-which must be closely coordinated with other aspects of the phage growth cycles . We show herein that there are multiple pathways available for the assembly of L5 recombination complexes, including the early synapsis of the attP and attB DNAs . This process is in contrast to the model for lambda integration and illustrates the different usage of molecular machineries to accomplish the same biological outcome.

Izv Akad Nauk Ser Biol, 2000 May-Jun, (3), 361 - 7
{Protist plankton from the Khanka lake}; Ladygina VP et al.; We present data on studies of protists in Lake Khanka in late August-early September . A total of 99 species of protists have been found, of them 14 phytoflagellates, 14 zooflagellates, five sarcodins, two heliozoans, 62 infusoria, and two suctorians . Small infusoria from the class of Kinetophragminophora, order Oligotrichida, as well as suborder Tetrahymenina were most numerous . The density of many protist species in Lake Khanka exceeds markedly that in several other high-productivity water reservoirs . Most of the protists belong to bacteriovores (bacteriophages) . Many of the found species have high density . This is evidence of the production of bacterial plankton in the lake . In view of very high water turbidity in the lake, we assume that the total production of phytoplankton is not that high . In this connection, an abundance of protists (protist plankton) indicates is an indication for the existence of an additional source of organic matter in Lake Khanka.

J Virol, 2000 Jul, 74(14), 6643 - 7
Recovery of pathogenic measles virus from cloned cDNA; Takeda M et al.; Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models . On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F . Kobune et al., J . Virol . 64:700-705, 1990) . Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells . Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches . One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3 . The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F . Radecke et al., EMBO J . 14:5773-5784, 1995) on which B95a cells were overlaid . Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys . Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.

J Mol Biol, 2000 Jun 30, 300(1), 213 - 9
Design and evolution of artificial M13 coat proteins; Weiss GA et al.; Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein . M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus . The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation . ACP is a non-functional coat protein because it is not required for the production of phage particles . Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity . In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides . The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions . In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display .

RNA, 2000 Jun, 6(6), 880 - 9
RNA secondary structures of the bacteriophage phi6 packaging regions; Pirttimaa MJ et al.; Bacteriophage phi6 genome consists of three segments of double-stranded RNA . During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles . Only phi6 RNA is packaged, and each particle contains only one copy of each segment . An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments . In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments . Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons . Previously reported pac site deletion studies are also discussed . Each pac site possesses a unique architecture, that, however, contains common structural elements.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7196 - 201
Characterization of bacteriophage T4-coordinated leading- and lagging-strand synthesis on a minicircle substrate; Salinas F et al.; The DNA replication complex of bacteriophage T4 has been assembled as a single unit on a minicircle substrate with a replication fork that permits an independent measurement of the amount of DNA synthesis on both the leading and lagging strands . The assembled replisome consists of the T4 polymerase {gene product 43 (gp43)}, clamp protein (gp45), clamp loader (gp44/62), helicase (gp41), helicase accessory factor (gp59), primase (gp61), and single-stranded DNA binding protein (gp32) . We demonstrate that on the minicircle the synthesis of the leading and lagging strands are coordinated and that the C-terminal domain of the gp32 protein regulates this coordination . We show that the reconstituted replisome encompasses two coupled holoenzyme complexes and present evidence that this coupling might include a gp43 homodimer interaction.

Virology, 2000 Jun 5, 271(2), 321 - 33
Molecular architecture of bacteriophage T4 capsid: vertex structure and bimodal binding of the stabilizing accessory protein, Soc; Iwasaki K et al.; T4 encodes two dispensable proteins that bind to the outer surface of the mature capsid . Soc (9 kDa) stabilizes the capsid against extremes of alkaline pH and temperature, but Hoc (40 kDa) has no perceptible effect . Both proteins have been developed as display platforms . Their positions on the hexagonal surface lattice of gp23*, the major capsid protein, were previously defined by two-dimensional image averaging of negatively stained electron micrographs of elongated variant capsids . We have extended these observations by reconstructing cryo-electron micrographs of isometric capsids produced by a point mutant in gene 23, for both Hoc+.Soc+ and Hoc+.Soc- phages . The expected T = 13 lattice was observed, with a single Hoc molecule at the center of each gp23* hexamer . The vertices are occupied by pentamers of gp24*: despite limited sequence similarity with gp23*, the respective monomers are similar in size and shape, suggesting they may have the same fold . However, gp24* binds neither Hoc nor Soc; in situ, Soc is visualized as trimers at the trigonal points of the gp23* lattice and as monomers at the sites closest to the vertices . In solution, Soc is a folded protein ( approximately 10% alpha-helix and 50-60% beta sheet) that is monomeric as determined by analytic ultracentrifugation . Thus its trimerization on the capsid surface is imposed by a template of three symmetry-related binding sites . The observed mode of Soc binding suggests that it stabilizes the capsid by a clamping mechanism and offers a possible explanation for the phenotype of osmotic shock resistance .

Virology, 2000 Jun 5, 271(2), 298 - 306
Forced retroevolution of an RNA bacteriophage; Licis N et al.; The operator hairpin ahead of the replicase gene in RNA bacteriophage MS2 contains overlapping signals for binding the coat protein and ribosomes . Coat protein binding inhibits further translation of the gene and forms the first step in capsid formation . The hairpin sequence was partially randomized to assess the importance of this structure element for the bacteriophage and to monitor alternative solutions that would evolve on the passaging of mutant phages . The evolutionary reconstruction of the operator failed in the majority of mutants . Instead, a poor imitation developed containing only some of the recognition signals for the coat protein . Three mutants were of particular interest in that they contained double nonsense codons in the lysis reading frame that runs through the operator hairpin . The simultaneous reversion of two stop codons into sense codons has a very low probability of occurring . Therefore the phage solved the problem by deleting the nonsense signals and, in fact, the complete operator, except for the initiation codon of the replicase gene . Several revertants were isolated with activities ranging from 1% to 20% of wild type . The operator, long thought to be a critical regulator, now appears to be a dispensable element . In addition, the results indicate how RNA viruses can be forced to step back to an attenuated form .

