Bacteriophage

Proc Natl Acad Sci U S A, 2004 Mar 30, 101(13), 4396 - 400 Epub 2004 Mar 15.
The role of RNA polymerase sigma subunit in promoter-independent initiation of transcription; Zenkin N et al.; In bacteria, initiation of transcription depends on the RNA polymerase sigma subunit, which brings catalytically proficient RNA polymerase core to promoters by binding to specific DNA elements located upstream of the transcription start point . Here, we study sigma-dependent synthesis of a transcript that is used to prime replication of the single-stranded genome of bacteriophage M13 . We show that, in this system, sigma plays no role in DNA recognition, which is accomplished solely through RNA polymerase core interaction with DNA downstream of the transcription start point . However, sigma is required for full-sized transcript synthesis by allowing RNA polymerase core to escape into productive elongation . RNA polymerase sigma may play a similar role during replication primer synthesis in other bacterial mobile elements whose life cycle involves a single-stranded DNA stage.

Proc Natl Acad Sci U S A, 2004 Mar 30, 101(13), 4373 - 8
The arginine finger of bacteriophage T7 gene 4 helicase: role in energy coupling; Crampton DJ et al.; The DNA helicase encoded by gene 4 of bacteriophage T7 couples DNA unwinding to the hydrolysis of dTTP . The loss of coupling in the presence of orthovanadate (Vi) suggests that the gamma-phosphate of dTTP plays an important role in this mechanism . The crystal structure of the hexameric helicase shows Arg-522, located at the subunit interface, positioned to interact with the gamma-phosphate of bound nucleoside 5' triphosphate . In this respect, it is analogous to arginine fingers found in other nucleotide-hydrolyzing enzymes . When Arg-522 is replaced with alanine (gp4-R522A) or lysine (gp4-R522K), the rate of dTTP hydrolysis is significantly decreased . dTTPase activity of the altered proteins is not inhibited by Vi, suggesting the loss of an interaction between Vi and gene 4 protein . gp4-R522A cannot unwind DNA, whereas gp4-R522K does so, proportionate to its dTTPase activity . However, gp4-R522K cannot stimulate T7 polymerase activity on double-stranded DNA . These findings support the involvement of the Arg-522 residue in the energy coupling mechanism.

J Pediatr, 2004 Apr, 144(4), 524 - 6
Interleukin receptor-associated kinase (IRAK-4) deficiency associated with bacterial infections and failure to sustain antibody responses; Day N et al.; We previously described a girl with recurrent episodes of pneumococcal pneumonia with septicemia and other infections,(1) found to have interleukin-1 receptor-associated kinase 4 deficiency (IRAK-4) deficiency.(2) In this report, we show that our patient is unable to sustain antibody responses either to polysaccharide or protein antigens or to a neoantigen-bacteriophage.

Vaccine, 2004 Apr 16, 22(13-14), 1666 - 71
Genetic immunisation against hepatitis B using whole bacteriophage lambda particles; March JB et al.; Mice and rabbits have been vaccinated with whole bacteriophage lambda particles containing a DNA vaccine expression cassette under the control of the CMV promoter (enhanced green fluorescent protein {lambda-EGFP} or hepatitis B surface antigen {lambda-HBsAg}) . Mice were vaccinated twice intramuscularly (i.m.) with 5x10(9) of lambda-EGFP phage (containing 250 ng DNA) and exhibited specific anti-EGFP responses 28 days post-vaccination . Rabbits were vaccinated i.m . with 4x10(10) of lambda-HBsAg phage (2 microg DNA) or recombinant HBsAg protein . Following two vaccinations with lambda-HBsAg, one out of four rabbits exhibited high level anti-HBsAg responses (comparable to those seen using the recombinant HBsAg protein) . Following a third vaccination with lambda-HBsAg, all four rabbits showed similar high level responses which have not decreased after more than 6 months . High anti-phage responses were observed in all animals following the first immunization with lambda-HBsAg, indicating that a high antibody titre against the phage carrier did not prevent a subsequent immune response against the DNA vaccine component . Compared to results in mice using equivalent lambda-HBsAg doses, anti-HBsAg responses were much higher in rabbits, which could indicate a swamping effect in mice . Since phage lambda DNA is approximately 50 kb in size (tenfold larger than most plasmid vectors used for naked DNA immunisation), a comparable dose of phage lambda DNA given as intact phage particles actually delivers tenfold less vaccine DNA on a per gene copy (molar) basis . Thus the efficiency of the technique may be even higher than the data at first suggests.

J Mol Biol, 2004 Apr 23, 338(2), 229 - 40
Crystal structure of the excisionase-DNA complex from bacteriophage lambda; Sam MD et al.; The excisionase (Xis) protein from bacteriophage lambda is the best characterized member of a large family of recombination directionality factors that control integrase-mediated DNA rearrangements . It triggers phage excision by cooperatively binding to sites X1 and X2 within the phage, bending DNA significantly and recruiting the phage-encoded integrase (Int) protein to site P2 . We have determined the co-crystal structure of Xis with its X2 DNA-binding site at 1.7A resolution . Xis forms a unique winged-helix motif that interacts with the major and minor grooves of its binding site using an alpha-helix and an ordered beta-hairpin (wing), respectively . Recognition is achieved through an elaborate water-mediated hydrogen-bonding network at the major groove interface, while the preformed hairpin forms largely non-specific interactions with the minor groove . The structure of the complex provides insights into how Xis recruits Int cooperatively, and suggests a plausible mechanism by which it may distort longer DNA fragments significantly . It reveals a surface on the protein that is likely to mediate Xis-Xis interactions required for its cooperative binding to DNA.

Mol Microbiol, 2004 Apr, 52(2), 501 - 13
Initial cos cleavage of bacteriophage lambda concatemers requires proheads and gpFI in vivo; Sippy J et al.; The development of bacteriophage lambda and double-stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly . Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process . For example, lambda phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging . Similar observations have been made for herpes simplex 1 virus . In the case of lambda, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting . We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut . When lambda proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model . Phage 21 is a sister phage of lambda, and although its capsid proteins share approximately 60% residue identity with lambda's, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI . Models for the role of proheads and gpFI in cos cutting are discussed.

FEBS Lett, 2004 Apr 9, 563(1-3), 135 - 40
Insights into the structure of human cytomegalovirus large terminase subunit pUL56; Savva CG et al.; Terminases are a class of proteins which catalyze the generation of unit-length genomes during DNA packaging . These essential proteins are conserved throughout the herpesviruses and many double-stranded DNA bacteriophages . We have determined the structure of the large terminase subunit pUL56 of human cytomegalovirus, a highly pathogenic virus, to 2.6 nm resolution . Image analysis of purified pUL56 suggests that the molecule exists as a dimer formed by the association of two ring-like structures positioned on top of each other and connected by a pronounced density on one side . The 3D reconstruction of pUL56 provides first structural insights into the active protein.

Virology, 2004 Apr 25, 322(1), 82 - 92
Bacteriophage P4 Vis protein is needed for prophage excision; Cali S et al.; Upon infection of its host Escherichia coli, satellite bacteriophage P4 can integrate its genome into the bacterial chromosome by Int-mediated site-specific recombination between the attP and the attB sites . The opposite event, excision, may either occur spontaneously or be induced by a superinfecting P2 helper phage . In this work, we demonstrate that the product of the P4 vis gene, a regulator of the P4 late promoters P(LL) and P(sid), is needed for prophage excision . This conclusion is supported by the following evidence: (i) P4 mutants carrying either a frameshift mutation or a deletion of the vis gene were unable to excise both spontaneously or upon P2 phage superinfection; (ii) expression of the Vis protein from a plasmid induced P4 prophage excision; (iii) excision depended on a functional integrase (Int) protein, thus suggesting that Vis is involved in the formation of the excision complex, rather than in the excision recombination event per se; (iv) Vis protein bound P4 DNA in the attP region at two distinct boxes (Box I and Box II), located between the int gene and the attP core region, and caused bending of the bound DNA . Furthermore, we mapped by primer extension the 5' end of the int transcript and found that ectopic expression of Vis reduced its signal intensity, suggesting that Vis is also involved in negative regulation of the int promoter.

Structure (Camb), 2004 Apr, 12(4), 583 - 92
The crystal structure of the UvsW helicase from bacteriophage T4; Sickmier EA et al.; In bacteriophage T4, the WXY system repairs DNA damage by a process that involves homologous recombination . This system comprises three proteins, the RecA-like recombination protein UvsX, a recombination mediator protein UvsY, and a helicase UvsW . Here we report the 2.0 A resolution crystal structure of the N-terminal two domains of the UvsW helicase (UvsWNF; residues 1-282) . The structure reveals a typical helicase RecA-like domain linked to a small N-terminal alpha/beta domain that likely binds the nucleic acid substrate . The missing C-terminal portion of UvsW almost certainly corresponds to the second RecA-like domain typically found in monomeric helicases . The putative substrate binding domain is unique within the known helicase structures, and it resembles the novel "double-wing" DNA binding domain from the phage T4 MotA transcription factor that mediates the expression of T4 middle genes . The functional implications of this homology for the role of UvsW in T4 DNA metabolism are discussed.

Structure (Camb), 2004 Apr, 12(4), 569 - 81
Secondary structure switching in Cro protein evolution; Newlove T et al.; We report the solution structure of the Cro protein from bacteriophage P22 . Comparisons of its sequence and structure to those of lambda Cro strongly suggest an alpha-to-beta secondary structure switching event during Cro evolution . The folds of P22 Cro and lambda Cro share a three alpha helix fragment comprising the N-terminal half of the domain . However, P22 Cro's C terminus folds as two helices, while lambda Cro's folds as a beta hairpin . The all-alpha fold found for P22 Cro appears to be ancestral, since it also occurs in cI proteins, which are anciently duplicated paralogues of Cro . PSI-BLAST and transitive homology analyses strongly suggest that the sequences of P22 Cro and lambda Cro are globally homologous despite encoding different folds . The alpha+beta fold of lambda Cro therefore likely evolved from its all-alpha ancestor by homologous secondary structure switching, rather than by nonhomologous replacement of both sequence and structure.

J Bacteriol, 2004 Apr, 186(8), 2266 - 74
Transgenic expression of RecA of the spirochetes Borrelia burgdorferi and Borrelia hermsii in Escherichia coli revealed differences in DNA repair and recombination phenotypes; Putteet-Driver AD et al.; After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agent Borrelia burgdorferi, we characterized the functional activities of RecA of B . burgdorferi, as well as RecA of the relapsing fever spirochete Borrelia hermsii and the free-living spirochete Leptospira biflexa, in a recA mutant of Escherichia coli . As a control, E . coli RecA was expressed from the same plasmid vector . DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C . Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth by red gam mutant bacteriophage lambda . Overall, we found that transgenic cells with recA genes of B . burgdorferi, B . hermsii, and L . biflexa had approximately equivalent activities in promoting homologous recombination in the lacZ duplication assay, but cells with B . burgdorferi recA and, most notably, B . hermsii recA were significantly less capable than cells with L . biflexa recA or E . coli recA in responding to DNA damage or in facilitating plaque formation in the phage assay . The comparatively poor function of Borrelia recA in the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR in E . coli.

BMC Biotechnol . 2004 Apr 01;4(1):6.
A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes; van Wyngaardt W et al.; BACKGROUND: Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy . Most existing antibody repertoires are derived from human immunoglobulin genes . Genes from other species can, however, also be used . Because of the way in which gene conversion introduces diversity, the naive antibody repertoire of the chicken can easily be accessed using only two sets of primers . RESULTS: With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains . Synthetically randomised complementarity determining regions are included in some of the heavy chains . Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire . Affinities of three different antibody fragments were determined using surface plasmon resonance . Two were in the low nanomolar and one in the subnanomolar range . To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles . Virus antibodies were detected in a competitive ELISA . CONCLUSION: The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens . It has the potential to provide monoclonal reagents with applications in research and diagnostics . For in vitro applications, naive phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.

EMBO J, 2004 Apr 7, 23(7), 1483 - 93 Epub 2004 Apr 01.
Crystallographic snapshots of a replicative DNA polymerase encountering an abasic site; Hogg M et al.; Abasic sites are common DNA lesions, which are strong blocks to replicative polymerases and are potentially mutagenic when bypassed . We report here the 2.8 A structure of the bacteriophage RB69 replicative DNA polymerase attempting to process an abasic site analog . Four different complexes were captured in the crystal asymmetric unit: two have DNA in the polymerase active site whereas the other two molecules are in the exonuclease mode . When compared to complexes with undamaged DNA, the DNA surrounding the abasic site reveals distinct changes suggesting why the lesion is so poorly bypassed: the DNA in the polymerase active site has not translocated and is therefore stalled, precluding extension . All four molecules exhibit conformations that differ from the previously published structures . The polymerase incorporates dAMP across the lesion under crystallization conditions, indicating that the different conformations observed in the crystal may be part of the active site switching reaction pathway.

Virology, 2004 Apr 10, 321(2), 217 - 21
Novel and deviant Walker A ATP-binding motifs in bacteriophage large terminase-DNA packaging proteins; Mitchell MS et al.; Bacteriophage terminases constitute a very interesting class of viral-coded multifunctional ATPase "motors" that apparently drive directional translocation of DNA into an empty viral capsid . A common Walker A motif and other conserved signatures of a critical ATPase catalytic center are identified in the N-terminal half of numerous large terminase proteins . However, several terminases, including the well-characterized lambda and SPP1 terminases, seem to lack the classic Walker A in the N-terminus . Using sequence alignment approaches, we discovered the presence of deviant Walker A motifs in these and many other phage terminases . One deviation, the presence of a lysine at the beginning of P-loop, may represent a 3D equivalent of the universally conserved lysine in the Walker A GKT/S signature . This and other novel putative Walker A motifs that first came to light through this study help define the ATPase centers of phage and viral terminases as well as elicit important insights into the molecular functioning of this fundamental motif in biological systems.

Med Hypotheses, 2004, 62(4), 493 - 8
Bacteriophages in autoimmune disease and other inflammatory conditions; Riley PA; There are several autoimmune diseases and other inflammatory conditions where an infectious aetiology is suggested by the epidemiology, clinical course and pathological findings . Many candidate bacteria and viruses have been considered as potential aetiological agents but mostly without firm proof . Bacteriophages are viruses that infect bacteria and may be found wherever bacteria are located, but would not be detected unless specifically sought . They have not previously been considered to be pathogens . Bacteriophages are immunogenic and therefore could play a role in the pathogenesis of autoimmune and other inflammatory diseases by acting as antigens on epithelial surfaces, bound to antibody as immune complexes, through molecular mimicry or possibly as superantigens.

Proteins, 2004 May 1, 55(2), 339 - 50
Data-based model and parameter evaluation in dynamic transcriptional regulatory networks; Cavelier G et al.; Finding the causality and strength of connectivity in transcriptional regulatory networks from time-series data will provide a powerful tool for the analysis of cellular states . Presented here is the design of tools for the evaluation of the network's model structure and parameters . The most effective tools are found to be based on evolution strategies . We evaluate models of increasing complexity, from lumped, algebraic phenomenological models to Hill functions and thermodynamically derived functions . These last functions provide the free energies of binding of transcription factors to their operators, as well as cooperativity energies . Optimization results based on published experimental data from a synthetic network in Escherichia coli are presented . The free energies of binding and cooperativity found by our tools are in the same physiological ranges as those experimentally derived in the bacteriophage lambda system . We also use time-series data from high-density oligonucleotide microarrays of yeast meiotic expression patterns . The algorithm appropriately finds the parameters of pairs of regulated regulatory yeast genes, showing that for related genes an overall reasonable computation effort is sufficient to find the strength and causality of the connectivity of large numbers of them .

J Mol Biol, 2004 Apr 9, 337(5), 1109 - 22
The phiX174 protein J mediates DNA packaging and viral attachment to host cells; Bernal RA et al.; Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA . Many viruses, including the Microviridae bacteriophages phiX174, G4, and alpha3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome . The phiX174 DNA-binding protein, J, is 13 amino acid residues longer than the alpha3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus . Chimeric phiX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging . However, chimeric alpha3 and G4 phages, containing the phiX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species . In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein . The structure of alpha3 packaged with phiX174 J protein was determined to 3.5A resolution and compared with the previously determined structures of phiX174 and alpha3 . The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type alpha3 virion proteins . The amino-terminal region of the phiX174 J protein, which is missing from wild-type alpha3 virions, is mostly disordered in the alpha3 chimera . The differences observed between solution properties of wild-type phiX174, wild-type alpha3, and alpha3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein . When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid . In addition, the properties of the phiX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection . Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection.

Cancer Gene Ther, 2004 May, 11(5), 363 - 70
Fusion protein from RGD peptide and Fc fragment of mouse immunoglobulin G inhibits angiogenesis in tumor; Li J et al.; Targeting tumor vasculature represents an interesting approach for the treatment of solid tumors . The alpha v beta 3 integrins have been found to be specifically associated with angiogenesis in tumors . By using bacteriophage display technology, Ruoslahti et al found that a group of peptides containing the RGD (Arg-Gly-Asp) motif have high-binding affinity to the alpha v beta 3 integrins in tumors . In this study, we designed a fusion protein containing the RGD sequence and the Fc fragment of mouse IgG in order to target the Fc portion of IgG to the tumor vasculature to elicit an antiangiogenesis immune response . In vivo angiogenesis and tumor studies demonstrated that the fusion protein (RGD/mFc) inhibited tumor angiogenesis and tumor growth and improved overall survival . This approach may generate new therapeutic agents for solid tumor treatment.

J Biol Chem, 2004 May 28, 279(22), 23384 - 93 Epub 2004 Mar 23.
The linker region between the helicase and primase domains of the gene 4 protein of bacteriophage T7 . Role in helicase conformation and activity; Lee SJ et al.; The gene 4 protein of bacteriophage T7 provides both helicase and primase activities . The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for template-directed oligoribonucleotide synthesis . A 26 amino acid linker region (residues 246-271) connects the two domains and is essential for the formation of functional hexamers . In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4 . The in vivo screening revealed Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function . Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize . In the presence of Mg2+, all of the altered proteins oligomerize . However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation . Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA . Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required . The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.

J Biol Chem, 2004 May 21, 279(21), 22190 - 7 Epub 2004 Mar 24.
Effect of single-stranded DNA-binding proteins on the helicase and primase activities of the bacteriophage T7 gene 4 protein; He ZG et al.; Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities . Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions . To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA . Wild-type gp2.5 and E . coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP . Neither protein inhibits the binding of gp4 to single-stranded DNA . The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-Delta26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA . Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E . coli SSB protein is highly inhibitory at relative low concentrations.

Nat Rev Microbiol, 2004 Feb, 2(2), 166 - 73
Population and evolutionary dynamics of phage therapy; Levin BR et al.; Following a sixty-year hiatus in western medicine, bacteriophages (phages) are again being advocated for treating and preventing bacterial infections . Are attempts to use phages for clinical and environmental applications more likely to succeed now than in the past? Will phage therapy and prophylaxis suffer the same fates as antibiotics--treatment failure due to acquired resistance and ever-increasing frequencies of resistant pathogens? Here, the population and evolutionary dynamics of bacterial-phage interactions that are relevant to phage therapy and prophylaxis are reviewed and illustrated with computer simulations.

Protein Expr Purif, 2004 May, 35(1), 126 - 30
Expression and purification of wild type and mutant forms of the yeast mitochondrial core RNA polymerase, Rpo41; Matsunaga M et al.; The mitochondrial RNA polymerase (mtRNAP) from Saccharomyces cerevisiae (yeast) is composed of two nuclear encoded proteins, the core RNA polymerase (Rpo41) and the mitochondrial transcription factor (Mtf1) . Although Rpo41 is strikingly similar to the single subunit RNAPs from the T7 and T3 bacteriophage (T7RNAP), the core mtRNAP requires Mtf1 for accurate transcription from a linear promoter-containing DNA template, while T7RNAP does not require any other additional factors for promoter selectivity . The fact that the mtRNAP requires an additional promoter utilization factor makes it an excellent model system for the analysis of the transitions that occur during transcription initiation . However, large-scale purification of the 153 kDa Rpo41 has only been reported from yeast cells, or as a recombinant from baculovirus, both sources requiring extensive purification with poor yields . We have developed a His-tagged Rpo41 expression construct suitable for rapid purification of large amounts of soluble Rpo41 from bacterial cells . Transcriptionally active forms of both wild type and point mutants of Rpo41 can be purified by a combination of batch ion exchange chromatography to remove nucleic acids and nickel affinity chromatography . An additional advantage of the isolation of Rpo41 from bacterial cells is the absence of its associated specificity factor Mtf1 . This allows analysis of combinations of mutant forms of both components of the mtRNAP holoenzyme.

Plant Cell, 2004 Apr, 16(4), 1047 - 59 Epub 2004 Mar 22.
Enod40, a short open reading frame-containing mRNA, induces cytoplasmic localization of a nuclear RNA binding protein in Medicago truncatula; Campalans A et al.; In eukaryotes, diverse mRNAs containing only short open reading frames (sORF-mRNAs) are induced at specific stages of development . Their mechanisms of action may involve the RNA itself and/or sORF-encoded oligopeptides . Enod40 genes code for highly structured plant sORF-mRNAs involved in root nodule organogenesis . A novel RNA binding protein interacting with the enod40 RNA, MtRBP1 (for Medicago truncatula RNA Binding Protein 1), was identified using a yeast three-hybrid screening . Immunolocalization studies and use of a MtRBP1-DsRed2 fluorescent protein fusion showed that MtRBP1 localized to nuclear speckles in plant cells but was exported into the cytoplasm during nodule development in enod40-expressing cells . Direct involvement of the enod40 RNA in MtRBP1 relocalization into cytoplasmic granules was shown using a transient expression assay . Using a (green fluorescent protein)/MS2 bacteriophage system to tag the enod40 RNA, we detected in vivo colocalization of the enod40 RNA and MtRBP1 in these granules . This in vivo approach to monitor RNA-protein interactions allowed us to demonstrate that cytoplasmic relocalization of nuclear proteins is an RNA-mediated cellular function of a sORF-mRNA.

J Struct Biol, 2004 Apr-May, 146(1-2), 72 - 8
Recruitment of host ATP-dependent proteases by bacteriophage lambda; Kobiler O et al.; Upon infection of a bacterial cell, the temperate bacteriophage lambda executes a regulated temporal program with two possible outcomes: (1) Cell lysis and virion production or (2) establishment of a dormant state, lysogeny, in which the phage genome (prophage) is integrated into the host chromosome . The prophage is replicated passively as part of the host chromosome until it is induced to resume the lytic cycle . In this review, we summarize the evidence that implicates every known ATP-dependent protease in the regulation of specific steps in the phage life cycle . The proteolysis of specific regulatory proteins appears to fine-tune phage gene expression . The bacteriophage utilizes multiple proteases to irreversibly inactivate specific regulators resulting in a temporally regulated program of gene expression . Evolutionary forces may have favored the utilization of overlapping protease specificities for differential proteolysis of phage regulators according to different phage life styles.

Bioinformatics, 2004 Mar 22, 20(5), 629 - 35 Epub 2004 Jan 22.
PHIRE, a deterministic approach to reveal regulatory elements in bacteriophage genomes; Lavigne R et al.; MOTIVATION: In silico genome analysis of bacteriophage genomes focuses mainly on gene discovery and functional assignment . The search for regulatory elements contained within these genome sequences is often based on prior knowledge of other genomic elements or on learning algorithms of experimentally determined data, potentially leading to a biased prediction output . The PHage In silico Regulatory Elements (PHIRE) program is a standalone program in Visual Basic . It performs an algorithmic string-based search on bacteriophage genome sequences to uncover and extract subsequence alignments hinting at regulatory elements contained within these genomes, in a deterministic manner without any prior experimental or predictive knowledge . RESULTS: The PHIRE program was tested on known phage genomes with experimentally verified regulatory elements . PHIRE was able to extract phage regulatory sequences correctly for bacteriophages T7, T3, YeO3-12 and lambda, based solely on the genome sequence . For 11 bacteriophages, new predictions of conserved phage-specific putative regulatory elements were made, further corroborating this approach . AVAILABILITY: Freely available for academic use . Commercial users should contact the corresponding author.

Gene, 2004 Mar 31, 329, 115 - 24
Distribution of minigenes in the bacteriophage lambda chromosome; Oviedo NA et al.; The bar loci in the chromosome of bacteriophage lambda inhibit phage vegetative growth in bacteria defective for peptidyl-tRNA hydrolase (Pth) . Expression of the bar regions results in accumulation of peptidyl-tRNA, inhibition of protein synthesis, and arrest of mutant cell growth . These effects have been ascribed to the expression of two-codon ORFs present in translatable sequences named 'minigenes' in the lambda bar regions . To investigate the nature, frequency, and distribution of minigenes in the phage genome, we conducted a survey of their location in lambda DNA . A short-fragment random genomic DNA library was constructed for the identification of clones inhibitory of Pth-defective cells (bar-like phenotype) . Three new bar-like minigenes were identified in the library but only one was on the sense strand and it had a rare initiation codon . This result contrasted with the in silico identification of over a hundred putative minigenes using an ad hoc computer program on both strands of lambda DNA . Unlike bar constructs, most of the toxic constructed clones were also toxic to wild-type bacteria, thus suggesting a different inhibition mechanism . Sequence analysis of these cloned inserts showed that they harbored minigenes, mini-ORFs, gene starts, gene ends, or combinations thereof . Our data suggest that minigene-like sequences may, at least partly, account for toxicity in wild-type cells . We propose that clustering of minigenes at gene ends may play a role in gene expression . Other minigenes identified in silico were non-toxic . It is still an open question what the in vivo function of these and toxic minigenes might be.

Biochem Biophys Res Commun, 2004 Apr 9, 316(3), 755 - 62
The relationship between palindrome avoidance and intragenic codon usage variations: a Monte Carlo study; Fuglsang A; Several studies have shown that codon usage within genes varies, as it seems dependent on both codon context and codon position within the gene . Given that palindromes in addition often are avoided in genomes, this study aimed at finding out if intragenic variations in codon usage may be a way to control the amount and location of palindromes . A Monte Carlo algorithm was written which resampled the codons in genes while keeping the amino acid sequence of the translation product constant . On the resampled sequences, palindromes were counted and their intragenic positions mapped . Escherichia coli K12 uses type II restriction-modification systems and displays pronounced codon usage phenomena . Using this as a reference organism it was clearly shown that the number of palindromes in genes is generally lower than the amount of palindromes in resampled genes; thus, the succession of codons seems to be a way to decrease the number of palindromes . The intragenic position of palindromes in resampled sequences, however, was largely equal to the position in the native genes, so codon usage phenomena are unlikely to be a way to control the intragenic position of palindromes . The analysis was repeated on two bacteriophages and gave similar same results, even though the virus genomes are much smaller . Studies on the endosymbionts Buchnera sp . APS and Wigglesworthia sp., which seemingly have no type II restriction-modification systems, showed that in these species there is only weak evidence for codon usage acting to control the number of palindromes.

J Mol Biol, 2004 Apr 2, 337(4), 905 - 15
Very fast folding and association of a trimerization domain from bacteriophage T4 fibritin; Guthe S et al.; The foldon domain constitutes the C-terminal 30 amino acid residues of the trimeric protein fibritin from bacteriophage T4 . Its function is to promote folding and trimerization of fibritin . We investigated structure, stability and folding mechanism of the isolated foldon domain . The domain folds into the same trimeric beta-propeller structure as in fibritin and undergoes a two-state unfolding transition from folded trimer to unfolded monomers . The folding kinetics involve several consecutive reactions . Structure formation in the region of the single beta-hairpin of each monomer occurs on the submillisecond timescale . This reaction is followed by two consecutive association steps with rate constants of 1.9(+/-0.5)x10(6)M(-1)s(-1) and 5.4(+/-0.3)x10(6)M(-1)s(-1) at 0.58 M GdmCl, respectively . This is similar to the fastest reported bimolecular association reactions for folding of dimeric proteins . At low concentrations of protein, folding shows apparent third-order kinetics . At high concentrations of protein, the reaction becomes almost independent of protein concentrations with a half-time of about 3 ms, indicating that a first-order folding step from a partially folded trimer to the native protein (k=210 +/- 20 s(-1)) becomes rate-limiting . Our results suggest that all steps on the folding/trimerization pathway of the foldon domain are evolutionarily optimized for rapid and specific initiation of trimer formation during fibritin assembly . The results further show that beta-hairpins allow efficient and rapid protein-protein interactions during folding.

J Biol Chem, 2004 May 21, 279(21), 21957 - 65 Epub 2004 Mar 16.
Role of protein-protein interactions during herpes simplex virus type 1 recombination-dependent replication; Nimonkar AV et al.; Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity . It also serves as a means to replicate genomic termini . We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A . V., and Boehmer, P . E . (2003) Proc . Natl . Acad . Sci . U . S . A . 100, 10201-10206) . In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme . Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42) . Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8) . Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions . Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8) . Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis . In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1 . These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.

J Biol Chem, 2004 May 21, 279(21), 22218 - 27 Epub 2004 Mar 15.
Distribution, sequence homology, and homing of group I introns among T-even-like bacteriophages: evidence for recent transfer of old introns; Sandegren L et al.; Self-splicing group I introns are being found in an increasing number of bacteriophages . Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG) . The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations . We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns . A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a "full set" of these introns . Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages . The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations . Several of the introns can home to closely related intronless phages during mixed infections . However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site . The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing . These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages.

J Am Chem Soc, 2004 Mar 24, 126(11), 3454 - 60
Ligating DNA with DNA; Sreedhara A et al.; Cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation . A commonly used enzyme for this reaction is bacteriophage T4 DNA ligase, which requires ATP as the energy source to catalyze the otherwise unfavorable formation of a phosphodiester bond . Using in vitro selection, we have isolated a DNA sequence that catalyzes the ligation of DNA in the absence of protein enzymes . We have used the action of two catalytic DNAs, an ATP-dependent self-adenylating deoxyribozyme (AppDNA) and a self-ligating deoxyribozyme, to create a ligation system that covalently joins oligonucleotides via the formation of a 3',5'-phosphodiester linkage . The two-step process is conducted in separate reaction vessels wherein the products of deoxyribozyme adenylation are purified before their use as substrates for deoxyribozyme ligation . The final ligation step of the deoxyribozyme-catalyzed sequence of reactions mimics the final step of the T4 DNA ligase reaction . The initial rate constant (k(obs)) of the optimized deoxyribozyme ligase was found to be 1 x 10(-)(4) min(-)(1) . Under these conditions, the ligase deoxyribozyme promotes DNA ligation at least 10(5)-fold faster than that generated by a simple DNA template . The self-ligating deoxyribozyme has also been reconfigured to generate a trans-acting construct that joins separate DNA oligonucleotides of defined sequence . However, the sequence requirements of the AppDNA and that of the 3' terminus of the deoxyribozyme ligase limit the range of sequences that can be ligated.

Biochemistry, 2004 Mar 23, 43(11), 2987 - 95
Use of a multiple-enzyme/multiple-reagent assay system to quantify activity levels in samples containing mixtures of matrix metalloproteinases; Rasmussen FH et al.; Matrix metalloproteinases (MMPs) are a family of enzymes that are up-regulated in many diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA) . Here we report on a novel technique that can be used to simultaneously measure activity levels for a panel of enzymes, such as the MMPs . The technique, termed the multiple-enzyme/multiple-reagent assay system (MEMRAS), relies on the use of reagents such as substrates with varying selectivity profiles against a group of enzymes . When reaction rates are measured by following a change in fluorescence with time, for mixtures of enzymes, an equation with unknown concentrations for each activity is generated for each reagent used . Simultaneously solving the set of equations leads to a solution for the unknown concentrations . We have applied this mathematical technique to measure activity levels for mixtures of MMPs such as collagenase 3 and gelatinase A . In addition, because we were most interested in determining collagenase 3 levels as a potential biological marker for OA, we developed highly selective substrates for this enzyme by using results found in previous bacteriophage substrate-mapping experiments . Some of the best substrates tested have specific activities for collagenase 3 that are 37,000-, 17,000-, 90-, and 200-fold selective over stromelysin 1, collagenase 1, and gelatinases A and B, respectively.

