J Bacteriol, 1999 Jul, 181(14), 4391 - 6 Lysis and lysis inhibition in bacteriophage T4: rV mutations reside in the holin t gene; Dressman HK et al.; Upon infecting populations of susceptible host cells, T-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37 degrees C after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells . The timing of lysis depends upon gene t and upon one or more rapid-lysis (r) genes whose inactivation prevents lysis inhibition . t encodes a holin that mediates the movement of the T4 endolysin though the inner cell membrane to its target, the cell wall . The rI protein has been proposed to sense superinfection . Of the five reasonably well characterized r genes, only two, rI and rV, are clearly obligatory for lysis inhibition . We show here that rV mutations are alleles of t that probably render the t protein unable to respond to the lysis inhibition signal . The tr alleles cluster in the 5' third of t and produce a strong r phenotype, whereas conditional-lethal t alleles produce the classical t phenotype (inability to lyse) and other t alleles produce additional, still poorly understood phenotypes . tr mutations are dominant to t+, a result that suggests specific ways to probe T4 holin function. Rev Med Virol, 1997 Jul, 7(2), 107 - 122 Capsid assembly and DNA packaging in herpes simplex virus; Homa FL et al.; The genome of HSV-1 contains 80-85 open reading frames . Genetic and biochemical evidence suggests that at least 39 of these genes encode proteins that are components of the HSV-1 virion . The architecture of the HSV-1 virion consists of a trilaminar lipid envelope, an amorphous layer known as the tegument, a capsid shell, and a DNA-containing core . The capsid is an icosahedral shell whose major morphological features are 162 capsomers . It is composed of a major capsid protein called VP5 and three less abundant proteins, VP19C, VP23 and VP26 . VP5 is the structural subunit of all 162 capsomers while VP19C and VP23 are located in the space between the capsomers . In addition to the structural proteins, capsid assembly involves participation of the HSV-1-encoded protease and the scaffolding protein, preVP22a . DNA packaging involves participation of DNA, empty capsids, and at least seven additional HSV-1-encoded proteins . Considerable advances have been made in understanding the structure of the capsid shell, largely as the result of applying cryoelectron microscopy techniques . Use of recombinant baculoviruses has allowed for a detailed analysis of the proteins required for capsid assembly . More recently, an in vitro system has been developed which has aided in defining the assembly pathway by identifying intermediates in the assembly of intact capsids . The in vitro system has identified a fragile roundish procapsid which matures into the polyhedral capsid in a transition similar to that undergone by bacteriophage proheads . This review is a summary of our present knowledge with respect to the structure and assembly of the HSV-1 capsid and what is known about the seven genes involved in DNA packaging . Proteins, 1999 Aug 1, 36(2), 217 - 27 Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides; Nuttall SD et al.; We have designed and engineered the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) variable (V-like) domain to produce a human-based protein scaffold for peptide display . First, to test whether the CTLA-4 CDR-like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14-residue intra-disulfide-linked neuropeptide . Upon expression as periplasmic-targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild-type V-domain were produced . These mutations in CTLA-4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation-dependent anti-CTLA-4 monoclonal antibody showed that the V-domain framework remained correctly folded . Secondly, to develop a system for library selection, we displayed both wild-type and mutated CTLA-4 proteins on the surface of fd-bacteriophage as fusions with the geneIII protein . CTLA-4 displayed on phage bound specifically to immobilized CD80-Ig and CD86-Ig and in one-step panning enriched 5,000 to 2,600-fold respectively over wild-type phage . Bacteriophage displaying CTLA-4 with somatostatin in CDR3 (CTLA-4R-Som3) specifically bound somatostatin receptors on transfected CHO-K1 cells pre-incubated with 1 microg/ml tunicamycin to remove receptor glycosylation . Binding was specific, as 1 microM somatostatin successfully competed with CTLA-4R-Som3 . CTLA-4R-Som3 also activated as well as binding preferentially to non-glycosylated receptor subtype Sst4 . The ability to substitute CDR-like loops within CTLA-4 will enable design and construction of more complex libraries of single V-like domain binding molecules . Proteins 1999;36:217-227 . Biotechnol Bioeng, 1999 Aug 5, 64(3), 373 - 6 Activity losses among T4 lysozyme charge variants after adsorption to colloidal silica; Bower CK et al.; Enzyme structure and function depend to some extent on enzyme net charge and charge location . Altering the charge of even a single residue may affect the interaction between enzyme and substrate such that all catalytic activity is lost . In this study we investigated the effect of net charge and charge location on the enzymatic activity of synthetic mutants of bacteriophage T4 lysozyme in the presence of colloidal silica . Enzymatic activity decreased upon adsorption, and these changes were variant-specific . Results were interpreted with reference to differences in adsorbed enzyme structure and orientation, and electrostatic effects . By exploring the effects of enzyme charge on adsorption, it may be possible to gain a better understanding of how enzyme structure influences adsorption and function at an interface . Mol Gen Genet, 1999 Jun, 261(4-5), 762 - 9 Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants; Wegrzyn A et al.; We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria . We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants . On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host . However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size . On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis . We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain . A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated . The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent) . We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 8046 - 51 Stability of the mitochondrial genome requires an amino-terminal domain of yeast mitochondrial RNA polymerase; Wang Y et al.; Mitochondrial RNA (mtRNA) polymerases are related to bacteriophage RNA polymerases, but contain a unique amino-terminal extension of unknown origin and function . In addition to harboring mitochondrial targeting information, we show here that the amino-terminal extension of yeast mtRNA polymerase is required for a mtDNA maintenance function that is separable from the known RNA polymerization activity of the enzyme . Deletion of 185 N-terminal amino acids from the enzyme results in a temperature-sensitive mitochondrial petite phenotype, characterized by increased instability and eventual loss of the mitochondrial genome . Mitochondrial transcription initiation in vivo is largely unaffected by this mutation and expression of just the amino-terminal portion of the protein in trans partially suppresses the mitochondrial defect, indicating that the amino-terminal extension of the enzyme harbors an independent functional domain that is required for mtDNA replication and/or stability . These results suggest that amino-terminal extensions present in mtRNA polymerases comprise functional domains that couple additional activities to the transcription process in mitochondria. J Mol Biol, 1999 Jul 2, 290(1), 1 - 7 Raman optical activity of filamentous bacteriophages: hydration of alpha-helices; Blanch EW et al.; We report the first observations of vibrational Raman optical activity (ROA) on intact viruses . Specifically, ROA spectra of the filamentous bacteriophages Pf1, M13 and IKe in aqueous solution were measured in the range approximately 600-1800 cm-1 . On account of its ability to probe directly the chiral elements of biomolecular structure, ROA has provided a new perspective on the solution structures of these well-studied systems . The ROA spectra of all three are dominated by signatures of helical elements in the major coat proteins, as expected from pre-existing data . The helical elements generate strong sharp positive ROA bands at approximately 1300 and 1342 cm-1in H2O solution, but in2H2O solution the approximately 1342 cm-1bands disappear completely . The spectra are similar to those of polypeptides under conditions that produce