J Mol Biol, 2000 Jun 2, 299(2), 447 - 62
Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp . 9 degrees N-7; Rodriguez AC et al.; The 2.25 A resolution crystal structure of a pol alpha family (family B) DNA polymerase from the hyperthermophilic marine archaeon Thermococcus sp . 9 degrees N-7 (9 degrees N-7 pol) provides new insight into the mechanism of pol alpha family polymerases that include essentially all of the eukaryotic replicative and viral DNA polymerases . The structure is folded into NH(2)- terminal, editing 3'-5' exonuclease, and polymerase domains that are topologically similar to the two other known pol alpha family structures (bacteriophage RB69 and the recently determined Thermococcus gorgonarius), but differ in their relative orientation and conformation.The 9 degrees N-7 polymerase domain structure is reminiscent of the "closed" conformation characteristic of ternary complexes of the pol I polymerase family obtained in the presence of their dNTP and DNA substrates . In the apo-9 degrees N-7 structure, this conformation appears to be stabilized by an ion pair . Thus far, the other apo-pol alpha structures that have been determined adopt open conformations . These results therefore suggest that the pol alpha polymerases undergo a series of conformational transitions during the catalytic cycle similar to those proposed for the pol I family . Furthermore, comparison of the orientations of the fingers and exonuclease (sub)domains relative to the palm subdomain that contains the pol active site suggests that the exonuclease domain and the fingers subdomain of the polymerase can move as a unit and may do so as part of the catalytic cycle . This provides a possible structural explanation for the interdependence of polymerization and editing exonuclease activities unique to pol alpha family polymerases.We suggest that the NH(2)-terminal domain of 9 degrees N-7 pol may be structurally related to an RNA-binding motif, which appears to be conserved among archaeal polymerases . The presence of such a putative RNA- binding domain suggests a mechanism for the observed autoregulation of bacteriophage T4 DNA polymerase synthesis by binding to its own mRNA . Furthermore, conservation of this domain could indicate that such regulation of pol expression may be a characteristic of archaea . Comparion of the 9 degrees N-7 pol structure to its mesostable homolog from bacteriophage RB69 suggests that thermostability is achieved by shortening loops, forming two disulfide bridges, and increasing electrostatic interactions at subdomain interfaces .

J Mol Biol, 2000 Jun 2, 299(2), 337 - 49
Changes in the 17 bp spacer in the P(R) promoter of bacteriophage lambda affect steps in open complex formation that precede DNA strand separation; McKane M et al.; Tau plots and temperature-shift experiments were used to determine which step in the formation of transcriptionally-competent open complexes is affected by changing the length of the 17 bp spacer separating the -10 and -35 consensus regions of the P(R) promoter of bacteriophage lambda . Abortive initiation assays at 37 degrees C indicate that the primary effect of insertion of a base-pair, thereby increasing spacer length to 18 bp, is a decrease in k(f), the rate constant for conversion from closed (RP(c)) to open (RP(o)) complexes, by approximately a factor of 4 . The mutation did not significantly affect K(B), the equilibrium constant for formation of closed complexes, and decreased K(B)k(f) by a factor of 3 . Deletion of a bp to create a 16 bp spacer had a much greater effect, decreasing the measured value of k(f) by a factor of about 25 to 30, and K(B)k(f) by a factor of 7 to 8 . When the values of the parameters for the deletion mutant were corrected for incomplete occupancy of RP(o) at equilibrium, the effects of the deletion were even greater . In particular, the corrected value of K(B)k(f) was about 15 times lower than the corresponding value for two promoters with wild-type spacing . Based on temperature shift experiments, the changes in spacer length did not affect the equilibrium at 20 degrees C between RP(i), a stable intermediate in which DNA strands are not separated, and RP(o) . Although differential sensitivity of single-stranded bases to KMnO(4) indicated that in about 20% of the open complexes at 20 degrees C the DNA strands are not fully separated (RP(o1)), the distribution between these complexes and RP(o2) (DNA strands fully separated) was also not affected significantly by changes in spacer length . Thus, changes in spacer length primarily affect k(2), the rate constant for conversion of RP(c) to RP(i), which corresponds to a nucleation of DNA strand-separation . Application of published data and/or algorithms for determining effects of nucleotide sequence on twist angle or rise at individual bp steps does not provide a simple explanation of the difference in promoter strength between P(R) derivatives with 16 bp spacing and those with 18 bp spacing .

J Mol Biol, 2000 May 26, 299(1), 53 - 73
Genomic sequence and analysis of the atypical temperate bacteriophage N15; Ravin V et al.; N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli . While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres . Here, we describe the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens . The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms . The left arm contains a long cluster of structural genes that are closely related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB . The right arm likewise contains a mixture of apparently phage- and plasmid-derived genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases . The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized.

Mol Gen Genet, 2000 May, 263(4), 619 - 24
Core-binding specificity of bacteriophage integrases; Gottfried P et al.; The site-specific recombination systems of bacteriophages lambda and HK022 share the same mechanism and their integrase proteins show strong homology . Nevertheless the integrase protein of each phage can only catalyze recombination between its own att sites . Previous work has shown that the specificity determinants in the att sites are located within the sequences that bind the integrase to the core of att . DNA fragments that carry attL and attR sites of each phage were challenged with each of the two integrases and the DNA-protein complexes were examined by the gel-retardation technique . The results show that each integrase can form higher-order DNA-protein complexes only with its cognate att sites, suggesting that differences in the mode of binding to the core are responsible for the specificity difference between the two integrases.

Mol Gen Genet, 2000 May, 263(4), 592 - 600
The bacteriophage D108 Ner repressor binds a conformationally distinct operator; Kukolj G et al.; The Ner protein encoded by the transposable coliphage D108, an 8.6 kDa lambda Cro-like repressor, binds to an operator spanning 50 bp of DNA . The distinguishing features of this operator are two perfect 11-bp inverted repeats (5'-CCGTGAGCTAC-3') that are separated by an 8-bp AT-rich spacer . Hyperreactivity of the ner operator to potassium permanganate and the hydroxyl radical indicate that the AT-rich spacer assumes a variant conformation consistent with a bend . Using an electrophoretic mobility shift assay, we demonstrated that Ner does not display significant affinity for a single 11-bp site . Furthermore, DNase I protection analysis and circular-permutation binding assays reveal that alterations in the length and sequence of the AT-rich spacer that separates the 11-bp inverted repeats significantly alter Ner-operator interactions, and demonstrate that the intrinsically bent ner operator is conformationally altered upon protein binding.

Arch Biochem Biophys, 2000 May 15, 377(2), 324 - 33
The amino-terminal region of the long-chain fatty acid transport protein FadL contains an externally exposed domain required for bacteriophage T2 binding; Cristalli G et al.; The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in beta-structure and span the outer membrane multiple times to form a long-chain fatty acid specific channel . Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin-sensitive sites, both predicted to be in externally exposed loops of FadL . Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I-A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395 . Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops . A peptide corresponding to the amino-terminal region of FadL (FadL28-160) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino-proximal extracellular loop of the protein confers T2 binding activity . Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed . According to this model, FadL spans the outer membrane 20 times as antiparallel beta-strands . The 20 antiparallel beta-strands are presumed to form a beta-barrel specific for long-chain fatty acids . On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL28-160 peptide, the predicted extracellular regions between beta-strands 1 and 2 and beta-strands 3 and 4 are expected to contribute to a domain of the protein required for long-chain fatty acid and bacteriophage T2 binding . The first trypsin-sensitive site (R93) lies between predicted beta-strands 3 and 4 while the second (R384) is between beta-strands 17 and 18 . The trypsin-resistant region of FadL is predicted to contain 13 antiparallel beta-strands and contribute to the long-chain fatty acid specific channel.