Genetics, 2004 Jan, 166(1), 19 - 24
Drift increases the advantage of sex in RNA bacteriophage Phi6; Poon A et al.; The pervasiveness of sex and recombination remains one of the most enigmatic problems in evolutionary biology . According to many theoretical models, recombination can increase the rate of adaptation by restoring genetic variation . However, the potential for genetic drift to generate conditions that produce this outcome has yet to be studied experimentally . We have designed and performed an experiment that reveals the effects of drift on existing genetic variation by minimizing the influence of variation on beneficial mutation rate . Our experiment was conducted in populations of RNA bacteriophage Phi6 initiated from a common source population at varying bottleneck sizes . The segmented genome of this virus results in genetic exchange between viruses that co-infect the same host cell . In response to selection for growth in a high-temperature environment, sexual lines outperformed their asexual counterparts on average . The advantage of sex attenuated with increasing effective population size, implying that the rate of adaptation was limited by clonal interference among segments caused by drift . This is the first empirical evidence that the advantage of sex during adaptation increases with the intensity of drift.

J Mol Biol, 2004 Mar 26, 337(3), 513 - 9
De novo folding of membrane proteins: an exploration of the structure and NMR properties of the fd coat protein; Im W et al.; De novo folding simulations of the major pVIII coat protein from filamentous fd bacteriophage, using a newly developed implicit membrane generalized Born model and replica-exchange molecular dynamics, are presented and discussed . The quality of the predicted structures, judged by comparison of the root-mean-square deviations of a room temperature ensemble of conformations from the replica-exchange simulations and experimental structures from both solid-state NMR in lipid bilayers and solution-phase NMR on the protein in micelles, was quite good, reinforcing the general quality of the folding simulations . The transmembrane helical segment of the protein was well defined in comparison with experiment and the amphipathic helical fragment remained at the membrane/aqueous phase boundary while undergoing significant conformational flexibility due to the loop connecting the two helical segments of the protein . Additional comparisons of computed solid-state NMR properties, the 15N chemical shift and 15N-1H dipolar coupling constants, showed semi-quantitative agreement with the corresponding measurements . These findings suggest an emerging potential for the de novo investigation of integral membrane peptides and proteins and a mechanism to assist experimental approaches to the characterization and structure determination of these important systems.

Cell, 2004 Feb 6, 116(3), 351 - 3
Active-site dynamics in RNA polymerases; Landick R; New crystal structures of transcription complexes formed by bacteriophage T7 RNA polymerase reveal a nucleotide-addition cycle driven by active-site conformational changes similar to those observed in DNA polymerases, and suggest provocative hypotheses for the more complex multisubunit RNA polymerases of free-living organisms.

Exp Parasitol, 2004 Jan-Feb, 106(1-2), 37 - 44
Trypanosoma brucei: a first-generation CRE-loxP site-specific recombination system; Barrett B et al.; The bacteriophage CRE-loxP system of DNA recombination is widely used to manipulate segments of the genomes of mice and other eukaryotes for the purpose of studying the regulation and functions of their genes . Since this recombination system could have similar applications in analyzing the genomes of trypanosomatids, we assessed the action of CRE recombinase on its loxP DNA recognition sites in Trypanosoma brucei after inserting tetracycline-regulated CRE and two 34-bp loxP sites into the T . brucei genome . We found that when loxP sites flank in a direct orientation the transcription termination sequence (1.1 kb) of the T . brucei GPEET/PAG3 locus, CRE recombinase deletes this termination sequence, permitting transcription and subsequent expression of a downstream reporter gene for the green fluorescent protein (GFP) . Thus, the CRE-loxP system is highly efficient in T . brucei, but the experimental results also indicate that a better way than the existing tetracycline-regulated system is required to completely silence expression of CRE in the T . brucei genome when it is not needed before the full range of CRE-loxP applications currently used in mice can be exploited in African trypanosomes.

Virus Res, 2004 Apr, 101(1), 93 - 100
Self-assembly of double-stranded RNA bacteriophages; Poranen MM et al.; Double-stranded RNA viruses infecting bacterial hosts belong to the Cystoviridae family . Bacteriophage phi6 is one of the best characterized dsRNA viruses and shares structural as well as functional similarities with other well-studied eukaryotic dsRNA viruses (e.g . L-A, rotavirus, bluetongue virus, and reovirus) . The assembly pathway of the enveloped, triple-layered phi6 virion has been well documented and can be divided into four distinct steps which are (1) procapsid formation, (2) genome encapsidation and replication, (3) nucleocapsid surface shell assembly, and (4) envelope formation . In this review, we focus primarily on the procapsid and nucleocapsid assembly for which in vitro systems have been established . The in vitro assembly systems have been instrumental in revealing assembly intermediates and conformational changes that are common to phi6 and phi8, two cystoviruses with negligible sequence homology . Two viral enzymes, the packaging NTPase (P4) and the RNA-dependent RNA polymerase (P2), were found essential for the nucleation step . The nucleation complex contains one or more tetramers of the major procapsid protein (P1) and is further stabilized by protein P4 . Interaction of P1 and P4 during assembly is accompanied by an additional folding of their respective polypeptide chains . The in vitro assembled procapsids were shown to selectively package and replicate the genomic ssRNA . Furthermore, in vitro assembly of infectious nucleocapsids has been achieved in the case of phi6 . The in vitro studies indicate that the nucleocapsid coat protein (P8) assembles around the polymerase complex in a template-assisted manner . Implications for the assembly of other dsRNA viruses are also presented.

Virus Res, 2004 Apr, 101(1), 83 - 92
Packaging, replication and recombination of the segmented genome of bacteriophage Phi6 and its relatives; Mindich L; The genomes of bacteriophage Phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented dsRNA genomes into preformed polyhedral structures called procapsids or inner cores . The packaging requires hydrolysis of NTPs and takes place in the order S:M:L . Minus strand synthesis begins after the completion of the plus strand packaging . The packaging and replication reactions can be studied in vitro with purified components . A model has been presented that proposes that the program of serially dependent packaging is determined by the conformational changes at the surface of the procapsid due to the amount of RNA packaged at each step . The in vitro packaging and replication system has facilitated the application of reverse genetics and the study of recombination in the family of Cystoviridae.

Virus Res, 2004 Apr, 101(1), 45 - 55
RNA-dependent RNA polymerases of dsRNA bacteriophages; Makeyev EV et al.; Genome replication and transcription of riboviruses are catalyzed by an RNA-dependent RNA polymerase (RdRP) . RdRPs are normally associated with other virus- or/and host-encoded proteins that modulate RNA polymerization activity and template specificity . The polymerase complex of double-stranded dsRNA viruses is a large icosahedral particle (inner core) containing RdRP as a minor constituent . In phi6 and other dsRNA bacteriophages from the Cystoviridae family, the inner core is composed of four virus-specific proteins . Of these, protein P2, or Pol subunit, has been tentatively identified as RdRP by sequence comparisons, but the role of this protein in viral RNA synthesis has not been studied until recently . Here, we overview the work on the Pol subunits of phi6 and related viruses from the standpoints of function, structure and evolution.

Mol Microbiol, 2004 Mar, 51(6), 1719 - 28
Switching the polarity of a bacteriophage integration system; Smith MC et al.; During lysogenic growth many temperate bacteriophage genomes are integrated into the host's chromosome and efficient integration and excision are therefore an essential part of the phage life cycle . The Streptomyces phage phiC31 encodes an integrase related to the resolvase/invertases and is evolutionarily and mechanistically distinct from the integrase of phage lambda . We show that during phiC31 integration the polarity of the recombination sites, attB and attP, is dependent on the sequences of the two base pairs (bp) where crossover occurs . A loss or switch in polarity of the recombination sites can occur by mutation of this dinucleotide, leading to incorrectly joined products . The properties of the mutant sites implies that phiC31 integrase interacts symmetrically with the substrates, which during synapsis can align apparently freely in either of two alternative forms that lead to correct or incorrect joining of products . Analysis of the topologies of the reaction products provided evidence that integrase can synapse and activate strand exchange even when recombinant products cannot form due to mismatches at the crossover site . The topologies of the recombination products are complex and indicative of multiple pathways to product formation . The efficiency of integration of a phiC31 derivative, KC859, into an attB site with switched polarity was assayed in vivo and shown to be no different from integration into a wild-type attB . Thus neither the host nor KC859 express a factor that influences the alignment of the recombination sites at synapsis.

Appl Environ Microbiol, 2004 Mar, 70(3), 1506 - 13
Optimization of procedures for counting viruses by flow cytometry; Brussaard CP; The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems . However, the methods used in flow cytometric analyses of viruses have not been consistent to date . A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here . The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested . A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined . The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80 degrees C . Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 x 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80 degrees C, and cooled for 5 min prior to analysis . The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80 degrees C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.

Bioorg Med Chem Lett, 2004 Mar 22, 14(6), 1389 - 93
Dual surface selection methodology for the identification of thrombin binding epitopes from hotspot biased phage-display libraries; Rajagopal S et al.; Protein libraries biased towards amino-acid residues found at so-called 'hotspots' were incorporated into the beta-sheet region of the thermostable variant (HTB1) of the B1 domain of the immunoglobulin (IgG) binding protein G and expressed as gene 3 fusions on M13 bacteriophage . The HTB1 library (2.2 x 10(9)) variants with a minimal 12 amino acid basis set were selected for binding IgG, to ensure structural conservation, and subsequently to thrombin to evolve a thrombin-binding function . We believe that this dual surface selection strategy will have great utility in evolving new bi-functional proteins without compromising structure . Furthermore the discrete beta-sheet epitopes identified by our methodology will lend itself to small-molecule mimicry of beta-sheets.

Electrophoresis, 2004 Mar, 25(6), 785 - 9
Diffusion constant in gel electrophoresis at high fields; Krawczyk MJ et al.; We present new measurements of the diffusion constant D in standard (slab-gel) electrophoresis of DNA at fields up to 10 V/cm . Molecules investigated are bacteriophages: T4 of length 173 kbp and lambda of length 48.5 kbp cut by restriction enzyme HindIII . We show, that D increases with the molecule length for electric field E above 5 V/cm . The results are interpreted within the geometration model.

Electrophoresis, 2004 Mar, 25(6), 779 - 84
Analysis of proteins stained by Alexa dyes; Huang S et al.; Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes . To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained . The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination . The Alexa-stained proteins are observed directly in gels by illumination with an ultraviolet transilluminator . The test multimolecular particle is bacteriophage T7 . The protein capsid of T7 is a multimolecular complex that has both external and internal proteins . SDS-PAGE of Alexa-stained bacteriophage T7 produces fluorescent capsid proteins each of which usually comigrates with an unstained protein . However, one Alexa-induced modification of protein migration was observed by SDS-PAGE . Mass spectrometry shows that the protein with modified migration is the major protein of the outer shell of the T7 capsid . The procedures used are generally applicable . The distribution of Alexa staining among T7 capsid proteins depends on the size of the dye molecule used . The larger the dye molecule is, the greater the preference for external proteins.

Trends Biochem Sci, 2004 Mar, 29(3), 127 - 35
Chromatin remodeling by RNA polymerases; Studitsky VM et al.; Chromatin packages DNA tightly into the eukaryotic nucleus and maintains its proper functioning . Recent studies suggest the existence of two distinct mechanisms of progression of RNA polymerases through chromatin . The first is characteristic of eukaryotic RNA polymerase III, bacteriophage RNA polymerases, and probably ATP-dependent chromatin remodeling complexes . In this mechanism, nucleosomes are translocated without release of the octamer into solution . By contrast, transcription by RNA polymerase II (Pol II) involves displacement of one H2A-H2B dimer . Nucleosomes can present a barrier for transcribing Pol II that can be regulated in vivo . Analysis of the mechanisms of transcription through chromatin should provide important information about mechanisms of chromatin remodeling and gene regulation at the level of transcript elongation.

J Mol Biol, 2004 Mar 12, 337(1), 105 - 13
Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis; Suzuki H et al.; The FliF ring is the base for self-assembly of the bacterial flagellum and the FliF/FliG ring complex is the core of the rotor of the flagellar motor . We report the structures of these two ring complexes obtained by electron cryomicroscopy and single-particle image analysis at 22A and 25A resolution, respectively . Direct comparison of these structures with the flagellar basal body made by superimposing the density maps on the central section reveals many interesting features, such as how the mechanically stable connection between the ring and the rod is formed, how directly FliF domains are involved in the near axial density of the basal body forming the proximal end of the central channel for a potential gating mechanism, some indication of flexibility in the connection of FliF and FliG, and structural and functional similarities to the head-to-tail connectors of bacteriophages.

Proteins, 2004 Mar 1, 54(4), 681 - 92
Probing the alpha-complementing domain of E . coli beta-galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition; Poussu E et al.; Protein structure-function relationships can be studied by using linker insertion mutagenesis, which efficiently identifies essential regions in target proteins . Bacteriophage Mu in vitro DNA transposition was used to generate an extensive library of pentapeptide insertion mutants within the alpha-complementing domain 1 of Escherichia coli beta-galactosidase, yielding mutants at 100% efficiency . Each mutant contained an accurate 15-bp insertion that translated to five additional amino acids within the protein, and the insertions were distributed essentially randomly along the target sequence . Individual mutants (alpha-donors) were analyzed for their ability to restore (by alpha-complementation) beta-galactosidase activity of the M15 deletion mutant (alpha-acceptor), and the data were correlated to the structure of the beta-galactosidase tetramer . Most of the insertions were well tolerated, including many of those disrupting secondary structural elements even within the protein's interior . Nevertheless, certain sites were sensitive to mutations, indicating both known and previously unknown regions of functional importance . Inhibitory insertions within the N-terminus and loop regions most likely influenced protein tetramerization via direct local effects on protein-protein interactions . Within the domain 1 core, the insertions probably caused either lateral shifting of the polypeptide chain toward the protein's exterior or produced more pronounced structural distortions . Six percent of the mutant proteins exhibited temperature sensitivity, in general suggesting the method's usefulness for generation of conditional phenotypes . The method should be applicable to any cloned protein-encoding gene .

Proteins, 2004 Apr 1, 55(1), 187 - 97
Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1; Nuttall SD et al.; The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains . The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units . We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage . Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum . Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops . These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E . coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M) . One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops . The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific . Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications .

J Bacteriol, 2004 Mar, 186(6), 1606 - 13
A subassembly of R27-encoded transfer proteins is dependent on TrhC nucleoside triphosphate-binding motifs for function but not formation; Gilmour MW et al.; The transfer of plasmid DNA molecules between bacterial cells is achieved by a large array of conjugative transfer proteins which assemble into both cytoplasmic and membrane-associated complexes . TrhC is a membrane-associated protein that is required for the transfer of the IncHI1 resistance plasmid R27 . Homologous proteins are encoded in all known conjugative systems, and each contains characteristic nucleoside triphosphate (NTP)-binding domains . An assembly of R27-encoded proteins was previously visualized by use of a TrhC-green fluorescent protein fusion, which appeared as discrete membrane-associated fluorescent foci . We have utilized this experimental system to determine the requirements for assembly of this TrhC-associated protein complex, and we found that 12 of the other 18 R27 transfer proteins are required for focus formation . An individual focus possibly represents a subassembly comprised of some or all of these transfer proteins . These data support the notion that the transfer apparatus is a multicomponent structure . In contrast, substitutions and deletions within TrhC NTP-binding motifs had minor effects on focus formation, but these mutations did affect plasmid transfer and bacteriophage susceptibility . These results indicate that TrhC requires intact NTP-binding motifs to function during conjugative transfer but that these motifs are not essential for the assembly of TrhC into a complex with other transfer proteins.

J Bacteriol, 2004 Mar, 186(6), 1591 - 7
H protein of bacteriophage 16-3 and RkpM protein of Sinorhizobium meliloti 41 are involved in phage adsorption; Putnoky P et al.; The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3 . In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants . A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis . A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3 . The nucleotide sequences of the h gene as well as a host range mutant allele were also established . In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption . Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.

Plant J, 2004 Mar, 37(6), 889 - 96
Conditional, recombinase-mediated expression of genes in plant cell cultures; Joubes J et al.; In plant cells, overexpression of critical genes can be hampered by deleterious effects on development that results in a counterselection of transgenic cells harboring the gene of interest . Inducible expression systems have been reported, but many of them show unwanted leaky expression . To circumvent this potential problem, a novel inducible system was developed based on two previously characterized systems: the CRE-loxP site-specific recombination system of bacteriophage P1 and the subcellular targeting of proteins by a mammalian glucocorticoid receptor (GR) . By fusing the receptor domain of the rat GR to the carboxyl terminus of the CRE recombinase, a double-lock conditional transcriptional induction system was created that is highly useful to overexpress genes whose expression may block transgenic regeneration . Furthermore, because the designed vector utilizes the GATEWAY recombination technology, cloning was restriction- and ligation-free, thus rendering the vector suitable for high-throughput research . The system was tested in Nicotiana tabacum bright yellow-2 (BY-2) cells and its efficiency was demonstrated for the controlled overexpression of the gus reporter gene and a mutant allele of the A-type cyclin-dependent kinase (CDKA), which is known to be a potent inhibitor of the cell cycle.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 588 - 90 Epub 2004 Feb 25.
Production, crystallization and preliminary X-ray crystallographic studies of the bacteriophage phi 12 packaging motor; Mancini EJ et al.; The hexameric ATPase P4 from bacteriophage phi 12 is responsible for packaging single-stranded genomic precursors into the viral procapsid . P4 was overexpressed in Escherichia coli and purified . Crystals of native and selenomethionine-derivatized P4 have been obtained that belong to space group I222, with half a hexamer in the asymmetric unit and unit-cell parameters a = 105.0, b = 130.5, c = 158.9 A . A second crystal form of different morphology can occur in the same crystallization drop . The second form belongs to space group P1, with four hexamers in the asymmetric unit and unit-cell parameters a = 114.9, b = 125.6, c = 153.9 A, alpha = 90.1, beta = 91.6, gamma = 90.4 degrees . Synchrotron X-ray diffraction data have been collected for the I222 and P1 crystal forms to 2.0 and 2.5 A resolution, respectively.

Nephrol Dial Transplant, 2004 May, 19(5), 1298 - 301 Epub 2004 Feb 19.
Genotyping: a new application for the spent dialysate in peritoneal dialysis; Gillerot G et al.; BACKGROUND: The dialysate of patients on peritoneal dialysis (PD) is used to determine the concentration of growth factors and cytokines, and as a source of resident peritoneal cells for subsequent culture experiments . We hypothesized that the cells contained in spent dialysate samples obtained at the time of the peritoneal equilibration test (PET) and subsequently stored may represent a source of DNA from a given PD patient . METHODS: We characterized a protocol of DNA extraction from dialysates obtained in PD patients after a long dwell during the initial PET and kept frozen up to several years . After amplification of the source DNA by strand displacement using the bacteriophage phi 29 DNA polymerase, we performed polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for the Glu298Asp polymorphism of ENOS to demonstrate the suitability of the extracted DNA for genotyping . RESULTS: A significant amount of DNA (mean yield 12 microg/ml dialysate) was extracted from frozen dialysate samples . The extraction yield was not influenced by the duration of storage at -20 degrees C . Following amplification, the DNA extracted from the dialysate was used successfully for genotyping the Glu298Asp polymorphism of ENOS, as demonstrated by parallel analyses using DNA extracted from the peripheral blood and sequencing . CONCLUSIONS: These results demonstrate that the dialysis effluent obtained at the time of the initial PET and stored at -20 degrees C is a reliable source of DNA that can be used subsequently for PCR amplification, RFLP analysis and sequencing.

Biopolymers, 2003, 71(6), 675 - 85
Polar residue tagging of transmembrane peptides; Melnyk RA et al.; Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains . However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity . We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble . Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented . Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states . Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state . Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments . The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes .

Eur Biophys J, 2004 Oct, 33(6), 497 - 505 Epub 2004 Feb 26.
Helical packaging of semiflexible polymers in bacteriophages; Metzler R et al.; We investigate multilayered helical packaging of double-stranded DNA, or of a general polymer chain with persistence length lb, into an ideal, inert cylindrical container, reaching densities slightly below close packaging . We calculate the free energy as a function of the packaged length, based on the energies for bending, twisting, the suffered entropy loss, and the electrostatic energy in a Debye-Huckel model . In the absence of charges on the packaged polymer, a critical packaging force can be determined, similar to the mechanism involved in DNA unzipping models . When charges are taken into consideration, in the final packaging state the charges which are chemically distant become geometrically close, and therefore a steep rise is seen in the free energy . We argue that due to the extremely ordered and almost closely packaged final state the actual packaging geometry does not influence the behaviour of the free energy, pointing towards a certain universality of this state of the polymer . Our findings are compared to a recent simulations study, showing that the model is sensitive to the screening length.

Nat Biotechnol, 2004 Mar, 22(3), 321 - 5 Epub 2004 Feb 08.
Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase; Kim DH et al.; Small interfering RNAs (siRNA) are potent reagents for directed post-transcriptional gene silencing and a major new genetic tool for investigating mammalian cells . When synthetic siRNAs are used for gene silencing, the costs can be substantial because of variations in siRNA efficacies . An alternative to chemically synthesized siRNAs are siRNAs produced by bacteriophage T7 RNA polymerase . We found that siRNAs synthesized from the T7 RNA polymerase system can trigger a potent induction of interferon alpha and beta in a variety of cell lines . Surprisingly, we also found very potent induction of interferon alpha and beta by short single-stranded RNAs (ssRNAs) transcribed with T3, T7 and Sp6 RNA polymerases . Analyses of the potential mediators of this response revealed that the initiating 5' triphosphate is required for interferon induction . We describe here an improved method for T7 siRNA synthesis that alleviates the interferon response while maintaining full efficacy of the siRNAs.

J Virol, 2004 Mar, 78(6), 2711 - 21
Presence of an encephalomyocarditis virus internal ribosome entry site sequence in avian infectious bronchitis virus defective RNAs abolishes rescue by helper virus; Dove B et al.; Avian infectious bronchitis virus (IBV) defective RNAs (D-RNAs) have been used for the expression of heterologous genes in a helper-virus-dependent expression system . The heterologous genes were expressed under the control of an IBV transcription-associated sequence (TAS) derived from gene 5 of IBV Beaudette . However, coronavirus D-RNA expression vectors display an inherent instability following serial passage with helper virus, resulting in the eventual loss of the heterologous genes . The use of the picornavirus encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence to initiate gene translation was investigated as an alternative method to the coronavirus-mediated TAS-controlled heterologous gene expression system . IBV D-RNAs containing the chloramphenicol acetyltransferase (CAT) reporter gene, under EMCV IRES control, were assessed for IRES-mediated CAT protein translation . CAT protein was detected from T7-derived IBV D-RNA transcripts in a cell-free protein synthesis system and in situ in avian chick kidney (CK) cells following T7-derived D-RNA synthesis from a recombinant fowlpox virus expressing the bacteriophage T7 DNA-dependent RNA polymerase . However, CAT protein was not detected in CK cells from IRES-containing IBV D-RNAs, in which the IRES-CAT construct was inserted at two different positions within the D-RNA, in the presence of helper IBV . Northern blot analysis demonstrated that the IRES-containing D-RNAs were not rescued on serial passage with helper virus, indicating that the EMCV IRES sequence had a detrimental effect on IBV D-RNA rescue.

J Theor Biol, 2004 Mar 21, 227(2), 229 - 37
Bistability and switching in the lysis/lysogeny genetic regulatory network of bacteriophage lambda; Tian T et al.; Bistability and switching are two important aspects of the genetic regulatory network of lambda phage . Positive and negative feedbacks are key regulatory mechanisms in this network . By the introduction of threshold values, the developmental pathway of lambda phage is divided into different stages . If the protein level reaches a threshold value, positive or negative feedback will be effective and regulate the process of development . Using this regulatory mechanism, we present a quantitative model to realize bistability and switching of lambda phage based on experimental data . This model gives descriptions of decisive mechanisms for different pathways in induction . A stochastic model is also introduced for describing statistical properties of switching in induction . A stochastic degradation rate is used to represent intrinsic noise in induction for switching the system from the lysogenic pathway to the lysis pathway . The approach in this paper represents an attempt to describe the regulatory mechanism in genetic regulatory network under the influence of intrinsic noise in the framework of continuous models.

Biotechniques, 2004 Feb, 36(2), 296 - 300, 302
Pathotyping of Newcastle disease virus with a filamentous bacteriophage; Ramanujam P et al.; A filamentous phage bearing the peptide sequence TLTTKLY was isolated from a heptapeptide phage display library against a velogenic Newcastle disease virus (NDV) . In order to investigate the potential of this specific phage as an immunological reagent in virus pathotyping, an enzyme-linked immunosorbent assay (ELISA)-based method was developed . This method can differentiate the velogenic strains from the mesogenic and lentogenic strains . An equilibrium-binding assay in solution showed that the interactions between the phage and all the NDV strains gave rise to two widely differing dissociation constants (Kdrel) . Based upon the first Kdrel values, NDV strains can be classified into two groups; the first comprises the velogenic strains, and the second consists of the mesogenic and lentogenic strains . These results indicate a high degree of correlation between the binding affinities and pathotyping of NDV strains using the TLTTKLY phage.

Protein Eng Des Sel, 2004 Jan, 17(1), 13 - 20
A native-like artificial protein from antisense DNA; Fischer N et al.; We describe the creation of folded chimaeric proteins by combining a designed polypeptide segment (bait) derived from a beta-sheet of a human antibody variable domain with random polypeptide segments encoded by human cDNA fragments . The repertoire of polypeptides was displayed on the surface of filamentous bacteriophage and folded polypeptides were selected by proteolysis . One of these, 2a6, was readily expressed in the Escherichia coli cytoplasm as a soluble and protease-resistant protein and could be purified after heating the bacterial lysate to 90 degrees C . Soluble 2a6 is dimeric and its CD spectrum is consistent with components of both alpha and beta structure . 2a6 cooperatively and reversibly unfolds by heat or urea with a folding energy of 11.4 kcal mol(-1) for the transition between folded dimer and unfolded monomer and its refolding steps proceed without the formation of detectable aggregates . Its stability and folding properties are therefore typical of native proteins . Sequence analysis revealed that the cDNA segment in 2a6 was recruited from the antisense strand of a human gene, suggesting that antisense sequences can provide a reservoir for the evolution of soluble and stable proteins.

J Biol Chem, 2004 Apr 30, 279(18), 19217 - 29 Epub 2004 Feb 25.
Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase; Choi JY et al.; The N-2 atom of guanine (G) is susceptible to modification by various carcinogens . Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease(-) (T7(-)) . RT and T7(-) effectively bypassed N(2)-methyl(Me)G and readily extended primers but were strongly blocked by N(2)-ethyl(Et)G, N(2)-isobutylG, N(2)-benzylG, and N(2)-methyl(9-anthracenyl)G . Steady-state kinetics of single nucleotide incorporation by RT and T7(-) showed a decrease of 10(3) in k(cat)/K(m) for dCTP incorporation opposite N(2)-MeG and a further large decrease opposite N(2)-EtG . Misincorporation frequency was increased 10(2)-10(3)-fold by a Me group and another approximately 10(3)-fold by an Et group . dATP was preferentially incorporated opposite bulky N(2)-alkylG molecules . N(2)-MeG attenuated the pre-steady-state kinetic bursts with RT and T7(-), and N(2)-EtG eliminated the bursts . Large elemental effects with thio-dCTP(alphaS) were observed with N(2)-EtG (6- and 72-fold decreases) but were much less with N(2)-MeG, indicating that the N(2)-Et group may affect the rate of the chemistry step (phosphodiester bond formation) . Similar values of K(d(dCTP)) and K(d(DNA)) and k(off) rates of DNA substrates from RT and T7(-) indicate that ground-state binding and dissociation rates are not considerably affected by the bulk . We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions.

J Am Chem Soc, 2004 Mar 3, 126(8), 2414 - 20
Measurements of side-chain 13C-13C residual dipolar couplings in uniformly deuterated proteins; Vogeli B et al.; 13C-only spectroscopy was used to measure multiple residual (13)C-(13)C dipolar couplings (RDCs) in uniformly deuterated and (13)C-labeled proteins . We demonstrate that (13)C-start and (13)C-observe spectra can be routinely used to measure an extensive set of the side-chain residual (13)C-(13)C dipolar couplings upon partial alignment of human ubiquitin in the presence of bacteriophages Pf1 . We establish that, among different broadband polarization transfer schemes, the FLOPSY family can be used to exchange magnetization between a J coupled network of spins while largely decoupling dipolar interactions between these spins . An excellent correlation between measured RDCs and the 3D structure of the protein was observed, indicating a potential use of the (13)C-(13)C RDCs in the structure determination of perdeuterated proteins.

Virology, 2004 Feb 20, 319(2), 185 - 9
In vivo recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides; Oppenheim AB et al.; We demonstrate that the bacteriophage lambda Red functions efficiently recombine linear DNA or single-strand oligonucleotides (ss-oligos) into bacteriophage lambda to create specific changes in the viral genome . Point mutations, deletions, and gene replacements have been created . While recombineering with oligonucleotides, we encountered other mutations accompanying the desired point mutational change . DNA sequence analysis suggests that these unwanted mutations are mainly frameshift deletions introduced during oligonucleotide synthesis.

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2788 - 93 Epub 2004 Feb 18.
Portability and fidelity of RNA-repair systems; Schwer B et al.; Yeast tRNA ligase (Trl1) is an essential enzyme that converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO(4), 3'-5' phosphodiester at the splice junction . Trl1 also catalyzes splicing of HAC1 mRNA during the unfolded protein response . Trl1 performs three reactions: the 2',3'-cyclic phosphate of the proximal RNA fragment is hydrolyzed to a 3'-OH, 2'-PO(4) by a cyclic phosphodiesterase; the 5'-OH of the distal RNA fragment is phosphorylated by a GTP-dependent polynucleotide kinase; and the 3'-OH, 2'-PO(4), and 5'-PO(4) ends are then sealed by an ATP-dependent RNA ligase . The removal of the 2'-PO(4) at the splice junction is catalyzed by the essential enzyme Tpt1, which transfers the RNA 2'-PO(4) to NAD(+) to form ADP-ribose 1"-2"-cyclic phosphate . Here, we show that the bacteriophage T4 enzymes RNA ligase 1 and polynucleotide kinase/phosphatase can fulfill the tRNA and HAC1 mRNA splicing functions of yeast Trl1 in vivo and bypass the requirement for Tpt1 . These results attest to the portability of RNA-repair systems, notwithstanding the significant differences in the specificities, mechanisms, and reaction intermediates of the individual yeast and T4 enzymes responsible for the RNA healing and sealing steps . We surmise that Tpt1 and its unique metabolite ADP-ribose 1"-2"-cyclic phosphate do not play essential roles in yeast independent of the tRNA-splicing reaction . Our finding that one-sixth of spliced HAC1 mRNAs in yeast cells containing the T4 RNA-repair system suffered deletion of a single nucleotide at the 3' end of the splice-donor site suggests a model whereby the yeast RNA-repair system evolved a requirement for the 2'-PO(4) for RNA ligation to suppress inappropriate RNA recombination.