alpha-helical conformations . Our present results, together with results from other studies, suggest that the positive approximately 1342 cm-1ROA bands are generated by a highly hydrated form of alpha-helix, and that the positive approximately 1300 cm-1bands originate in alpha-helix in a more hydrophobic environment . The presence of significant amounts of highly hydrated helical sequences accords with the known flexibility of these viruses . Differences of spectral detail for Pf1, M13 and IKe demonstrate that ROA is sensitive to subtle variations of conformation and hydration within the major coat proteins, with M13 and IKe possibly containing more non-helical structure than Pf1 . The ROA spectra of Pf1 at temperatures above and below that at which a structural transition is known to occur (approximately 10 degrees C) reveal little difference in the protein conformation between the two forms, but there are indications of changes in DNA structure . Biochemistry, 1999 Jun 29, 38(26), 8367 - 76 NMR structure and functional studies of the Mu repressor DNA-binding domain; Ilangovan U et al.; The repressor protein of bacteriophage Mu establishes and maintains lysogeny by shutting down transposition functions needed for phage DNA replication . It interacts with several repeated DNA sequences within the early operator, preventing transcription from two divergent promoters . It also directly represses transposition by competing with the MuA transposase for an internal activation sequence (IAS) that is coincident with the operator and required for efficient transposition . The transposase and repressor proteins compete for the operator/IAS region using homologous DNA-binding domains located at their amino termini . Here we present the solution structure of the amino-terminal DNA-binding domain from the repressor protein determined by heteronuclear multidimensional nuclear magnetic resonance spectroscopy . The structure of the repressor DNA-binding domain provides insights into the molecular basis of several temperature sensitive mutations and, in combination with complementary experiments using flourescence anisotropy, surface plasmon resonance, and circular dichroism, defines the structural and biochemical differences between the transposase and repressor DNA-binding modules . We find that the repressor and enhancer domains possess similar three-dimensional structures, thermostabilities, and intrinsic affinities for DNA . This latter result suggests that the higher affinity of the full-length repressor relative to that of the MuA transposase protein originates from cooperative interactions between repressor protomers and not from intrinsic differences in their DNA-binding domains . In addition, we present the results of nucleotide and amino acid mutagenesis which delimits the minimal repressor DNA-binding module and coarsely defines the nucleotide dependence of repressor binding. Biochemistry, 1999 Jun 22, 38(25), 8094 - 101 Steady-state kinetic characterization of RB69 DNA polymerase mutants that affect dNTP incorporation; Yang G et al.; The function of six highly conserved residues (Arg482, Lys483, Lys486, Lys560, Asn564, and Tyr567) in the fingers domain of bacteriophage RB69 DNA polymerase (RB69 gp43) were analyzed by kinetic studies with mutants in which each of these residues was replaced with Ala . Our results suggest that Arg482, Lys486, Lys560, and Asn564 contact the incoming dNTP during the nucleotidyl transfer reaction as judged by variations in apparent Km and kcat values for dNTP incorporation by these mutants compared to those for the exonuclease deficient parental polymerase under steady-state conditions . On the basis of our studies, as well as on the basis of the crystal structure of RB69 gp43, we propose that a conformational change in the fingers domain, which presumably occurs prior to polymerization, brings the side chains of Arg482, Lys486, Lys560, and Asn564 into the vicinity of the primer-template terminus where they can contact the triphosphate moiety of the incoming dNTP . In particular, on the basis of structural studies reported for the "closed" forms of two other DNA polymerases and from the kinetic studies reported here, we suggest that (i) Lys560 and Asn564 contact the nonbonding oxygens of the alpha and beta phosphates, respectively, and (ii) both Arg482 and Lys486 contact the gamma phosphate oxygens of the incoming dNTP of RB69 gp43 prior to the nucleotidyl transfer reaction . We also found that Ala substitutions at each of these four RB69 gp43 sites could incorporate dGDP as a substrate, although with markedly reduced efficiency compared to that with dGTP . In contrast in the parental exo- background, the K483A and Y567A substituted enzymes could not use dGDP as a substrate for primer extension . These results, taken together, are consistent with the putative roles of the four conserved residues in RB69 gp43 as stated above. Biochemistry, 1999 Jun 15, 38(24), 7696 - 709 Sliding clamp of the bacteriophage T4 polymerase has open and closed subunit interfaces in solution; Alley SC et al.; The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (beta clamp), and yeast (PCNA) are required for processive DNA synthesis by their cognate DNA polymerases . The X-ray crystal structures of all three of these clamps have been shown to be closed, circular complexes . This paper reports investigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation, fluorescence, and hydrodynamic modeling . Mutants of gp45 with inter- and intrasubunit disulfide bonds were created to alter the solution structure of gp45, with additional mutagenesis used to investigate the importance of the proline-rich loop region found between the two domains of each gp45 monomer . The wild-type gp45 trimer assembles from monomers cooperatively with a dissociation constant of 0.21 microM2 and values between 0.088 and 0 . 32 microM2 for the mutants . Velocity ultracentrifugation experiments showed that wild-type gp45 possesses a sedimentation coefficient strongly dependent on concentration, typical of asymmetric or elongated molecules, that when extrapolated to zero concentration yields a sedimentation coefficient of 4.0 S . The loop and the disulfide mutants exhibited sedimentation coefficients with little concentration dependence, typical of symmetric or spherical molecules, that when extrapolated to zero concentration yielded sedimentation coefficients of 4.4-4.8 S . The lower sedimentation coefficient in the former case is consistent with wild-type gp45 being more asymmetric or elongated than the mutant forms . Fluorescence-resonance energy-transfer experiments were used to measure the distance between two amino acids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interface . For an intrasubunit disulfide mutant, the distance between these two amino acids was determined to be 19 A (14 A in the X-ray crystal structure), consistent with a closed complex . For the mutants without intrasubunit disulfides, the efficiency of fluorescence-resonance energy transfer was in accord with a model of gp45 being an open complex composed of two closed subunit interfaces and a third open interface separated by a distance of 35-38 A . The collective data supplemented with hydrodynamic modeling were consistent with gp45 subunit separation achieved within the plane of the gp45 ring. J Bacteriol, 1999 Jul, 181(13), 3967 - 73 Regulation of podJ expression during the Caulobacter crescentus cell cycle; Crymes WB Jr et al.; The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of the Caulobacter crescentus cell cycle . Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally . In contrast, hyperexpression of podJ results in a lethal cell division defect . Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA . Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter . Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site . Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors. J Bacteriol, 1999 Jul, 181(13), 4050 - 61 Site-specific recombination of temperate Myxococcus xanthus phage Mx8: genetic elements required for integration; Magrini V et al.; Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination . The Mx8 int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome . When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3' ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M . xanthus genome . Although Int-mediated site-specific recombination occurs between attP and either attB1 (within trnD1) or attB2 (within trnD2), the attP x attB1 reaction is highly favored and often is accompanied by a deletion between attB1 and attB2 . The int gene is the only Mx8 gene required in trans for attP x attB recombination . The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state . All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate attP x attB recombination efficiently . The attP core lies within the int coding sequence, and the product of integration is a prophage in which the 3' end of int is replaced by host sequences . The prophage intX gene is predicted to encode an integrase with a different C terminus. Electrophoresis, 1999 Jun, 20(6), 1162 - 70 Mutation detection using fluorescent enzyme mismatch cleavage with T4 endonuclease VII; Babon JJ et al.; Mutation detection techniques are often limited by sensitivity, ease of use and short fragment lengths . Enzyme mismatch cleavage (EMC) is a technique capable of rapidly scanning 1 kbp fragments of DNA for mutations . It relies on the ability of a bacteriophage resolvase enzyme, T4 endonuclease VII, to cleave DNA at single base pair mismatches and small heteroduplex loops . Originally the process was performed using radioactively labeled DNA and the results analysed after denaturing polyacrylamide gel electrophoresis and autoradiography . However, access to systems capable of detecting fluorescent species migrating through a gel and the widespread availability of fluorescently tagged primers have greatly improved upon the original technique . A number of mutations were detected using fluorescent EMC and the results compared to performing the technique using radiolabeled DNA . Fluorescent EMC detected the presence, position and number of mutations in DNA fragments as large as 1 kbp . The fluorescent method was found to have advantages over the original method in its ease of use, increase in signal-to-noise ratio and the ability to multiplex samples by labeling DNA fragments with different fluorophores . This improvement on an already established method provides a sensitive, robust technique for mutation detection. Pac Symp Biocomput . 1999;:532-41. Similarity between the sequences of taxol-selected peptides and the disordered loop of the anti-apoptotic protein, Bcl-2; Rodi DJ et al.; The anti-cancer drug taxol is known to bind to and induce the polymerization of tubulin and has recently been shown to bind to the anti-apoptotic protein Bcl-2, but not to its homolog, Bcl-XL . Libraries of random peptides displayed on the surface of a bacteriophage were screened to select those exhibiting affinity for taxol . The sequences of these peptides were compared to sequences of proteins involved in mitosis and apoptosis . No significant similarities were detected between the sequences of tubulins and the taxol-selected peptides . However, a high level of similarity exists between the selected peptides and the disordered loop of Bcl-2 . Conversely, there was little similarity between the sequences of the selected peptides and Bcl-XL . These results indicate that peptides displayed on the surface of a bacteriophage can mimic the ligand-binding behavior of a disordered protein loop and that comparison of the sequences of affinity-selected peptides with protein sequences can be predictive for ligand binding. J Biol Chem, 1999 Jun 25, 274(26), 18341 - 50 Functional and physical interaction between WRN helicase and human replication protein A; Brosh RM Jr et al.; The human premature aging disorder Werner syndrome (WS) is associated with a large number of symptoms displayed in normal aging . The WRN gene product, a DNA helicase, has been previously shown to unwind short DNA duplexes (</=53 base pairs) in a reaction stimulated by single-stranded DNA-binding proteins . We have studied the helicase activity of purified WRN protein on a variety of DNA duplex substrates to characterize the unwinding properties of the enzyme in greater detail . WRN helicase can catalyze unwinding of long duplex DNA substrates up to 849 base pairs in a reaction dependent on human replication protein A (hRPA) . Escherichia coli SSB and bacteriophage T4 gene 32 protein (gp32) completely failed to stimulate WRN helicase to unwind long DNA duplexes indicating a specific functional interaction between WRN and hRPA . So far, there have been no reports of any physical interactions between WRN helicase and other proteins . In support of the functional interaction, we demonstrate a direct interaction between WRN and hRPA by coimmunoprecipitation of purified proteins . The physical and functional interaction between WRN and hRPA suggests that the two proteins may function together in vivo in a pathway of DNA metabolism such as replication, recombination, or repair. Exp Eye Res, 1999 Jun, 68(6), 679 - 84 Epitope discovery using bacteriophage display: the minimum epitope of an anti-IRBP antibody; Tighe PJ et al.; The purpose of this study was to determine, using random peptide library (RPL) technologies, the minimal epitope requirements of the mouse monoclonal anti-interphotoreceptor-retinoid-binding protein antibody, H3B5 . This previously characterized antibody is used as an example to examine whether RPL's offer a relatively easy and rapid route to obtaining detailed epitope mapping data.A pentadecamer random peptide library (RPL) displayed on the major coat protein (gene 8) of filamentous bacteriophage (F88-4-15) was used as a target for selection by the anti-IRBP monoclonal antibody, H3B5 . Three rounds of library selection were performed, and 90 of the resultant RPL clones were examined for affinity to H3B5 by enzyme-linked immunosorbent assay (ELISA) . DNA sequencing of ELISA positive clones provided sequence of the region encoding the random peptide.After three rounds of selection of the RPL, 76.7% of clones examined interacted with H3B5, 17.7% did not show significant binding and 6.6% bound to control antibody also . The essential elements of the peptide epitope were determined by sequence comparison of 24 clones to be the four amino-acid sequence (Aspartic or glutamic acid)-Proline-Arginine-(Leucine, Isoleucine or Valine) . This motif {(D/E) PR (L/I/V)} is in agreement, but at greater resolution, than previous synthetic peptide studies where the motif AASEDPRL was identified . Other motifs were found which bound to H3B5 but did not share primary structure similarities (peptidomimetics) . Selection from a RPL has rapidly defined the minimal requirements for the H3B5 epitope in fine detail . Such a process offers great potential for investigating antibody-antigen interactions and core sequences of an epitope, and enables the identification of motifs in other proteins which may be recognized by the antibody, providing information on possible cross-reactivity . J Gen Virol, 1999 Jun, 80 ( Pt 6), 1463 - 9 Vaccinia virus-bacteriophage T7 expression vector for complementation analysis of late gene processes; Eckert D et al.; A vaccinia virus-bacteriophage T7 RNA polymerase hybrid transient expression vector has been developed for complementation analysis of late gene functions in vaccinia virus . The conditionally defective virus ts21 was modified to express the bacteriophage T7 RNA polymerase . The derived virus, vtsT7, was conditionally defective in viral late gene expression but produced high levels of a target protein under the control of a T7 promoter at non-permissive temperatures . The level of beta-galactosidase expression under the control of a T7 promoter was slightly lower in vtsT7 infections than those with the prototypical T7 RNA polymerase vector vTF7.3 . However, the levels of expression for the human immunodeficiency virus envelope gene, a protein which undergoes post-translational modification, was slightly higher in vtsT7 infections, suggesting that some proteins may be expressed better in the absence of vaccinia virus late gene expression . Infections using vtsT7 at a low m.o.i . at 39 degrees C resulted in the accumulation of high molecular mass, non-linear replicative intermediates of vaccinia virus DNA replication and high levels of expression of a transfected gene proximal to a T7 promoter . The virus vtsT7 provides a means for the analysis of potential trans-acting factors participating in vaccinia virus late processes such as resolution of DNA replicative intermediates. Nucleic Acids Res, 1999 Jul 1, 27(13), 2777 - 84 An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications; Haapa S et al.; Transposons are mobile genetic elements and have been utilized as essential tools in genetics over the years . Though highly useful, many of the current transposon-based applications suffer from various limitations, the most notable of which are: (i) transposition is performed in vivo, typically species specifically, and as a multistep process; (ii) accuracy and/or efficiency of the in vivo or in vitro transposition reaction is not optimal; (iii) a limited set of target sites is used . We describe here