J Bacteriol, 2000 Jul, 182(13), 3761 - 6
Cloning, expression, and purification of the K5 capsular polysaccharide lyase (KflA) from coliphage K5A: evidence for two distinct K5 lyase enzymes; Clarke BR et al.; The Escherichia coli K5 capsular polysaccharide {-4)-betaGlcA-(1, 4)-alphaGlcNAc-(1-} is a receptor for the capsule-specific bacteriophage K5A . Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface . The bacteriophage K5A lyase gene (kflA) was cloned and sequenced . The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E . coli SEBR 3282 . There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time . Southern blot analysis revealed that kflA was not present on the chromosome of the E . coli strains examined . In contrast, elmA was present in a subset of E . coli strains . Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF(L)) located 5' of the endosialidase gene of the E . coli K1 capsule-specific bacteriophage K1E . The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related . The deduced polypeptide sequence of ORF(L) in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF(L) is a truncated remnant of KflA . The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA . A (His)(6)-KflA fusion protein was overexpressed in E . coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture . The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.

Mol Microbiol, 2000 Jun, 36(5), 1124 - 34
Antisense RNA-dependent transcription termination sites that modulate lysogenic development of satellite phage P4; Briani F et al.; In the lysogenic state, bacteriophage P4 prevents the expression of its own replication genes, which are encoded in the left operon, through premature transcription termination . The phage factor responsible for efficient termination is a small, untranslated RNA (CI RNA), which acts as an antisense RNA and controls transcription termination by pairing with two complementary sequences (seqA and seqC) located within the leader region of the left operon . A Rho-dependent termination site, timm, was previously shown to be involved in the control of P4 replication gene expression . In the present study, by making use of phage PhiR73 as a cloning vector and of suppressor tRNAGly as a reporter gene, we characterized two additional terminators, t1 and t4 . Although transcription termination at neither site requires the Rho factor, only t1 has the typical structure of a Rho-independent terminator . t1 is located between the PLE promoter and the cI gene, whereas t4 is located between cI and timm . Efficient termination at t1 requires the CI RNA and the seqA target sequence; in vitro, the CI RNA enhanced termination at t1 in the absence of any bacterial factor . A P4 mutant, in which the t1 terminator has been deleted, can still lysogenize both Rho+ and Rho- strains and exhibits increased expression of CI RNA . These data indicate that t1 and the Rho-dependent timm terminators are not essential for lysogeny . t1 is involved in CI RNA autoregulation, whereas t4 appears to be the main terminator necessary to prevent expression of the lytic genes in the lysogenic state.

J Immunol, 2000 Jun 15, 164(12), 6221 - 9
Analysis of the diversity of a sheep antibody repertoire as revealed from a bacteriophage display library; Charlton KA et al.; We have applied bacteriophage display technology to construct and analyze the diversity of an IgG library of >1 x 108 clones from an adult sheep immunized against the hapten atrazine . We have identified eight new VH gene families (VH2-VH9) and five new Vkappa gene families (VkappaV-VkappaIX) . The heavy and kappa light chain variable region gene loci were found to be far more diverse than previously thought.

J Cell Biochem, 2000 May, 78(2), 251 - 63
Binding motifs of CBP2 a potential cell surface target for carcinoma cells; Sauk JJ et al.; Previously we have shown (Hebert et al . {1999} J . Cell Biochem . 73:248-258) that among many cell lines the CBP2 gene product, Hsp47, eludes its retention receptor, erd2P, resulting in the appearance of Hsp47 on the cell surface associated with the tetraspanin protein CD9 . Since Hsp47 possesses a highly restricted binding cleft, random peptide display libraries were used to characterize peptides binding to Hsp47 and then to target this protein on carcinoma cell lines in vitro . Comparison of the clones obtained from panning revealed little specific homology based on sequence alone . To determine whether carcinoma cells expressing Hsp47 could selectively take up the selected bacteriophages, traditional immunofluorescence and confocal microscopy were employed . These studies revealed that phage-displaying Hsp47 binding peptides bound to cell lines expressing Hsp47 and that the peptides were rapidly taken up to a location coincident with Hsp47 staining . These observations were confirmed by cytometric analyses . These data indicate that CBP2 product may provide a molecular target for chemotherapy and/or imaging of malignancies .

EMBO J, 2000 Jun 1, 19(11), 2671 - 80
One protein from two open reading frames: mechanism of a 50 nt translational bypass; Herr AJ et al.; Translating ribosomes bypass a 50 nt coding gap in order to fuse the information found in the two open reading frames (ORFs) of bacteriophage T4 gene 60 . This study investigates the underlying mechanism by focusing on the competition between initiation of bypassing and termination at the end of the first ORF . While nearly all ribosomes initiate bypassing, no more than 50% resume translation in the second ORF . Two previously described cis-acting stimulatory signals are critical for favoring initiation of bypassing over termination . Genetic analysis of these signals supports a working model in which the first (a stem-loop structure at the junction between the first ORF and the coding gap) interferes with decoding in the A-site, and the second (a stretch of amino acids in the nascent peptide encoded by the first ORF) destabilizes peptidyl-tRNA-mRNA pairing.

J Mol Biol, 2000 Jun 9, 299(3), 573 - 84
Large conformational changes in the maturation of a simple RNA virus, nudaurelia capensis omega virus (NomegaV); Canady MA et al.; An assembly intermediate of a small, non-enveloped RNA virus has been discovered that exhibits striking differences from the mature virion . Virus-like particles (VLPs) of Nudaurelia capensis omega virus (NomegaV), a T=4 icosahedral virus infecting Lepidoptera insects, were produced in insect cells using a baculovirus vector expressing the coat protein . A procapsid form was discovered when NomegaV VLPs were purified at neutral pH conditions . These VLPs were fragile and did not undergo the autoproteolytic maturation that occurs in the infectious virus . Electron cryo-microscopy (cryoEM) and image analysis showed that, compared with the native virion, the VLPs were 16% larger in diameter, more rounded, porous, and contained an additional internal domain . Upon lowering the pH to 5.0, the VLP capsids became structurally indistinguishable from the authentic virion and the subunits autoproteolyzed . The NomegaV protein subunit coordinates, which were previously determined crystallographically, were modelled into the 28 A resolution cryoEM map of the procapsid . The resulting pseudo-atomic model of the NomegaV procapsid demonstrated the large rearrangements in quaternary and tertiary structure needed for the maturation of the VLPs and presumably of the virus . Based on this model, we propose that electrostatically driven rearrangements of interior helical regions are responsible for the large conformational change . These results are surprising because large structural rearrangements have not been found in the maturation of any other small RNA viruses . However, similarities of this conformational change to the maturational processes of more complex DNA viruses (e.g . bacteriophages and herpesvirus) and to the swelling of simple plant viruses suggest that structural changes in icosahedral viruses, which are integral to their function, have similar strategies and perhaps mechanisms .