J Bacteriol, 2004 Mar, 186(5), 1503 - 17
Genomic and genetic analysis of Bordetella bacteriophages encoding reverse transcriptase-mediated tropism-switching cassettes; Liu M et al.; Liu et al . recently described a group of related temperate bacteriophages that infect Bordetella subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science 295: 2091-2094, 2002) . Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the mtd (major tropism determinant) gene, which determines specificity for receptors on host bacteria . In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability . Forty-nine coding sequences were identified . Of these coding sequences, bbp36 contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and bpm encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations . Morphological and sequence analysis suggests that these Bordetella phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes . Bordetella bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection.

Biophys Chem, 2004 Feb 15, 107(3), 255 - 62
Line shape analyses for water 17O NMR quintet observed in a bacteriophage Pf1 solution at different temperatures; Mao XA et al.; Partially resolved 17O NMR quintet was observed in a filamentous bacteriophage Pf1 solution at 70 degrees C with a quadrupole splitting approximately 100 Hz . As the temperature decreased, the resolution was reduced but the line shapes were still indicative of residual quadrupole splitting . Line shape analyses were performed using the quadrupolar relaxation theory for spin 5/2 . The contribution to the residual quadrupole splitting from the electric field gradients stemming from the phage filaments, which were oriented in the magnet, was taken into account . As a result, the observed 17O spectra at different temperatures were simulated and the hydration number of the phage DNA was determined.

Mol Divers, 2004, 8(1), 35 - 50
Effective combinatorial strategy to increase affinity of carbohydrate binding by peptides; Landon LA et al.; The Thomsen-Friedenreich antigen, a carcinoma-associated disaccharide involved in carcinoma cell homotypic aggregation and increased metastatic potential, has clinical value as a prognostic indicator and a marker of metastasized cells . Hence, it can reasonably be predicted that antigen-binding macromolecules are valuable clinical in vivo diagnostic/therapeutic targeting agents . Recently, we have selected first-generation antigen-binding peptides from a random peptide bacteriophage display library and have applied combinatorial affinity maturation to select functionally-maturated peptides, which target cultured carcinoma cells and inhibit carcinoma cell aggregation . In the current study we hypothesize that a targeted search of sequence space surrounding the antigen-binding consensus sequence will select unpredictable amino acid sequences in the non-consensus portions of the peptides, leading to increased affinity for the carbohydrate and greater solubility in physiological buffers . This comprehensive in vitro analysis demonstrates that preferential evolution of the amino-terminal sequence of the peptides occurred, which correlated, in structure/function studies, with the acquisition of maturated function . The maturated peptides are more soluble than the earlier peptides . Studies of peptide binding to the disaccharide indicate that two maturated peptides (P-30-1, F03) have higher affinity for the antigen and bind with higher intensity to the surface of cultured human carcinoma cells than the first-generation peptides . The results support our hypothesis that affinity maturation can improve carbohydrate binding by peptides and have theoretical importance as the first report of maturation of carbohydrate-binding affinity in a small, soluble peptide.

J Virol, 2004 Mar, 78(5), 2637 - 41
Identification of peptides that inhibit the DNA binding, trans-activator, and DNA replication functions of the human papillomavirus type 11 E2 protein; Deng SJ et al.; Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library . Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro . These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPV-associated diseases.

Mol Biol Evol, 2004 Apr, 21(4), 746 - 59 Epub 2004 Feb 12.
A new group of tyrosine recombinase-encoding retrotransposons; Goodwin TJ et al.; A wide variety of novel tyrosine recombinase (YR)-encoding retrotransposons were identified using data emerging from the various eukaryotic genome sequencing projects . Although many of these elements are clearly members of the previously described DIRS group of YR retrotransposons, a substantial number, including elements from a variety of fungi and animals, belong to a distinct and previously unrecognized group . We refer to these latter elements as the Ngaro group after a representative from zebrafish . Like the members of the DIRS group, Ngaro elements encode proteins bearing reverse transcriptase (RT) and ribonuclease H (RH) domains similar to those of long terminal repeat (LTR) retrotransposons . Phylogenetic analyses based on alignments of RT/RH and YR domains, however, indicate that Ngaro and DIRS are anciently diverged groups . Differences in coding capacity also support the distinction between the two groups . For instance, we found that DIRS elements all encode a protein domain which is similar in sequence to the DNA methyltransferases of certain bacteriophages, whereas this domain is absent from all Ngaro elements . Together, the Ngaro and DIRS groups of YR retrotransposons contain elements with an astonishing diversity in structures, with variations in the nature of the associated repeat sequences and in the arrangement and complement of coding regions . In addition they contain elements with some surprising features, such as spliceosomal introns and long overlapping open reading frames.

Structure (Camb), 2004 Feb, 12(2), 307 - 16
The structural basis for RNA specificity and Ca2+ inhibition of an RNA-dependent RNA polymerase; Salgado PS et al.; The RNA-dependent RNA polymerase of bacteriophage phi6 transcribes mRNA from the three segments of the dsRNA viral genome . We have cocrystallized RNA oligonucleotides with the polymerase, revealing the mode of binding of RNA templates . This binding is somewhat different from that previously seen for DNA oligomers, leading to additional RNA-protein hydrogen bonds, consistent with a preference for RNA . Activation of the RNA/polymerase complex by the addition of substrate and Mg2+ initiates a single round of reaction within the crystal to form a dead-end complex that partially collapses within the enzyme active site . By replacing Mg2+ with Ca2+, we have been able to capture the inhibited complex which shows distortion that explains the structural basis for the inhibition of such polymerases by Ca2+.

J Invest Dermatol, 2004 Jan, 122(1), 103 - 10
Characterization of the anti-BP180 autoantibody reactivity profile and epitope mapping in bullous pemphigoid patients; Di Zenzo G et al.; Bullous pemphigoid is a subepidermal bullous disease of skin and mucosae associated with autoantibodies to BP180 . To characterize the humoral response to BP180, we generated a random BP180 epitope library displayed on lambda bacteriophage . After validation of the library by epitope mapping of three BP180-specific monoclonal antibodies, 15 novel or known BP180 epitopes were identified using 10 bullous pemphigoid serum samples . Fifty-seven bullous pemphigoid and 81 control sera were then assayed against the selected epitopes . Thirty-one out of 57 (54%) bullous pemphigoid sera reacted with at least an additional antigenic site other than the NC16A, within the extracellular (37%) and intracellular (28%) domains of BP180 . In addition, the reactivity with extracellular epitopes of BP180 contained within the residue stretches 508-541 and 1331-1404 appeared to be related to the presence of both skin and mucosal involvement . Finally, a preliminary analysis of the epitope pattern in the disease course indicated that bullous pemphigoid patients exhibit a specific reactivity pattern, and that binding to intracellular epitopes of BP180, in addition to NC16A, may be detectable at an early clinical stage . Our findings provide novel insights into the pathophysiology of bullous pemphigoid and show the potential of the utilized approach as a tool for a rapid diagnosis of bullous pemphigoid patients and their management.

Mar Biotechnol (NY), 2001 Jun, 3(Supplement 1), S185 - 95
Bacteriophage lambda and plasmid pUR288 transgenic fish models for detecting in vivo mutations; Winn RN et al.; We adapted transgenic rodent mutation assays based on fish carrying bacteriophage lambda and plasmid pUR288 vectors to address the needs for improved methods to assess health risks from exposure to environmental mutagens and also to establish new animal models to study in vivo mutagenesis . The approach entails separating the vectors from fish genomic DNA and then shuttling them into specialized strains of E . coli bacteria to analyze spontaneous and induced mutations in either lacI and cII or lacZ mutational targets . Fish exhibited low frequencies of spontaneous mutants comparable to the sensitivity of transgenic rodent models . Mutations detected after treating fish with chemical mutagens showed concentration-dependent, tissue-specific, and time-dependent relationships . Spontaneous and induced mutational spectra also were consistent with the specificity of known mutagens, further supporting the utility of transgenic fish for studies of in vivo mutagenesis.

J Appl Genet, 2004, 45(1), 111 - 20
Bacteriophage contamination: is there a simple method to reduce its deleterious effects in laboratory cultures and biotechnological factories?
Los M, Czyz A, Sell E, Wegrzyn A, Neubauer P, Wegrzyn G.
Infection of bacterial cultures by bacteriophages as well as prophage induction in the host cells are serious problems in both research and biotechnological laboratories . Generally, prevention strategies (like good laboratory/factory hygiene, sterilisation, decontamination and disinfection) are necessary to avoid bacteriophage contamination . However, it is well known that no matter how good the laboratory/factory practice and hygiene are, bacteriophage infections occur from time to time . The use of immunised or resistant bacterial strains against specific phages may be helpful, but properties of the genetically modified strains resistant to phages are often worse (from the point of view of a researcher or a biotechnological company) than those of the parental, phage-sensitive strains . In this article we review recent results that may provide a simple way to minimise deleterious effects of bacteriophage infection and prophage induction . It appears that low bacterial growth rates result in a significant inhibition of lytic development of various bacteriophages . Moreover, spontaneous prophage induction is less frequent in slowly growing bacteria.

Nucleic Acids Res, 2004 Feb 10, 32(3), 1083 - 90 Print 2004.
Interactions among CII protein, RNA polymerase and the lambda PRE promoter: contacts between RNA polymerase and the -35 region of PRE are identical in the presence and absence of CII protein; Marr MT et al.; The DNA recognition sequence for the transcriptional activator, CII protein, which is critical for lysogenization by bacteriophage lambda, overlaps the -35 region of the P(RE) promoter . Data presented here show that activation by CII does not change the pattern of cleavage of the -35 region of P(RE) by iron (S)-1-(p-bromoacetamidobenzyl)-EDTA (Fe-BABE) conjugated to the sigma subunit of RNA polymerase (RNAP) . Thus, the overall interaction between sigma and the -35 region of P(RE) is not significantly altered by CII . Therefore, the effects of the activator on RNAP binding to the promoter and formation of open complexes do not reflect a large-scale qualitative change in the nature of the interaction between RNAP and promoter DNA . The ability of CII to stimulate lysogenization is reduced in the presence of plasmid-borne rpoA variants encoding alanine substitutions at several positions in the C-terminal domain of the alpha subunit . However, it has not been possible to identify residues that directly affect the interaction between the activator and RNA polymerase.

Genes Dev, 2004 Feb 1, 18(3), 344 - 54
Cooperativity in long-range gene regulation by the lambda CI repressor; Dodd IB et al.; Effective repression of cI transcription from PRM by the bacteriophage lambda CI repressor requires binding sites (OL) located 2.4 kb from the promoter . A CI tetramer bound to OL1.OL2 interacts with a tetramer bound near PRM (OR1.OR2), looping the intervening DNA . We previously proposed that in this CI octamer:DNA complex, the distant OL3 operator and the weak OR3 operator overlapping PRM are juxtaposed so that a CI dimer at OL3 can cooperate with a CI dimer binding to OR3 . Here we show that OL3 is necessary for effective repression of PRM and that the repressor at OL3 appears to interact specifically with the repressor at OR3 . The OL3-CI-OR3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators . The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation . The results establish the lambda system as a powerful tool for examining long-range gene regulatory interactions in vivo.

J Biol Chem, 2004 Apr 30, 279(18), 18288 - 95 Epub 2004 Feb 10.
DNA-thumb interactions and processivity of T7 DNA polymerase in comparison to yeast polymerase eta; Cannistraro VJ et al.; The replicative polymerase of bacteriophage T7 is structurally and mechanistically well characterized . The crystal structure of T7 DNA polymerase or gene 5 protein complexed to its processivity factor, Escherichia coli thioredoxin, a primer-template, and a dideoxynucleotide reveals how this enzyme interacts with the 3'-end of the primer-template, but does not show how thioredoxin confers processivity to the polymerase . In the crystal structure highly conserved amino acids Asn(335) and Ser(338) of the thumb subdomain of T7 DNA polymerase are seen to interact with phosphates 7 and 8 of the DNA template strand . Results with a mutant T7 DNA polymerase in which aliphatic residues are substituted for these amino acids and experiments with different length and methylphosphonate-modified primer-templates demonstrate that these interactions are essential for processive synthesis and d(A.T)(n) tract bypass . Our data with methylphosphonate-modified DNA suggests that thioredoxin confers processivity to T7 DNA polymerase in part by causing an interaction with the phosphate backbone or minor groove of DNA . Residues Asn(335) and Ser(338) may also function with a nearby helix-loop-helix motif located at residues 339-372 to enclose the DNA during processive synthesis . Our results suggest that this structure must be held close to the DNA by ionic interactions to function . These interactions also allow for DNA sliding but physically block the passage of a 3T bulge in the template . In contrast, yeast polymerase eta, a polymerase that non-mutagenically repairs cis-syn thymidine dimers, allows the same bulge to slide past its thumb subdomain during synthesis . A relaxed thumb interaction with the DNA could account for the notably low processivity of polymerase eta.

J Biol Chem, 2004 Apr 16, 279(16), 16736 - 43 Epub 2004 Feb 09.
Visualizing the assembly and disassembly mechanisms of the MuB transposition targeting complex; Greene EC et al.; MuB, a protein essential for replicative DNA transposition by the bacteriophage Mu, is an ATPase that assembles into a polymeric complex on DNA . We used total internal reflection fluorescence microscopy to observe the behavior of MuB polymers on single molecules of DNA . We demonstrate that polymer assembly is initiated by a stochastic nucleation event . After nucleation, polymer assembly occurs by a mechanism involving the sequential binding of small units of MuB . MuB that bound to A/T-rich regions of the DNA assembled into large polymeric complexes . In contrast, MuB that bound outside of the A/T-rich regions failed to assemble into large oligomeric complexes . Our data also show that MuB does not catalyze multiple rounds of ATP hydrolysis while remaining bound to DNA . Rather, a single ATP is hydrolyzed, then MuB dissociates from the DNA . Finally, we show that "capping" of the enhanced green fluorescent protein-MuB polymer ends with unlabeled MuB dramatically slows, but does not halt, dissociation . This suggests that MuB dissociation occurs through both an end-dependent mechanism and a slower mechanism wherein subunits dissociate from the polymer interior.

J Biol Chem, 2004 Apr 30, 279(18), 19035 - 45 Epub 2004 Feb 09.
Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork; Ma Y et al.; Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand . After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis . In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis . The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/loading protein gp59 . Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions . We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32 . Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32 . These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly . In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity . The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork . The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.

Methods Mol Biol, 2004, 257, 135 - 54
Using the lambdaN peptide to tether proteins to RNAs; Baron-Benhamou J et al.; Proteins interacting with messenger RNAs (mRNAs) affect their nuclear processing, export, translation efficiency, stability, or cytoplasmic localization . Such RNA-binding proteins are often modular, containing RNA-binding domain(s) and other functional modules . To analyze the function of such proteins independent of their normal RNA-binding domains or to introduce effector modules to defined RNA-binding regions, a number of tethering approaches have been developed, often based on the use of large proteins and their specifically interacting RNA sequences . Here we report the use of a versatile system to tether proteins to mRNAs . The 22 amino acid RNA-binding domain of the lambda bacteriophage antiterminator protein N (lambdaN-(1-22) or lambdaN peptide) is used to tag the protein of interest, and its specific 19 nt binding site (boxB) is inserted into the target RNA recruiting the properties of the fusion protein to the RNA . The major advantage of this system derives from the small size of the peptide and its target sequence, which facilitates cloning and its use for biochemical experiments and diminishes possible interferences with the fused protein . The chapter illustrates the use of this system to create dedicated mRNA-specific factors involved in processes, such as mRNA translation and nonsense-mediated mRNA decay.

Mikrobiologiia, 2003 Nov-Dec, 72(6), 785 - 91
{The cotransduction of pET system plasmids by mutants of T4 and RB43 bacteriophages}; Tianiashin VI et al.; The study of the cotransduction of the plasmid pairs pET-3a-pLysE and pET-3a-pLysS by the mutant phage T4alc7 showed that the antibiotic resistance markers of the plasmids were cotransduced with a high frequency . The analysis of the plasmid DNA of cotransductants and cotransformants showed that the mutant phage T4alc7 can be used for obtaining the monomeric and oligomeric forms of plasmids and for the cotransduction of two-plasmid overproduction systems into E . coli strains . The plaque mutants RB43-03 and RB43-13 derived from bacteriophage RB43 were found to be able to cotransduce the antibiotic resistance markers of pET-3a and pLysE plasmids.

J Biol Chem, 2004 Apr 16, 279(16), 16591 - 7 Epub 2004 Feb 05.
The affinity of GXXXG motifs in transmembrane helix-helix interactions is modulated by long-range communication; Melnyk RA et al.; Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer . To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer) . In both constructs, the GXXXG motif is necessary for TM-TM association . Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXGVXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold . Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity . Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and approximately 18 A in structural space), work in concert to control TM-TM affinity synergistically.

Biochem Biophys Res Commun, 2004 Mar 5, 315(2), 376 - 80
Transcription reinitiation properties of bacteriophage T7 RNA polymerase; Ferrari R et al.; We have analyzed the kinetics of transcription initiation and reinitiation in vitro by one of the simplest and best characterized transcription machineries, bacteriophage T7 RNA polymerase (T7 RNAP) . We used a short transcription unit with T7-specific promoter and terminator elements as a template, and a heparin challenge assay to distinguish the first transcription cycle from the subsequent ones . When present at sub-saturating concentrations with respect to template DNA, T7 RNAP could find its promoter and initiate the first transcription cycle in less than 1min . Reinitiation under the same conditions proceeded more slowly, with only three new transcription cycles being completed in 10min; after that time, reinitiation practically ceased . When the polymerase was in large excess over template DNA, however, reinitiation proceeded linearly for longer times, at a rate of 1cycle/min . Our data suggest that polymerase recycling represents a critical step in T7 RNAP transcription, and that such a step may become rate-limiting for transcription at sub-saturating polymerase concentrations.

J Biol Chem, 2004 Apr 23, 279(17), 17473 - 82 Epub 2004 Feb 04.
A concerted mechanism for the suppression of a folding defect through interactions with chaperones; Doyle SM et al.; Specific amino acid substitutions confer a temperature-sensitive-folding (tsf) phenotype to bacteriophage P22 coat protein . Additional amino acid substitutions, called suppressor substitutions (su), relieve the tsf phenotype . These su substitutions are proposed to increase the efficiency of procapsid assembly, favoring correct folding over improper aggregation . Our recent studies indicate that the molecular chaperones GroEL/ES are more effectively recruited in vivo for the folding of tsf:su coat proteins than their tsf parents . Here, the tsf:su coat proteins are studied with in vitro equilibrium and kinetic techniques to establish a molecular basis for suppression . The tsf:su coat proteins were monomeric, as determined by velocity sedimentation analytical ultracentrifugation . The stability of the tsf:su coat proteins was ascertained by equilibrium urea titrations, which were best described by a three-state folding model, N <--> I <--> U . The tsf:su coat proteins either had stabilized native or intermediate states as compared with their tsf coat protein parents . The kinetics of the I <--> U transition showed a decrease in the rate of unfolding and a small increase in the rate of refolding, thereby increasing the population of the intermediate state . The increased intermediate population may be the reason the tsf:su coat proteins are aggregation-prone and likely enhances GroEL-ES interactions . The N --> I unfolding rate was slower for the tsf:su proteins than their tsf coat parents, resulting in an increase in the native state population, which may allow more competent interactions with scaffolding protein, an assembly chaperone . Thus, the suppressor substitution likely improves folding in vivo through increased efficiency of coat protein-chaperone interactions.

J Biol Chem, 2004 Apr 16, 279(16), 16136 - 43 Epub 2004 Feb 05.
T7 lysozyme represses T7 RNA polymerase transcription by destabilizing the open complex during initiation; Stano NM et al.; Bacteriophage T7 lysozyme binds to T7 RNA polymerase and inhibits transcription initiation and the transition from initiation to elongation . We have investigated each step of transcription initiation to determine where T7 lysozyme has the most effect . Stopped flow and equilibrium DNA binding studies indicate that T7 lysozyme does not inhibit the formation of the preinitiation open complex (open complex in the absence of initiating nucleotide) . T7 lysozyme, however, does prevent the formation of a fully open initiation complex (open complex in the presence of the initiating nucleotide) . This is consistent with the results that in the presence of T7 lysozyme the rate of G ladder RNA synthesis is about 5-fold slower and the GTP Kd is about 2-fold higher, but T7 lysozyme does not inhibit the initial rate of RNA synthesis with a premelted bulge-6 promoter (bubble from -4 to +2) . Neither the RNA synthesis rate nor the extent of promoter opening is restored by increasing the initiating nucleotide concentration, indicating that T7 lysozyme represses transcription by interfering with the formation of a stable and a fully open initiation bubble or by altering the structure of the DNA in the initiation complex . As a consequence of the unstable initiation bubble and/or the inhibition of the conformational changes in the N-terminal domain of T7 RNAP, T7 lysozyme causes an increased production of abortive products from 2- to 5-mer that delays the transition from the initiation to the elongation phase.

Mol Microbiol, 2004 Feb, 51(4), 949 - 62
The high-resolution functional map of bacteriophage SPP1 portal protein; Isidro A et al.; An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur . A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6 . Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function . Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus . The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule . Most of the mutations fell in classes 3 and 4 . This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence . The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.

Funct Integr Genomics, 2004 Jul, 4(3), 188 - 95 Epub 2004 Feb 05.
Calculating biological behaviors of epigenetic states in the phage lambda life cycle; Zhu XM et al.; The biology and behavior of bacteriophage lambda regulation have been the focus of classical investigations of molecular control of gene expression . Both qualitative and quantitative aspects of this behavior have been systematically characterized experimentally . Complete understanding of the robustness and stability of the genetic circuitry for the lysis-lysogeny switch remains an unsolved puzzle . It is an excellent test case for our understanding of biological behavior of an integrated network based on its physical, chemical, DNA, protein, and functional properties . We have used a new approach to non-linear dynamics to formulate a new mathematical model, performed a theoretical study on the phage lambda life cycle, and solved the crucial part of this puzzle . We find a good quantitative agreement between the theoretical calculation and published experimental observations in the protein number levels, the lysis frequency in the lysogen culture, and the lysogenization frequency for mutants of O(R) . We also predict the desired robustness for the lambda genetic switch . We believe that this is the first successful example in the quantitative calculation of robustness and stability of the phage lambda regulatory network, one of the simplest and most well-studied regulatory systems.

Nucleic Acids Res, 2004 Feb 03, 32(2), 834 - 41 Print 2004.
Role of the RNA polymerase alpha subunits in CII-dependent activation of the bacteriophage lambda pE promoter: identification of important residues and positioning of the alpha C-terminal domains; Kedzierska B et al.; The bacteriophage lambda CII protein stimulates the activity of three phage promoters, p(E), p(I) and p(aQ), upon binding to a site overlapping the -35 element at each promoter . Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) to demonstrate that one alphaCTD binds near position -41 at p(E), whilst the other alphaCTD binds further upstream . The alphaCTD bound near position -41 is oriented such that its 261 determinant is in close proximity to sigma(70) . The location of alphaCTD in CII-dependent complexes at the p(E) promoter is very similar to that found at many activator-independent promoters, and represents an alternative configuration for alphaCTD at promoters where activators bind sites overlapping the -35 region . We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of alphaCTD is involved in stimulation of the p(E) promoter by CII, and this was confirmed by in vitro transcription assays . We also show that whereas the K271E substitution in alphaCTD results in a drastic decrease in CII-dependent activation of p(E), the p(I) and p(aQ) promoters are less sensitive to this substitution, suggesting that the role of alphaCTD at the three lysogenic promoters may be different.

Biopolymers, 2004 Feb 15, 73(3), 348 - 55
Packaging double-helical DNA into viral capsids; LaMarque JC et al.; DNA packaging in bacteriophage P4 has been examined using a molecular mechanics model with a reduced representation containing one pseudoatom per turn of the double helix . The model is a discretized version of an elastic continuum model . The DNA is inserted piecewise into the model capsid, with the structure being reoptimized after each piece is inserted . Various optimization protocols were investigated, and it was found that molecular dynamics at a very low temperature (0.3 K) produces the optimal packaged structure . This structure is a concentric spool, rather than the coaxial spool that has been commonly accepted for so many years . This geometry, which was originally suggested by Hall and Schellman in 1982 (Biopolymers Vol . 21, pp . 2011-2031), produces a lower overall elastic energy than coaxial spooling .

Nucleic Acids Res, 2004 Jan 30, 32(2), 653 - 60 Print 2004.
Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase; Eastberg JH et al.; T4 phage polynucleotide kinase (PNK) displays 5'-hydroxyl kinase, 3'-phosphatase and 2',3'-cyclic phosphodiesterase activities . The enzyme phosphorylates the 5' hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonucleotide substrates of various lengths and sequences . In these structures, the 5' ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer . Depending on the ssDNA length, the first two or three nucleotide bases are well ordered . Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases . The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5'-GT DNA end than for a 5'-TG end . The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique substrate molecule.

J Virol, 2004 Feb, 78(4), 2114 - 20
Evolutionary potential of an RNA virus; Makeyev EV et al.; RNA viruses are remarkably adaptable to changing environments . This is medically important because it enables pathogenic viruses to escape the immune response and chemotherapy and is of considerable theoretical interest since it allows the investigation of evolutionary processes within convenient time scales . A number of earlier studies have addressed the dynamics of adapting RNA virus populations . However, it has been difficult to monitor the trajectory of molecular changes in RNA genomes in response to selective pressures . To address the problem, we developed a novel in vitro evolution system based on a recombinant double-stranded RNA bacteriophage, phi 6, containing a beta-lactamase (bla) gene marker . Carrier-state bacterial cells are resistant to ampicillin, and after several passages, they become resistant to high concentrations of another beta-lactam antibiotic, cefotaxime, due to mutations in the virus-borne bla gene . We monitored the changes in bla cDNAs induced by cefotaxime selection and observed an initial explosion in sequence variants with multiple mutations throughout the gene . After four passages, a stable, homogeneous population of bla sequences containing three specific nonsynonymous mutations was established . Of these, two mutations (E104K and G238S) have been previously reported for beta-lactamases from cefotaxime-resistant bacterial isolates . These results extend our understanding of the molecular mechanisms of viral adaptation and also demonstrate the possibility of using an RNA virus as a vehicle for directed evolution of heterologous proteins.

J Virol, 2004 Feb, 78(4), 1623 - 35
Dramatic effects of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside on the genome structure, packaging, and egress of guinea pig cytomegalovirus; Nixon DE et al.; The halogenated benzimidazoles BDCRB (2-bromo-5,6-dichloro-1-beta-D-riborfuranosyl benzimidazole riboside) and TCRB (2,5,6-trichloro-1-beta-D-riborfuranosyl benzimidazole riboside) were the first compounds shown to inhibit cleavage and packaging of herpesvirus genomes . Both inhibit the formation of unit length human cytomegalovirus (HCMV) genomes by a poorly understood mechanism (M . R . Underwood et al., J . Virol . 72:717-715, 1998; P . M . Krosky et al., J . Virol . 72:4721-4728, 1998) . Because the simple genome structure of guinea pig cytomegalovirus (GPCMV) provides a useful model for the study of herpesvirus DNA packaging, we investigated the effects of BDCRB on GPCMV . GPCMV proved to be sensitive to BDCRB (50% inhibitory concentration = 4.7 microM), although somewhat less so than HCMV . In striking contrast to HCMV, however, a dose of BDCRB sufficient to reduce GPCMV titers by 3 logs (50 microM) had no effect on the quantity of GPCMV genomic DNA that was formed in infected cells . Electron microscopy revealed that this DNA was in fact packaged within intranuclear capsids, but these capsids failed to egress from the nucleus and failed to protect the DNA from nuclease digestion . The terminal structure of genomes formed in the presence of BDCRB was also altered . Genomes with ends lacking a terminal repeat at the right end, which normally exist in an equimolar ratio with those having one copy of the repeat at the right end, were selectively eliminated by BDCRB treatment . At the left end, BDCRB treatment appeared to induce heterogeneous truncations such that 2.7 to 4.9 kb of left-end-terminal sequences were missing . These findings suggest that BDCRB induces premature cleavage events that result in truncated genomes packaged within capsids that are permeable to nuclease . Based on these and other observations, we propose a model for duplication of herpesvirus terminal repeats during the cleavage and packaging process that is similar to one proposed for bacteriophage T7 (Y . B . Chung, C . Nardone, and D . C . Hinkle, J . Mol . Biol . 216:939-948, 1990).

Arch Virol, 2004 Feb, 149(2), 365 - 77 Epub 2003 Oct 09.
Expression of a foot-and-mouth disease virus immunodominant epitope by a filamentous bacteriophage vector; Kim YJ et al.; We described the construction of a recombinant filamentous phage displaying on its surface the immunodominant site of VP1 protein of foot-and-mouth disease virus (FMDV) . The coding sequence was inserted at the amino-terminus of the major coat protein pVIII via a spacer . The hybrid phage proved to be antigenic as it was recognized by polyclonal and monoclonal anti FMDV sera . In two experiments involving immunisation of guinea-pigs with the recombinant phage, a low antibody response was generated . This suggests a possible role for phage displayed peptides in inducing anti FMDV immunity and the possibility of further development is discussed.

Arch Virol, 2004 Feb, 149(2), 241 - 59 Epub 2003 Nov 04.
Characterization of Plys-proximal morphogenetic genes of transposable bacteriophage Mu; Siboo IR et al.; Late during the bacteriophage Mu lytic cycle, Mu DNA must be matured and packaged from its dispersed integration sites in the host DNA in order to produce progeny virions . Whereas control of late gene transcription in Mu is becoming well understood, less is known about the phage morphogenetic process . To investigate the latter, we cloned and sequenced a approximately 4.3-kb region of the phage DNA beginning just upstream of the leftmost late promoter Plys . Previous mapping of amber mutations had located the lysis (lys) and proposed DNA maturation genes D and E in this region . When the DNA sequence was analyzed, seven potential open reading frames were found . DNA sequence analysis of amber mutations in genes D and E identified the sixth and seventh open reading frames as D and E, respectively . Cloning and expression of this region enabled production of cell-free protein extracts that specifically recognize the phage-encoded packaging sequence (pac), a characteristic exhibited by phage maturation enzymes . In addition, the E protein was found to share homology with the large subunit of many phage DNA maturation enzymes . These results support the hypothesis that D and E encode subunits of the Mu DNA maturation enzyme.

J Biol Chem, 2004 Apr 2, 279(14), 13412 - 7 Epub 2004 Jan 22.
Transcription termination by phage HK022 Nun is facilitated by COOH-terminal lysine residues; Kim HC et al.; The 109-amino acid Nun protein of prophage HK022 excludes superinfecting bacteriophage lambda by blocking transcription elongation on the lambda chromosome . Multiple interactions between Nun and the transcription elongation complex are involved in this reaction . The Nun NH(2)-terminal arginine-rich motif binds BOXB sequence in nascent lambda transcripts, whereas the COOH terminus binds RNA polymerase and contacts DNA template . Nun Trp(108) is required for interaction with DNA and transcription arrest . We analyzed the role of the adjacent Lys(106) and Lys(107) residues in the Nun reaction . Substitution of the lysine residues with arginine (K106R/K107R) had no effect on transcription arrest in vitro or in vivo . Nun K106A/K107A was partially active, whereas Nun K106D/K107D was defective in vitro and failed to exclude lambda . All mutants bound RNA polymerase and BOXB . In contrast to Nun K106R/K107R and K106A/K107A, Nun K106D/K107D did not cross-link DNA template . These results suggest that transcription arrest is facilitated by electrostatic interactions between positively charged Nun residues Lys(106) and Lys(107) and negatively charged DNA phosphate groups . These may assist intercalation of Trp(108) into template.