a genetic analysis methodology that is based on bacteriophage Mu DNA transposition and circumvents such limitations . The Mu transposon tool is composed of only a few components and utilizes a highly efficient and accurate in vitro DNA transposition reaction with a low stringency of target preference . The utility of the Mu system in functional genetic analysis is demonstrated using restriction analysis and genetic footprinting strategies . The Mu methodology is readily applicable in a variety of current and emerging transposon-based techniques and is expected to generate novel approaches to functional analysis of genes, genomes and proteins. J Mol Biol, 1999 Jun 25, 289(5), 1219 - 30 Topological selectivity of a hybrid site-specific recombination system with elements from Tn3 res/resolvase and bacteriophage P1 loxP/Cre; Kilbride E et al.; In order to investigate the functions of the parts of the Tn 3 recombination site res, we created hybrid recombination sites by placing the loxP site for Cre recombinase adjacent to the "accessory" resolvase-binding sites II and III of res . The efficiency and product topology of in vitro recombination by Cre between two of these hybrid sites were affected by the addition of Tn 3 resolvase . The effects of resolvase addition were dependent on the relative orientation and spacing of the elements of the hybrid sites . Substrates with sites II and III of res close to loxP gave specific catenated or knotted products (four-noded catenane, three-noded knot) when resolvase and Cre were added together . The product topological complexity increased when the length of the spacer DNA segment between loxP and res site II was increased . Similar resolvase-induced effects on Cre recombination product topology were observed in reactions of substrates with loxP sites adjacent to full res sites . The results demonstrate that the res accessory sites are sufficient to impose topological selectivity on recombination, and imply that intertwining of two sets of accessory sites defines the simple catenane product topology in normal resolvase-mediated recombination . They are also consistent with current models for the mechanism of catalysis by Cre . J Mol Biol, 1999 Jun 18, 289(4), 815 - 26 Inhibition of Escherichia coli RNA polymerase by bacteriophage T7 gene 2 protein; Nechaev S et al.; The 64 amino acid residue product of bacteriophage T7 gene 2 (gp2) binds the Escherichia coli RNA polymerase and inhibits transcription . We localized the gp2 binding site to within 53 amino acid residues in the functionally dispensable region of the RNA polymerase beta' subunit . We investigated the effect of gp2 on transcription at a -10/-35 promoter and at an "extended -10" promoter . Our results indicate that binding of gp2 to the sigma70holoenzyme (Esigma70) prevents promoter recognition at -10/-35 promoters . Once open promoter complexes are formed, however, Esigma70transcription is resistant to gp2, since gp2 can no longer bind RNA polymerase . Surprisingly, transcription inhibition by gp2 is both sigma and promoter-specific . gp2 has little effect on Esigma70transcription from an extended -10 promoter, which does not depend on sigma70region 4 interactions with the -35 promoter box for its activity . gp55-dependent phage T4 late promoter transcription is also resistant to gp2 . From these results, we conclude that the interaction of the sigma70region 4 with the -35 consensus promoter element is the primary target of gp2 inhibition . J Mol Biol, 1999 Jun 18, 289(4), 747 - 75 The topological mechanism of phage lambda integrase; Crisona NJ et al.; Bacteriophage lambda integrase (Int) is a versatile site-specific recombinase . In concert with other proteins, it mediates phage integration into and excision out of the bacterial chromosome . Int recombines intramolecular sites in inverse or direct orientation or sites on separate DNA molecules . This wide spectrum of Int-mediated reactions has, however, hindered our understanding of the topology of Int recombination . By systematically analyzing the topology of Int reaction products and using a mathematical method called tangles, we deduce a unified model for Int recombination . We find that, even in the absence of (-) supercoiling, all Int reactions are chiral, producing one of two possible enantiomers of each product . We propose that this chirality reflects a right-handed DNA crossing within or between recombination sites in the synaptic complex that favors formation of right-handed Holliday junction intermediates . We demonstrate that the change in linking number associated with excisive inversion with relaxed DNA is equally +2 and -2, reflecting two different substrates with different topology but the same chirality . Additionally, we deduce that integrative Int recombination differs from excisive recombination only by additional plectonemic (-) DNA crossings in the synaptic complex: two with supercoiled substrates and one with relaxed substrates . The generality of our results is indicated by our finding that two other members of the integrase superfamily of recombinases, Flp of yeast and Cre of phage P1, show the same intrinsic chirality as lambda Int . Structure Fold Des, 1999 Mar 15, 7(3), 289 - 96 The three-dimensional structure of a DNA translocating machine at 10 A resolution; Valpuesta JM et al.; BACKGROUND: Head-tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process . Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions . RESULTS: Recombinant bacteriophage phi29 connectors have been crystallized in two-dimensional aggregates . An average projection image and a three-dimensional map have been obtained at 8 A and 10 A resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals . The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass . The three-dimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail . At the junction of the two domains, 12 smaller appendages are visualized . A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through . CONCLUSIONS: The propeller-like structure of the phi29 connector strengthens the notion of the connector rotating during DNA packaging . The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation. Vet Res, 1999 Mar-Jun, 30(2-3), 157 - 79 Pathogenic diversity of Escherichia coli and the emergence of 'exotic' islands in the gene stream; Dozois CM et al.; Escherichia coli is a highly adaptive bacterial species that is both a member of the commensal intestinal flora and a versatile pathogen associated with numerous types of intestinal and systemic infections in humans and other animals . The spectrum of diseases caused by E . coli is due to the acquisition of specific virulence genes harbored on plasmids, bacteriophages, or within distinct DNA segments termed pathogenicity islands (PAIs) that are absent from the genomes of commensal E . coli strains . PAIs are likely to have been transferred horizontally and may have integrated into the E . coli chromosome through bacteriophage or plasmid integration or transposition . The contribution of intergenic inheritance to the adaptation and evolution of E . coli, types of PAIs associated with different groups of pathogenic E . coli and approaches to identify unique sequence islands (USIs), some of which might confer pathogenicity, in E . coli and other bacteria are presented. J Bacteriol, 1999 Jun, 181(12), 3688 - 94 Structural and functional roles of the surface-exposed loops of the beta-barrel membrane protein OmpA from Escherichia coli; Koebnik R; The N-terminal domain of the OmpA protein from Escherichia coli, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel beta-barrel whose eight transmembrane beta-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops . This protein domain serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins . In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations . All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology . This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops . The shortening of surface-exposed loops did not reduce the thermal stability of the protein . However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3 . Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation . An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest beta-structured integral membrane protein known to date . These results represent a further step toward the development of artificial outer membrane proteins. Brain Res Mol Brain Res, 1999 Jun 8, 69(2), 209 - 22 Characterization of a promoter for the human glial cell line-derived neurotrophic factor gene; Baecker PA et al.; To address the regulation of glial cell line-derived neurotrophic factor (GDNF) gene expression, we have isolated 5' extended cDNAs, cloned the human GDNF gene, and characterized the promoter . GDNF-encoding 5' extended cDNAs containing a novel exon were isolated via reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from human fetal kidney and adult skeletal muscle . The GDNF gene was isolated from a human genomic library in a P1 bacteriophage vector . Analysis of the 5' flanking sequence revealed a promoter that lacks a CCAAT-box motif and is GC rich . Consensus binding sites for a variety of transcription factors have been identified in the promoter . Promoter/reporter plasmids have been constructed by fusion of the promoter and a portion of exon I to a luciferase gene . The promoter/reporter construct and a number of promoter deletions were transiently transfected into two human cell lines known to express GDNF . Multiple enhancer and silencer regions were revealed as well as a minimal promoter sufficient for basal transcription . Finally, a RT-PCR assay, specific for transcripts originating from this GDNF promoter, was developed and used to show that this promoter is active in vivo . The results suggest GDNF is regulated in a complex manner . J Mol Biol, 1999 Jun 4, 289(2), 249 - 60 Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4; Lin H et al.; Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead . Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes . Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies . The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4 . Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites . In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro.The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues) . As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site . Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly internalizes terminase regions within the portal in the packasome complex . Both similarities and differences are seen in comparison to analogous sites which have been identified in phages T3 and lambda . J Virol, 1999 Jul, 73(7), 5714 - 21 Packaging-competent capsids of a herpes simplex virus temperature-sensitive mutant have properties similar to those of in vitro-assembled procapsids; Rixon FJ et al.; Newcomb and coworkers (W . W . Newcomb, F . L . Homa, D . R . Thomsen, F . P . Booy, B . L . Trus, A . C . Steven, J . V . Spencer, and J . C . Brown, J . Mol . Biol . 263:432-446, 1996; W . W . Newcomb, F . L . Homa, D . R . Thomsen, Z . Ye, and J . C . Brown, J . Virol . 68:6059-6063, 1994) have recently described an in vitro herpes simplex virus (HSV) capsid assembly product which, because of certain parallels between its properties and those of bacteriophage proheads, they have designated the procapsid . As in their bacteriophage counterparts, there are marked differences between the structures of the two types of particle, and conversion from the procapsid to the capsid form requires extensive reconfiguration of the subunits . This reconfiguration occurs spontaneously upon extended in vitro incubation . One of the distinctive features of the HSV procapsids is that, unlike mature capsids, they are unstable and disassemble upon storage at 2 degrees C . Using a mutant of HSV type 1 (ts1201), which has a lesion in the protease responsible for maturational cleavage of the scaffolding protein, we have demonstrated that capsids present within cells infected at nonpermissive temperatures are also cryosensitive and disappear if the cells are incubated at 0 degrees C . This suggests that ts1201 capsids may resemble procapsids in structure . However, ts1201 capsids remain cryosensitive following extended incubation at an elevated temperature and, therefore, do not appear to undergo the spontaneous reconfiguration seen with in vitro-assembled procapsids . The lesion in ts1201 is reversible, and capsids formed at the nonpermissive temperature can undergo maturational cleavage and go on to form infectious virions following downshift to permissive temperatures . The sensitivity of ts1201 capsids to low temperatures is closely correlated with the cleavage status of the scaffolding protein, suggesting that proteolysis may act to trigger their conversion to the stable form . The experiments described here provide the firmest evidence yet that the procapsid has a biologically relevant role in the virus life cycle. Mol Microbiol, 1999 May, 32(4), 715 - 27 Characterization of the dual start motif of a class II holin gene; Barenboim M et al.; Holins are small membrane proteins that, at a genetically programmed time in a bacteriophage infective cycle, allow bacteriolytic enzymes, or endolysins, to escape to the periplasm and to attack the cell wall . Most holins fall into two sequence classes, I and II, based on the number of potential transmembrane domains (three for class I and two for class II) . The prototype class I holin gene, S lambda, has a dual start motif and encodes not only the effector holin, Slambda105, but also an inhibitor, Slambda107, with a Met-Lys ...extension at the terminus . The prototype class II holin gene of phage 21, S 21, begins with the motif Met-Lys-Ser-Met ..., and a potential RNA secondary structure overlaps the Shine-Dalgarno sequence . Here, we demonstrate that (i) two protein products are elaborated from S 21, S2171 and S2168; (ii) the shorter product is required for lysis; (iii) the longer product, S2171, inhibits S 21 function; and (iv) the Lys-2 residue is important for the inhibitor function . Moreover, the RNA stem-loop structure is involved in the downregulation of S2171 synthesis . However, our results suggest that, in S 21, different segments of the single consensus Shine-Dalgarno sequence serve the two translational starts . These results show that the dual start motifs of class II holin genes are functionally homologous to those of class I holin genes. Curr Biol, 1999 Jun 3, 9(11), 569 - 78 Localization and anchoring of mRNA in budding yeast; Beach DL et al.; BACKGROUND: Eukaryotic cells localize selected mRNAs to a region of the cell as a means to sequester proteins . Signals within the 3' untranslated region (3' UTR) facilitate mRNA localization by both actin and microtubule cytoskeletal systems . Recently, an mRNA in the yeast Saccharomyces cerevisiae, ASH1, was shown to coalesce into a discrete particle that is maintained at the bud tip . Mutations in five genes, SHE1-SHE5, cause defects in particle formation and/or localization of the ASH1 transcript . Factors at the destination of the mRNA transport remain to be identified . RESULTS: We have developed a system to label mRNA in living yeast with green fluorescent protein (GFP) and follow the dynamics of mRNA movement and localization . Constitutively expressing an ASH1 mRNA containing the bacteriophage MS2 coat-protein binding site adjacent to the ASH1 3' UTR allowed us to visualize ASH1 mRNA with an MS2-coat-protein-GFP fusion protein (together denoted 'gRNAASH1') . The gRNAASH1 was restricted to the bud tip in small to large budded cells, migrated to the bud neck prior to cell separation and then rapidly relocalized to the incipient site of bud growth . It also localized to regions of polarized growth during mating . In cells lacking Bud6p/Aip3p or Bnilp/She5p, which are involved in polarity establishment and actin organization, gRNAASH1 migrated to the bud but failed to remain at the bud tip . These studies reveal discrete transport and anchoring steps in mRNA localization . CONCLUSIONS: The ASH1 mRNA was maintained at sites of polarized growth throughout the vegetative and mating cell cycles . Bud6p/Aip3p and Bni1p/She5p are required to maintain the transcript at the cortical bud cap. Eur J Immunol, 1999 May, 29(5), 1581 - 6 A prototype pathogen bound ex vivo to human erythrocyte complement receptor 1 via bispecific monoclonal antibody complexes is cleared to the liver in a mouse model; Nardin A et al.; Immune complexes (IC) bound to the primate erythrocyte (E) complement receptor (CR1) are cleared from the circulation of primates and localized to the liver . IC can be bound to E CR1 either via C3b opsonization or with cross-linked mAb complexes (heteropolymers, HP) which contain an mAb specific for CR1 and a mAb specific for a prototype pathogen . The long-term goal of our work is to apply the HP to the treatment of human diseases associated with blood-borne pathogens . Therefore we have investigated the feasibility of a non-primate model by studying clearance in mice of bacteriophage phiX174 bound via HP to primate E . E-HP-phiX174 complexes were prepared in vitro and infused into the circulation of mice under conditions allowing short term survival of E in the circulation . By radioimmunoassays and flow cytometry, we found that phiX174 is removed from E and