J Clin Microbiol, 2000 Jun, 38(6), 2374 - 7
Isolation of Acanthamoeba-specific antibodies from a bacteriophage display library; Khan NA et al.; Acanthamoeba causes opportunistic eye infections in humans, which can lead to severe keratitis and may ultimately result in blindness . Current methods for identifying this organism rely on culture and microscopy . In this paper, we describe the isolation of antibody fragments that can be used for the unequivocal identification of Acanthamoeba . A bacteriophage antibody display library was used to isolate antibody fragments that bind specifically to Acanthamoeba . Individual clones were studied by enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence . Four antibody clones that specifically bind to Acanthamoeba spp . were identified.

Biochem Biophys Res Commun, 2000 Jun 7, 272(2), 477 - 9
From a short amino acidic sequence to the complete gene; Minambres B et al.; A useful strategy directed to the isolation of a required gene with a high GC content is reported . Using a degenerate oligonucleotide probe, deduced from the amino terminus of a protein, it is possible to obtain a fragment of DNA containing its encoding gene by PCR amplification . Furthermore, the cloning of a desired gene can be accomplished in two steps by using an oligonucleotide deduced (i) from an internal sequence, (ii) from a consensus sequence, or (iii) from a DNA sequence adjacent to a disrupting element (transposon, insertion sequence, cassette) . This method, which could be applied to a bacteriophage, plasmid, or cosmid genomic library, has been successfully used for cloning several genes from different biological systems .

J Biochem (Tokyo), 2000 Jun, 127(6), 1057 - 63
Affinity selection of DNA-binding proteins displayed on bacteriophage lambda; Zhang Y et al.; Two transcription factors, human ATF1, its DNA-binding domain (ATF1BD), and the DNA-binding domain (GAL4BD) of the yeast GAL4 protein, were displayed on the surface of bacteriophage lambda vectors and efficiently selected by DNA fragments immobilized in microtiter wells . The DNA-binding proteins are fused to the carboxy terminus of the tail protein gpV and head protein gpD of the vectors, lambdafoo and lambdafooDc, respectively . After a single round of affinity selection, the fusion phages were successfully enriched 60- to 4,000-fold over the vector phages . Further, the GAL4BD fusion phages were enriched 5- and 15-fold by affinity selection using specific DNA as probes over nonspecific DNA when expressed on lambdafooDc and lambdafoo, respectively . The ATF1BD fusion phages were also sequence-specifically enriched greater than 4-fold when displayed on lambdafoo . These results suggest that the lambdafoo display system is useful for in vitro studying of protein-DNA interactions and may be applied to screening of DNA-binding protein from complex cDNA libraries through DNA-binding affinity.

Mol Biol Evol, 2000 Jun, 17(6), 942 - 50
Big-benefit mutations in a bacteriophage inhibited with heat; Bull JJ et al.; High temperature inhibits the growth of the wild-type bacteriophage phiX174 . Three different point mutations were identified that each recovered growth at high temperature . Two affected the major capsid protein (residues F188 and F242), and one affected the internal scaffolding protein (B114) . One of the major capsid mutations (F242) is located in a beta strand that contacts B114 in the procapsid during viral maturation, whereas the other capsid mutation (F188) is involved in subunit interactions at the threefold axis of symmetry . Selective coefficients of these mutations ranged from 13.9 to 3.8 in the inhibitory, hot environment, but all mutations reduced fitness at normal temperature . The selective effect of one of the mutations (F242) was evaluated at high temperature in four different genetic backgrounds and exhibited epistasis of diminishing returns: as log fitness of the background genotype increased from -0.1 to 4.1, the fitness boost provided by the F242 mutation decreased from 3.9 to 0 . 8 . These results support a model in which viral fitness is bounded by an upper limit and the benefit of a mutation is scaled according to the remaining opportunity for fitness improvement in the genome.

Exp Parasitol, 2000 Mar, 94(3), 180 - 9
Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage; Hong SJ et al.; We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB) . The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure . The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6 . Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths . cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST . The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids . The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%) . The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs . The apparent K(m) value of a recombinant enzyme was 0.78 mM . Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane . The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel . These findings indicate that adult P . westermani has a class sigma GST .

Placenta, 2000 Mar-Apr, 21 Suppl A, S106 - 12
Phage display: a molecular tool for the generation of antibodies--a review; Schmitz U et al.; Phage display comprises an array of methods, which can be used to display proteins on bacteriophages . We present in this article a summary of techniques, which can be used to express antibody libraries on bacteriophages . Since many researchers are more familiar with the conventional hybridoma technique for production of monoclonal antibodies we describe analogies and differences between these two techniques, both of which are used to reach comparable scientific objectives . We focus on the features which are specific to phage display techniques rather than for hybridoma techniques . These comprise the freedom to choose other animals than the mouse for immunization, the enormously large sample (up to 10(9) clones) which can be drawn and immortalized from a single immunized animal and the opportunity to enhance affinity of isolated antibodies by in vitro affinity maturation . The panning techniques, which are used to enrich specifically binding phage antibodies from the huge libraries are briefly summarized.

Biochemistry, 2000 Jun 6, 39(22), 6652 - 9
A thioredoxin from the hyperthermophilic archaeon Methanococcus jannaschii has a glutaredoxin-like fold but thioredoxin-like activities; Lee DY et al.; A thioredoxin homologue (Mj0307) from the hyperthermophilic archaeon Methanococcus jannaschii (MjTRX) was cloned, produced in E . coli, and compared to the thioredoxin from E . coli (ETRX) . The secondary structure profile of MjTRX obtained by NMR spectroscopy shows that it has four beta-sheets and three alpha-helices arranged in betaalphabetaalphabetabetaalpha, similar to that of glutaredoxin . However, MjTRX supports the growth of T7 bacteriophage in E . coli and is weakly reduced by the thioredoxin reductase from E . coli, indicating that MjTRX is functionally closer to a thioredoxin than a glutaredoxin . MjTRX has higher specific insulin reductase activity than ETRX and retained its full activity over 4 days at 95 degrees C, whereas ETRX lost its activity in 150 min . The standard state redox potential of MjTRX is about -277 mV, which is the lowest value thus far known among redox potentials of the thioredoxin superfamily . This indicates that the lower redox potential is necessary in keeping catalytic disulfide bonds reduced in the cytoplasm and in coping with oxidative stress in an anaerobic hyperthermophile.