J Virol Methods, 2004 Mar 15, 116(2), 209 - 11
A simple method for cloning the complete begomovirus genome using the bacteriophage phi29 DNA polymerase; Inoue-Nagata AK et al.; The bacteriophage phiDNA polymerase amplifies circular DNA in a rolling circle amplification mechanism . This characteristic was applied to amplify and clone the complete circular DNA genome of a begomovirus . Total DNA extracted from infected tissue was used as the template of an amplification reaction using the commercial kit TempliPhi (Amersham Biosciences) . The amplified DNA could be used for direct sequencing and was cloned after digestion with a single cutting restriction endonuclease . The use of this enzyme simplified the cloning steps and increased the cloning efficiency of the complete genome of a circular plant DNA virus.

J Immunol Methods, 2004 Jan, 284(1-2), 119 - 32
Bivalent antibody phage display mimics natural immunoglobulin; Lee CV et al.; We report the development of a system for displaying bivalent antibody fragments on M13 bacteriophage in a manner that effectively mimics the binding behavior of natural antibodies . In the "bivalent display" format, two copies of antigen binding sites are displayed on the coat of a single phage particle . Bivalent display was first achieved by the insertion of a dimerization domain, consisting of an IgG1 hinge region and a homodimerizing GCN4 leucine zipper, between a Fab and the C-terminal domain of the M13 gene-3 minor coat protein . In a phagemid-based display system, the resulting "Fab'-zip-phage" particles display bivalent Fabs that resemble natural IgGs . An important functional consequence of bivalent display is an avidity effect, which results in a greatly reduced off-rate for phage bound to immobilized antigen . The avidity effect improved the capture and retention of bivalent Fab'-zip-phage relative to monovalent Fab-phage both with antigen immobilized on plates and with cell surface antigen . To examine the requirements for bivalent display on phage, we systematically trimmed down the dimerization domain and found that a single cysteine was sufficient to confer the same avidity effect conferred by the complete dimerization domain . Bivalent antibody phage display should be useful for many applications . In particular, the technology should aid in the production of antibodies against difficult antigens, and also, in selections that require dimerization for activity.

J Immunol, 2004 Feb 1, 172(3), 1777 - 85
Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects; Storni T et al.; DNA rich in nonmethylated CG motifs (CpGs) greatly facilitates induction of immune responses against coadministered Ags . CpGs are therefore among the most promising adjuvants known to date . Nevertheless, CpGs are characterized by two drawbacks . They have unfavorable pharmacokinetics and may exhibit systemic side effects, including splenomegaly . We show in this study that packaging CpGs into virus-like particles (VLPs) derived from the hepatitis B core Ag or the bacteriophage Qbeta is a simple and attractive method to reduce these two problems . CpGs packaged into VLPs are resistant to DNase I digestion, enhancing their stability . In addition, and in contrast to free CpGs, packaging CpGs prevents splenomegaly in mice, without affecting their immunostimulatory capacity . In fact, vaccination with CpG-loaded VLPs was able to induce high frequencies of peptide-specific CD8(+) T cells (4-14%), protected from infection with recombinant vaccinia viruses, and eradicated established solid fibrosarcoma tumors . Thus, packaging CpGs into VLPs improves both their immunogenicity and pharmacodynamics.

FEMS Immunol Med Microbiol, 2004 Jan 15, 40(1), 21 - 6
Bacteriophage-mediated nucleic acid immunisation; Clark JR et al.; Whole bacteriophage lambda particles, containing reporter genes under the control of the cytomegalovirus promoter (P(CMV)), have been used as delivery vehicles for nucleic acid immunisation . Following intramuscular injection of mice with lambda-gt11 containing the gene for hepatitis B surface antigen (HBsAg), anti-HBsAg responses in excess of 150 mIU ml(-1) were detected . When isolated peritoneal macrophages were incubated with whole lambda particles containing the gene for green fluorescent protein (GFP) under the control of P(CMV), GFP antigen was detected on the macrophage surface 8 h later . Results suggested that direct targeting of antigen-presenting cells by bacteriophage 'vaccines' may occur, leading to enhanced immune responses compared to naked DNA delivery . Bacteriophage DNA vaccines offer several advantages: they do not contain antibiotic resistance genes, they offer a large cloning capacity (approximately 15 kb), the DNA is protected from environmental degradation, they offer the potential for oral delivery, and large-scale production is cheap, easy and extremely rapid.

Mol Cell, 2004 Jan 16, 13(1), 45 - 53
Structure of a ternary transcription activation complex; Jain D et al.; The cI protein of bacteriophage lambda (lambdacI) activates transcription by binding a DNA operator just upstream of the promoter and interacting with the RNA polymerase sigma subunit domain 4 (sigma(4)) . We determined the crystal structure of the lambdacI/sigma(4)/DNA ternary complex at 2.3 A resolution . There are no conformational changes in either protein, which interact through an extremely small interface involving at most 6 amino acid residues . The interactions of the two proteins stabilize the binding of each protein to the DNA . The results provide insight into how activators can operate through a simple cooperative binding mechanism but affect different steps of the transcription initiation process.

J Microsc, 2004 Feb, 213(Pt 2), 172 - 9
Terminal protein-induced stretching of bacteriophage phi29 DNA; Wang H et al.; Stretching of DNA molecules helps to resolve detail during the fluorescence microscopy of both single DNA molecules and single DNA-protein complexes . To make stretching occur, intricate procedures of specimen preparation and manipulation have been developed in previous studies . By contrast, the present study demonstrates that conventional procedures of specimen preparation cause DNA stretching to occur, if the specimen is the double-stranded DNA genome of bacteriophage phi29 . Necessary for this stretching is a protein covalently bound at both 5' termini of phi29 DNA molecules . Some DNA molecules are attached to a cover glass only at the two ends . Others are attached at one end only with the other end free in solution . The extent of stretching varies from approximately 50% overstretched to approximately 50% understretched . The understretched DNA molecules are internally mobile to a variable extent . In addition to stretching, some phi29 DNA molecules also undergo assembly to form both linear and branched concatemers observed by single-molecule fluorescence microscopy . The assembly also requires the terminal protein . The stretched DNA molecules are potentially useful for observing DNA biochemistry at the single molecule level.

J Microsc, 2004 Feb, 213(Pt 2), 101 - 9
Fluorescence microscopy of colour-tagged nanoparticles that are undergoing thermal motion; Wang H et al.; To bypass limitations of conventional biochemical analysis, single-particle biochemical analysis is used . To improve single-particle biochemical analysis, procedures are needed to keep nanometre-sized particles in focus while the particles are undergoing thermal motion . A simple, inexpensive procedure is developed here for keeping particles in focus during the continuous observing/discriminating/recording of two different particles, both of which are undergoing thermal motion . This procedure concentrates the particles in a plane of solution that is in focus when the cover glass surface is in focus . An essential component of the procedure is the addition of molten, low-melt agarose to the specimen . Motionless binding to glass is inhibited by inclusion of anti-stick additives in the specimen . Both carrier protein (gelatin) and non-ionic detergent (Triton X-100) are anti-stick additives successfully used here . Intact bacteriophages T3 and T7 are used as model particles, in anticipation of the use of the procedures developed here for the analysis of the assembly of bacteriophages . Observing/discriminating/recording of colour-tagged bacteriophages T3 and T7 is achieved at video frame rate with image splitting to discriminate colours.

Biochemistry, 2004 Jan 27, 43(3), 782 - 90
Electrochemical studies of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of bacteriophage lambda protein phosphatase; Reiter TA et al.; Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large superfamily of metallophosphoesterases, including serine/threonine protein phosphatases, purple acid phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11 . Members of this family share several common characteristics, including a common phosphoesterase motif, secondary structural fold (betaalphabetaalphabeta), and metal ligand environment, and often accommodate a dinuclear metal center . The identity of the active site metals often differs between family members . Despite the extensive spectroscopic studies of several family members, only the standard redox potential of porcine purple acid phosphate (PAP) has been measured . In this report, we investigate the redox properties of another member of this protein family . The standard redox potentials of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of lambdaPP were determined from anaerobic redox titration experiments . Two different S = 5/2, mono-Fe3+ lambdaPP species were identified: the first with an E/D approximately 0.17, g = 8.9 and 4.8, and an Eo' approximately +130 mV; the second with E/D approximately 0.05, g = 6.7, 5.9, and 4.4, and an Eo' approximately +120 mV . The first and second mono-Fe3+ species are thought to represent Fe present in the M2 and M1 sites, respectively . The addition of Zn2+ to mono-Fe3+ lambdaPP results in a decrease in both mono-Fe3+ species and the appearance of a new S = 5/2, Fe(3+)-Zn2+ species (E/D approximately 0.02, g = 5.9, and an Eo' > +175 mV) . The Fe-Fe lambdaPP titration revealed an S = 1/2, Fe(3+)-Fe2+ (g < 2) species with an Eo' > +128 mV . These results suggest that the active site of lambdaPP supports a high oxidation potential for both metal sites and may indicate an equally oxidizing active site for other member metallophosphoesterases.

J Biol Chem, 2004 Mar 26, 279(13), 12067 - 75 Epub 2004 Jan 17.
Bacteriophage T4 32 protein is required for helicase-dependent leading strand synthesis when the helicase is loaded by the T4 59 helicase-loading protein; Jones CE et al.; In the bacteriophage T4 DNA replication system, T4 gene 59 protein binds preferentially to fork DNA and accelerates the loading of the T4 41 helicase . 59 protein also binds the T4 32 single-stranded DNA-binding protein that coats the lagging strand template . Here we explore the function of the strong affinity between the 32 and 59 proteins at the replication fork . We show that, in contrast to the 59 helicase loader, 32 protein does not bind forked DNA more tightly than linear DNA . 32 protein displays a strong binding polarity on fork DNA, binding with much higher affinity to the 5' single-stranded lagging strand template arm of a model fork, than to the 3' single-stranded leading strand arm . 59 protein promotes the binding of 32 protein on forks too short for cooperative binding by 32 protein . We show that 32 protein is required for helicase-dependent leading strand DNA synthesis when the helicase is loaded by 59 protein . However, 32 protein is not required for leading strand synthesis when helicase is loaded, less efficiently, without 59 protein . Leading strand synthesis by wild type T4 polymerase is strongly inhibited when 59 protein is present without 32 protein . Because 59 protein can load the helicase on forks without 32 protein, our results are best explained by a model in which 59 helicase loader at the fork prevents the coupling of the leading strand polymerase and the helicase, unless the position of 59 protein is shifted by its association with 32 protein.

J Biol Chem, 2004 Apr 16, 279(16), 16581 - 90 Epub 2004 Jan 16.
Crystal structure of the Mor protein of bacteriophage Mu, a member of the Mor/C family of transcription activators; Kumaraswami M et al.; Transcription from the middle promoter, Pm, of bacteriophage Mu requires the phage-encoded activator protein Mor and bacterial RNA polymerase . Mor is a sequence-specific DNA-binding protein that mediates transcription activation through its interactions with the C-terminal domains of the alpha and sigma subunits of bacterial RNA polymerase . Here we present the first structure for a member of the Mor/C family of transcription activators, the crystal structure of Mor to 2.2-A resolution . Each monomer of the Mor dimer is composed of two domains, the N-terminal dimerization domain and C-terminal DNA-binding domain, which are connected by a linker containing a beta strand . The N-terminal dimerization domain has an unusual mode of dimerization; helices alpha1 and alpha2 of both monomers are intertwined to form a four-helix bundle, generating a hydrophobic core that is further stabilized by antiparallel interactions between the two beta strands . Mutational analysis of key leucine residues in helix alpha1 demonstrated a role for this hydrophobic core in protein solubility and function . The C-terminal domain has a classical helix-turn-helix DNA-binding motif that is located at opposite ends of the elongated dimer . Since the distance between the two helix-turn-helix motifs is too great to allow binding to two adjacent major grooves of the 16-bp Mor-binding site, we propose that conformational changes in the protein and DNA will be required for Mor to interact with the DNA . The highly conserved glycines flanking the beta strand may act as pivot points, facilitating the conformational changes of Mor, and the DNA may be bent.

Biochemistry, 2004 Jan 20, 43(2), 393 - 404
Evaluating the contribution of base stacking during translesion DNA replication; Reineks EZ et al.; Despite the nontemplating nature of the abasic site, dAMP is often preferentially inserted opposite the lesion, a phenomenon commonly referred to as the "A-rule" . We have evaluated the molecular mechanism accounting for this unique behavior using a thorough kinetic approach to evaluate polymerization efficiency during translesion DNA replication . Using the bacteriophage T4 DNA polymerase, we have measured the insertion of a series of modified nucleotides and have demonstrated that increasing the size of the nucleobase does not correlate with increased insertion efficiency opposite an abasic site . One analogue, 5-nitroindolyl-2'-deoxyriboside triphosphate, was unique as it was inserted opposite the lesion with approximately 1000-fold greater efficiency compared to that for dAMP insertion . Pre-steady-state kinetic measurements yield a kpol value of 126 s(-1) and a Kd value of 18 microM for the insertion of 5-nitroindolyl-2'-deoxyriboside triphosphate opposite the abasic site . These values rival those associated with the enzymatic formation of a natural Watson-Crick base pair . These results not only reiterate that hydrogen bonding is not necessary for nucleotide insertion but also indicate that the base-stacking and/or desolvation capabilities of the incoming nucleobase may indeed play the predominant role in generating efficient DNA polymerization . A model accounting for the increase in catalytic efficiency of this unique nucleobase is provided and invokes pi-pi stacking interactions of the aromatic moiety of the incoming nucleobase with aromatic amino acids present in the polymerase's active site . Finally, differences in the rate of 5-nitroindolyl-2'-deoxyriboside triphosphate insertion opposite an abasic site are measured between the bacteriophage T4 DNA polymerase and the Klenow fragment . These kinetic differences are interpreted with regard to the differences in various structural components between the two enzymes and are consistent with the proposed model for DNA polymerization.

Science, 2004 Jan 9, 303(5655), 213 - 7
Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires; Mao C et al.; We report a virus-based scaffold for the synthesis of single-crystal ZnS, CdS, and freestanding chemically ordered CoPt and FePt nanowires, with the means of modifying substrate specificity through standard biological methods . Peptides (selected through an evolutionary screening process) that exhibit control of composition, size, and phase during nanoparticle nucleation have been expressed on the highly ordered filamentous capsid of the M13 bacteriophage . The incorporation of specific, nucleating peptides into the generic scaffold of the M13 coat structure provides a viable template for the directed synthesis of semiconducting and magnetic materials . Removal of the viral template by means of annealing promoted oriented aggregation-based crystal growth, forming individual crystalline nanowires . The unique ability to interchange substrate-specific peptides into the linear self-assembled filamentous construct of the M13 virus introduces a material tunability that has not been seen in previous synthetic routes . Therefore, this system provides a genetic toolkit for growing and organizing nanowires from semiconducting and magnetic materials.

Appl Environ Microbiol, 2004 Jan, 70(1), 527 - 34
Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage; Oda M et al.; A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7 . The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid . The DNA fragment around soc was amplified by PCR and sequenced . The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage . GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively . Fusion of GFP to SOC did not change the host range of PP01 . On the contrary, the binding affinity of the recombinant phages to the host cell increased . However, the stability of the recombinant phages in alkaline solution decreased . Adsorption of the GFP-labeled PP01 phages to the E . coli cell surface enabled visualization of cells under a fluorescence microscope . GFP-labeled PP01 phage was not only adsorbed on culturable E . coli cells but also on viable but nonculturable or pasteurized cells . The coexistence of insensitive E . coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E . coli O157:H7 . After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4 degrees C, E . coli O157:H7 cells could be visualized by fluorescence microscopy . The GFP-labeled PP01 phage could be a rapid and sensitive tool for E . coli O157:H7 detection.

Protein Expr Purif, 2004 Feb, 33(2), 304 - 10
Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase; Loscha K et al.; In Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks . It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 {DnaG(434-581)} that interact with the hexameric DnaB helicase . Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E . coli strain . This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage lambda-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR . A TOCSY spectrum of a 2mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579 . This was consistent with sequence conservation among most-closely related primases . Linewidths in a NOESY spectrum of a 0.5mM sample in 10mM phosphate, pH 6.05, 0.1M NaCl, recorded at 36 degrees C, indicated the protein to be monomeric . Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a=b=142.2A, c=192.1A, and diffracted beyond 2.7A resolution with synchrotron radiation.

Protein Expr Purif, 2004 Feb, 33(2), 166 - 75
Identification, cloning, and expression of bacteriophage T5 dnk gene encoding a broad specificity deoxyribonucleoside monophosphate kinase (EC 2.7.4.13); Mikoulinskaia GV et al.; The nucleotide sequence corresponding to 13-19.5% of the bacteriophage T5 genome in early region C was determined (GenBank AY 140897) . One of the five major single-stranded interruptions (nicks) of bacteriophage T5 DNA was identified at 18.5% . The sequenced region was annotated and the putative functions of some open reading frames were proposed by comparison with databases . The dnk gene, encoding a deoxyribonucleoside monophosphate kinase, was identified using a previously defined N-terminal amino acid sequence . The gene was cloned and expressed in Escherichia coli, the enzyme was purified to homogeneity with high yield using two alternative methods, and the recombinant deoxyribonucleoside monophosphate kinase was found to have the same activity and specificity as the native enzyme.

J Biol Chem, 2004 Mar 19, 279(12), 11081 - 7 Epub 2004 Jan 05.
Factors that influence selection of coding resumption sites in translational bypassing: minimal conventional peptidyl-tRNA:mRNA pairing can suffice; Herr AJ et al.; This study investigates bypassing initiated from codons immediately 5' of a stop codon . The mRNA slips and is scanned by the peptidyl-tRNA for a suitable landing site, and standard decoding resumes at the next 3' codon . This work shows that landing sites with potentially strong base pairing between the peptidyl-tRNA anticodon and mRNA are preferred, but sites with little or no potential for Watson-Crick or wobble base pairing can also be utilized . These results have implications for re-pairing in ribosomal frameshifting . Shine-Dalgarno sequences in the mRNA can alter the distribution of landing sites observed . The bacteriophage T4 gene 60 nascent peptide, known to influence take-off in its native context, imposes stringent P-site pairing requirements, thereby limiting the number of suitable landing sites.

Cancer Chemother Pharmacol, 2004 Apr, 53(4), 279 - 87 Epub 2003 Dec 24.
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-{(methylamino)carbonyl}hydrazine (VNP40101M): I . Direct inhibition of O6-alkylguanine-DNA alkyltransferase (AGT) by electrophilic species generated by decomposition; Penketh PG et al.; PURPOSE: To investigate the interaction of the electrophilic species generated by the decomposition of the antineoplastic prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-{(methylamino)carbonyl}hydrazine (VNP40101M) on the ability of O(6)-alkylguanine-DNA alkyltransferase (AGT) to repair alkylated O(6)-chloroethylguanine and/or N(1),O(6)-ethanoguanine DNA lesions . MATERIALS AND METHODS: The contributions of inhibitory electrophilic species generated from VNP40101M towards AGT was assessed using analogues that selectively generated either the chloroethylating or the carbamoylating components of VNP40101M . The activity of AGT was determined from the inhibition of crosslink formation from O(6)-chloroethylguanine and/or N(1),O(6)-ethanoguanine lesions . The half-lives of sulfonylhydrazine derivatives and isocyanates were measured using an acidification assay which gives a change in absorbance proportional to the release or consumption of small quantities of protons . RESULTS: Both of the reactive components produced by VNP40101M directly inactivated cloned human AGT; the carbamoylating moiety (IC(50) about 13 micro M) was approximately seven- to eight-fold more potent than the alkylating component(s) (IC(50) about 100 micro M) . These inhibitory actions were moderated by the addition of naked T5 bacteriophage DNA . Thus, AGT bound to DNA was markedly more resistant than free AGT to these electrophilic species . DNA also blocked the spontaneous loss of AGT activity which occurred upon incubation of this protein under mild conditions . CONCLUSIONS: The reaction of AGT with the methyl isocyanate generated from the decomposition of VNP40101M increased the net number of crosslinks generated by VNP40101M compared to a sulfonylhydrazine prodrug that formed the equivalent alkylating species in the absence of the cogeneration of methyl isocyanate . These actions may be of significance to the antineoplastic activity of VNP40101M.

Nat Biotechnol, 2004 Jan, 22(1), 31 - 6
Old dogma, new tricks--21st Century phage therapy; Thiel K; As antibiotic resistant bacteria threaten a public health crisis, biotechnology is turning to bacteriophages, nature's tiniest viruses . But can phage therapy overcome its historical baggage?

Nucleic Acids Res . 2004 Jan 02;32(1):e6.
Improved full-length cDNA production based on RNA tagging by T4 DNA ligase; Clepet C et al.; Second-strand cDNA priming is a central problem for full-length characterization of transcripts . A new strategy using bacteriophage T4 DNA ligase and partially degenerate adapters is proposed for grafting a sequence tag to the end of polyribonucleotides . Based on this RNA tagging system and previously described protocols, a new method for full-length cDNA production has been implemented . Validation of the method is shown in Arabidopsis thaliana by the construction of a full-length cDNA library and the analysis of 154 clones and by 5'-RACE-PCR run on a documented experimental system.

Acta Pharm Hung, 2003, 73(2), 97 - 102
{Photodynamic inactivation of porphyrin sensitized T7 phages: efficiency and mechanism of action}; Egyeki M et al.; We investigated the efficiency and the mechanism of action of two glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 bacteriophage . Both types of porphyrins sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different . Our result suggests that both type I and type II reaction play a role in the virus inactivation . Optical melting studies revealed structural changes in the protein part but not in the DNA of the photo-chemically treated nucleoprotein complex . Polymerase chain reaction (PCR) analysis failed to demonstrate any DNA damage.

Vet Immunol Immunopathol, 2004 Jan, 97(1-2), 39 - 51
Cloning of porcine scFv antibodies by phage display and expression in Escherichia coli; Li F et al.; Oligonucleotide primers were designed for recovery of Ig H, kappa and lambda transcripts from porcine splenic cDNA . The products were cloned and scFvs constructed in a phage display vector . E . coli HB2151 was transformed with the constructs and upon induction, scFvs of the predicted molecular weight could be detected in culture supernatants by Western blotting and immunoprecipitation with anti-pig IgG . Bacteriophage displaying a swine scFv were diluted into an excess of phage carrying a human anti-thyroglobulin scFv and then panned on plastic coated with anti-pig IgG . Rapid enrichment of phage carrying the porcine scFv through three rounds of selection demonstrated the successful display of an authentically folded pig Ig at the viral surface . The data demonstrate that phage display techniques can be applied successfully to porcine Igs and that expression of recombinant pig scFvs in bacteria can be achieved . These techniques offer the opportunity to generate authentic porcine monoclonal reagents for basic and applied studies.

J Biol Chem, 2004 Mar 5, 279(10), 8991 - 8 Epub 2003 Dec 29.
Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin; Papanikolopoulou K et al.; The folding of beta-structured, fibrous proteins is a largely unexplored area . A class of such proteins is used by viruses as adhesins, and recent studies revealed novel beta-structured motifs for them . We have been studying the folding and assembly of adenovirus fibers that consist of a globular C-terminal domain, a central fibrous shaft, and an N-terminal part that attaches to the viral capsid . The globular C-terminal, or "head" domain, has been postulated to be necessary for the trimerization of the fiber and might act as a registration signal that directs its correct folding and assembly . In this work, we replaced the head of the fiber by the trimerization domain of the bacteriophage T4 fibritin, termed "foldon." Two chimeric proteins, comprising the foldon domain connected at the C-terminal end of four fiber shaft repeats with or without the use of a natural linker sequence, fold into highly stable, SDS-resistant trimers . The structural signatures of the chimeric proteins as seen by CD and infrared spectroscopy are reported . The results suggest that the foldon domain can successfully replace the fiber head domain in ensuring correct trimerization of the shaft sequences . Biological implications and implications for engineering highly stable, beta-structured nanorods are discussed.

Biophys J, 2004 Jan, 86(1 Pt 1), 58 - 66
Sensitivity of OR in phage lambda; Bakk A et al.; We investigate the sensitivity of the right operator in bacteriophage lambda . In particular, the system is probed in the three different regulatory protein concentration-regimes: 1), lysogen (CI dominates); 2), during induction (CI and Cro at comparable concentrations); and 3), after induction (Cro dominates) . Systematic perturbations of the protein-operator binding energies show in a lysogen that the activity (production rate) at promoter PRM is robust to variations, in contrast to PR, where the sensitivity is high . Both promoters, however, show large sensitivity in regimes 2 and 3 . In all regimes we identify several suppressors, meaning that for a given large perturbation (+/-2 kcal/mol) of one binding energy, there exist compensating perturbation(s) that restore the wild-type activity.

Eur J Biochem, 2004 Jan, 271(1), 135 - 45
Mutations in the hydrophobic core and in the protein-RNA interface affect the packing and stability of icosahedral viruses; Lima SM et al.; The information required for successful assembly of an icosahedral virus is encoded in the native conformation of the capsid protein and in its interaction with the nucleic acid . Here we investigated how the packing and stability of virus capsids are sensitive to single amino acid substitutions in the coat protein . Tryptophan fluorescence, bis-8-anilinonaphthalene-1-sulfonate fluorescence, CD and light scattering were employed to measure urea- and pressure-induced effects on MS2 bacteriophage and temperature sensitive mutants . M88V and T45S particles were less stable than the wild-type forms and completely dissociated at 3.0 kbar of pressure . M88V and T45S mutants also had lower stability in the presence of urea . We propose that the lower stability of M88V particles is related to an increase in the cavity of the hydrophobic core . Bis-8-anilinonaphthalene-1-sulfonate fluorescence increased for the pressure-dissociated mutants but not for the urea-denatured samples, indicating that the final products were different . To verify reassembly of the particles, gel filtration chromatography and infectivity assays were performed . The phage titer was reduced dramatically when particles were treated with a high concentration of urea . In contrast, the phage titer recovered after high-pressure treatment . Thus, after pressure-induced dissociation of the virus, information for correct reassembly was preserved . In contrast to M88V and T45S, the D11N mutant virus particle was more stable than the wild-type virus, in spite of it also possessing a temperature sensitive growth phenotype . Overall, our data show how point substitutions in the capsid protein, which affect either the packing or the interaction at the protein-RNA interface, result in changes in virus stability.

J Bacteriol, 2004 Jan, 186(1), 1 - 7
The preferred substrate for RecA-mediated cleavage of bacteriophage 434 repressor is the DNA-bound dimer; Pawlowski DR et al.; Induction of a lysogen of a lambdoid bacteriophage usually involves RecA-stimulated autoproteolysis of the bacteriophage repressor protein . Previous work on the phage repressors showed that the monomeric form of the protein is the target of RecA . Our previous work indicated that in the case of bacteriophage 434, virtually none of the repressor is present as a monomer in vivo . Hence, if the repressor in a lysogen is present as a dimer, how can RecA-stimulated autoproteolysis play a role in bacteriophage induction? We examined this question by determining the rate of RecA-stimulated 434 repressor cleavage as a function of repressor concentration and added DNA . Our results show that binding of 434 repressor to a specific DNA binding site dramatically increases the velocity of repressor autocleavage compared to the velocity of cleavage of the monomer and concentration-induced dimer . DNA binding-deficient hemidimers formed between the intact repressor and its C-terminal domain fragment have a lower rate of cleavage than DNA-bound dimers . These results show that the DNA-bound 434 repressor dimer, which is the form of the repressor that is required for its transcriptional regulatory functions, is the preferred form for RecA-stimulated autocleavage . We also show that the rate of repressor autocleavage is influenced by the sequence of the bound DNA . Kinetic analysis of the autocleavage reaction indicated that the DNA sequence influences the velocity of 434 repressor autocleavage by affecting the affinity of the repressor-DNA complex for RecA, not the chemical cleavage step . Regardless of the mechanism, the finding that the presence and precise sequence of DNA modulate the autocleavage reaction shows that DNA allosterically affects the function of 434 repressor.

J Biomol Tech, 2003 Jun, 14(2), 143 - 8
TempliPhi: A sequencing template preparation procedure that eliminates overnight cultures and DNA purification; Reagin MJ et al.; Preparing plasmid templates for DNA sequencing is the most time-consuming step in the sequencing process . Current template preparation methods rely on a labor-intensive, multistep procedure that takes up to 24 h and produces templates of varying quality and quantity . The TempliPhi DNA Sequencing Template Amplification Kit eliminates the requirement for extended bacterial growth prior to sequencing and saves laboratory personnel hands-on time by eliminating the centrifugation and transfer steps currently required by older preparatory methods . In addition, costly purification filters and columns are not necessary, as amplified product can be added directly to a sequencing reaction . Starting material can be any circular template from a colony, culture, glycerol stock, or plaque . Based on rolling circle amplification and employing bacteriophage Phi29 DNA polymerase, the method can produce 3-5 microg of template directly from a single bacterial colony in as little as 4 h . Implementation of these procedures in a laboratory or core sequencing facility can decrease cost on tips, plates, and other plasticware, while at the same time increase throughput.

Biochemistry, 2003 Dec 23, 42(50), 14957 - 67
Differential modes of recognition in N peptide-boxB complexes; Austin RJ et al.; N proteins from bacteriophages lambda, P22, and phi21 modulate transcription elongation by binding nascent "boxB" mRNA hairpins . This RNA recognition is mediated by N-terminal arginine-rich peptide sequences capable of interacting with their cognate boxB RNA targets . Here, we have analyzed the affinity and specificity of the peptide-RNA interactions that modulate this transcriptional switch . To do this, we constructed a series of peptides based on the wild-type lambda, P22, and phi21 N protein binding domains ranging from 11 to 22 residues and analyzed their interactions with the leftward and rightward boxB RNA hairpin targets for all three phage . Binding constant (K(d)) values were determined using RNA hairpins labeled with 2-aminopurine (2AP) and monitoring the fluorescence change as peptide was added . K(d)'s demonstrate that lambda and P22 N peptides bind to their cognate boxB targets with high specificity and show equal affinities for their leftward and rightward hairpins . Surprisingly, phi21 shows very little specificity for its cognate targets . Lambda and P22 N peptides exhibit differential modes of recognition with specificity conferred by their amino- and carboxy-terminal modules, respectively . We have generated a reciprocal matrix of substituted peptides to examine the contributions of individual residues to specificity . Amino acid coupling analysis supports a binding model where the Arg8 residue of lambda peptide acts as a conformational hot spot, anchoring the induced loop fold of its boxB hairpin target.