cleared from the circulation coincident with loss of HP and CR1, and that the majority of cleared phiX174 is localized to the liver . Through the use of HP constructed with Fab' fragments, we verified the requirement for the Fc portion of the mAb in clearance; inhibition of C3 activation delayed clearance, suggesting a role for complement . The present findings in the mouse confirm previous observations in the non-human primate model. J Biol Chem, 1999 Jun 11, 274(24), 16813 - 8 The Doc toxin and Phd antidote proteins of the bacteriophage P1 plasmid addiction system form a heterotrimeric complex; Gazit E et al.; The toxin (Doc) and antidote (Phd) proteins of the plasmid addiction system of bacteriophage P1 were purified as a complex . Cocrystals of the complex contained a 2:1 molar ratio of Phd:Doc as assayed by dye binding following SDS-polyacrylamide gel electrophoresis and as determined by amino acid analysis . Gel filtration and analytical ultracentrifugation revealed that the two addiction proteins interact in solution to form a P2D trimer composed of one Doc and two Phd molecules . These results support a model in which Phd inhibits the toxic activity of Doc by direct binding . Circular dichroism experiments showed that changes in secondary structure accompany formation of the heterotrimeric complex, raising the possibility that Phd may act by an allosteric mechanism . Studies of Phd and Doc molecules labeled with fluorescent energy donor and acceptor groups gave an equilibrium dissociation constant of about 0.8 microM(2) and a very short, sub second half-life of complex dissociation . As a consequence, low concentrations of free Doc toxin are likely to be present both transiently and in the steady state in cells containing the Phd antidote, making mechanisms of single-hit Doc toxicity improbable. Int J Food Microbiol, 1999 Mar 1, 47(1-2), 43 - 50 The use of a fluorescent bacteriophage assay for detection of Escherichia coli O157:H7 in inoculated ground beef and raw milk; Goodridge L et al.; The objective of this study was to develop a fluorescent bacteriophage assay (FBA) for the detection of E . coli O157:H7 in ground beef and raw milk . The FBA is a two step assay that combines immunomagnetic separation, to separate the target organism from mixed culture, with a highly specific fluorescently stained bacteriophage to label the E . coli O157:H7 cells . When used in conjunction with flow cytometry, the FBA was able to detect 2.2 CFU/g of artificially contaminated ground beef following a 6 h enrichment . Between 10(1) and 10(2) CFU/ml of artificially contaminated raw milk were detectable after a 10 h enrichment step . The results show that the FBA is potentially useful as a rapid technique for the preliminary detection of E . coli O157:H7 in food. J Immunol Methods, 1999 Apr 22, 224(1-2), 89 - 99 Mapping of the minimal domain encoding a conformational epitope by lambda phage surface display: factor VIII inhibitor antibodies from haemophilia A patients; Kuwabara I et al.; Haemophilia A patients who receive repeated transfusion of fVIII concentrates often develop inhibitor alloantibodies, resulting in reduced efficacy of the therapy . Determination of fVIII epitopes for the alloantibodies is essential for an understanding of their inhibitory effect on blood coagulation . Random fragments of fVIII displayed on lambda phage particles were selected using two patient plasmas immobilized onto the surface of a microtiter plate . A set of clones defined the minimal domain that consisted of 157 amino acid residues including cysteine at both boundaries . The minimal domain absorbed most of the binding activities of the plasmas to fVIII, suggesting that the domain contains a major determinant for the plasmas . Site-directed mutagenesis and chemical denaturation of the domain confirmed that a tertiary structure formed by the disulfide bridge was recognized by the antibodies . The epitope domain defined overlaps with fVIII binding sites to vWf and phospholipid, and may play an important role in blood coagulation . Thus, the bacteriophage lambda surface display may be useful for mapping the minimal folding domain of various protein antigens that contain a conformational epitope. J Mol Biol, 1999 Jun 11, 289(3), 503 - 16 Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate; Jones JM et al.; PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication . MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome . However, this fork lacks the single-stranded DNA needed to load DnaB . Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA . The helicase activity of PriA could then open the duplex to create the DnaB binding site . In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template . PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction . Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro . This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication . Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks . As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication . Biophys J, 1999 Jun, 76(6), 3267 - 77 Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids; Thuman-Commike PA et al.; Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid . To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid . In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA . Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits . To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids . The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein . Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein . Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid. Epidemiol Infect, 1999 Apr, 122(2), 337 - 41 Molecular typing of Escherichia coli O157:H7 (H-) isolates from cattle in Japan; Akiba M et al.; A total of 77 Escherichia coli O157:H7 (H-) isolates from cattle in Japan were investigated by molecular biological methods . Most of these isolates (43 isolates) possessed the stx-2 gene, but not stx1 . Fifteen bacteriophage types and 50 pulsed-field gel electrophoresis (PFGE) profiles were observed . One isolate was indistinguishable from the human outbreak strain by these methods . This indicates that cattle must be considered as a possible source of human E . coli O157:H7 infection in Japan. Nucleic Acids Res, 1999 Jun 15, 27(12), 2494 - 502 Analysis of the cooperative thermal unfolding of the td intron of bacteriophage T4; Brion P et al.; The thermal stability of folded transcripts of the td intron of bacteriophage T4 that carried up to three base substitutions was investigated by temperature gradient gel electrophoresis (TGGE) and UV melting . The unfolding of this autocatalytic group I intron is endothermic and entropically driven . Although the effects of mutations in base pairs follow in most cases the expected order G-C>A-U>G.U>A.C, the extent of global destabilization varies strongly according to the helix in which substitutions are located . Effects are more pronounced in the P7 helix which forms, together with the P3 helix, the central pseudoknot of group I introns . The stability of the tertiary fold was also monitored as a function of ionic concentration and of the nature of the ion . At low ionic strength, the stabilizing effect of divalent ions is independent of the nature of the ion . However, with increasing ionic concentration, stabilization is most pronounced for Mg2+and less for Mn2+with Ca2+having intermediate effects . Ammonium ions stabilize folding with a similar slope, but at concentrations about 400 times higher than divalent ions . The apparent enthalpic change associated with the tertiary structure thermal unfolding increases strongly with increasing concentrations of divalent ions . A similar increase is observed with the monovalent ammonium ions . However, in the presence of NH4+ions, the apparent enthalpy peaks at 2.0 M and decreases beyond. FEBS Lett, 1999 Apr 30, 450(1-2), 89 - 94 Spontaneous rearrangements in RNA sequences; Chetverina HV et al.; The ability of RNAs to spontaneously rearrange their sequences under physiological conditions is demonstrated using the molecular colony technique, which allows single RNA molecules to be detected provided that they are amplifiable by the replicase of bacteriophage Qbeta . The rearrangements are Mg2+-dependent, sequence-non-specific, and occur both in trans and in cis at a rate of 10(-9) h(-1) per site . The results suggest that the mechanism of spontaneous RNA rearrangements differs from the transesterification reactions earlier observed in the presence of Qbeta replicase, and have a number of biologically important implications. J Biol Chem, 1999 Jun 4, 274(23), 16431 - 6 RNA polymerase-specific nucleosome disruption by