Biochemistry, 2000 May 30, 39(21), 6401 - 9
DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process . Nucleotide hydrolysis is not required; Picha KM et al.; Many helicases assemble into ring-shaped hexamers and bind DNA in their central channel . This raises the question as to how the DNA gets into the central channel to form a topologically linked complex . We have used the presteady-state stopped-flow kinetic method and protein fluorescence changes to investigate the mechanism of single-stranded DNA (ssDNA) binding to the bacteriophage T7 helicase-primase, gp4A' . We have found that the kinetics of 30-mer ssDNA binding to a preformed gp4A' hexamer in the presence of both Mg-dTMP-PCP and Mg-dTTP are similar, indicating that Mg-dTTP binding is sufficient and hydrolysis is not necessary for efficient DNA binding . Multiple transient changes in gp4A' fluorescence revealed a four-step mechanism for DNA binding with Mg-dTTP . These transient changes were analyzed by global fitting and kinetic simulation to determine the intrinsic rate constants of this four-step mechanism . The initial steps, including the bimolecular encounter of the DNA with the helicase and a subsequent conformational change, were fast . We propose that these initial steps of DNA binding occur at a readily accessible site, which is likely to be on the outside of the hexamer ring . The binding of the 30-mer ssDNA at this loading site is followed by slower conformational changes that allow the DNA to transit into the central channel of gp4A' via a ring-opening or threading pathway.

Cancer Res, 2000 May 15, 60(10), 2584 - 8
Effects of Thomsen-Friedenreich antigen-specific peptide P-30 on beta-galactoside-mediated homotypic aggregation and adhesion to the endothelium of MDA-MB-435 human breast carcinoma cells; Glinsky VV et al.; Both the ability of malignant cells to form multicellular aggregates via homotypic or heterotypic aggregation and their adhesion to the endothelium are important if not critical during early stages of cancer metastasis . The tumor-associated carbohydrate Thomsen-Friedenreich antigen (T antigen) and beta-galactoside binding lectins (galectins) have been implicated in tumor cell adhesion and tissue invasion . In this study, we demonstrate the involvement of T antigen in both homotypic aggregation of MDA-MB-435 human breast carcinoma cells and their adhesion to the endothelium . The T antigen-specific peptide P-30 (HGRFILPWWYAFSPS) selected from a bacteriophage display library was able to inhibit spontaneous homotypic aggregation of MDA-MB-435 cells up to 74% in a dose-dependent manner . Because T antigen has beta-galactose as a terminal sugar, the expression profile of beta-galactoside-binding lectins (galectins) in MDA-MB-435 cells was studied . Our data indicated the abundant expression of {35S}methionine/cysteine-labeled galectin-1 and galectin-3 in this cell line, which suggested possible interactions between galectins and T antigen . As revealed by laser confocal microscopy, both galectin-1 and galectin-3 also participate in the adhesion of the MDA-MB-435 cells to the endothelium . We observed the clustering of galectin-3 on endothelial cells at the sites of the contact with tumor cells, consistent with its possible interaction with T antigen on cancer cells The galectin-1 signal, however, strongly accumulated at the sites of cell-cell contacts predominantly on tumor cells . The T antigen-specific P-30 significantly (50%) inhibited this adhesion, which indicated that T antigen participates in the adhesion of MDA-MB-435 breast cancer cells to the endothelium . The ability of synthetic P-30 to inhibit both the spontaneous homotypic aggregation of MDA-MB-435 cells and their adhesion to the endothelium (>70 and 50%, respectively) suggests its potential functional significance for antiadhesive therapy of cancer metastasis.

Acta Biochim Pol, 1999, 46(4), 879 - 84
Translational frameshift sites within bacteriophage lambda genes rexA and cI; Hayes S et al.; Phage lambda's cI-rexA-rexB operon displays an intriguing example of regulation by an unexplained mechanism of polarity . We have identified three potential -1 translational frameshift sites and present a model for translational frameshift suppression by lambda's CI repressor as a mechanism of regulating operon polarity, implying an additional role for CI self-regulation.

Biochemistry, 2000 May 23, 39(20), 6157 - 63
Membrane assembly of the bacteriophage Pf3 major coat protein; Meijer AB et al.; The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle . The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain . The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm . A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane . In this approach, the fluorescence probe N-{(iodoacetyl)amino}ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein . The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes . We subsequently studied the fluorescence characteristics at the different positions in the protein . We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide . The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane . A nearly identical result was seen previously for the membrane-bound M13 coat protein . On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein . DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface . Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface . These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.

Biochemistry, 2000 May 9, 39(18), 5573 - 85
Regulation of rho-dependent transcription termination by NusG is specific to the Escherichia coli elongation complex; Pasman Z et al.; To terminate transcription in E . coli, Rho protein binds an RNA loading site on the nascent transcript, translocates 5'--> 3' along the RNA in an ATP-driven process, and, upon reaching the transcription elongation complex, brings about RNA release . Thus, the Rho-dependent termination process can be viewed, in part, as a kinetic competition between the rate of transcript elongation by RNA polymerase (RNAP) and the rate of Rho translocation along the nascent transcript . In the context of this model, NusG, which is an essential E . coli protein, regulates Rho-dependent termination in an apparently paradoxical way, increasing the rate of transcription elongation of E . coli RNAP in the absence of Rho while also shifting the sites of Rho-dependent termination upstream on the template . Here we investigate the regulation of Rho-dependent termination by NusG . Analytical ultracentrifugation was used to establish the existence of a stable complex of NusG and Rho and to demonstrate a stoichiometry of one NusG monomer per Rho hexamer . Surface plasmon resonance was used to examine the kinetics of the formation and dissociation of the NusG-Rho complex, yielding an association rate constant (k(on)) of 2.8 (+/-0.8) x 10(5) M(-)(1) s(-)(1), a dissociation rate constant (k(off)) of 3.9 (+/-0.7) x 10(-)(3) s(-)(1), and a calculated equilibrium (dissociation) constant (K(d)) of 1.5 (+/-0.3) x 10(-)(8) M . The apparent stability of the NusG-Rho complex is insensitive to changes in salt (potassium acetate) concentration between 0.05 and 0.15 M . The translocation and transcription termination activities of Rho at saturating NusG concentrations were, however, both sensitive to salt concentration over this range, suggesting that these activities do not directly reflect the stability of the NusG-Rho complex . Rho-dependent termination could be demonstrated for transcription complexes in which E . coli RNAP had been substituted by either bacteriophage SP6 or T7 RNAP . NusG, however, was not active in transcription termination assays with either of these phage RNAPs . Thus, we conclude that NusG modulates Rho-dependent termination by interacting specifically with the RNAP of the E . coli elongation complex to render the complex more susceptible to the termination activity of Rho.