J Environ Qual, 2003 Nov-Dec, 32(6), 2046 - 53
Sorption of MS2 bacteriophage to layered double hydroxides: effects of reaction time, pH, and competing anions; You Y et al.; Batch sorption and column breakthrough studies were conducted to investigate the potential of layered double hydroxides (LDHs) to remove bacteriophage MS2 from contaminated waters . All four of the LDHs evaluated in this study had very high retention capacities for MS2 . Sorption results showed that MS2 could be completely removed from 5.2 x 10(2) plaque-forming units (pfu)/mL solution by Mg-Al LDH 2 (i.e., 2:1 Mg to Al ratio LDH), with the highest sorption capacity observed in this study of 1.51 x 10(10) pfu/g . Attachment of MS2 to LDHs was a rapid process and reached quasi-equilibrium after a 1-h reaction time . Within the pH range studied (pH 4-9), Mg-Al LDH 2 showed high sorption potential for MS2 at all pH values but sorption decreased slightly with increasing solution pH . Background solution anions influenced virus sorption, with SO4(2-) and HPO4(2-) decreasing sorption significantly whereas the presence of NO3- had little effect on the attachment of MS2 to Mg-Al LDH 2 . The addition of another virus (phiX174) only caused a slight decrease in the retention of MS2 by Mg-Al LDH 2, suggesting that there was insignificant competitive sorption between MS2 and phiX174 on LDH surfaces . Results from column experiments indicate that there was no MS2 breakthrough from columns packed with Mg-Al LDH 2-coated sand, suggesting complete MS2 retention at the virus concentration tested . The high mass recovery by beef extract solution revealed that the removal of viruses by the LDH was due to sorption of MS2 to LDH surfaces, rather than inactivation.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15748 - 53 Epub 2003 Dec 12.
Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants; Costantino N et al.; Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos) . Such recombination is possible because bacteriophage lambda-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20-70 bases . This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms . The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome . In E . coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence . In so doing, MMR can reduce the apparent recombination frequency by >100-fold . In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation . Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence.

BMC Evol Biol . 2003 Dec 10;3(1):24.
Evolution of phage with chemically ambiguous proteomes; Bacher JM et al.; BACKGROUND: The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution . The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem . The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality . RESULTS: The bacteriophage Qbeta initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan . After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan . Seven mutations were fixed throughout the phage in two independent lines of descent . None of the mutations changed a tryptophan residue . CONCLUSIONS: A relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins . These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon . It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates.

J Biol Chem, 2004 Mar 12, 279(11), 9978 - 86 Epub 2003 Dec 08.
Dimerization of TonB is not essential for its binding to the outer membrane siderophore receptor FhuA of Escherichia coli; Koedding J et al.; FhuA belongs to a family of specific siderophore transport systems located in the outer membrane of Escherichia coli . The energy required for the transport process is provided by the proton motive force of the cytoplasmic membrane and is transmitted to FhuA by the protein TonB . Although the structure of full-length TonB is not known, the structure of the last 77 residues of a fragment composed of the 86 C-terminal amino acids was recently solved and shows an intertwined dimer (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A . (2001) J . Biol . Chem . 276, 27535-27540) . We analyzed the ability of truncated C-terminal TonB fragments of different lengths (77, 86, 96, 106, 116, and 126 amino acid residues, respectively) to bind to the receptor FhuA . Only the shortest TonB fragment, TonB-77, could not effectively interact with FhuA . We have also observed that the fragments TonB-77 and TonB-86 form homodimers in solution, whereas the longer fragments remain monomeric . TonB fragments that bind to FhuA in vitro also inhibit ferrichrome uptake via FhuA in vivo and protect cells against attack by bacteriophage Phi80.

Appl Environ Microbiol, 2003 Dec, 69(12), 7499 - 506
Experimental examination of bacteriophage latent-period evolution as a response to bacterial availability; Abedon ST et al.; For obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time) . This trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny . Here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria . Theory suggests that higher bacterial densities should select for shorter phage latent periods . Consistently, we have found that higher host densities (>/== approximately 10(7) bacteria/ml) can enrich stocks of phage RB69 for variants that display shorter latent periods than the wild type . One such variant, dubbed sta5, displays a latent period that is approximately 70 to 80% of that of the wild type-which is nearly as short as the RB69 eclipse period-and which has a corresponding burst size that is approximately 30% of that of the wild type . We show that at higher host densities (>/== approximately 10(7) bacteria/ml) the sta5 phage can outcompete the RB69 wild type, though only under conditions of direct (same-culture) competition . We interpret this advantage as corresponding to slightly faster sta5 population growth, resulting in multifold increases in mutant frequency during same-culture growth . The sta5 advantage is lost, however, given indirect (different-culture) competition between the wild type and mutant or given same-culture competition but at lower densities of phage-susceptible bacteria (</= approximately 10(6) bacteria/ml) . From these observations we suggest that phage displaying very short latent periods may be viewed as specialists for propagation when bacteria within cultures are highly prevalent and transmission between cultures is easily accomplished.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15440 - 5 Epub 2003 Dec 02.
Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides; Smith HO et al.; We have improved upon the methodology and dramatically shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides . As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of bacteriophage X174 (5386 bp) from a single pool of chemically synthesized oligonucleotides . The procedure involves three key steps: (i) . gel purification of pooled oligonucleotides to reduce contamination with molecules of incorrect chain length, (ii) . ligation of the oligonucleotides under stringent annealing conditions (55 degrees C) to select against annealing of molecules with incorrect sequences, and (iii) . assembly of ligation products into full-length genomes by polymerase cycling assembly, a nonexponential reaction in which each terminal oligonucleotide can be extended only once to produce a full-length molecule . We observed a discrete band of full-length assemblies upon gel analysis of the polymerase cycling assembly product, without any PCR amplification . PCR amplification was then used to obtain larger amounts of pure full-length genomes for circularization and infectivity measurements . The synthetic DNA had a lower infectivity than natural DNA, indicating approximately one lethal error per 500 bp . However, fully infectious X174 virions were recovered after electroporation into Escherichia coli . Sequence analysis of several infectious isolates verified the accuracy of these synthetic genomes . One such isolate had exactly the intended sequence . We propose to assemble larger genomes by joining separately assembled 5- to 6-kb segments; approximately 60 such segments would be required for a minimal cellular genome.

J Virol Methods, 2004 Jan, 115(1), 19 - 30
A simple mathematical formula for stoichiometry quantification of viral and nanobiological assemblage using slopes of log/log plot curves; Shu D et al.; In nanotechnology, biomolecular assemblies serve not only as model systems for the construction of nanodevices, but they can also be used directly as templates for the formation of nanostructures . Biological nano-building blocks can either be isolated as complete functional units from living cells or viruses (biological "Top down" approach) or formed by biomolecular assembly from recombinant or synthetic components ("Bottom up" approach) . In both cases, rational design of nanostructures requires knowledge of the stoichiometry of the biological structures, which frequently occur as multimers, i.e., the morphological complex is composed of multiple copies of one or more macromolecules . In this paper, a method is described for the stoichiometric quantification of molecules in bio-nanostructures . The method is based on using dilution factors and relative concentrations rather than absolute quantities, which are often difficult to determine, especially in short-lived assembly intermediates . The approach exploits the fact that the larger the stoichiometry of the component is, the more dramatic is the influence of the dilution factor (decrease in concentration) on the reaction . We established and used the method to determine the stoichiometry of components of bacterial virus phi29 . The log of dilution factors was plotted against the log of reaction yield . The stoichiometry Z was determined with the equation Z=-1.58+2.4193T-0.001746T(2) {T in (0,1000), or 90 degree angle alpha in (0 degrees, 89.9 degrees )}, where T is the slope of the curve (tangent of 90 degree angle alpha, which is the angle between the x-axis and the concentration dependent curve) . Z can also be determined from a standard table given in this report . With the bacteriophage phi29 in vitro assembly system, up to 5x10(8) infectious virions per ml can be assembled from 11 purified components, giving our method a sensitivity of nine orders of magnitude . We confirmed the stoichiometries of phi29 components that were determined previously with microscopic approaches . The described method also responded to programmed stoichiometry changes, which were generated by assembling the phi29 DNA packaging motor from modified pRNA (DNA-packaging RNA) molecules forming a trimer of dimers or a dimer of trimers, instead of the wild-type hexamer.

Structure (Camb), 2003 Dec, 11(12), 1557 - 67
The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold; Rafferty JB et al.; Holliday junction resolution performed by a variety of structure-specific endonucleases is a key step in DNA recombination and repair . It is believed that all resolvases carry out their reaction chemistries in a similar fashion, utilizing a divalent cation to facilitate the hydrolysis of the phosphodiester backbone of the DNA, but their architecture varies . To date, with the exception of bacteriophage T4 endonuclease VII, each of the known resolvase enzyme structures has been categorized into one of two families: the integrases and the nucleases . We have now determined the structure of the Escherichia coli RusA Holliday junction resolvase, which reveals a fourth structural class for these enzymes . The structure suggests that dimer formation is essential for Mg(2+) cation binding and hence catalysis and that like the other resolvases, RusA distorts its Holliday junction target upon binding . Key residues identified by mutagenesis experiments are well positioned to interact with the DNA.

Nucleic Acids Res, 2003 Dec 15, 31(24), 7059 - 69
Tracking EcoKI and DNA fifty years on: a golden story full of surprises; Loenen WA; 1953 was a historical year for biology, as it marked the birth of the DNA helix, but also a report by Bertani and Weigle on 'a barrier to infection' of bacteriophage lambda in its natural host, Escherichia coli K-12, that could be lifted by 'host-controlled variation' of the virus . This paper lay dormant till Nobel laureate Arber and PhD student Dussoix showed that the lambda DNA was rejected and degraded upon infection of different bacterial hosts, unless it carried host-specific modification of that DNA, thus laying the foundations for the phenomenon of restriction and modification (R-M) . The restriction enzyme of E.coli K-12, EcoKI, was purified in 1968 and required S-adenosylmethionine (AdoMet) and ATP as cofactors . By the end of the decade there was substantial evidence for a chromosomal locus hsdK with three genes encoding restriction (R), modification (M) and specificity (S) subunits that assembled into a large complex of >400 kDa . The 1970s brought the message that EcoKI cut away from its DNA recognition target, to which site the enzyme remained bound while translocating the DNA past itself, with concomitant ATP hydrolysis and subsequent double-strand nicks . This translocation event created clearly visible DNA loops in the electron microscope . EcoKI became the archetypal Type I R-M enzyme with curious DNA translocating properties reminiscent of helicases, recognizing the bipartite asymmetric site AAC(N6)GTGC . Cloning of the hsdK locus in 1976 facilitated molecular understanding of this sophisticated R-M complex and in an elegant 'pas de deux' Murray and Dryden constructed the present model based on a large body of experimental data plus bioinformatics . This review celebrates the golden anniversary of EcoKI and ends with the exciting progress on the vital issue of restriction alleviation after DNA damage, also first reported in 1953, which involves intricate control of R subunit activity by the bacterial proteasome ClpXP, important results that will keep scientists on the EcoKI track for another 50 years to come.

Eur J Biochem, 2003 Dec, 270(24), 4846 - 58
Solution structure and stability of the full-length excisionase from bacteriophage HK022; Rogov VV et al.; Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction . As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28-->Ser was used in this work . This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry . The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49) . These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase . Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor . Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution . The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization . Such an isomerization has been previously observed and characterized only in eukaryotic proteins.

J Colloid Interface Sci, 2004 Jan 1, 269(1), 251 - 4
Competitive adsorption of bacteriophage T4 lysozyme stability variants at hydrophilic glass surfaces; Lee WK et al.; The competitive adsorption behavior exhibited by the wild-type T4 lysozyme and two of its structural stability variants was studied by 125I radioisotope labeling . The mutant lysozymes were produced by substitution of the isoleucine residue at position 3 in the wild type with a tryptophan residue, resulting in a protein with lower structural stability, or with a cysteine residue, resulting in a protein with higher structural stability . Adsorption kinetics were recorded for binary protein mixtures in contact with a clean glass surface, in which one variant had been radiolabeled and the other had not . All pair permutations were tested . The kinetic data show that in instances in which exchange reactions between adsorbed protein and dissolved protein occur, they occur such that more stable variants are removed from the surface by less stable variants . The less stable proteins thus exhibited an advantage in competitive adsorption over the more stable proteins, in these tests.

Mol Microbiol, 2004 Jan, 51(1), 203 - 13
Evidence for dynamic clustering of carboxy-terminal aromatic amino acids in TonB-dependent energy transduction; Ghosh J et al.; Escherichia coli uses the proton motive force of the cytoplasmic membrane and TonB protein to energize the active transport of iron-siderophores and vitamin B12 across the outer membrane . TonB shuttles between the cytoplasmic and outer membranes, presumably during the course of energy transduction . Previous results indicated that the carboxy-terminal 65 amino acids of TonB are essential for both its outer membrane association and activity . A highly conserved region (residues 199-216) within this domain, predicted to be an amphipathic alpha-helix, was the initial focus of this study . Scanning mutagenesis indicated that only the aromatic residues F202, W213 and Y215 were individually important for activity . When the crystal structure of a dimeric TonB carboxy-terminus subsequently became available, we observed that two additional aromatic residues outside that region, F180 and F230, were potentially engaged in end-on hydrophobic interactions with the three residues identified previously . Changing these five aromatic residues individually to alanine reduced TonB activity . Surprisingly, however, each substitution exhibited a unique phenotypic profile with respect to ability to support {55Fe}-ferrichrome transport, sensitivity to colicins B, D, Ia and M or sensitivity to bacteriophage phi80 . The phenotypic results suggested that the carboxy-terminus of TonB was a flexible and dynamic domain that could interact specifically with different ligands or transporters, perhaps through the aromatic residues . The possibility of interactions among all the aromatic residues was tested using double-mutant cycle analysis . All possible combinations of alanine substitutions were constructed, with the result that TonB containing any double-alanine substitution was inactive in the phenotypic assays, while retaining the ability to associate with the outer membrane . This synergistic, rather than additive, effect of the double mutants suggested that, consistent with the flexibility suggested by analysis of the single substitutions, all the aromatic residues might be capable of interacting with one another . A means of reconciling these results with the crystal structure is presented.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2337 - 41 Epub 2003 Nov 27.
Order and disorder in crystals of hexameric NTPases from dsRNA bacteriophages; Mancini EJ et al.; The packaging of genomic RNA in members of the Cystoviridae is performed by P4, a hexameric protein with NTPase activity . Across family members such as Phi6, Phi8 and Phi13, the P4 proteins show low levels of sequence identity, but presumably have similar atomic structures . Initial structure-determination efforts for P4 from Phi6 and Phi8 were hampered by difficulties in obtaining crystals that gave ordered diffraction . Diffraction from crystals of full-length P4 showed a variety of disorder and anisotropy . Subsequently, crystals of Phi13 P4 were obtained which yielded well ordered diffraction to 1.7 A . Comparison of the packing arrangements of P4 hexamers in different crystal forms and analysis of the disorder provides insights into the flexibility of this family of proteins, which might be an integral part of their biological function.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2060 - 4 Epub 2003 Nov 27.
Crystallization and preliminary analysis of a dsDNA bacteriophage capsid intermediate: Prohead II of HK97; Wikoff WR et al.; HK97 Prohead II is an early intermediate in the maturation of HK97, a T = 7 dsDNA-tailed bacteriophage related to bacteriophage lambda . Previously, selected capsid-protein genes of HK97 were expressed in Escherichia coli and spontaneously assembled to form an icosahedral capsid that followed a maturation pathway closely similar to the authentic virion . The crystal structure of the mature HK97 capsid (Head II) made in this way was reported at 3.5 A resolution . Additional high-resolution structures of intermediates are needed to understand the maturation mechanism . The crystal structure of expressed Prohead II will elucidate the early steps of HK97 assembly . Crystals of the Prohead II mutant W336F were grown in 0.1 M HEPES pH 7.5, 0.2 M CaCl(2) and 2-3% PEG 4000 at a Prohead II concentration of 16.5 mg ml(-1) . It was not possible to grow high-quality crystals of wild-type Prohead II . Diffraction was observed to 5 A resolution from these crystals on beamline 14BM-C at the Advanced Photon Source and data were collected to 5.5 A with a completeness of 77% . The space group was P2(1)3, with unit-cell parameter a = 707.0 A and four particles in the unit cell . The particles are on the body diagonals of the cubic cell, with icosahedral threefold axes coincident with crystallographic threefold axes . Self-rotation function and locked-rotation function analysis determined the particle orientation and a one-dimensional R-factor search along the body diagonal indicated that the particle centers were close to (1/4, 1/4, 1/4) and symmetry-related positions . Molecular-replacement averaging and phase extension are under way.

J Mol Biol, 2003 Dec 12, 334(5), 885 - 99
The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 A resolution; Helgstrand C et al.; The HK97 bacteriophage capsid is a unique example of macromolecular catenanes: interlocked rings of covalently attached protein subunits . The chain mail organization of the subunits stabilizes a particle in which the maximum thickness of the protein shell is 18A and the maximum diameter is 550A . The electron density has the appearance of a balloon illustrating the extraordinary strength conferred by the unique subunit organization . The refined structure shows novel qualities of the HK97 shell protein, gp5 that, together with the protease gp4, guides the assembly and maturation of the virion . Although gp5 forms hexamers and pentamers and the subunits exist in different structural environments, the tertiary structures of the seven protein molecules in the viral asymmetric unit are closely similar . The interactions of the subunits in the shell are exceptionally complex with each subunit interacting with nine other subunits . The interactions of the N-terminus released after gp5 cleavage appear important for organization of the loops that become crosslinked to the core of a neighboring subunit at the maturation . A comparison with a model of the Prohead II structure revealed that the surfaces of non-covalent contact between the monomers that build up hexamers/pentamers are completely redefined during maturation.

Biochem Biophys Res Commun, 2003 Dec 12, 312(2), 494 - 9
Balancer-Cre transgenic mouse germ cells direct the incomplete resolution of a tri-loxP-targeted Cyp1a1 allele, producing a conditional knockout allele; Uno S et al.; To generate conditional alleles, genes are commonly engineered to contain recognition sites for bacteriophage recombinases, such as Cre recombinase . When such motifs (lox sites) flank essential gene sequences, and provided that Cre recombinase is expressed, Cre recombinase will excise the flanked sequence-creating a conditional knockout allele . Targeted conditional alleles contain a minimum of three lox sites . It would be desirable to have Cre recombinase perform partial resolution (i.e., recombination some of the time between only the two lox sites flanking the marker gene) . Here we report use of the commercially available Balancer2-Cre transgenic mouse line to carry out this function from a tri-loxP-site-containing cytochrome p450 1A1 (Cyp1a1) targeted allele . Such incomplete resolution of this complex locus occurred progressively with age in germ cells of male mice; the conditional Cyp1a1 gene was recovered in offspring from mice containing the targeted Cyp1a1 allele and the Cre recombinase transgene . Removal of the marker gene resulted in a conditional Cyp1a1 allele whose expression was indistinguishable from that of the wild-type allele.

Mol Cell, 2003 Nov, 12(5), 1113 - 23
The crystal structure of the bifunctional primase-helicase of bacteriophage T7; Toth EA et al.; Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA . The lynchpin of the T7 replication system is a bifunctional primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand . We have determined a 3.45 A crystal structure of the T7 primase-helicase that shows an articulated arrangement of the primase and helicase sites . The crystallized primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed primase domains . This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity . The double-jointed structure of the primase-helicase permits a free range of motion for the primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.

J Biol Chem, 2004 Feb 13, 279(7), 6077 - 86 Epub 2003 Nov 22.
Mutations in a conserved motif inhibit single-stranded DNA binding and recombination mediator activities of bacteriophage T4 UvsY protein; Bleuit JS et al.; The UvsY recombination mediator protein is critical for homologous recombination in bacteriophage T4 . UvsY uses both protein-protein and protein-DNA interactions to mediate the assembly of the T4 UvsX recombinase onto single-stranded (ss) DNA, forming presynaptic filaments that initiate DNA strand exchange . UvsY helps UvsX compete with Gp32, the T4 ssDNA-binding protein, for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions . The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood . The goal of this study was to isolate mutant forms of UvsY protein that are specifically defective in UvsY-ssDNA interactions, so that the contribution of this activity to recombination processes could be assessed independent of other factors . A conserved motif of UvsY found in other DNA-binding proteins was targeted for mutagenesis . Two missense mutants of UvsY were isolated in which ssDNA binding activity is compromised . These mutants retain self-association activity, and form stable associations with UvsX and Gp32 proteins in patterns similar to wild-type UvsY . Both mutants are partially, but not totally, defective in stimulating UvsX-catalyzed recombination functions including ssDNA-dependent ATP hydrolysis and DNA strand exchange . The data are consistent with a model in which UvsY plays bipartite roles in presynaptic filament assembly . Its protein-ssDNA interactions are suggested to moderate the destabilization of Gp32-ssDNA, whereas its protein-protein contacts induce a conformational change of the UvsX protein, giving UvsX a higher affinity for the ssDNA and allowing it to compete more effectively with Gp32 for binding sites.

Protein Sci, 2003 Dec, 12(12), 2732 - 47
C-terminal hydrophobic interactions play a critical role in oligomeric assembly of the P22 tailspike trimer; Gage MJ et al.; The tailspike protein from the bacteriophage P22 is a well characterized model system for folding and assembly of multimeric proteins . Folding intermediates from both the in vivo and in vitro pathways have been identified, and both the initial folding steps and the protrimer-to-trimer transition have been well studied . In contrast, there has been little experimental evidence to describe the assembly of the protrimer . Previous results indicated that the C terminus plays a critical role in the overall stability of the P22 tailspike protein . Here, we present evidence that the C terminus is also the critical assembly point for trimer assembly . Three truncations of the full-length tailspike protein, TSPDeltaN, TSPDeltaC, and TSPDeltaNC, were generated and tested for their ability to form mixed trimer species . TSPDeltaN forms mixed trimers with full-length P22 tailspike, but TSPDeltaC and TSPDeltaNC are incapable of forming similar mixed trimer species . In addition, mutations in the hydrophobic core of the C terminus were unable to form trimer in vivo . Finally, the hydrophobic-binding dye ANS inhibits the formation of trimer by inhibiting progression through the folding pathway . Taken together, these results suggest that hydrophobic interactions between C-terminal regions of P22 tailspike monomers play a critical role in the assembly of the P22 tailspike trimer.

Photochem Photobiol, 2003 Oct, 78(4), 349 - 54
Pyrimidine dimer formation and oxidative damage in M13 bacteriophage inactivation by ultraviolet C irradiation; Kurosaki Y et al.; The mechanism by which UV-C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South-Western blotting for pyrimidine dimers . The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions . With a decrease in M13 infectivity on irradiation, single-stranded circular genomic DNA (sc-DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc-DNA and linear-form DNA . However, the amount of sc-DNA remaining was not correlated with the survival of M13 . The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PP) increased as a function of irradiation dose . The decrease in M13 infectivity was highly correlated with the increase in CPD and (6-4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . 8-Oxo-7,8-dihydro-2'-deoxyguanosine did not form in the M13 genome after UV-C irradiation . Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide . Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV-C irradiation under these conditions . These results indicated that UV-C irradiation induced not only CPD and (6-4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity . The indirect effect of UV-C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV-C.

Cell Mol Life Sci, 2003 Nov, 60(11), 2356 - 70
Structure and morphogenesis of bacteriophage T4; Leiman PG et al.; Bacteriophage T4 is one of the most complex viruses . More than 40 different proteins form the mature virion, which consists of a protein shell encapsidating a 172-kbp double-stranded genomic DNA, a 'tail,' and fibers, attached to the distal end of the tail . The fibers and the tail carry the host cell recognition sensors and are required for attachment of the phage to the cell surface . The tail also serves as a channel for delivery of the phage DNA from the head into the host cell cytoplasm . The tail is attached to the unique 'portal' vertex of the head through which the phage DNA is packaged during head assembly . Similar to other phages, and also herpes viruses, the unique vertex is occupied by a dodecameric portal protein, which is involved in DNA packaging.

J Biol Chem, 2004 Jan 30, 279(5), 3472 - 83 Epub 2003 Nov 18.
Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution; Boldt JL et al.; The bacteriophage lambda integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates . Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates . We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40-60% of recombination substrates at the Holliday junction stage of the reaction . These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least . We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway . We have now identified new inhibitors of the excision pathway . One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides . This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre . This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases . We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution . This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.

J Mol Biol, 2003 Nov 28, 334(3), 349 - 61
Regulation of translation of the head protein of T4 bacteriophage by specific binding of EF-Tu to a leader sequence; Snyder L et al.; Recent evidence indicates that translation elongation factor Tu (EF-Tu) has a role in the cell in addition to its well established role in translation . The translation factor binds to a specific region called the Gol region close to the N terminus of the T4 bacteriophage major head protein as the head protein emerges from the ribosome . This binding was discovered because EF-Tu bound to Gol peptide is the specific substrate of the Lit protease that cleaves the EF-Tu between amino acid residues Gly59 and lle60, blocking phage development . These experiments raised the question of why the Gol region of the incipient head protein binds to EF-Tu, as binding to incipient proteins is not expected from the canonical role of EF-Tu . Here, we use gol-lacZ translational fusions to show that cleavage of EF-Tu in the complex with Gol peptide can block translation of a lacZ reporter gene fused translationally downstream of the Gol peptide that activated the cleavage . We propose a model to explain how binding of EF-Tu to the emerging Gol peptide could cause translation to pause temporarily and allow time for the leader polypeptide to bind to the GroEL chaperonin before translation continues, allowing cotranslation of the head protein with its insertion into the GroEL chaperonin chamber, and preventing premature synthesis and precipitation of the head protein . Cleavage of EF-Tu in the complex would block translation of the head protein and therefore development of the infecting phage . Experiments are presented that confirm two predictions of this model . Considering the evolutionary conservation of the components of this system, this novel regulatory mechanism could be used in other situations, both in bacteria and eukaryotes, where proteins are cotranslated with their insertion into cellular structures.

Eur J Biochem, 2003 Nov, 270(22), 4439 - 46
Disorder-order transition of lambda CII promoted by low concentrations of guanidine hydrochloride suggests a stable core and a flexible C-terminus; Datta AB et al.; The CII protein of bacteriophage lambda, which activates the synthesis of the lambda repressor, plays a key role in the lysis-lysogeny switch . CII has a small in vivo half-life due to its proteolytic susceptibility, and this instability is a key component for its regulatory role . The structural basis of this instability is not known . While studying guanidine hydrochloride-assisted unfolding of CII, we found that low concentrations of the chaotrope (50-500 microM) have a considerable effect on the structure of this protein . This effect is manifest in an increase in molar ellipticity, an enhancement of intrinsic tryptophan fluorescence intensity and a reduction in ANS binding . At low concentrations of guanidine hydrochloride CII is stabilized, as reflected in a significant decrease in the rate of proteolysis by trypsin and resistance to thermal aggregation, while the tetrameric nature of the protein is retained . Thus low concentrations of guanidine hydrochloride promote a more structured conformation of the CII protein . On the basis of these observations, a model has been proposed for the structure of CII wherein the protein equilibrates between a compact form and a proteolytically accessible form, in which the C-terminal region assumes different structures.

Reprod Biol Endocrinol . 2003 Nov 07;1(1):79.
Phage integrases for the construction and manipulation of transgenic mammals; Hollis RP et al.; Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites . Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule . Here we demonstrate the ability of the phiC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos . Transgenic early embryos and a mid-gestation mouse are reported . We also demonstrate the ability of the phiC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material . We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations . The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.

J Mol Biol, 2003 Nov 21, 334(2), 309 - 25
Improved Gō-like models demonstrate the robustness of protein folding mechanisms towards non-native interactions; Karanicolas J et al.; The use of simple theoretical models has provided a considerable contribution to our present understanding of the means by which proteins adopt their native fold from the plethora of available unfolded states . A common assumption in building computationally tractable models has been the neglect of stabilizing non-native interactions in the class of models described as "Go-like." The focus of this study is the characterization of the folding of a number of proteins via a Go-like model, which aims to map a maximal amount of information reflecting the protein sequence onto a "minimalist" skeleton . This model is shown to contain sufficient information to reproduce the folding transition states of a number of proteins, including topologically analogous proteins that fold via different transition states . Remarkably, these models also demonstrate consistency with the general features of folding transition states thought to be stabilized by non-native interactions . This suggests that native interactions are the primary determinant of most protein folding transition states, and that non-native interactions lead only to local structural perturbations . A prediction is also included for an asymmetrical folding transition state of bacteriophage lambda protein W, which has yet to be subjected to experimental characterization.

J Mol Biol, 2003 Nov 21, 334(2), 241 - 54
High-density functional display of proteins on bacteriophage lambda; Gupta A et al.; We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda . DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences . Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites . Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed . The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD . The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage . The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products . The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries . Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system . The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.

Structure (Camb), 2003 Nov, 11(11), 1445 - 51
Structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus; Doan DN et al.; Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus of the Arteriviridae family, genomically related to the coronaviruses . PRRSV is the causative agent of both severe and persistent respiratory disease and reproductive failure in pigs worldwide . The PRRSV virion contains a core made of the 123 amino acid nucleocapsid (N) protein, a product of the ORF7 gene . We have determined the crystal structure of the capsid-forming domain of N . The structure was solved to 2.6 A resolution by SAD methods using the anomalous signal from sulfur . The N protein exists in the crystal as a tight dimer forming a four-stranded beta sheet floor superposed by two long alpha helices and flanked by two N- and two C-terminal alpha helices . The structure of N represents a new class of viral capsid-forming domains, distinctly different from those of other known enveloped viruses, but reminiscent of the coat protein of bacteriophage MS2.

Structure (Camb), 2003 Nov, 11(11), 1339 - 48
Bacteriophage MS2: molecular weight and spatial distribution of the protein and RNA components by small-angle neutron scattering and virus counting; Kuzmanovic DA et al.; Small-angle neutron scattering (SANS) has been used to extend the structural characterization of the MS2 phage by examining its physical characteristics in solution . Specifically, the contrast variation technique was employed to determine the molecular weight of the individual components of the MS2 virion (protein shell and genomic RNA) and the spatial relationship of the genomic RNA to its protein shell . A consequence of this work was to evaluate a novel particle counting instrument, the integrated virus detection system (IVDS) that, in combination with SANS, has the potential to provide rapid quantitative physical characterization of unidentified viruses and phage.

Nucleic Acids Res, 2003 Nov 15, 31(22), 6502 - 8
Arginine/serine repeats are sufficient to constitute a splicing activation domain; Philipps D et al.; SR proteins are essential pre-mRNA splicing factors that have been shown to bind a number of exonic splicing enhancers where they function to stimulate the splicing of adjacent introns . Members of the SR protein family contain one or two N-terminal RNA binding domains, as well as a C-terminal arginine-serine (RS) rich domain . The RS domains mediate protein-protein interactions with other RS domain containing proteins and are essential for many, but not all, SR protein functions . Hybrid proteins containing an RS domain fused to the bacteriophage MS2 coat protein are sufficient to activate enhancer-dependent splicing in HeLa cell nuclear extract when bound to the pre-mRNA . Here we report progress towards determining the protein sequence requirements for RS domain function . We show that the RS domains from non-SR proteins can also function as splicing activation domains when tethered to the pre-mRNA . Truncation experiments with the RS domain of the human SR protein 9G8 identified a 29 amino acid segment, containing 26 arginine or serine residues, that is sufficient to activate splicing when fused to MS2 . We also show that synthetic domains composed solely of RS dipeptides are capable of activating splicing, although their potency is proportional to their size.

Appl Environ Microbiol, 2003 Nov, 69(11), 6628 - 33
Sampling natural viral communities from soil for culture-independent analyses; Williamson KE et al.; An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses . In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy {EFM}, and transmission electron microscopy {TEM}) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils . Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain . Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 x 10(8) g of dry soil(-1)), but specific soil-eluant combinations posed significant problems to enumeration by EFM . Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5 x 10(8) g of dry soil(-1)) that were about five times lower . Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.

