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