Acta Crystallogr D Biol Crystallogr, 2000 Jun, 56 ( Pt 6), 775 - 7
Overexpression, crystallization and preliminary X-ray crystallographic analysis of dihydrofolate reductase from bacteriophage T4; Oh YK et al.; Dihydrofolate reductase (DHFR) from bacteriophage T4 is a homodimer consisting of 193-residue subunits . It has been crystallized in the presence of the cofactor (NADPH) and an inhibitor (aminopterin) at 296 K using sodium chloride as precipitant . The crystals are tetragonal, belonging to the space group P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 61.14, c = 123.23 A under cryogenic conditions . The asymmetric unit contains a single subunit, with a corresponding V(m) of 2.65 A(3) Da(-1) and a solvent content of 53 . 6% . Native data have been collected from a crystal to 1.9 A resolution using synchrotron X-rays.

Biotechniques, 2000 May, 28(5), 904 - 8, 910, 912
Microcapillary reactors using solid-phase DNA sequencing for direct sample introduction into slab gels; Xu Y et al.; Solid-phase micro-reactors have been prepared in glass capillaries for DNA sequencing applications using slab gel electrophoresis, which consisted of a fused silica capillary (i.d . = 100 microns; o.d . = 365 microns; length = 15 cm; volume = 1.2 microL) that contained a covalently bound biotin molecule . With the addition of streptavidin to the capillary, an anchoring site was produced for the tethering of biotinylated DNA sequencing templates to the wall of the capillary . Using a four-lane, single dye primer chemistry sequencing strategy, the individual tracts were prepared in the capillaries using cycle sequencing (20 thermal cycles) on a PCR-generated lambda-bacteriophage template (about 1000 bp) . The dye label in this case was a fluorescent tag that displayed emission properties in the near-IR and could be processed on an automated sequencer . The read length was found to be 589 bases, which was determined primarily by the fractionating power of the gel . It was also found that the tethering system was very stable to typical cycle sequencing conditions, with the amount of tethered DNA lost amounting to 40% after 120 thermal cycles . The ability to use dye terminator chemistry was also investigated by using a near-IR dye-labeled terminator (ddGTP) . It was found that the quality of the ladder that was generated was comparable to that obtained in a conventional sample preparation format . However, ethanol precipitation was required before gel loading to remove excess terminator.

Virology, 2000 May 25, 271(1), 182 - 96
Characterization of murine coronavirus neutralization epitopes with phage-displayed peptides; Yu MW et al.; Phage-displayed peptide libraries were used to map immunologically relevant epitopes on the surface (S) glycoprotein of a neurotropic murine coronavirus (MHV-A59) . Three in vitro virus-neutralizing and in vivo protective mAbs against either continuous or discontinuous epitopes on the S glycoprotein were used to screen 12 different peptide libraries expressed on the pVIII major coat protein of the fd filamentous bacteriophage . Consensus sequences that matched short sequences within the S glycoprotein were identified . The sequence of a tight-binding, mAb-selected peptide suggested the location of a discontinuous epitope within the N-terminal S1 subunit . Several tightly binding phage were amplified and used directly as immunogens in BALB/c and C57BL/6 mice . Partial protection of C57BL/6 mice against a lethal acute virus infection was achieved with a phage preparation that displayed a linear epitope . Protection correlated with the presence of sufficient levels of specific antiviral antibodies recognizing the same immunodominant domain and 13-mer peptide, located within the C-terminal S2 subunit, as the selecting mAb . Thus, the direct use of phage-displayed peptides to evaluate protective antiviral immune responses complements their use to characterize antibody-binding epitopes . This is the first evaluation of protective immunization induced by mAb-selected phage-displayed peptides .

Mutat Res, 2000 Apr 28, 459(3), 237 - 42
Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver; Shevelev IV et al.; Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes . Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro . The extent of correction increases together with the rise in 3'-->5' exonuclease concentration . Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude . These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.

Wound Repair Regen, 2000 Mar-Apr, 8(2), 128 - 37
Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-beta1 mRNA and protein than normal skin and cells; Wang R et al.; Transforming growth factor-beta1 is a well-known fibrogenic cytokine produced by many types of cells including dermal fibroblasts . To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor-beta1 gene expression was evaluated in small skin samples . Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription-polymerase chain reaction technique was used . Quantitation of gene expression by reverse transcription-polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards . We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor-beta1 starting with a gene specific polymerase chain reaction product . After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen alpha1(I) cDNA with compatible ends was inserted into the polymerase chain reaction-DNA fragment . The recombinant cDNA was re-amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters . Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription-polymerase chain reaction together with the tissue or cellular RNA . After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide . The bands for internal standard and transforming growth factor-beta1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription-polymerase chain reaction . The number of mRNA molecules/cell was calculated . We examined the transforming growth factor-beta1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five-fold more transforming growth factor-beta1 mRNA than normal skin per unit of wet weight . We used this procedure to quantitate transforming growth factor-beta1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin . The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor-beta1 than normal cells (116 +/- 6 vs . 97 +/- 7, p = 0.017, n = 5) . These results were supported by Northern analysis for transforming growth factor-beta1 mRNA in the cells and enzyme-linked immunosorbent assay for TGF-beta1 protein in fibroblast-conditioned medium . In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor-beta1, which may be important in hypertrophic scar formation . The construction of the gene specific internal standard for reverse transcription-polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.

J Bacteriol, 2000 Jun, 182(11), 3165 - 74
Mutually exclusive utilization of P(R) and P(RM) promoters in bacteriophage 434 O(R); Xu J et al.; Establishment and maintenance of a lysogen of the lambdoid bacteriophage 434 require that the 434 repressor both activate transcription from the P(RM) promoter and repress transcription from the divergent P(R) promoter . Several lines of evidence indicate that the 434 repressor activates initiation of P(RM) transcription by occupying a binding site adjacent to the P(RM) promoter and directly contacting RNA polymerase . The overlapping architecture of the P(RM) and P(R) promoters suggests that an RNA polymerase bound at P(R) may repress P(RM) transcription initiation . Hence, part of the stimulatory effect of the 434 repressor may be relief of interference between RNA polymerase binding to the P(RM) promoter and to the P(R) promoter . Consistent with this proposal, we show that the repressor cannot activate P(RM) transcription if RNA polymerase binds at P(R) prior to addition of the 434 repressor . However, unlike the findings with the related lambda phage, formation of RNA polymerase promoter complexes at P(RM) and at P(R) apparently are mutually exclusive . We find that the RNA polymerase-mediated inhibition of repressor-stimulated P(RM) transcription requires the presence of an open complex at P(R) . Taken together, these results indicate that establishment of an open complex at P(R) directly prevents formation of an RNA polymerase-P(RM) complex.