Zh Obshch Biol, 2003 Sep-Oct, 64(5), 421 - 33
{Information value of various trinucleotides of some genetic systems}; Mamonova MA et al.; New method to reveal the sites in genomes obtaining the high information capacity is developed . A distribution of those sites of the length 3 among 16 viral genomes and 11 bacteriophages genomes has been studied . It is shown that some triplets with high information capacity occur in a family of relatively close genomes with the increased frequency . The molecular evolution aspects of a persistence of highly scored sites with respect to their information capacity among various genomes are discussed.

J Biol Chem, 2004 Jan 30, 279(5), 3239 - 44 Epub 2003 Nov 03.
Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase . A single-molecule view of the transcription cycle; Skinner GM et al.; A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time . To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP . By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules . After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed . Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt) . The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.

J Mol Biol, 2003 Nov 14, 334(1), 37 - 52
Defining the bacteriophage T4 DNA packaging machine: evidence for a C-terminal DNA cleavage domain in the large terminase/packaging protein gp17; Rentas FJ et al.; Double-stranded DNA packaging in bacteriophage T4 and other viruses occurs by translocation of DNA into an empty prohead by a packaging machine assembled at the portal vertex . Coordinated with this complex process is the cutting of concatemeric DNA to initiate and terminate DNA packaging and encapsidate one genome-length viral DNA . The catalytic site responsible for cutting, and the mechanisms by which cutting is precisely coordinated with DNA translocation remained as interesting open questions . Phage T4, unlike the phages with defined ends (e.g . lambda, T3, T7), packages DNA in a strictly headful manner, and exhibits no strict sequence specificity to initiate or terminate DNA packaging . Previous evidence suggests that the large terminase protein gp17, a key component of the T4 packaging machine, possesses a non-specific DNA cutting activity . A histidine-rich metal-binding motif, H382-X(2)-H385-X(16)-C402-X(8)-H411-X(2)-H414-X(15)-H430-X(5)-H436, in the C-terminal half of gp17 is thought to be involved in the terminase cleavage . Here, exhaustive site-directed mutagenesis revealed that none of the cysteine and histidine residues other than the H436 residue is critical for function . On the other hand, a cluster of conserved residues within this region, D401, E404, G405, and D409, are found to be critical for function . Biochemical analyses showed that the D401 mutants exhibited a novel phenotype, showing a loss of in vivo DNA cutting activity but not the DNA packaging activity . The functional nature of the critical residues and their disposition in the conserved loop region between two predicted beta-strands suggest that these residues are part of a metal-coordinated catalytic site that cleaves the phosphodiester bond of DNA substrate . The data suggest that the T4 terminase consists of at least two functional domains, an N-terminal DNA-translocating ATPase domain and a C-terminal DNA-cutting domain . Although the DNA recognition mechanisms may be distinct, it appears that T4 and other phage terminases employ a common catalytic paradigm for phosphodiester bond cleavage that is used by numerous nucleases.

J Mol Biol, 2003 Nov 14, 334(1), 13 - 23
SegG endonuclease promotes marker exclusion and mediates co-conversion from a distant cleavage site; Liu Q et al.; Bacteriophages T2 and T4 are closely related T-even phages . However, T4 genetic markers predominate in the progeny of mixed infections, a phenomenon termed marker exclusion . One region previously mapped where the frequency of T2 markers in the progeny is extremely low is located around gene 32 . Here, we describe SegG, a GIY-YIG family endonuclease adjacent to gene 32 of phage T4 that is absent from phage T2 . In co-infections with T2 and T4, cleavage in T2 gene 32 by T4-encoded SegG initiates a gene conversion event that results in replacement of T2 gene 32 markers with the corresponding T4 sequence . Interestingly, segG inheritance is limited, apparently because of the physical separation of its cleavage and insertion sites, which are 332 base-pairs apart . This contrasts with efficient inheritance of the phage T4 td group I intron and its endonuclease, I-TevI, for which the distance separating the I-TevI cleavage site and td insertion site is 23 base-pairs . Furthermore, we show that co-conversion tracts generated by repair of SegG and I-TevI double-strand breaks contribute to the localized exclusion of T2 markers . Our results demonstrate that the endonuclease activities of SegG and I-TevI promote the spread of these two endonucleases to progeny phage, consistent with their role as selfish genetic elements, and also provide a mechanism by which the genetic contribution of T2 markers to progeny phage is reduced.

J Mol Microbiol Biotechnol, 2003, 6(1), 57 - 66
Mutations in the N-terminus of the major coat protein (pVIII, gp8) of filamentous bacteriophage affect infectivity; Li Z et al.; Filamentous bacteriophages are nonlytic, male-specific bacteriophages which infect Escherichia coli carrying an F-episome . The molecular mechanism of infection remains elusive, including the role of the major coat protein pVIII . In order to evaluate the contributions of major coat protein pVIII in the process of infection, two phage display libraries were generated . One library consisted of random amino acids at positions 2, 4, 5, 8, 11 and 12 of the N-terminus of major coat protein pVIII . The second library was generated by randomizing these positions as well as position 1 . All these residues were previously shown to be exposed at the surface of the virions by being accessible to ligands . The infectivity of randomly selected mutant phages was analyzed . The present results demonstrate that phages modified at these positions can be correctly assembled and secreted into the exoplasm, although the efficiency was slightly lower than that of wild-type phage . Their infectivity varied greatly, and a general structural pattern underlying infectivity did not emerge . However, residual differences were observed between infectious and defective phage; in general, uncharged polar amino acids present at positions 5 and 11 of the N-terminus of pVIII reduced phage infectivity, whereas polar residues at position 8 facilitated infection . The first position of pVIII is remarkably critical for infection; when this alanine was substituted with other residues, most of the phages lost their infectivity . These results shed new light on the true complexity of random peptide pVIII phage display libraries .

Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 540 - 5
Virus particle detection by solid phase immunocapture and atomic force microscopy; Nettikadan SR et al.; A novel application of atomic force microscopy (AFM) in the rapid, label-free detection and identification of viruses is described . Multiplexed, miniaturized antibody domains were constructed using "ink-jet" protein arraying technology . The solid-phase affinity substrate termed the "ViriChip" was used in the immunocapture of bacteriophage fd, canine parvoviruses, and coxsackieviruses and analyzed by AFM . Immunocapture was found to be antibody-specific with a sensitivity of 10(8)pfu/ml in 30min . Virus binding was found to be linear for concentration between 10(8) and 10(10)pfu/ml and did not reach saturation through 4h.

Acta Crystallogr D Biol Crystallogr, 2003 Nov, 59(Pt 11), 2004 - 16 Epub 2003 Oct 23.
Twinned crystals and anomalous phasing; Dauter Z; Merohedral or pseudomerohedral twinning of crystals cannot be identified from inspection of the diffraction patterns . Several methods for the identification of twinning and the estimation of the twin fraction are suitable for macromolecular crystals and all are based on the statistical properties of the measured diffraction intensities . If the crystal twin fraction is estimated and is not too close to 0.5, the diffraction data can be detwinned; that is, related to the individual crystal specimen . However, the detwinning procedure invariably introduces additional inaccuracies to the estimated intensities, which substantially increase when the twin fraction approaches 0.5 . In some cases, a crystal structure can be solved with the original twinned data by standard techniques such as molecular replacement, multiple isomorphous replacement or multiwavelength anomalous diffraction . Test calculations on data collected from a twinned crystal of gpD, the bacteriophage lambda capsid protein, show that the single-wavelength anomalous diffraction (SAD) method can be used to solve its structure even if the data set corresponds to a perfectly twinned crystal with a twin fraction of 0.5.

J Gen Virol, 2003 Nov, 84(Pt 11), 2895 - 908
Genetic content and evolution of adenoviruses; Davison AJ et al.; This review provides an update of the genetic content, phylogeny and evolution of the family Adenoviridae . An appraisal of the condition of adenovirus genomics highlights the need to ensure that public sequence information is interpreted accurately . To this end, all complete genome sequences available have been reannotated . Adenoviruses fall into four recognized genera, plus possibly a fifth, which have apparently evolved with their vertebrate hosts, but have also engaged in a number of interspecies transmission events . Genes inherited by all modern adenoviruses from their common ancestor are located centrally in the genome and are involved in replication and packaging of viral DNA and formation and structure of the virion . Additional niche-specific genes have accumulated in each lineage, mostly near the genome termini . Capture and duplication of genes in the setting of a 'leader-exon structure', which results from widespread use of splicing, appear to have been central to adenovirus evolution . The antiquity of the pre-vertebrate lineages that ultimately gave rise to the Adenoviridae is illustrated by morphological similarities between adenoviruses and bacteriophages, and by use of a protein-primed DNA replication strategy by adenoviruses, certain bacteria and bacteriophages, and linear plasmids of fungi and plants.

Curr Opin Microbiol, 2003 Oct, 6(5), 506 - 11
Bacteriophage genomics; Hendrix RW; Comparative genomic studies of bacteriophages, especially the tailed phages, together with environmental studies, give a dramatic new picture of the size, genetic structure and dynamics of this population . Sequence comparisons reveal some of the detailed mechanisms by which these viruses evolve and influence the evolution of their bacterial and archaeal hosts . We see rampant horizontal exchange of sequences among genomes, mediated by both homologous and nonhomologous recombination . High frequency exchange among phages occupying similar ecological niches leads to a high degree of mosaic diversity in local populations . Horizontal exchange also takes place at lower frequency across the entire span of phage sequence space.

J Struct Biol, 2003 Sep, 143(3), 258 - 62
Repetitive versus monomeric antigen presentation: direct visualization of antibody affinity and specificity; Baschong W et al.; The concept of presenting antigens in a repetitive array to obtain high titers of specific antibodies is increasingly applied by using surface-engineered viruses or bacterial envelopes as novel vaccines . A case for this concept was made 25 years ago, when producing high-titer antisera against ordered arrays of gp23, the major capsid protein of bacteriophage T4 (Aebi et al., Proc . Natl . Acad . Sci . USA, 74 (1977) 5514-5518) . In view of the current interest in this concept we thought it useful to employ this system to directly visualize the dependence of antibody affinity and specificity on antigen presentation . We compared antibodies raised against T4 polyheads, a tubular variant of the bacteriophage T4 capsid, which have gp23 hexamers arranged in a crystalline lattice (gp23(repetitive)), with those raised against the hexameric gp23 subunits (gp23(monomeric)) . The labeling patterns of Fab-fragments prepared from these antibodies when bound to polyheads were determined by electron microscopy and image enhancement . Anti-gp23(repetitive) bound in a monospecific, stoichiometric fashion to the gp23 units constituting the polyhead surface . In contrast, anti-gp23(monomeric) decorated the polyhead surface randomly and with a 40-fold lower occupancy . These results concur with the difference in titers established by ELISA for the antisera against the repetitively displayed form of antigen (anti-gp23(repetitive)) and the randomly presented antigen (gp23(monomeric)), and they constitute a compelling visual documentation of the concept of repetitive antigen presentation to elicite a serotype-like immune response.

J Biol Chem, 2004 Jan 16, 279(3), 2012 - 9 Epub 2003 Oct 21.
A mutation in the yeast mitochondrial core RNA polymerase, Rpo41, confers defects in both specificity factor interaction and promoter utilization; Matsunaga M et al.; The yeast mitochondrial RNA polymerase (RNAP) is composed of the core RNAP, Rpo41, and the mitochondrial transcription factor, Mtf1 . Both are required for mitochondrial transcription, but how the two proteins interact to create a functional, promoter-selective holoenzyme is still unknown . Rpo41 is similar to the single polypeptide bacteriophage T7RNAP, which does not require additional factors for promoter-selective initiation but whose activity is modulated during infection by association with T7 lysozyme . In this study we used the co-crystal structure of T7RNAP and T7 lysozyme as a model to define a potential Mtf1 interaction surface on Rpo41, making site-directed mutations in Rpo41 at positions predicted to reside at the same location as the T7RNAP/T7 lysozyme interface . We identified Rpo41 mutant E1224A as having reduced interactions with Mtf1 in a two-hybrid assay and a temperature-sensitive petite phenotype in vivo . Although the E1224A mutant has full activity in a non-selective in vitro transcription assay, it is temperature-sensitive for selective transcription from linear DNA templates containing the 14S rRNA, COX2, and tRNAcys mitochondrial promoters . The tRNAcys promoter defect can be rescued by template supercoiling but not by addition of a dinucleotide primer . The fact that mutation of Rpo41 results in selective transcription defects indicates that the core RNAP, like T7RNAP, plays an important role in promoter utilization.

Biochem Cell Biol, 2003 Aug, 81(4), 307 - 15
The product of the bacteriophage lambda W gene: purification and properties; Murialdo H et al.; Gene W is one of the 10 genes that control the morphogenesis of the bacteriophage lambda head . The morpho genesis of the phage lambda head proceeds through the synthesis of an intermediate assembly called the prohead . This is an empty shell into which the bacteriophage DNA is introduced--packaged--by the phage enzyme DNA terminase . The product of W (gpW) acts after DNA packaging, but before the addition of another phage product, gene product FII, and before the addition of tails . The role of gpW is unknown . The structure of N- and C-tagged gpW has been previously determined by nuclear magnetic resonance (NMR) spectroscopy . Here we report some of the properties of the native protein . The purification of gpW to homogeneity, overproduced by a plasmid derivative, is described . To obtain large amounts of the protein, the ribosome-binding site had to be modified, showing that inefficient translation of the message is the main mechanism limiting W gene expression . The molecular weight of the protein is in close agreement to the value predicted from the DNA sequence of the gene, which suggests that it is not post-transcriptionally modified . It behaves as a monomer in solution . Radioactively labeled gpW is incorporated into phage particles in in vitro complementation, showing that gpW is a structural protein . The stage at which gpW functions and other circumstantial evidence support the idea that six molecules of gpW polymerize on the connector before the incorporation of six molecules of gpFII and before the tail attaches.

J Mol Biol, 2003 Oct 31, 333(4), 781 - 815
Evolution and classification of P-loop kinases and related proteins; Leipe DD et al.; Sequences and structures of all P-loop-fold proteins were compared with the aim of reconstructing the principal events in the evolution of P-loop-containing kinases . It is shown that kinases and some related proteins comprise a monophyletic assemblage within the P-loop NTPase fold . An evolutionary classification of these proteins was developed using standard phylogenetic methods, analysis of shared sequence and structural signatures, and similarity-based clustering . This analysis resulted in the identification of approximately 40 distinct protein families within the P-loop kinase class . Most of these enzymes phosphorylate nucleosides and nucleotides, as well as sugars, coenzyme precursors, adenosine 5'-phosphosulfate and polynucleotides . In addition, the class includes sulfotransferases, amide bond ligases, pyrimidine and dihydrofolate reductases, and several other families of enzymes that have acquired new catalytic capabilities distinct from the ancestral kinase reaction . Our reconstruction of the early history of the P-loop NTPase fold includes the initial split into the common ancestor of the kinase and the GTPase classes, and the common ancestor of ATPases . This was followed by the divergence of the kinases, which primarily phosphorylated nucleoside monophosphates (NMP), but could have had broader specificity . We provide evidence for the presence of at least two to four distinct P-loop kinases, including distinct forms specific for dNMP and rNMP, and related enzymes in the last universal common ancestor of all extant life forms . Subsequent evolution of kinases seems to have been dominated by the emergence of new bacterial and, to a lesser extent, archaeal families . Some of these enzymes retained their kinase activity but evolved new substrate specificities, whereas others acquired new activities, such as sulfate transfer and reduction . Eukaryotes appear to have acquired most of their kinases via horizontal gene transfer from Bacteria, partly from the mitochondrial and chloroplast endosymbionts and partly at later stages of evolution . A distinct superfamily of kinases, which we designated DxTN after its sequence signature, appears to have evolved in selfish replicons, such as bacteriophages, and was subsequently widely recruited by eukaryotes for multiple functions related to nucleic acid processing and general metabolism . In the course of this analysis, several previously undetected groups of predicted kinases were identified, including widespread archaeo-eukaryotic and archaeal families . The results could serve as a framework for systematic experimental characterization of new biochemical and biological functions of kinases.

J Bacteriol, 2003 Nov, 185(21), 6490 - 2
Host range of chlamydiaphages phiCPAR39 and Chp3; Everson JS et al.; The host range of phiCPAR39 is limited to four Chlamydophila species: C . abortus, C . caviae, C . pecorum, and C . pneumoniae . Chp3 (a newly discovered bacteriophage isolated from C . pecorum) shares three of these hosts (C . abortus, C . caviae, and C . pecorum) but can additionally infect Chlamydophila felis . The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species . Binding studies also show that phiCPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature.

J Bacteriol, 2003 Nov, 185(21), 6463 - 6
Nucleotide sequence of the integration site of the temperate bacteriophage 6220, which carries the Shiga toxin gene stx(1ox3); Koch C et al.; The integration site, attR, of the Shiga toxin-encoding phage 6220 (stx(1ox3)) has been determined . The phage integrates into the chromosome of its Escherichia coli host strain, CB6220, within a gene that is homologous to gene Z2577 and encodes an oxidoreductase . This new integration site was found in different Stx1ox3-producing enterohemorrhagic E . coli strains, which were analyzed by PCR.

J Bacteriol, 2003 Nov, 185(21), 6409 - 14
Analysis of specific binding involved in genomic packaging of the double-stranded-RNA bacteriophage phi6; Qiao X et al.; The genomes of bacteriophage phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus-strand transcripts of the segmented double-stranded-RNA genomes into preformed polyhedral structures called procapsids or inner cores . The packaging requires the hydrolysis of nucleoside triphosphates and takes place in the order segment S-segment M, segment L . Packaging is dependent upon unique sequences of about 200 nucleotides near the 5' ends of plus-strand transcripts of the three genomic segments . It appears that P1 is the determinant of the RNA binding sites . Directed mutation of P1 was used to locate regions that are important for genomic packaging . Specific binding of RNA to the exterior of the procapsid was dependent upon ATP, and a region that showed a high level of cross-linking to phage-specific RNA was located . Antibodies to peptide sequences were prepared, and their abilities to bind to the exterior of procapsids were determined . Sites sensitive to trypsin and to factor Xa were determined as well.

J Bacteriol, 2003 Nov, 185(21), 6325 - 30
Characterization of the integrase gene and attachment site for the Myxococcus xanthus bacteriophage Mx9; Julien B; Bacteriophage Mx9 is a temperate phage that infects Myxococcus xanthus . It lysogenizes the bacteria by integrating into the bacterial chromosome by site-specific recombination at one of two sites, attB1 or attB2 . Integration at attB1 results in deletion of DNA between the two attB sites . The attB2 site lies within the 5' region of the M . xanthus tRNA(Gly) gene . Mx9 integration requires a single protein, Int . Analysis of integration revealed that the phage attachment site (attP) is contained in the int gene and that upon integration, the 3' end of the int gene is altered . Plasmids containing fusions of the pilA or mgl promoter to lacZ integrated at either Mx9 attB site have higher levels of transcription than the same fusions integrated at the Mx8 attB site.

J Biol Chem, 2003 Dec 26, 278(52), 52333 - 9 Epub 2003 Oct 14.
A context-dependent ClpX recognition determinant located at the C terminus of phage Mu repressor; Defenbaugh DA et al.; The bacteriophage Mu immunity repressor is a conformationally sensitive sensor that can be interconverted between forms resistant to and sensitive to degradation by ClpXP protease . Protease-sensitive repressor molecules with an altered C-terminal sequence promote rapid degradation of the wild-type repressor by inducing its C-terminal end to become exposed . Here we determined that the last 5 C-terminal residues (CTD5) of the wild-type repressor contain the motif required for recognition by the ClpX molecular chaperone, a motif that is strongly dependent upon the context in which it is presented . Although attachment of the 11-residue ssrA degradation tag to the C terminus of green fluorescent protein (GFP) promoted its rapid degradation by ClpXP, attachment of 5-27 C-terminal residues of the repressor failed to promote degradation . Disordered peptides derived from 41 and 35 C-terminal residues of CcdA (CcdA41) and thioredoxin (TrxA35), respectively, activated CTD5 when placed as linkers between GFP and repressor C-terminal sequences . However, when the entire thioredoxin sequence was included as a linker to promote an ordered configuration of the TrxA35 peptide, the resulting substrate was not degraded . In addition, a hybrid tag, in which CTD5 replaced the 3-residue recognition motif of the ssrA tag, was inactive when attached directly to GFP but active when attached through the CcdA41 peptide . Thus, CTD5 is sufficient to act as a recognition motif but has requirements for its presentation not shared by the ssrA tag . We suggest that activation of CTD5 may require presentation on a disordered or flexible domain that confers ligand flexibility.

Curr Opin Pharmacol, 2003 Oct, 3(5), 557 - 62
Biosensor profiling of molecular interactions in pharmacology; Cooper MA; Techniques employed to profile the pharmacological properties of a molecule in vitro normally require some type of radio-, enzymatic- or fluorescent-labeling of the ligand and/or the receptor . In contrast, biosensor techniques do not require labeling, and they allow virtually any complex to be screened with minimal assay development . Scientists in both academia and industry are now using biosensors in areas that encompass almost all sectors of drug discovery, diagnostics and the life sciences . Assays have been developed for the analysis of small molecules, proteins, oligonucleotides, bacteriophage, viruses, bacteria and cells . In addition, novel biosensor applications are being developed for the predictive profiling of key pharmacokinetic parameters of a molecule (adsorption, distribution, metabolism, excretion and toxicity).

J Theor Biol, 2003 Nov 7, 225(1), 45 - 57
Risk management in biological evolution; Wagner A; I present a framework to study the evolution of traits that allow an organism to survive life-threatening but rare risks . Specifically, I am concerned with risks so rare that any one individual in a population may not experience the risk-causing event in its lifetime . A theory of rare risk management is virtually absent in evolutionary biology, although it is well developed in economics . This is surprising because of the great influence economics had on evolutionary biology, and because biology is full of examples for evolved risk management traits . They include the ability of bacteria to sporulate, of pathogens to survive antibiotic treatment, of temperate bacteriophages to enter a lytic life cycle, as well as traits that allow higher organisms to survive rare environmental disasters, such as sporadic wildfires and irregular flooding . I make predictions about the sustenance of risk management traits under two scenarios, one where the catastrophic events cause individual deaths, and another one where catastrophic events cause population extinction . A well-developed theory of risk management will not only predict the distribution of risk management traits, but may also serve other purposes, such as to reconstruct the spectrum of environments that an organism encountered in its evolutionary history from the record stored in its genome's memory.

Biosens Bioelectron, 2003 Oct 30, 19(1), 59 - 66
Nucleic acid purification using microfabricated silicon structures; Cady NC et al.; A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor . This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm . DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer . Positive pressure was used to move solutions through the device, removing the need for centrifugation steps . The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer . Additionally, the device removed approximately 87% of the protein from a cell lysate . Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor . The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.

Gene, 2003 Oct 2, 315, 145 - 52
Gene cloning, purification, and stoichiometry quantification of phi29 anti-receptor gp12 with potential use as special ligand for gene delivery; Guo S et al.; Bacterial virus phi29 is the most efficient in vitro DNA packaging system, with which up to 90% of the added DNA can be packaged into purified recombinant procapsid in vitro . The findings that phi29 virions can be assembled with the exclusive use of cloned gene products have bred a thought that phi29 has a potential to be a gene delivery vector since it is a nonpathogenic virus . gp12 of bacterial virus phi29 has been reported to be the anti-receptor that is responsible for binding the virus particle to the host cell . We cloned the gene coding gp12, overexpressed it in Escherichia coli, and purified the gene product to study the properties and functions of gp12 in virus assembly . According to SDS PloyAcrylamide Gel Electrophoresis (SDS-PAGE) analysis and N-terminal sequencing, recombinant gp12 isolated from E . coli had a molecular mass of 80 kDa, and 24 amino acids at N-terminal were cleaved after expression . The purified recombinant gp12 was incorporated into phi29 particles and converted the gp12-lacking assembly intermediates of phi29 into infectious virions in vitro . This purified protein gp12 was able to compete with infectious phi29 virions for binding to the host cell, thus inhibiting the infection by phi29 . Scanning Transmission Electron Microscopy (STEM) analysis and sedimentation studies revealed that recombinant gp12 products were assembled into biologically active dimers . Analysis of the dose-response curve showed that 12 dimeric gp12 complexes were assembled onto viral particles and that each virion contained 24 copies of gp12 molecules . The results provide a basis for future research into bacteriophage-host interaction by modifying the anti-receptor protein . The ultimate goal is to re-target the bacteriophage to new host cells for the purpose of gene delivery.

Gene, 2003 Oct 2, 315, 63 - 9
Mini-lambda: a tractable system for chromosome and BAC engineering; Court DL et al.; The bacteriophage lambda (lambda) recombination system Red has been used for engineering large DNA fragments cloned into P1 and bacterial artificial chromosomes (BAC or PAC) vectors . So far, this recombination system has been utilized by transferring the BAC or PAC clones into bacterial cells that harbor a defective lambda prophage . Here we describe the generation of a mini-lambda DNA that can provide the Red recombination functions and can be easily introduced by electroporation into any E . coli strain, including the DH10B-carrying BACs or PACs . The mini-lambda DNA integrates into the bacterial chromosome as a defective prophage . In addition, since it retains attachment sites, it can be excised out to cure the cells of the phage DNA . We describe here the use of the mini-lambda recombination system for BAC modification by introducing a selectable marker into the vector sequence of a BAC clone . In addition, using the mini-lambda, we create a single missense mutation in the human BRCA2 gene cloned in a BAC without the use of any selectable marker . The ability to generate recombinants very efficiently demonstrates the usefulness of the mini-lambda as a very simple mobile system for in vivo genome engineering by homologous recombination, a process named recombineering.

Virology, 2003 Sep 30, 314(2), 706 - 15
Temperature requirements for initiation of RNA-dependent RNA polymerization; Yang H et al.; To continue the molecular characterization of RNA-dependent RNA polymerases of dsRNA bacteriophages (Cystoviridae), we purified and biochemically characterized the wild-type (wt) and a temperature-sensitive (ts) point mutant of the polymerase subunit (Pol) from bacteriophage phi12 . Interestingly, initiation by both wt and the ts phi12 Pol was notably more sensitive to increased temperatures than the elongation step, the absolute value of the nonpermissive temperature being lower for the ts enzyme . Experiments with the Pol subunit of related cystovirus phi6 revealed a similar differential sensitivity of the initiation and elongation steps . This is consistent with the previous result showing that de novo initiation by RdRp from dengue virus is inhibited at elevated temperatures, whereas the elongation phase is relatively thermostable . Overall, these data suggest that de novo RNA-dependent RNA synthesis in many viral systems includes a specialized thermolabile state of the RdRp initiation complex.

FEMS Microbiol Lett, 2003 Sep 26, 226(2), 221 - 7
Different contributions of the outer and inner R-core residues of lipopolysaccharide to the recognition by spike H and G proteins of bacteriophage phiX174; Inagaki M et al.; The binding of spike H and G proteins of bacteriophage phiX174 with lipopolysaccharides (LPSs) were evaluated by a competitive enzyme-linked plate assay using the biotin-labeled LPS of Escherichia coli C, one of a host strain, and the non-labeled LPSs having different R-core polysaccharide lengths . H protein promptly decreased its affinity when some saccharide residues were truncated from the outer R-core . However, G protein showed significant affinity to the LPSs lacking all the residues of the outer R-core and some of the inner R-core . Thus, G protein rather than H protein well recognized the residues of the inner R-core of LPS.

Biotechnol Adv, 1994, 12(4), 625 - 33
The impact of biotechnology on the dairy industry; Coffey AG et al.; The review focusses on the use of genetic techniques to manipulate bacteria that are important to the dairy industry . Both classical and molecular approaches have been used to improve strains involved in yoghurt and cheese production . Examples are provided of methods for; increasing efficiency of substrate conversion, regulating the production of flavour enhancing metabolites, and developing starter cultures resistant to bacteriophage and bacteriocin attack . The possible applications of these systems are discussed.

Biotechnol Adv, 2001 Feb 1, 19(1), 1 - 33
Biotechnological applications of phage and cell display; Benhar I; In recent years, the use of surface-display vectors for displaying polypeptides on the surface of bacteriophage and bacteria, combined with in vitro selection technologies, has transformed the way in which we generate and manipulate ligands, such as enzymes, antibodies and peptides . Phage display is based on expressing recombinant proteins or peptides fused to a phage coat protein . Bacterial display is based on expressing recombinant proteins fused to sorting signals that direct their incorporation on the cell surface . In both systems, the genetic information encoding for the displayed molecule is physically linked to its product via the displaying particle . Using these two complementary technologies, we are now able to design repertoires of ligands from scratch and use the power of affinity selection to select those ligands having the desired (biological) properties from a large excess of irrelevant ones . With phage display, tailor-made proteins (fused peptides, antibodies, enzymes, DNA-binding proteins) may be synthesized and selected to acquire the desired catalytic properties or affinity of binding and specificity for in vitro and in vivo diagnosis, for immunotherapy of human disease or for biocatalysis . Bacterial surface display has found a range of applications in the expression of various antigenic determinants, heterologous enzymes, single-chain antibodies, and combinatorial peptide libraries . This review explains the basis of phage and bacterial surface display and discusses the contributions made by these two leading technologies to biotechnological applications . This review focuses mainly on three areas where phage and cell display have had the greatest impact, namely, antibody engineering, enzyme technology and vaccine development.

J Biol Chem, 2004 Jan 9, 279(2), 1343 - 50 Epub 2003 Oct 06.
Enzymatic mechanism of RNA translocation in double-stranded RNA bacteriophages; Lisal J et al.; Many complex viruses acquire their genome by active packaging into a viral precursor particle called a procapsid . Packaging is performed by a viral portal complex, which couples ATP hydrolysis to translocation of nucleic acid into the procapsid . The packaging process has been studied for a variety of viruses, but the mechanism of the associated ATPase remains elusive . In this study, the mechanism of RNA translocation in double-stranded RNA bacteriophages is characterized using rapid kinetic analyses . The portal complex of bacteriophage 8 is a hexamer of protein P4, which exhibits nucleotide triphosphatase activity . The kinetics of ATP binding reveals a two-step process: an initial, fast, second-order association, followed by a slower, first-order phase . The slower phase exhibits a high activation energy and has been assigned to a conformational change . ATP binding becomes cooperative in the presence of RNA . Steady-state kinetics of ATP hydrolysis, which proceeds only in the presence of RNA, also exhibits cooperativity . On the other hand, ADP release is fast and RNA-independent . The steady-state rate of hydrolysis increases with the length of the RNA substrate indicating processive translocation . Raman spectroscopy reveals that RNA binds to P4 via the phosphate backbone . The ATP-induced conformational change affects the backbone of the bound RNA but leaves the protein secondary structure unchanged . This is consistent with a model in which cooperativity is induced by an RNA link between subunits of the hexamers and translocation is effected by an axial movement of the subunits relative to one another upon ATP binding.

Transplant Proc, 2003 Sep, 35(6), 2372 - 3
New insights into the possible role of bacteriophages in transplantation; Gorski A et al.; Due to the increasing prevalence of drug-resistant bacterial infections in the "post-antibiotic era," bacteriophages (bacterial viruses, BP) may be useful to administer to transplant recipients without exposing them to an increased risk of rejection, which occurs consequent to some viral infections . Herein we present evidence that at least some coliphages (T4) do not pose such risk . Interestingly, they may produce immunosuppressive effects extending transplant survival . Our data suggest that BP may be used in clinical transplantation to treat drug-resistant bacterial infections and perhaps as an adjunct to immunosuppressive therapy.

Res Microbiol, 2003 Oct, 154(8), 547 - 52
A role for bacteriophage T4 rI gene function in the control of phage development during pseudolysogeny and in slowly growing host cells; Los M et al.; Although most studies on bacteriophages have been performed under laboratory conditions that are optimal for host cell growth, in nature, bacteria and bacteriophages coexist in different habitats . Here, by using different growth rates in carbon-limited chemostats, we investigated the development of phage T4 in its host Escherichia coli . Our results strongly suggest that T4 can form pseudolysogens not only when bacterial growth is completely inhibited, but also in growing host cells . The rI gene, previously known to be indispensable for lysis inhibition, seems to play an important role in optimization of phage development in slowly growing cells as well as during establishment and maintenance of pseudolysogeny.

Annu Rev Microbiol, 2003, 57, 301 - 22
Bacteriophage-induced modifications of host RNA polymerase; Nechaev S et al.; Bacteriophages have developed an impressive array of ingenious mechanisms to modify bacterial host RNA polymerase to make it serve viral needs . In this review we summarize the current knowledge about two types of host RNA polymerase modifications induced by double-stranded DNA phages: covalent modifications and modifications through RNA polymerase-binding proteins . We interpret the biochemical and genetic data within the framework of a structure-function model of bacterial RNA polymerase and viral biology.

J Cell Biochem, 2003 Oct 15, 90(3), 509 - 17
Combinatorial discovery of tumor targeting peptides using phage display; Landon LA et al.; Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents . Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available . Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting . Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage . In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro . Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems . To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal . Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals . In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs . In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells . In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences .

Chem Biol, 2003 Sep, 10(9), 847 - 58
Synthetic compound libraries displayed on the surface of encoded bacteriophage; Woiwode TF et al.; We describe a technology for attaching libraries of synthetic compounds to coat proteins of bacteriophage particles such that the identity of the chemical structure is encoded in the genome of the phage, analogous to peptides displayed on phage surfaces by conventional phage-display techniques . This format allows a library of synthetic compounds to be screened very efficiently as a single pool . Encoded phage serve as extremely robust reporters of the presence of each compound, providing exquisite sensitivity for identification of active compounds engaged in complex biological processes such as receptor-mediated endocytosis and transcytosis . To evaluate this approach, we constructed a library of 980 analogs of folic acid displayed on T7 phage, and demonstrated rapid identification of compounds that bind to folate receptor and direct endocytosis of associated phage particles into cells that express the targeted receptor.

Chem Biol, 2003 Sep, 10(9), 783 - 4
Bacteriophage that display small molecules; Fellouse F et al.; Technology has been developed to display small molecules on phage particles . This innovation enables the generation of libraries of phage-tagged compounds with novel properties that are well suited for in vivo assays.

Biol Cell, 2003 Sep, 95(6), 393 - 8
Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage; Messaoudi C et al.; Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance . However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis . Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage . In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio . Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.

Virology, 2003 Sep 15, 314(1), 1 - 8
Cleavage leads to expansion of bacteriophage P4 procapsids in vitro; Wang S et al.; Proteolytic cleavage of the structural proteins is an important part of the maturation process for most bacteriophages and other viruses . In the double-stranded DNA bacteriophages this cleavage is associated with DNA packaging, capsid expansion, and scaffold removal . To understand the role of protein cleavage in the expansion of bacteriophages P2 and P4, we have experimentally cleaved P4 procapsids produced by overexpression of the capsid and scaffolding proteins . The cleavage leads to particle expansion and scaffold removal in vitro . The resulting expanded capsid has a thin-shelled structure similar, but not identical, to that of mature virions.

J Mol Biol, 2003 Oct 10, 333(1), 59 - 73
Metal ion binding in the active site of the junction-resolving enzyme T7 endonuclease I in the presence and in the absence of DNA; Freeman AD et al.; Endonuclease I of bacteriophage T7 is a DNA junction-resolving enzyme . We have previously used crystallography to demonstrate the binding of two manganese ions into the active site that is formed by three carboxylate (Glu 20, Asp 55 and Glu 65) and a lysine residue (Lys 67) . Endonuclease I is active in the presence of magnesium, manganese, iron (II) and cobalt (II) ions, weakly active in the presence of nickel, copper (II) and zinc ions, and completely inactive in the presence of calcium ions . However, using calorimetry, we have observed the binding of two calcium ions to the free enzyme in a manner very similar to the binding of manganese ions . In the presence of iron (II) ions, we have obtained a cleavage of the continuous strands of a junction bound by endonuclease I, at sites close to (but not identical with) enzyme-induced hydrolysis . The results suggest that this arises from attack by locally generated hydroxyl radicals, arising from iron (II) ions bound into the active site . This therefore provides an indirect way of examining metal ion binding in the enzyme-junction complex . Ion binding in free protein (by calorimetry) and the enzyme-junction complex (iron-induced cleavage) have been studied in series of active-site mutants . Both confirm the importance of the three carboxylate ligands, and the lack of a requirement for Lys67 for the ion binding . Calorimetry points to particularly critical role of Asp55, as mutation completely abolishes all binding of both manganese and calcium ions.

Curr Opin Mol Ther, 2003 Aug, 5(4), 376 - 82
Site-specific genomic strategies for gene therapy; Portlock JL et al.; Like any disease treatment, gene therapy should be safe and efficacious . Safety can be addressed by directly correcting the defective gene itself or by ensuring that genomic integration of a transgene is site-specific . Unfortunately, it has proven difficult to achieve this level of safety without a concomitant loss in efficiency of gene correction or insertion . In this review, recent research attempts to achieve efficient site-specific gene therapy, including strategies using targeted gene conversion, adeno-associated virus vectors and site-specific bacteriophage recombinases are discussed . We believe that these approaches hold promise for site-specific, safe and efficient gene therapy.

Theor Appl Genet, 2003 Nov, 107(7), 1157 - 68 Epub 2003 Sep 25.
Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants; Zhang W et al.; After the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants . There are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays . We have tested the Cre/ lox system from bacteriophage P1 for its ability to precisely excise stably integrated marker genes from chromosomes in transgenic maize plants . Two strategies, crossing and autoexcision, have been tested and demonstrated . In the crossing strategy, plants expressing the Cre recombinase are crossed with plants bearing a transgene construct in which the selectable marker gene is flanked by directly repeated lox sites . Unlike previous reports in which incomplete somatic and germline excision were common, in our experiments complete somatic and germline marker gene excision occurred in the F(1) plants from most crosses with multiple independent Cre and lox lines . In the autoexcision strategy, the cre gene, under the control of a heat shock-inducible promoter, is excised along with the nptII marker gene . Our results show that a transient heat shock treatment of primary transgenic callus is sufficient for inducing cre and excising the cre and nptII genes . Genetic segregation and molecular analysis confirmed that marker gene removal is precise, complete and stable . The autoexcision strategy provides a way of removing the selectable marker gene from callus or other tissues such as embryos and kernels.

Biophys J, 2003 Oct, 85(4), 2606 - 18
Reversible and fast association equilibria of a molecular chaperone, gp57A, of bacteriophage T4; Ali SA et al.; The association of a molecular chaperone, gp57A, of bacteriophage T4, which facilitates formation of the long and short tail fibers, was investigated by analytical ultracentrifugation, differential scanning microcalorimetry, and stopped-flow circular dichroism (CD) to establish the association scheme of the protein . Gp57A is an oligomeric alpha-helix protein with 79 amino acids . Analysis of the sedimentation velocity data by direct boundary modeling with Lamm equation solutions together with a more detailed boundary analysis incorporating association schemes led us to conclude that at least three oligomeric species of gp57A are in reversible and fast association equilibria and that a 3(mer)-6(mer)-12(mer) model described the data best . On the other hand, differential scanning microcalorimetry revealed a highly reversible two-step transition of dissociation/denaturation, both of which accompanied decrease in CD at 222 nm . The melting curve analysis revealed that it is consistent with a 6(mer)-3(mer)-1(mer) model . The refolding/association kinetics of gp57A measured by stopped-flow CD was consistent with the interpretation that the bimolecular reaction from trimer to hexamer was preceded by a fast alpha-helix formation in the dead-time . Trimer or hexamer is likely the functional oligomeric state of gp57A.

Mol Microbiol, 2003 Oct, 50(1), 333 - 47
A biochemical analysis of the interaction of DNA gyrase with the bacteriophage Mu, pSC101 and pBR322 strong gyrase sites: the role of DNA sequence in modulating gyrase supercoiling and biological activity; Oram M et al.; Replication of bacteriophage Mu DNA, a process requiring efficient synapsis of the prophage ends, takes place within the confines of the Escherichia coli nucleoid . Critical to ensuring rapid synapsis is the function of the SGS, a strong gyrase site, located at the centre of the Mu genome . Replacement of the SGS by the strong gyrase sites from pSC101 or pBR322 fails to support efficient prophage replication . To probe the unique SGS properties we undertook a biochemical analysis of the interaction of DNA gyrase with the Mu SGS, pSC101 and pBR322 sites . In binding and cleavage assays the order of efficacy was pSC101 > Mu SGS >> pBR322 . However, in supercoiling assays the Mu SGS (cloned into pUC19) exhibited a strong enhancement of gyrase-catalysed supercoiling over pUC19 alone; the pSC101 site showed none and the pBR322 site gave a moderate improvement . Most striking was the Mu SGS-dependent increase in processivity of the gyrase reaction . This highly processive supercoiling coupled with efficient binding may account for the unique biological properties of the SGS . The results emphasize the importance of the DNA substrate as an active component in modulating the gyrase supercoiling reaction, and in determining the biological roles of specialized gyrase sites.

Mol Microbiol, 2003 Oct, 50(1), 129 - 43
Bacteriophage T4-encoded Stp can be replaced as activator of anticodon nuclease by a normal host cell metabolite; Amitsur M et al.; The bacterial tRNALys-specific anticodon nuclease is known as a phage T4 exclusion system . In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI . Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme . Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides . Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate . Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC . The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP . The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex . Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC . The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection.

Mol Microbiol, 2003 Oct, 50(1), 89 - 99
The molecular basis of co-operative DNA binding between lambda integrase and excisionase; Swalla BM et al.; Higher-order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA . Formation of such complexes, known as intasomes, is required for site-specific recombination catalysed by bacteriophage Lambda Integrase protein (Int) . Int-catalysed recombination is regulated by a second bacteriophage-encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration . To exert its effect, Xis binds co-operatively with Int, thereby inducing and stabilizing a DNA bend that alters the intasome structures formed during recombination . A rare int mutant, int 2268 ts, was reported (Enquist, L.W . and Weisberg, R.A . (1984) Mol Gen Genet 195: 62-69) to be more defective for excision than integration . Here, we have determined that this mutant Int protein contains an E47K substitution, and that the resultant excision-specific defect is due, at least in part, to destabilized interactions between Int and Xis . Analysis of several engineered substitutions at Int position 47 showed that a negatively charged residue is required for co-operative DNA binding between Int and Xis, and suggest that the Int-E47 residue may contact Xis directly . Substitutions at Int position 47 also affect co-operative binding among Int proteins at arm-type DNA sites, and thereby reduce the efficiency of both integration and excision . Collectively, these results suggest that a single surface of the Int amino-terminal domain mediates two alternate types of co-operative binding interactions.

Genetics, 2003 Sep, 165(1), 11 - 21
Genetics of cosQ, the DNA-packaging termination site of phage lambda: local suppressors and methylation effects; Wieczorek DJ et al.; The cos site of the bacteriophage lambda chromosome contains the sites required for DNA processing and packaging during virion assembly . cos is composed of three subsites, cosQ, cosN, and cosB . cosQ is required for the termination of chromosome packaging . Previous studies have shown cosQ mutations to be suppressed in three ways: by a local suppressor within cosQ; by an increase in the length of the lambda chromosome; and by missense mutations affecting the prohead's portal protein, gpB . In the first study reported here, revertants of a set of cosQ mutants were screened for suppressors, and cis-acting suppressors of cosQ mutations were studied; these included second-site cosQ point mutations, base-pair insertions within cosQ, and an additional genome-lengthening suppressor . The 7-bp-long cosQ, with the sequence 5'-GGGTCCT-3', coincides exactly with the recognition site for the EcoO109I restriction/methylation system, which has the consensus sequence 5'-PuGGNCCPy-3' . In a second study, EcoO109I methylation was found to strongly interfere with the residual cosQ function of leaky cosQ mutants . cis-acting suppressors that overcome methylation-associated defects, including a methylation-dependent suppressor, were also isolated . Models of cosQ suppression are presented.

Nat Struct Biol, 2003 Oct, 10(10), 812 - 9 Epub 2003 Sep 21.
Context and conformation dictate function of a transcription antitermination switch; Xia T et al.; In bacteriophage l, transcription elongation is regulated by the N protein, which binds a nascent mRNA hairpin (termed boxB) and enables RNA polymerase to read through distal terminators . We have examined the structure, energetics and in vivo function of a number of N-boxB complexes derived from in vitro protein selection . Trp18 fully stacks on the RNA loop in the wild-type structure, and can become partially or completely unstacked when the sequence context is changed three or four residues away, resulting in a recognition interface in which the best binding residues depend on the sequence context . Notably, in vivo antitermination activity correlates with the presence of a stacked aromatic residue at position 18, but not with N-boxB binding affinity . Our work demonstrates that RNA polymerase responds to subtle conformational changes in cis-acting regulatory complexes and that approximation of components is not sufficient to generate a fully functional transcription switch.

J Biol Chem, 2003 Dec 12, 278(50), 49839 - 49 Epub 2003 Sep 18.
Dissociative properties of the proteins within the bacteriophage T4 replisome; Trakselis MA et al.; DNA replication is a highly processive and efficient process that involves the coordination of at least eight proteins to form the replisome in bacteriophage T4 . Replication of DNA occurs in the 5' to 3' direction resulting in continuous replication on the leading strand and discontinuous replication on the lagging strand . A key question is how a continuous and discontinuous replication process is coordinated . One solution is to avoid having the completion of one Okazaki fragment to signal the start of the next but instead to have a key step such as priming proceed in parallel to lagging strand replication . Such a mechanism requires protein elements of the replisome to readily dissociate during the replication process . Protein trapping experiments were performed to test for dissociation of the clamp loader and primase from an active replisome in vitro whose template was both a small synthetic DNA minicircle and a larger DNA substrate . The primase, clamp, and clamp loader are found to dissociate from the replisome and are continuously recruited from solution . The effect of varying protein concentrations (dilution) on the size of Okazaki fragments supported the protein trapping results . These findings are in accord with previous results for the accessory proteins but, importantly now, identify the primase as dissociating from an active replisome . The recruitment of the primase from solution during DNA synthesis has also been found for Escherichia coli but not bacteriophage T7 . The implications of these results for RNA priming and extension during the repetitive synthesis of Okazaki fragments are discussed.

J Biol Chem, 2003 Dec 12, 278(50), 49828 - 38 Epub 2003 Sep 18.
The application of a minicircle substrate in the study of the coordinated T4 DNA replication; Yang J et al.; A reconstituted in vitro bacteriophage T4 DNA replication system was studied on a synthetic 70-mer minicircle substrate . This substrate was designed so that dGMP and dCMP were exclusively incorporated into the leading and the lagging strand, respectively . This design allows the simultaneous and independent measurement of the leading and lagging strand synthesis . In this paper, we report our results on the characterization of the 70-mer minicircle substrate . We show here that the minicircle substrate supports coordinated leading and lagging strand synthesis under the experimental conditions employed . The rate of the leading strand fork movement was at an average of approximately 150 nucleotides/s . This rate decreased to less than 30 nucleotides/s when the helicase was omitted from the reaction . These results suggest that both the holoenzyme and the primosome can be simultaneously assembled onto the minicircle substrate . The lagging strand synthesized on this substrate is of an average of 1.5 kb, and the length of the Okazaki fragments increased with decreasing {rNTPs} . The proper response of the Okazaki fragment size toward the change of the priming signal further indicates a functional replisome assembled on the minicircle template . The effects of various protein components on the leading and lagging strand synthesis were also studied . The collective results indicate that coordinated strand synthesis only takes place within certain protein concentration ranges . The optimal protein levels of the proteins that constitute the T4 replisome generally bracket the concentrations of the same proteins in vivo . Omission of the primase has little effect on the rate of dNMP incorporation or the rate of the fork movement on the leading strand within the first 30 s of the reaction . This inhibition only becomes significant at later times of the reaction and may be associated with the accumulation of single-stranded DNA leading to the collapse of active replisomes.

Clin Immunol, 2003 Sep, 108(3), 199 - 210
Improving selection of alphaIIbbeta3-binding phage antibodies with increased reactivity derived from immunized donors; Jacobin MJ et al.; Although many studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have demonstrated the frequent development of Abs directed against the alphaIIbbeta3 integrin, little is known about the induced anti-alphaIIbbeta3 autoantibodies at the molecular level . Phage display is a powerful technology for selecting and engineering mAbs expressed on the surface of filamentous bacteriophage . Combinatorial libraries of single-chain IgG were constructed from splenocytes from two patients with AITP and one patient with GT . In a previous study, activated platelets or alphaIIbbeta3-expressing CHO cells selection was performed to isolate human IgG anti-alphaIIbbeta3 binding fragments using combinatorial libraries created from the B cells of a GT and an AITP patient . However, we have experienced practical problems such as enrichment of truncated antibodies during selection . We decided to test prolonged treatments with elution agents after screening on the purified form of the alphaIIbbeta3 integrin activated with the RGD peptide . We obtained a higher percentage of clones with full-size antibody fragments as well as an enrichment of more specific alphaIIbbeta3-binding phage-Abs . Some of them, recognizing the activated form of the integrin, would be interesting to further study as potential diagnostic or therapeutic agents in acute coronary syndromes . Sequencing of selected phage-Abs revealed that they used different VH and VL genes with, for the majority of them, a high level of extensive hypermutations in the complementarity determining regions, indicating the diversity of the antigen-driven immune response that occurred in GT and AITP patients.

Ground Water, 2003 Sep-Oct, 41(5), 701 - 8
The effect of critical pH on virus fate and transport in saturated porous medium; Guan H et al.; Several viral transport experiments were conducted in a model aquifer 1 m long, using bacteriophages MS2 and phiX174 at various pH (4.6 to 8.3) conditions, to increase our understanding of virus behavior in ground water . The results indicate the existence of a critical pH at which the virus behavior changes abruptly . This is supported by data from field and batch experiments . The critical pH is determined to be 0.5 unit below the highest isoelectric point of the virus and porous medium . When water pH is below the critical pH, the virus has an opposite charge to at least one component of the porous medium, and is almost completely and irreversibly removed from the water . This suggests that electrostatic attraction at a subcritical water pH condition is an important factor controlling virus attenuation in ground water . The concept of critical pH can assist in the design of geologic barriers for preventing viral contamination in ground water.

Adv Protein Chem, 2003, 64, 301 - 23
Molecular mechanisms in bacteriophage T7 procapsid assembly, maturation, and DNA containment; Cerritelli ME et al.; Bacteriophage T7 is a double-stranded DNA bacteriophage that has attracted particular interest in studies of gene expression and regulation and of morphogenesis, as well as in biotechnological applications of expression vectors and phage display . We report here studies of T7 capsid assembly by cryoelectron microscopy and image analysis . T7 follows the canonical pathway of first forming a procapsid that converts into the mature capsid, but with some novel variations . The procapsid is a round particle with an icosahedral triangulation number of 7 levo, composed of regular pentamers and elongated hexamers . A singular vertex in the procapsid is occupied by the connector/portal protein, which forms 12-fold and 13-fold rings when overexpressed, of which the 12-mer appears to be the assembly-competent form . This vertex is the site of two symmetry mismatches: between the connector and the surrounding five gp 10 hexamers; and between the connector and the 8-fold cylindrical core mounted on its inner surface . The scaffolding protein, gp9, which is required for assembly, forms nubbin-like protrusions underlying the hexamers but not the pentamers, with no contacts between neighboring gp9 monomers . We propose that gp9 facilitates assembly by binding to gp10 hexamers, locking them into a morphogenically correct conformation . gp9 is expelled as the procapsid matures into the larger, thinner walled, polyhedral capsid . Several lines of evidence implicate the connector vertex as the site at which the maturation transformation is initiated: in vivo, maturation appears to be triggered by DNA packaging whereby the signal may involve interaction of the connector with DNA . In the mature T7 head, the DNA is organized as a tightly wound coaxial spool, with the DNA coiled around the core in at least four and perhaps as many as six concentric shells.

J Biol Chem, 2003 Dec 5, 278(49), 48627 - 32 Epub 2003 Sep 15.
TWINKLE Has 5' -> 3' DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein; Korhonen JA et al.; Mutations in TWINKLE cause autosomal dominant progressive external ophthalmoplegia, a human disorder associated with multiple deletions in the mitochondrial DNA . TWINKLE displays primary sequence similarity to the phage T7 gene 4 primase-helicase, but no specific enzyme activity has been assigned to the protein . We have purified recombinant TWINKLE to near homogeneity and demonstrate here that TWINKLE is a DNA helicase with 5' to 3' directionality and distinct substrate requirements . The protein needs a stretch of 10 nucleotides of single-stranded DNA on the 5'-side of the duplex to unwind duplex DNA . In addition, helicase activity is not observed unless a short single-stranded 3'-tail is present . The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5'-triphosphate, with UTP being the optimal substrate . DNA unwinding by TWINKLE is specifically stimulated by the mitochondrial single-stranded DNA-binding protein . Our enzymatic characterization strongly supports the notion that TWINKLE is the helicase at the mitochondrial DNA replication fork and provides evidence for a close relationship of the DNA replication machinery in bacteriophages and mammalian mitochondria.

Genes Dev, 2003 Sep 15, 17(18), 2334 - 45
Phage N4 RNA polymerase II recruitment to DNA by a single-stranded DNA-binding protein; Carter RH et al.; Transcription of bacteriophage N4 middle genes is carried out by a phage-coded, heterodimeric RNA polymerase (N4 RNAPII), which belongs to the family of T7-like RNA polymerases . In contrast to phage T7-RNAP, N4 RNAPII displays no activity on double-stranded templates and low activity on single-stranded templates . In vivo, at least one additional N4-coded protein (p17) is required for N4 middle transcription . We show that N4 ORF2 encodes p17 (gp2) . Characterization of purified gp2revealed that it is a single-stranded DNA-binding protein that activates N4 RNAPII transcription on single-stranded DNA templates through specific interaction with N4 RNAPII . On the basis of the properties of the proteins involved in N4 RNAPII transcription and of middle promoters, we propose a model for N4 RNAPII promoter recognition, in which gp2plays two roles, stabilization of a single-stranded region at the promoter and recruitment of N4 RNAPII through gp2-N4 RNAPII interactions . Furthermore, we discuss our results in the context of transcription initiation by mitochondrial RNA polymerases.

J Struct Biol, 2003 Aug, 143(2), 107 - 17
An elastic network model of HK97 capsid maturation; Kim MK et al.; The structure of the capsid of bacteriophage HK97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature . Typically the capsid assembles through polymerization and maturation processes . Maturation is composed of proteolytic cleavages to the precursor capsid (called Prohead II), expansion triggered by DNA packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links of neighboring subunits to create the mature capsid called Head II . We apply a coarse-grained elastic network interpolation (ENI) to generate a feasible pathway for conformational change from Prohead II to Head II . The icosahedral symmetry of the capsid structure offers a significant computational advantage because it is not necessary to consider the whole capsid structure but only an asymmetric unit consisting of one hexamer plus an additional subunit from an adjacent pentamer . We also analyze normal modes of the capsid structure using an elastic network model which is also subject to symmetry constraints . Using our model, we can visualize the smooth evolution of capsid expansion and revisit in more detail several interesting geometric changes recognized in early experimental works such as rigid body motion of two compact domains (A and P) with two refolding extensions (N-arm and E-loop) and track the approach of the two particular residues associated with isopeptide bonds that make hexagonal cross-links in Head II . The feasibility of the predicted pathway is also supported by the results of our normal mode analysis.

J Mol Biol, 2003 Sep 26, 332(4), 777 - 82
Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle; Weiss GA et al.; Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host . P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle . A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat . The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains . These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.

J Immunol Methods, 2003 Sep, 280(1-2), 139 - 55
Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine; Moghaddam A et al.; Use of phage display of recombinant antibodies and large repertoire naive antibody libraries for identifying antibodies of high specificity has been extensively reported . Nevertheless, there have been few reported antibodies to haptens that have originated from naive antibody libraries with potential use in diagnostics . We have used chain shuffling of lead single-chain fragment variable (scFv) antibodies, isolated from a naive antibody library, to screen for antibodies that specifically recognise the major metabolite of heroin, 6-monoacetylmorphine (6MAM) . The antibodies were identified by screening high-density colonies of Escherichia coli expressing soluble scFv antibody fragments without prior expression on bacteriophage (phage display) . The antibodies recognise 6MAM with affinities of 1-3x10(-7) M with no crossreactivity to morphine . These antibodies can potentially be used for developing a rapid immunoassay in drug-testing programs . To our knowledge, this is the first report of an antibody that distinguishes 6MAM from its de-acetylated form, morphine.

J Virol, 2003 Oct, 77(19), 10623 - 9
Significance in replication of the terminal nucleotides of the flavivirus genome; Khromykh AA et al.; Point mutations that resulted in a substitution of the conserved 3'-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication . Similarly, substitutions of the conserved 3'-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively . The same preference for cytidine in the 3'-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis . The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of phi6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3'-penultimate cytidines and a 3'-terminal pyrimidine . A previously untested substitution of the conserved pentanucleotide at the top of the 3'-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.

J Vet Diagn Invest, 1999 Nov, 11(6), 497 - 504
Development of a ssRNA internal control reagent for an infectious bursal disease virus reverse transcription/polymerase chain reaction-restriction fragment length polymorphism diagnostic assay; Smiley JR et al.; Infectious bursal disease virus (IBDV), family Birnaviradae, is the etiologic agent of a commercially important, globally distributed, contagious, immunosuppressive disease of young chickens . A restriction enzyme-compatible ssRNA internal control was developed for an IBDV reverse transcription/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) diagnostic assay . An 841-bp bacteriophage-lambda DNA fragment was directionally ligated to 3' and 5' oligonucleotide linkers containing the IBDV RT/PCR target primer sequences . A pGEM-3Zf (+) transcription vector containing the internal control construct was used in an in vitro transcription reaction to produce ssRNA . After RT and PCR amplification, the transcripts produced an 882-bp cDNA product, larger than, co-amplifiable with, and free of the restriction sites used to prepare RFLP patterns of the 743-bp IBDV cDNA target product . The limit of detection of the transcripts in the RT/PCR test is 3.2 femtograms . With the internal control, a test inhibition rate of 7.7% (20/261) was determined for the IBDV RT/PCR assay . By identifying inhibited tests, the assay was improved through a reduction in the number of false-negative results.

Orig Life Evol Biosph, 2003 Feb, 33(1), 95 - 108
Searching for the advantages of virus sex; Turner PE; Sex (genetic exchange) is a nearly universal phenomenon in biological populations . But this is surprising given the costs associated with sex . For example, sex tends to break apart co-adapted genes, and sex causes a female to inefficiently contribute only half the genes to her offspring . Why then did sex evolve? One famous model poses that sex evolved to combat Muller's ratchet, the mutational load that accrues when harmful mutations drift to high frequencies in populations of small size . In contrast, the Fisher-Muller Hypothesis predicts that sex evolved to promote genetic variation that speeds adaptation in novel environments . Sexual mechanisms occur in viruses, which feature high rates of deleterious mutation and frequent exposure to novel or changing environments . Thus, confirmation of one or both hypotheses would shed light on the selective advantages of virus sex . Experimental evolution has been used to test these classic models in the RNA bacteriophage phi6, a virus that experiences sex via reassortment of its chromosomal segments . Empirical data suggest that sex might have originated in phi6 to assist in purging deleterious mutations from the genome . However, results do not support the idea that sex evolved because it provides beneficial variation in novel environments . Rather, experiments show that too much sex can be bad for phi6; promiscuity allows selfish viruses to evolve and spread their inferior genes to subsequent generations . Here I discuss various explanations for the evolution of segmentation in RNA viruses, and the added cost of sex when large numbers of viruses co-infect the same cell.

J Biol Chem, 2003 Nov 28, 278(48), 48084 - 91 Epub 2003 Sep 08.
RNA packaging device of double-stranded RNA bacteriophages, possibly as simple as hexamer of P4 protein; Kainov DE et al.; Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids) . This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis . At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors . In the case of the double-stranded RNA bacteriophage phi 6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4 . In the present study, the purified P4 proteins from two other cystoviruses, phi 8 and phi 13, were characterized and compared with phi 6 P4 . All three proteins are hexameric, single-stranded RNA-stimulated NTPases with alpha/beta folds . Using a direct motor assay, we found that phi 8 and phi 13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of phi 6 P4 requires association with the procapsid . This difference is explained by the intrinsically high affinity of phi 8 and phi 13 P4s for nucleic acids . The unidirectional translocation results in RNA helicase activity . Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.

Anal Biochem, 2003 Oct 1, 321(1), 65 - 70
Detection of protein-protein interactions on SiO2/Si surfaces by spectroscopic ellipsometry; Kodera S et al.; We have applied spectroscopic ellipsometry to sensitive detection of specific protein-protein interactions on SiO2/Si substrates . First, the change of ellipticity of the reflected polarized light (600-1100 nm) was correlated with the thickness of the protein layer immobilized on SiO2/Si surfaces by measuring monomeric (myoglobin) and homotetrameric (hemoglobin) proteins with a similar monomer size . Protein-protein interactions were then measured with the antigen/antibody and cell-surface receptor/ligand systems; in each system either of the two proteins was bound to SiO2/Si substrates . Consequently, significant ellipticity changes were observed only for the cases where the interactions were specific . A specific antibody binding was also detectable with an antigen displayed on the surface of bacteriophage particles . These results show the usefulness of spectroscopic ellipsometry for sensitive detection of protein-protein interactions and its applicability to a detection method for the protein-based biochips to be developed in the future.

Biochem Biophys Res Commun, 2003 Sep 26, 309(3), 695 - 701
Interaction of hepatitis C virus NS5A with La protein revealed by T7 phage display; Houshmand H et al.; Although the hepatitis C virus (HCV) genome is synthesized by the virus-encoded RNA-dependent RNA polymerase NS5B, other viral and cellular factors are assumed to be required for template-specific initiation and regulation of RNA-synthesis . The cellular protein La, which normally associates with RNA polymerase III transcripts, also interacts with the 5'- and 3'-untranslated regions of several RNA viruses, including HCV . To investigate whether other viral gene products may be involved in this interaction, we constructed an HCV cDNA expression library in bacteriophage T7 allowing portions of the HCV polyprotein to be displayed on the phage surface . Screening of the phage library against La resulted in selection of clones displaying the N-terminal region of HCV NS5A . Co-precipitation of full-length and truncated forms of recombinant NS5A with La revealed that the N-terminal region of NS5A was both necessary and sufficient for binding to La . Although this region of NS5A is essential for HCV replication, the role of the NS5A-La interaction in the infected cell remains to be established.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2003 Aug, 11(4), 355 - 8
{Construction and significance of directional expression cDNA library from myeloid leukemia cell line U937}; Chen G et al.; To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted . The first and second strand of cDNA were synthesized through reverse transcription . After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated . Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector . The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration . The recombinants were examined by color selection . In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I . The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed . The average size of exogenous insert in the recombinants was about 1.7 kb . It is concluded that the constructed cDNA library can be used to screen target clones.

J Biol Chem, 2003 Nov 14, 278(46), 45864 - 81 Epub 2003 Sep 03.
Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity; Yen TY et al.; Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion . To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme . The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol . Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381 . The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage . Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site . A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified . Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.