J Bacteriol, 2000 Jun, 182(11), 3111 - 6
Proteolysis of bacteriophage lambda CII by Escherichia coli FtsH (HflB); Shotland Y et al.; FtsH (HflB) is a conserved, highly specific, ATP-dependent protease for which a number of substrates are known . The enzyme participates in the phage lambda lysis-lysogeny decision by degrading the lambda CII transcriptional activator and by its response to inhibition by the lambda CIII gene product . In order to gain further insight into the mechanism of the enzymatic activity of FtsH (HflB), we identified the peptides generated following proteolysis of the phage lambda CII protein . It was found that FtsH (HflB) acts as an endopeptidase degrading CII into small peptides with limited amino acid specificity at the cleavage site . beta-Casein, an unstructured substrate, is also degraded by FtsH (HflB), suggesting that protein structure may play a minor role in determining the products of proteolysis . The majority of the peptides produced were 13 to 20 residues long.

Sb Lek, 1998, 99(4), 455 - 64
{The effect of low-frequency electromagnetic fields on living organisms}; Strasak L et al.; This report studies effect of alternating low-frequency electromagnetic fields (Bm = 5-21.5 mT, f = 50 Hz, duration of exposure t = 0-24 min) on viability of bacteria Escherichia coli . We have shown that the growth of bacteria is impaired the electromagnetic field . Their ability to form colonies on a solid medium decreases in dependence on magnitude of magnetic field and on duration of exposure . The growth curve is influenced by the electromagnetic field as well . Effects of electromagnetic fields are independent of biological age in first four hours of their growth . We have found no morphological changes in bacterial systems in electromagnetic field by optical microscope . Viability of bacteria is bigger in a liquid medium and less in a solid medium . Bacteriophage BF 23 attach less to bacteria influenced by electromagnetic field . And finally, magnetic field did not make induction of production of bacteriophage . This effect indicates, that magnetic field did not damage DNA of exposed bacteria.

J Mol Biol, 2000 May 19, 298(5), 807 - 15
Topology of the components of the DNA packaging machinery in the phage phi29 prohead; Ibarra B et al.; Chromosome condensation inside dsDNA viral particles is a complex process requiring the coordinated action of several viral components . The similarity of the process in different viral systems has led to the suggestion that there is a common underlying mechanism for DNA packaging, in which the portal vertex or connector plays a key role . We have studied the topology of the packaging machinery using a number of antibodies directed against different domains of the connector . The charged amino-terminal, the carboxyl-terminal, and the RNA binding domain are accessible areas in the connector assembled into the prohead, while the domains corresponding to the 12 large appendages of the connector are buried inside the prohead . Furthermore, while the antibodies against the carboxyl and amino-terminal do not affect the packaging reaction, incubation of proheads with antibodies against the RNA binding domain abolishes the packaging activity . The comparison of the three-dimensional reconstructions of bacteriophage phi29 proheads with proheads devoid of their specific pRNA by RNase treatment shows that this treatment removes structural elements of the distal vertex of the portal structure, suggesting that the pRNA required for packaging is located at the open gate of the channel in the narrow side of the connector .

Biochemistry, 2000 May 16, 39(19), 5642 - 52
Phosphorothioate substitution can substantially alter RNA conformation; Smith JS et al.; Phosphorothioate substitution-interference experiments, routinely used to stereospecifically identify phosphoryl oxygen sites that participate in RNA-ligand binding and RNA-directed catalysis, rest in their interpretation on the untested assumption that substitution does not alter the conformation of the modified molecule from its biologically active state . Using NMR spectroscopy, we have tested this assumption by determining the structural effect of stereospecific phosphorothioate substitution at five positions in an RNA hairpin containing the binding site for bacteriophage MS2 capsid protein . At most sites, substitution has little or no effect, causing minor perturbations in the phosphate backbone and increasing the stacking among nucleotides in the hairpin loop . At one site, however, phosphorothioate substitution causes an unpaired adenine necessary for formation of the capsid protein-RNA complex to loop out of the RNA helix into the major groove . These results indicate that phosphorothioate substitution can substantially alter the conformation of RNA at positions of irregular secondary structure, complicating the use of substitution-interference experiments to study RNA structure and function.

Cancer Lett, 2000 Jun 1, 154(1), 29 - 37
DNA breakage by resveratrol and Cu(II): reaction mechanism and bacteriophage inactivation; Ahmad A et al.; Resveratrol (3,4',5-trihydroxy stilbene) is a phytoalexin and a polyphenolic compound present in human dietary material such as peanuts, mulberries, grapes and red wine . It is widely considered to possess cardiovascular protective properties and has also been shown to be chemopreventive against various stages of chemically induced carcinogenesis . It has recently been shown that resveratrol induces strand breakage in DNA in the presence of copper ions . In this paper, we have shown that resveratrol catalyzes the reduction of Cu(II) to Cu(I), which is accompanied by the formation of 'oxidized product(s)' of resveratrol, which in turn also appear to catalyze the reduction of Cu(II) . Strand scission by the resveratrol-Cu(II) system was found to be biologically active as assayed by bacteriophage inactivation . The results are discussed in relation to the putative chemopreventive mechanism of resveratrol.

J Virol, 2000 Jun, 74(11), 5266 - 72
Expression of hepatitis B virus X protein does not alter the accumulation of spontaneous mutations in transgenic mice; Madden CR et al.; Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma . Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens, although the mechanism for this cofactor role remains unknown . The ability of HBx to inhibit DNA repair in transiently transfected cell lines suggests one possible pathway . In the present study, primary hepatocytes isolated from transgenic mice that possess the HBV X gene under the control of the human alpha-1-antitrypsin regulatory region (ATX mice) were found to be deficient in their ability to conduct unscheduled DNA synthesis in response to UV-induced DNA damage . In order to measure the impact of HBx expression on DNA repair in vivo, double-transgenic mice that express HBx and possess a bacteriophage lambda transgene were sacrificed at 30, 90, and 240 days of age . Mutation frequency was determined for high-molecular-weight liver DNA of ATX and control mice by functional analysis of the lambda transgene . Expression of HBx did not significantly increase the accumulation of spontaneous mutations . These results are consistent with previous studies of HBx transgenic mice in which no effect of HBx on liver histology was apparent . This new animal model provides a powerful system in which to investigate the in vivo cooperation between HBx expression and environmental carcinogens.