Virology, 2003 Sep 1, 313(2), 622 - 8
Toxicity of the bacteriophage lambda cII gene product to Escherichia coli arises from inhibition of host cell DNA replication; Kedzierska B et al.; The bacteriophage lambda cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the "lysis-versus-lysogeny" decision during phage development . The CII protein is highly toxic to the host, Escherichia coli, when overproduced . However, the molecular mechanism of this toxicity is not known . Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E . coli cells . The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase alpha subunit . Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA(+) or rpoA341 hosts, could be relieved by overexpression of the E . coli dnaB and dnaC genes . In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E . coli strains contain mutations near dnaC . We conclude that the DNA replication machinery may be a target for the toxic activity of CII.

Biotechniques, 2003 Aug, 35(2), 368 - 75
Real-time PCR provides improved detection and titer determination of bacteriophage; Edelman DC et al.; The plaque assay is the traditional method for the quantification of bacteriophage, particularly for lambda cloning vectors . Unfortunately, this technique is fraught with procedural difficulties, and the quality of the data obtained from this "gold standard" assay may be inaccurate due to the subjective interpretation of the results . The application of quantitative real-time PCR (QPCR) technology can address these issues and be a more accurate platform to evaluate phage growth conditions and quantify viral titers in phage preparations . QPCR, with an improved primer set specific for lambda phage and coupled with fluorescent dye detection of PCR products, was used to detect and quantify phages in lysates with no prior DNA purification . Phages were detected below one plaque-forming unit, and at least 89 viral copies were detected from a purified DNA sample . When unknown concentrations of various phage preparations were assessed using QPCR, they were attained more efficiently, with greater sensitivity and precision, and the method produced more accurate quantitative data spanning a wider linear range than those obtained by the plaque assay (six logs vs . one log, respectively) . Finally, QPCR for the detection of phage has multiple applications, including conventional cloning and in alternative fields of study such as environmental sciences.

Microbiology, 2003 Sep, 149(Pt 9), 2443 - 53
Development of the Micromonospora carbonacea var . africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp; Alexander DC et al.; Micromonospora carbonacea var . africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome . Sequence analysis of a 4.4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP) . Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp . by conjugation from Escherichia coli . Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome . The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3' end of the tRNA . Integration of pMLP1-based plasmids in M . carbonacea var . africana caused a loss of the pMLP1 phage . Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site . Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.

J Mol Biol, 2003 Sep 12, 332(2), 415 - 22
Visualization by cryo-electron microscopy of genomic RNA that binds to the protein capsid inside bacteriophage MS2; Koning R et al.; The icosahedrally symmetrized structure of bacteriophage MS2 as determined by cryo-electron microscopy (EM) reveals the presence of genomic RNA that attaches to coat-protein dimers . Earlier X-ray diffraction studies revealed similar interactions between the unique operator hairpin of the MS2 genomic RNA and the coat-protein dimer . This observation leads us to conclude that not only the operator, but also many other RNA sequences in the genome of MS2, are able to bind to the coat-protein dimer . A substantial number of potential coat-protein-dimer binding sites are present in the genome of MS2 that can account for the observed RNA densities in the EM map . Moreover, it appears that these stem-loop structures are able to bind in a similar fashion to the coat protein dimer as the wild-type operator hairpin . The EM map also shows additional density between the potential operator-binding sites, linking the RNA stem-loops together to form an icosahedral network around the 3 and 5-fold axes . This RNA network is bound to the inside of the MS2 capsid and probably influences both capsid stability and formation, supporting the idea that capsid formation and RNA packaging are intimately linked to each other.

Science, 2003 Aug 29, 301(5637), 1235 - 8
Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder; van Oijen AM et al.; We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules . Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA . By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle . The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.

J Biol Chem, 2003 Nov 7, 278(45), 44139 - 46 Epub 2003 Aug 27.
Qbeta replicase discriminates between legitimate and illegitimate templates by having different mechanisms of initiation; Ugarov VI et al.; Qbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs (RQ RNAs) in vitro . Here we characterize template properties of the 5' and 3' fragments obtained by cleaving one of such RNAs at an internal site . We unexpectedly found that, besides the 3' fragment, Qbeta replicase can copy the 5' fragment and a number of its variants, although they lack the initiator region of RQ RNA . This copying can occur as a 3'-terminal elongation or through de novo initiation . In contradistinction to RQ RNA and its 3' fragment, initiation on these templates occurs without regard to the 3'-terminal or internal oligo(C) clusters, is GTP-independent, and does not result in a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid . The results suggest that, although Qbeta replicase can initiate and elongate on a variety of RNAs, only some of them are recognized as legitimate templates . GTP-dependent initiation on a legitimate template drives the enzyme to a "closed" conformation that may be important for keeping the template and the complementary nascent strand unannealed, without which the exponential replication is impossible . Triggering the GTP-dependent conformational transition at the initiation step could serve as a discriminative feature of legitimate templates providing for the high template specificity of Qbeta replicase.

Genetika, 2003 Jul, 39(7), 914 - 26
{Transduction of plasmid antibiotic resistance determinants with pseudo-T-even bacteriophages}; Taniashin VI et al.; Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied . It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction . Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained . These mutants facilitated transduction experiments in some cases . Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied . The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated . The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E . coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E . coli 802 . The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed . It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II . Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system.

Plant J, 2003 Sep, 35(5), 665 - 73
A novel procedure for the localization of viral RNAs in protoplasts and whole plants; Zhang F et al.; Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants . However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively . A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants . The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp) . A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts . However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm . Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions . Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells . Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation . Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins . In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves . This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.

Mol Genet Genomics, 2003 Nov, 270(3), 201 - 15 Epub 2003 Aug 23.
The gastrointestinal tract as the portal of entry for foreign macromolecules: fate of DNA and proteins; Palka-Santini M et al.; The gastrointestinal tract (GIT) of mammals is the main portal of entry for foreign DNA and proteins . We have documented the fate of orally administered DNA or protein in the GIT of the mouse . The gene for the Green Fluorescent Protein (GFP) (4.7 kb) and the genomes of bacteriophage M13 (7.25 kb) and adenovirus type 2 (Ad2; 35.9 kb) were used as test DNAs . Persistence of these DNAs in the GIT was monitored by Southern hybridization and fluorescent in situ hybridization (FISH) or by PCR . For studies on proteins, recombinant glutathione-S-transferase was fed to mice . Survival of the protein in the GIT was then assessed by Western blotting . Depending on feeding schedules and food regimens, but irrespective of mouse strain or DNA length, fragments of the GFP gene or other DNAs were detectable for up to 18 h after feeding by Southern blot analysis . The GFP DNA could be visualized by FISH in cecal epithelia . A high fiber diet reduced the time required for food to pass through the GIT, and foreign DNA was cleared more rapidly . A high fat diet or complexing of the foreign DNA with protamine or lipofectin did not extend DNA persistence times . Undegraded GST protein was detected only in foregut contents up to 30 min after feeding . At 15 and 30 min post feeding, trace amounts of GST were found in extracts of the kidney . The GIT is constantly exposed to highly recombinogenic fragments of foreign DNA and to intact foreign proteins . Our data have implications for studies on carcinogenesis and mutagenesis, and on the pathogenicity of infectious proteins such as prions.

Nat Struct Biol, 2003 Oct, 10(10), 849 - 55 Epub 2003 Aug 24.
Structure of the bacteriophage T4 DNA adenine methyltransferase; Yang Z et al.; DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence . We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy . T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a beta-hairpin that is conserved in the family of GATC-related MTase orthologs . Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site . The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.

Biochim Biophys Acta, 2003 Aug 25, 1628(3), 177 - 85
Structure-function relationships in nucleosomal arrays containing linker histone H5; Sanchez MA et al.; To study the structural and functional changes accompanying the integration of histone H5 into the nucleosome structure, linear DNA species have been employed with a terminal promoter for bacteriophage T7 RNA polymerase followed by tandem repeats of a 207-bp nucleosome positioning sequence . The oligonucleosomes assembled from 12-repeat DNA and saturating amounts of core histone octamer plus histone H5 are compacted, in the presence of 1 mM free magnesium ions, to the level of the 30-nm fiber . Under these ionic conditions the efficiency in RNA synthesis and the size distribution of RNA chains obtained with this template are the same as those corresponding to the template without H5, indicating that the 30-nm fiber stabilized by H5 does not impair RNA elongation . Therefore, under our experimental conditions, incorporation of one molecule of histone H5 per nucleosome does not affect elongation of RNA even when a folded structure is produced . However, elongation is inhibited by binding of an excess of H5.

Nucleic Acids Res, 2003 Sep 1, 31(17), 4965 - 72
Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase I; Tsujikawa L et al.; A DNA template containing a single ethyl phosphotriester was replicated in vitro by the bacteriophage T4 DNA polymerase and by Escherichia coli DNA polymerase I (DNA pol I) . Escherichia coli DNA pol I bypassed the lesion efficiently, but partial inhibition was observed for T4 DNA polymerase . The replication block produced by the ethyl phosphotriester was increased at low dNTP concentrations and for a mutant T4 DNA polymerase with an antimutator phenotype, increased proofreading activity, and reduced ability to bind DNA in the polymerase active center . These observations support a model in which an ethyl phosphotriester impedes primer elongation by T4 DNA polymerase by decreasing formation of the ternary DNA polymerase-DNA-dNTP complex . When primer elongation is not possible, proofreading becomes the favored reaction . Apparent futile cycles of nucleotide incorporation and proofreading, the idling reaction, were observed at the site of the lesion . The replication block was overcome by higher dNTP concentrations . Thus, ethyl phosphotriesters may be tolerated in vivo by the up-regulation of dNTP biosynthesis that occurs during the cellular checkpoint response to blocked DNA replication forks.

Free Radic Biol Med, 2003 Sep 1, 35(5), 495 - 503
Low levels of endogenous oxidative damage cluster levels in unirradiated viral and human DNAs; Sutherland BM et al.; Ionizing radiation induces bistranded DNA damage clusters-two or more oxidized bases, abasic, sites or strand breaks on opposing strands within a few helical turns-but it is not known if clusters are also formed in unirradiated DNA in solution or in unirradiated cultured human cells . The frequencies of endogenous oxidized purine clusters (recognized by Escherichia coli Fpg protein), oxidized pyrimidine clusters (recognized by Nth protein), and abasic clusters (cleavage by Nfo protein) were determined using quantitative gel electrophoresis, electronic imaging, and number average length analysis . Methods of DNA isolation and storage were found to affect cluster levels significantly . In bacteriophage T7 DNA prepared using stringent conditions, the frequencies of these clusters were <1/Mbp . In DNA from unirradiated human 28SC monocytes, the levels of such clusters were, at most, a few per gigabase pair.

Water Sci Technol, 2003, 47(12), 163 - 8
Removal of MS2 bacteriophage using membrane technologies; Hu JY et al.; Removals of MS2 bacteriophage virus using different membrane materials under different operating pressures were investigated . The results obtained in this study suggested that a better log removal in terms of MS2 bacteriophage virus could be achieved using Polyamide RO membrane under the optimum operating pressure of 100 psi . It is further noted that variable MS2 influent concentration levels resulted in corresponding variable log removals of the bacteriophages by the Polyamide RO membrane . The presence of MS2 bacteriophage virus in the effluent could possibly be due to leakage of bacteriophages through the membranes structure . Investigations using SEM and AFM showed that there were gaps or pores present in the membrane structure which were sufficiently large for the MS2 viruses to pass through.

Nat Struct Biol, 2003 Sep, 10(9), 688 - 93 Epub 2003 Aug 17.
Three-dimensional structure of bacteriophage T4 baseplate; Kostyuchenko VA et al.; The baseplate of bacteriophage T4 is a multiprotein molecular machine that controls host cell recognition, attachment, tail sheath contraction and viral DNA ejection . We report here the three-dimensional structure of the baseplate-tail tube complex determined to a resolution of 12 A by cryoelectron microscopy . The baseplate has a six-fold symmetric, dome-like structure approximately 520 A in diameter and approximately 270 A long, assembled around a central hub . A 940 A-long and 96 A-diameter tail tube, coaxial with the hub, is connected to the top of the baseplate . At the center of the dome is a needle-like structure that was previously identified as a cell puncturing device . We have identified the locations of six proteins with known atomic structures, and established the position and shape of several other baseplate proteins . The baseplate structure suggests a mechanism of baseplate triggering and structural transition during the initial stages of T4 infection.

RNA, 2003 Sep, 9(9), 1108 - 22
Novel "anti-reverse" cap analogs with superior translational properties; Jemielity J et al.; Synthetic analogs of the 5'-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover . Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m(7)Gp(3)G . Unfortunately, because of the presence of a 3'-OH on both the m(7)Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation . Previously we designed and synthesized two "anti-reverse" cap analogs (ARCAs), m(7)3'dGp(3)G and m(2)(7,3'-)(O)Gp(3)G, that cannot be incorporated in the reverse orientation because of modifications at the C3' position of m(7)Guo . In the present study, we have synthesized seven new cap analogs modified in the C2' and C3' positions of m(7)Guo and in the number of phosphate residues, m(2)(7,2'-)(O)Gp(3)G, m(7)2'dGp(3)G, m(7)2'dGp(4)G, m(2)(7,2'-)(O)Gp(4)G, m(2)(7,3'-)(O)Gp(4)G, m(7)Gp(5)G, and m(2)(7,3'-)(O)Gp(5)G . These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA . The results indicate that modifications at C2', like those at C3', prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs.

Eur J Biochem, 2003 Sep, 270(17), 3543 - 54
Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70; Nuttall SD et al.; The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody . In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length . Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection . VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1) . Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation . Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies . As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed . High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

J Virol, 2003 Sep, 77(17), 9613 - 21
Point mutations in exon I of the herpes simplex virus putative terminase subunit, UL15, indicate that the most conserved residues are essential for cleavage and packaging; Przech AJ et al.; The herpes simplex virus UL15 and UL28 genes are believed to encode two subunits of the terminase involved in cleavage and packaging of viral genomes . Analysis of the UL15 protein sequence and its herpesvirus homologues revealed the presence of 20 conserved regions . Twelve of the twenty regions conserved among herpesviruses are also conserved in terminases from DNA bacteriophage . Point mutations in UL15 were designed in four conserved regions: L120N (CR1), Q205E (CR2), Q251E (CR3), G263A (CR3), and Y285S (CR4) . Transfection experiments indicated that each mutant gene could produce stable UL15 protein at wild-type levels; however, only one mutant (Q251E) was able to complement the UL15-null virus . Each mutation was introduced into the viral genome by marker transfer, and all mutants except Q251E were unable to form plaques on Vero cells . Furthermore, failure to form plaques on Vero cells correlated with a defect in cleavage and packaging . Immunofluorescence experiments indicated that in cells infected with all mutant viruses the UL15 protein could be detected and was found to localize to replication compartments . Although wild-type and mutant Q251E were able to produce A, B, and C capsids, the rest of the mutants were only able to produce B capsids, a finding consistent with their defects in cleavage and packaging . In addition, all mutant UL15 proteins retained their ability to interact with B capsids . Therefore, amino acid residues 120, 205, 263, and 285 are essential for the cleavage and packaging process rather than for association with capsids or localization to replication compartments.

J Appl Microbiol, 2003, 95(3), 536 - 44
Influence of groundwater characteristics on the survival of enteric viruses; Gordon C et al.; AIMS: This study was undertaken to further understand the processes affecting the persistence of enteric viruses in groundwater . METHODS AND RESULTS: Varying temperature, oxygen and nutrient levels were tested in the presence and absence of groundwater micro-organisms to determine which of the factors tested had dominant influence on the decay of Escherichia coli, the bacteriophage MS2, poliovirus and coxsackievirus . The results indicated that the most influential factor affecting the decay of the viruses and E . coli was the presence of groundwater micro-organisms . The results also implied that temperature, the presence of oxygen and nutrient levels indirectly influence viruses and E . coli decay by influencing the activity of the groundwater micro-organisms . CONCLUSIONS: E . coli and the viruses displayed maximum decay under aerobic conditions, at 28 degrees C without the addition of nutrients in the presence of groundwater micro-organisms . SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that if the mode of action of the groundwater micro-organisms could be determined then the decay of viral pathogens in recharged waters may be more easily predicted.

Anal Bioanal Chem, 2003 Nov, 377(5), 834 - 42 Epub 2003 Aug 07.
Label-free screening of bio-molecular interactions; Cooper MA; The majority of techniques currently employed to interrogate a biomolecular interaction require some type of radio- or enzymatic- or fluorescent-labelling to report the binding event . However, there is an increasing awareness of novel techniques that do not require labelling of the ligand or the receptor, and that allow virtually any complex to be screened with minimal assay development . This review focuses on three major label-free screening platforms: surface plasmon resonance biosensors, acoustic biosensors, and calorimetric biosensors . Scientists in both academia and industry are using biosensors in areas that encompass almost all areas drug discovery, diagnostics, and the life sciences . The capabilities and advantages of each technique are compared and key applications involving small molecules, proteins, oligonucleotides, bacteriophage, viruses, bacteria, and cells are reviewed . The role of the interface between the biosensor surface (in the case of SPR and acoustic biosensors) and the chemical or biological systems to be studied is also covered with attention to the covalent and non-covalent coupling chemistries commonly employed.

J Clin Microbiol, 2003 Aug, 41(8), 3777 - 83
Presence of activatable Shiga toxin genotype (stx(2d)) in Shiga toxigenic Escherichia coli from livestock sources; Gobius KS et al.; Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1000-fold by the elastase present in mouse or human intestinal mucus . We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx(2d) . The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c vha), stx(2c vhb), or stx(2d EH250) . Subsequently, the stx(2c vha) and stx(2c vhb) operons were screened for the absence of a PstI site in the stx(2A) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype . Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d) . The complete nucleotide sequences of seven representative stx(2d) operons were determined . Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting . stx(2d) isolates were induced for the production of bacteriophages carrying stx . Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons . RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) operons were distinct . The STEC strains carrying these operons were isolated from retail ground beef . Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.

Nucleic Acids Res Suppl, 2002, (2), 207 - 8
In vitro selection of N peptide-binding RNA on a quartz-crystal microbalance; Murakawa A et al.; We report here an in vitro selection of a hairpin loop-RNA bound to N Peptide from bacteriophage lambda on a 27 MHz quartz-crystal microbalance (QCM) to study an interaction between an RNA structure and an RNA-binding peptide . The N Peptide was immobilized on a QCM electrode and specific RNAs binding to the peptide were selected from a GNRNA pentaloop-randomized RNA library with in situ monitoring of selection processes using a QCM . After the 5th round selection, the consensus sequences including a GCGCA loop were obtained from the pentaloop library . This RNA sequence was different from a binding site of a native N Protein (boxB RNA).

Genome, 2003 Aug, 46(4), 573 - 9
Modes of reproduction in Australian populations of Hypericum perforatum L . (St . John's wort) revealed by DNA fingerprinting and cytological methods; Mayo GM et al.; Hypericum perforatum L . (St . John's wort) is widely used in homeopathic medicine, but has also become a serious weed in Australia and many other countries . Reproduction in H . perforatum was investigated using markers based on restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) . Between two Australian populations, plants displayed 14 polymorphisms from a total of 22 scorable RFLP markers when genomic DNA was probed with M13 bacteriophage, but individuals within each population exhibited identical RFLP fingerprints . Ninety-four percent of the progeny of four crosses made between the two populations exhibited identical fingerprint and ploidy level to the maternal parent, and probably originated apomictically . Seven seedlings with recombinant RFLP or AFLP fingerprints were found from a total of 121 progeny . Both molecular marker techniques detected the same recombinants from a subset of screened progeny . Cytological analysis showed that the seven recombinants comprised three tetraploids (2n = 4x = 32), three hexaploids (2n = 6x = 48), and one aneuploid (2n - 1 = 31), which suggested that the level of normal reduced embryo sacs was only 2.5% . These results are discussed in relation to the management of invasive populations, and the implications for plant breeding and production of St . John's wort for medicinal purposes.

J Biol Chem, 2003 Oct 24, 278(43), 41749 - 55 Epub 2003 Jul 30.
Bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase): evidence for two distinct stages of methylation under single turnover conditions; Malygin EG et al.; We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant . T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA << 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-l-homocysteine (AdoHcy)) . We propose that the single turnover reaction proceeds in two stages . First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (kmeth1 = 0.21 s-1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination . In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (kmeth2 = 0.023 s-1) . We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand . The hemimethylated duplex 5'-GATC/5'-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions . The first stage (kmeth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation . The slower second stage (kmeth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain . Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions . When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced.

FEMS Microbiol Lett, 2003 Jul 29, 224(2), 225 - 9
Detection of bacteriophage VSH-1 svp38 gene in Brachyspira spirochetes; Stanton TB et al.; VSH-1 is a mitomycin C-inducible, non-lytic, phage-like agent that packages random 7.5-kb fragments of the Brachyspira hyodysenteriae genome . VSH-1 is the first recognized mechanism for gene transfer between B . hyodysenteriae cells . To analyze the distribution of VSH-1 among spirochetes, a 344-bp probe for gene svp38, encoding the VSH-1 major head protein, was amplified by polymerase chain reaction and used in Southern blot hybridizations with genomic DNA from various spirochete genera . The svp38 probe hybridized to a 40-kb SalI-SmaI fragment of the B . hyodysenteriae B78(T) chromosome, indicating VSH-1 DNA insertion into the chromosome at a unique site . Restriction endonuclease digested DNAs of 27 spirochete strains representing six Brachyspira species (B . hyodysenteriae, B . innocens, B . pilosicoli, B . murdochii, B . intermedia, B . alvinipulli) contained a single fragment hybridizing with the svp38 probe . DNAs from spirochete species of the genera Treponema, Spirochaeta, Borrelia, and Leptospira did not hybridize with the probe . VSH-1-like agents appear to be widely distributed among Brachyspira species and, as has been demonstrated for B . hyodysenteriae, may serve as useful gene transfer agents for those other species.

Virology, 2003 Jul 20, 312(1), 233 - 44
Establishment and maintenance of HSV latent infection is mediated through correct splicing of the LAT primary transcript; Kang W et al.; To study the effect of the splicing of HSV-1 latency-associated transcript (LAT) on viral latency, we constructed two mutant viruses (FHlambda+ and FHlambda-) in which the 168-bp HpaI-HpaI fragment within the 2-kb LAT intron was replaced by a 447-bp bacteriophage lambda sequence . The lambda DNA was inserted in opposite orientations in FHlambda+ and FHlambda- . The mutation in FHlambda+ disrupted the splicing of LAT primary transcript and altered both LAT exon and intron, whereas the mutation in FHlambda- virus preserved the wild-type splice sites and the wild-type exon . Quantitative PCR analysis revealed that during latency there was a reduction in the number of viral genomes in mouse trigeminal ganglia infected with FHlambda+ but not in those infected with FHlambda- . The decrease in the latent genome numbers was not due to a defect in viral replication during the acute stage of infection . Furthermore, trigeminal ganglia from mice latently infected with FHlambda+ displayed a slower reactivation kinetics compared to those infected with the parental strain . To elucidate the mechanism, we examined the antiapoptotic properties of these LAT constructs . A plasmid containing the pHlambda+ construct was found to be less protective for cells against apoptosis than plasmid containing the wild-type or pHlambda- constuct . These results suggest that the splicing of LAT primary transcript, and thus the correctly spliced exon product, play an important role in promoting the establishment and/or maintenance of viral latency.

Mol Microbiol, 2003 Aug, 49(4), 1145 - 54
An intramolecular disulphide bond reduces the efficacy of a lipoprotein plasma membrane sorting signal; Robichon C et al.; To study lipoprotein sorting in Escherichia coli, we devised a novel screen in which sensitivity or resistance to bacteriophage T5 and colicin M reflects the membrane localization of the bacteriophage T5-encoded lipoprotein Llp, which inactivates the outer membrane (OM) T5 receptor (FhuA) . When processed by lipoprotein signal peptidase, Llp has a serine at position +2, immediately after the fatty acylated N-terminal cysteine . As predicted by the '+2 lipoprotein sorting rule' that determines the localization of lipoproteins in the cell envelope, Llp is located in the OM . However, contrary to expectations, when serine +2 was replaced by aspartate, the canonical plasma membrane lipoprotein retention signal, Llp was still > or =40% targeted to the OM and protected cells against colicin M and phage T5 . OM association of this Llp derivative was abolished when a peptide spacer was inserted between the aspartate and the rest of Llp or when the formation of an intramolecular disulphide bond in Llp was prevented by substituting one or other of the cysteines involved . Furthermore, analysis of a MalE-Llp hybrid protein with or without a lipid moiety demonstrated that fatty acylation of Llp is essential for its OM association and for protection against colicin M and bacteriophage T5 . These data suggest (i) that phage-encoded Llp uses the endogenous E . coli Lol pathway for lipoprotein sorting to the OM and (ii) that the conformation of a lipoprotein can affect its sorting within the cell envelope.

Mol Microbiol, 2003 Aug, 49(4), 1043 - 9
The pair of arginine codons AGA AGG close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating peptidyl-tRNAArg4; Olivares-Trejo JJ et al.; To analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives . The lambda int gene has a high frequency of rare ATA, AGA and AGG codons; two of them (AGA AGG) located at positions 3 and 4 of the int open reading frame (ORF) . Escherichia coli pth (rap) cells, which are defective in peptidyl-tRNA hydrolase (Pth) activity, are more susceptible to the inhibitory effects of int expression as compared with wild-type cells . Cell growth and Int protein synthesis were enhanced by overexpression of Pth and tRNAArg4 cognate to AGG and AGA but not of tRNAIle2a specific for ATA . The increase of Int protein synthesis also takes place when the rare arginine codons AGA and AGG at positions 3 and 4 are changed to common arginine CGT or lysine AAA codons but not to rare isoleucine ATA codons . In addition, overexpression of int in Pth defective cells provokes accumulation of peptidyl-tRNAArg4 in the soluble fraction . Therefore, cell growth and Int synthesis inhibition may be due to ribosome stalling and premature release of peptidyl-tRNAArg4 from the ribosome at the rare arginine codons of the first tandem, which leads to cell starvation for the specific tRNA.

Nucleic Acids Res . 2003 Aug 1;31(15):e82.
General plasmids for producing RNA in vitro transcripts with homogeneous ends; Walker SC et al.; In vitro transcripts of bacteriophage RNA polymerases (RNAPs), such as T7 RNAP, often suffer from a considerable degree of 3'-end heterogeneity and, with certain promoter sequences, 5'-end heterogeneity . For some applications, this transcript heterogeneity poses a significant problem . A potential solution is to incorporate ribozymes into the transcripts at the 5'- and/or 3'-end of the target RNA sequence . This approach has been used quite widely but has required the generation of new transcription vectors or PCR-derived templates for each new RNA to be studied . To overcome this limitation, we have created two general plasmids for producing homogeneous RNA transcripts: one encodes a 3'- hepatitis delta virus (HDV) ribozyme and the other, used in combination with a two-step PCR, allows the production of double {5'-hammerhead (HH) and 3'-HDV} ribozyme constructs . A choice of cloning and run-off transcription linearisation restriction enzyme sites ensures that virtually any RNA sequence can be cloned and transcribed from these plasmids . For all the RNA sequences tested, good yields of transcript were obtained . These plasmids provide the tools for the simple, rapid creation of new RNA-coding plasmids to produce milligram quantities of homogeneous in vitro transcripts for all applications.

Nucleic Acids Res . 2003 Aug 1;31(15):e81.
Combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method; Zhang XM et al.; Recombinogenic engineering or recombineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases . We report here a general method for using recombineering to combine overlapping bacterial artificial chromosomes (BACs) to build larger, unified BACs . In order to test the feasibility of using recombineering to combine two large DNA fragments (>20 kb), we constructed a unified BAC containing the full-length tyrosinase-related protein-1 (Tyrp-1) gene from two library-derived BACs, one containing the 5' regulatory elements and the other containing the 3' coding exons . This was achieved using a two-step homologous recombination method enabled by the bacteriophage lambda Red proteins . In the first step, retrieval, a large DNA fragment (approximately 22 kb) was retrieved from one of the original BACs . In the second step, recombination, the retrieved DNA fragment was inserted into the second original BAC to form the unified BAC containing all the desired Tyrp-1 sequence . To further demonstrate the general applicability of our approach, an additional DNA fragment (approximately 20 kb) was inserted into the unified BAC downstream of the coding region . This method should prove very useful for enabling BAC manipulation in a variety of scenarios.

Nucleic Acids Res . 2003 Aug 1;31(15):e80.
A simple two-step, 'hit and fix' method to generate subtle mutations in BACs using short denatured PCR fragments; Yang Y et al.; The bacteriophage lambda recombination system has proven to be a valuable tool for engineering bacterial artificial chromosomes (BAC) . Due to its high efficiency, subtle alterations in the BACs can be generated using oligonucleotides as targeting vectors . Since no selection marker is used, recombinant clones are identified utilizing a selective PCR screening method . However, occasionally the selective PCR screening is not feasible . We describe here a two-step 'hit and fix' method that can be reliably used for generating any subtle alteration in BACs using short denatured PCR fragments as targeting vectors . In the first step of this method, 6-20 nucleotides are changed around the base where the mutation has to be generated . In the second step, these altered nucleotides are reverted to the original sequence and simultaneously a subtle alteration is introduced . Since in each step several nucleotides are changed, PCR primers specific for such alterations can be designed . This two-step method provides a simple and efficient tool for generating subtle alterations in BACs that can be very valuable for functional analysis of genes.

J Mol Biol, 2003 Aug 8, 331(2), 417 - 31
Three-dimensional structure of penicillium chrysogenum virus: a double-stranded RNA virus with a genuine T=1 capsid; Caston JR et al.; Although double-stranded (ds) RNA viruses are a rather diverse group, they share general architectural principles and numerous functional features . All dsRNA viruses, from the mammalian reoviruses to the bacteriophage phi6, including fungal viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA . This ubiquitous capsid consists of 120 protein subunits in a so-called T=2 organization . The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA viruses . We have used cryo-electron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26A resolution of the Penicillium chrysogenum virus (PcV) capsid . In contrast to all previous studies of dsRNA viruses, PcV capsid is an authentic T=1 capsid with 60 equivalent protein subunits . This T=1 capsid is built with the largest structural protein (110 kDa) . Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis . Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits . The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles . Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues . These results open new perspectives when considering the structural organization of dsRNA virus capsids.

J Mol Biol, 2003 Aug 8, 331(2), 361 - 73
The structure of the receptor-binding domain of the bacteriophage T4 short tail fibre reveals a knitted trimeric metal-binding fold; Thomassen E et al.; Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres . After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipo-polysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube . The short tail fibres consist of a parallel, in-register, trimer of gene product 12 (gp12).X-ray crystallography at 1.5A resolution of a protease-stable fragment of gp12 generated in the presence of zinc chloride reveals the structure of the C-terminal receptor-binding domain . It has a novel "knitted" fold, consisting of three extensively intertwined monomers . It reveals a metal-binding site, containing a zinc ion coordinated by six histidine residues in an octahedral conformation . We also suggest an LPS-binding region.

Mol Cell, 2003 Jul, 12(1), 187 - 98
A conformational switch controls the DNA cleavage activity of lambda integrase; Aihara H et al.; The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements . We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond . In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis . A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA . Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site . The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule . The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.

Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9292 - 5 Epub 2003 Jul 24.
Osmotic pressure inhibition of DNA ejection from phage; Evilevitch A et al.; Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes . We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure . In the particular case of bacteriophage lambda, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres . Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.

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