J Biol Chem, 2000 May 12, 275(19), 14541 - 9
Overproduction in Escherichia coli and characterization of yeast replication factor C lacking the ligase homology domain; Gomes XV et al.; Eukaryotic replication factor C (RF-C) is a heteropentameric complex that is required to load the replication clamp proliferating cell nuclear antigen onto primed DNA . Saccharomyces cerevisiae RF-C is encoded by the genes RFC1-RFC5 . The RFC1 gene was cloned under control of the strong inducible bacteriophage T7 promoter, yet induction did not yield detectable Rfc1p . However, a truncated form of RFC1 deleted for the coding region for amino acids 3-273, rfc1-DeltaN, did allow overproduction . The other four RFC genes were cloned into the latter plasmid to yield a single plasmid that overproduced RF-C to moderate levels . Overproduction of the complex was further enhanced when the Escherichia coli argU gene encoding the rare arginine tRNA was also overproduced . The enzyme thus produced in E . coli was purified to homogeneity through three column steps, including a proliferating cell nuclear antigen affinity column . This enzyme, as well as the enzyme purified from yeast, is prone to aggregation and inactivation, and therefore, light scattering was used to determine conditions stabilizing the enzyme and preventing aggregation . Broad-range carrier ampholytes at about 0.05% were found to be most effective . In some assays, the Rfc1-DeltaN containing RF-C from E . coli showed an increased activity compared with the full-length enzyme from yeast, likely because the latter enzyme exhibits significant nonspecific binding to single-stranded DNA . Replacement of RFC1 by rfc1-DeltaN in yeast shows essentially no phenotype with regard to DNA replication, damage susceptibility, telomere length maintenance, and intrachromosomal recombination.

J Biomol Struct Dyn, 2000 Apr, 17(5), 843 - 56
Structural equilibria in RNA as revealed by 19F NMR; Arnold JR et al.; We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage . The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure . Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form . This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration . Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined . Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl . A similar effect is observed with Sr2+, but not Ca2+ or Ba2+ . Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions . The overall, ensemble average, hairpin conformation is therefore salt-dependent . Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium . The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure . The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.

Protein Sci, 2000 Apr, 9(4), 647 - 54
Mutational analysis of the major coat protein of M13 identifies residues that control protein display; Weiss GA et al.; We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA . 2000 . High copy display of large proteins on phage for functional selections . J Mol Biol 296:487-495) . Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus . Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display . These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat . Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein . These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat . The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.

Mol Microbiol, 2000 Apr, 36(2), 437 - 46
Repair of double-strand breaks by incorporation of a molecule of homologous DNA; Lai YT et al.; An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double-strand breaks in the T7 genome . The efficiency of double-strand break repair was markedly increased by DNA molecules ('donor' DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending approximately 1 kb on either side of the break site . Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited . When the donor DNA molecules were labelled with 32P, repaired genomes incorporated label only near the site of the double-strand break . When repair was carried out with unlabelled donor DNA and {32P}-dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome . Repair was performed using donor DNA that had adjacent BamHI and PstI sites . When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double-strand break . These observations are most consistent with a model for double-strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.

Mol Microbiol, 2000 Apr, 36(2), 424 - 36
Specificity determinants for bacteriophage Hong Kong 022 integrase: analysis of mutants with relaxed core-binding specificities; Cheng Q et al.; The integrase (Int) proteins encoded by bacteriophages HK022 and lambda catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites . However, the Int proteins of HK022 and lambda are unable to catalyse recombination between non-cognate att sites . The att sites of both phages contain weak binding sites for Int, known as 'core-type' sites . Negatively acting nucleotide determinants associated with specific core sites (lambda B', HK022 B', HK022 C) are responsible for the barrier to non-cognate recombination . In this study, we used challenge phages to demonstrate that the lambda and HK022 Ints cannot bind to core sites containing non-cognate specificity determinants in vivo . We isolated mutants of the HK022 Int, which bind the lambda B' core site . Two mutants, D99N and D99A, have changed a residue in the core-binding (CB) domain, which may be directly contacting the core site DNA . We suggest that binding to the lambda B' site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the lambda B' site . We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine lambda att sites.

J Appl Microbiol, 2000 May, 88(5), 860 - 9
Development of a genetically modified bacteriophage for use in tracing sources of pollution; Daniell TJ et al.; Bacteriophage are frequently used as biotracers to identify the source of water pollutants . Genetic manipulation of bacteriophage M13mp18 has been used to enhance this technique by creating a library in which each recombinant bacteriophage genome contains a unique identification sequence . Techniques that identify a recombinant bacteriophage by the presence of the identification sequence, including polymerase chain reaction, restriction site polymorphism and plaque hybridization, have been developed . Recombinant bacteriophage can be used to test a large number of suspected sources simultaneously . The identification sequence also eliminates confusion with natural bacteriophage present in water samples . The performance of the modified bacteriophage and the techniques were assessed in simulated field trials on a restricted site carried out under a consent for environmental release of a genetically modified organism . The techniques were also field tested at sites in northwest England using wild-type M13 bacteriophage.

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5375 - 80
Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes; Endy D et al.; We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order . We used the simulation to compute the fitness of more than 10(5) mutants . We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase . Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates . Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two . Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type . Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively "nonessential" genes will be needed to improve the T7 simulation . In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology . Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested.

FEBS Lett, 2000 Apr 28, 472(2-3), 217 - 20
Bacteriophage and host mutants causing the rolling-circle lambda DNA replication early after infection; Konopa G et al.; There are two modes of bacteriophage lambda DNA replication during its lytic development in Escherichia coli cells . The circle-to-circle (theta) replication predominates at early stages of the phage growth, whereas rolling-circle (sigma) replication occurs late after infection to produce long concatemers that serve as substrates for packaging of lambda DNA into phage proheads . The mechanism regulating the switch from theta to sigma replication remains unknown . Our previous genetic studies indicated that the bacteriophage lambda Pts1piA66 mutant cannot replicate at 43 degrees C in the wild-type E . coli host, but it can replicate in the dnaA46(ts) mutant . Density shift experiments suggested that the parental DNA molecules of the infecting phage enter sigma replication . Here, using electron microscopy, we demonstrate that as soon as 5 min after infection of the dnaA46(ts) mutant by the lambdaPts1piA66 phage at 43 degrees C, the sigma replication intermediates are highly predominant over theta replication intermediates, contrary to the wild-type conditions (wild-type bacteria infected with the lambdaP(+) phage) . The initiation of replication of the lambdaPts1piA66 mutant at 43 degrees C was strongly inhibited in the dnaA(+) host, as demonstrated by electron microscopy and by pulse-labeling of the phage-derived plasmid replicon . Implications for the mechanism of the regulation of the switch from theta to sigma replication mode are discussed.

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