transcription in vivo; Sathyanarayana UG et al.; The nucleosomal chromatin structure within genes is disrupted upon transcription by RNA polymerase II . To determine whether this disruption is caused by transcription per se as opposed to the RNA polymerase source, we engineered the yeast chromosomal HSP82 gene to be exclusively transcribed by bacteriophage T7 RNA polymerase in vivo . Interestingly, we found that a fraction of the T7-generated transcripts were 3' end processed and polyadenylated at or near the 3' ends of the hsp82 and the immediately downstream CIN2 genes . Surprisingly, the nucleosomal structure of the T7-transcribed hsp82 gene remained intact, in marked contrast to the disrupted structure generated by much weaker, basal level transcription of the wild type gene by RNA polymerase II under non-heat shock conditions . Therefore, disruption of chromatin structure by transcription is dependent on the RNA polymerase source . We propose that the observed RNA polymerase dependence for transcription-induced nucleosome disruption may be related either to the differential recruitment of chromatin remodeling complexes, the rates of histone octamer translocation and nucleosome reformation during polymerase traversal, and/or the degree of transient torsional stress generated by the elongating polymerase. J Biol Chem, 1999 Jun 4, 274(23), 16141 - 6 Folding and stability of mutant scaffolding proteins defective in P22 capsid assembly; Greene B et al.; Bacteriophage P22 scaffolding subunits are elongated molecules that interact through their C termini with coat subunits to direct icosahedral capsid assembly . The soluble state of the subunit exhibits a partially folded intermediate during equilibrium unfolding experiments, whose C-terminal domain is unfolded (Greene, B., and King, J . (1999) J . Biol . Chem . 274, 16135-16140) . Four mutant scaffolding proteins exhibiting temperature-sensitive defects in different stages of particle assembly were purified . The purified mutant proteins adopted a similar conformation to wild type, but all were destabilized with respect to wild type . Analysis of the thermal melting transitions showed that the mutants S242F and Y214W further destabilized the C-terminal domain, whereas substitutions near the N terminus either destabilized a different domain or affected interactions between domains . Two mutant proteins carried an additional cysteine residue, which formed disulfide cross-links but did not affect the denaturation transition . These mutants differed both from temperature-sensitive folding mutants found in other P22 structural proteins and from the thermolabile temperature-sensitive mutants described for T4 lysozyme . The results suggest that the defects in these mutants are due to destabilization of domains affecting the weak subunit-subunit interactions important in the assembly and function of the virus precursor shell. Biochemistry, 1999 May 25, 38(21), 6943 - 52 Interaction of bacteriophage lambda protein phosphatase with Mn(II): evidence for the formation of a {Mn(II)}2 cluster; Rusnak F et al.; The interaction of bacteriophage lambda protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry . Reconstitution of bacteriophage lambda protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein . This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms . The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2 . The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster . EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G . Temperature-dependent studies indicated that these features arose from an excited state . Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity . The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites . These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases. Electrophoresis, 1999 Apr-May, 20(4-5), 653 - 9 Replication-induced protein synthesis and its importance to proteomics; Humphery-Smith I; Replication-induced protein synthesis (RIPS) can occur following the passage of the replisome due to transcription initiated by RNA polymerase in association with: (i) negative supercoiling trailing the replisome / replication fork, (ii) hemimethylation prior to the action of dam methylase, (iii) transient derepression following passage of the replisome/replication fork and prior to renewed synthesis of the repressor gene-product, and (iv) 'sliding clamp' accessory DNA-binding proteins binding to the lagging strand DNA duplex to retard rotational upstream propagation of supercoils . The latter include subunits of DNA polymerase III in Escherichia coli and gp45 in T4 bacteriophage . By far the most convincing evidence for the existence of RIPS comes from the pulse of protein synthesis which follows the passage of the replisome in late T4 bacteriophage, the dynamics of replication in Escherichia coli, recent results from cDNA high-density expression arrays in yeast and the workings of the lac-operon . More circumstantial evidence is provided by 'leaky' or 'aberrant' protein expression in genetic systems where attempts have been made to turn off protein synthesis by molecular means . In higher vertebrates, RIPS may have a potentially important role in explaining the mechanisms by which thymic and peripheral immune self-tolerance is established, either directly through antigen presentation on dendritic cells or through the presentation of peptides derived from T-cells . The latter model is preferred, as young T-cells will have recently divided and will be dying in large numbers near the antigen-presenting dendritic cells in the thymus . The functional utility of RIPS would appear to be linked to both facilitating cellular metabolism and an improved survival during stress . RIPS, as a potentially universal molecular phenomenon, presents proteomics with numerous challenges and opportunities, both technical and commercial. FEMS Immunol Med Microbiol, 1999 May, 24(1), 57 - 62 The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-O157 verotoxin-producing E . coli; Currie CG et al.; A mouse monoclonal antibody specific for the R3 lipopolysaccharide core type of Escherichia coli was used to determine the core type of E . coli O157:H7 and other non-O157 verotoxin-producing E . coli strains . Lipopolysaccharide extracts from 28 clinical isolates were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting and all were found to have the R3 core . None of the core lipopolysaccharide from the strains tested reacted with the control R1 and R2 specific monoclonal antibodies . A common core type between all the verotoxin-producing E . coli strains tested may be significant when considering the immune response to these bacteria, and to the receptor for the VT bacteriophage. Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6025 - 30 Making artificial antibodies: a format for phage display of combinatorial heterodimeric arrays; Gao C et al.; The gene VII protein (pVII) and gene IX protein (pIX) are associated closely on the surface of filamentous bacteriophage that is opposite of the end harboring the widely exploited pIII protein . We developed a phagemid format wherein antibody heavy- and light-chain variable regions were fused to the amino termini of pVII and pIX, respectively . Significantly, the fusion proteins interacted to form a functional Fv-binding domain on the phage surface . Our approach will be applicable to the display of generic peptide and protein libraries that can form combinatorial heterodimeric arrays . Consequently, it represents a first step toward artificial antibodies and the selection of novel biological activities. J Biol Chem, 1999 May 28, 274(22), 15305 - 14 Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins; Hang Q et al.; The terminase enzyme from bacteriophage lambda is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid . We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage lambda and, in particular, the nucleoprotein complexes involved in these processes . These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging . Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA subunits . We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate . Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step . Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme . The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo . These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor . The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA . Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme . The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies. Biopolymers, 1998, 48(2-3), 181 - 95 Protein-RNA recognition; De Guzman RN et al.; The x-ray structure of the glutamine aminoacyl tRNA synthetase bound to its cognate tRNA(Gln) and ATP was reported by Steitz and co-workers in 1989, providing the first high resolution structure of a protein-RNA complex . Since then, high resolution structures have been reported for RNA complexes with five other tRNA synthetases, the elongation factor Tu, the bacteriophage MS2 coat protein, the human spliceosomal U1A and U2B"-U1A' proteins, and the HIV-1 nucleocapsid protein . Although the number of high resolution structures of protein-RNA complexes are rather small, some general themes have begun to emerge regarding the nature and mechanisms of protein-RNA recognition. Nature, 1999 May 6, 399(6731), 80 - 3 Structural basis for initiation of transcription from an RNA polymerase-promoter complex; Cheetham GM et al.; Although the single-polypeptide-chain RNA polymerase from bacteriophage T7 (T7RNAP), like other RNA polymerases, uses the same mechanism of polymerization as the DNA polymerases, it can also recognize a specific promoter sequence, initiate new RNA chains from a single nucleotide, abortively cycle the synthesis of short transcripts, be regulated by a transcription inhibitor, and terminate transcription . As T7RNAP is homologous to the Pol I family of DNA polymerases, the differences between the structure of T7RNAP complexed to substrates and that of the corresponding DNA polymerase complex provides a structural basis for understanding many of these functional differences . T7RNAP initiates RNA synthesis at promoter sequences that are conserved from positions -17 to +6 relative to the start site of transcription . The crystal structure at 2.4 A resolution of T7RNAP complexed with a 17-base-pair promoter shows that the four base pairs closest to the catalytic active site have melted to form a transcription bubble . The T7 promoter sequence is recognized by interactions in the major groove between an antiparallel beta-loop and bases . The amino-terminal domain is involved in promoter recognition and DNA melting . We have also used homology modelling of the priming and incoming nucleoside triphosphates from the T7 DNA-polymerase ternary complex structure to explain the specificity of T7RNAP for ribonucleotides, its ability to initiate from a single nucleotide, and the abortive cycling at the initiation of transcription. Genetika, 1999 Jan, 35(1), 101 - 4 {DNA-fingerprinting of representatives of Bovinae subfamilies using the telomere markers (TTAGGG)4}; Semenova SK et al.; The (TTAGGG)4 oligonucleotide homologous to telomeric tandem repeats of human chromosomes was used for the first time as a multilocus hybridization probe for the analysis of genome variability in the two genera (Bos and Bison) of the Bovinae subfamily . DNA profiles for cattle, banteng, aurochs, and bison were obtained . Hybridization spectra were represented by the discrete individual- and species-specific bands characterized by codominant inheritance . For comparison, DNA profiles of the same samples obtained using the bacteriophage M13 DNA probe are presented . The usefulness of the microsatellite examined for the testing of pedigrees, description of intra- and interbreed variability as well as for determining relationships and the origins of the species of the Bovinae subfamily is discussed. J Mol Biol, 1999 May 21, 288(5), 899 - 909 Domain architecture of the bacteriophage phi29 connector protein; Valle M et al.; Viral connectors are essential components of the DNA packaging machinery . They interact with nucleic acids and other viral components to translocate DNA inside the viral head . We have attempted to locate the different structural and functional domains of the phage Phi29 connector using a combination of approaches to generate different antigenic probes . Complexes of native connectors with either monoclonal or monospecific antibodies were studied by immunoelectron microscopy and image averaging methods . The data were merged in a model of the connector domain structure at 2-3 nm resolution . This epitope mapping provides a general outline of the folding architecture of the connector polypeptide, following a complicated threading that places the amino and carboxyl-terminals in close alignment in the narrower domain at 2-3 nm from the top of the connector . The appendages are built up by a long and highly immunogenic sequence (amino acid residues 153 to 206) . The RNA binding domain forms part of the top of the narrow conical area of the connector, a flexible region that undergoes structural changes during viral morphogenesis . The DNA binding domain is located not far away, 2-3 nm below, in the outer side of the narrow conical part . The precise location of the functional domains of the connector, as well as their relative positions provide the first experimental framework for understanding the connector function . J Mol Biol, 1999 May 14, 288(4), 649 - 57 Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability; Holliger P et al.; Infection of Escherichia coli by filamentous bacteriophages is mediated by the minor phage coat protein g3p and involves two distinct cellular receptors, the F' pilus and the periplasmic protein TolA . Recently we have shown that the two receptors are contacted in a sequential manner, such that binding of TolA by the N-terminal domain g3p-D1 is conditional on a primary interaction of the second g3p domain D2 with the F' pilus . In order to better understand this process, we have solved the crystal structure of the g3p-D1D2 fragment (residues 2-217) from filamentous phage fd to 1.9 A resolution and compared it to the recently published structure of the same fragment from the related Ff phage M13 . While the structure of individual domains D1 and D2 of the two phages are very similar (rms<0.7 A), there is comparatively poor agreement for the overall D1D2 structure (rms>1.2 A) . This is due to an apparent movement of domain D2 with respect to D1, which results in a widening of the inter-domain groove compared to the structure of the homologous M13 protein . The movement of D2 can be described as a rigid-body rotation around a hinge located at the end of a short anti-parallel beta-sheet connecting domains D1 and D2 . Structural flexibility of at least parts of the D1D2 structure was also suggested by studying the thermal unfolding of g3p: the TolA binding site on D1, while fully blocked by D2 at 37 degrees C, becomes accessible after incubation at temperatures as low as 45 degrees C . Our results support a model for the early steps of phage infection whereby exposure of the coreceptor binding site on D1 is facilitated by a conformational change in the D1D2 structure, which in vivo is induced by binding to the F' pilus on the host cell and which can be mimicked in vitro by thermal unfolding . J Mol Biol, 1999 May 14, 288(4), 595 - 608 The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus phiX174; Dokland T et al.; An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174 . Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion . The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution . This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively . The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation . The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability . We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein . We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly . A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching . J Mol Biol, 1999 May 7, 288(3), 291 - 304 Post-transcriptional control of bacteriophage T4 gene 25 expression: mRNA secondary structure that enhances translational initiation; Nivinskas R et al.; Secondary structure of the mRNA in the translational initiation region is an important determinant of translation efficiency . However, the secondary structures that enhance or facilitate translation initiation are rare . We have previously proposed that such structure may exist in the case of bacteriophage T4 gene 25 translational initiation region, which contains three potential Shine-Dalgarno sequences (SD1, SD2, and SD3) with a spacing of 8, 17, and 27 nucleotides from the initiation codon of this gene, respectively . We now present results that clearly demonstrate the existence of a hairpin structure that includes SD1 and SD2 sequences and brings the SD3, the most typical of these Shine-Dalgarno sequences, to a favourable spacing with the initiation codon of gene 25.Using a phage T7 expression system, we show that mutations that prevent the formation of hairpin structure or eliminate the SD3 sequence result in a decreased level of gp25 synthesis . Double mutation in base-pair V restores the level of gene 25 expression that was decreased by either of the two mutations (C-to-G and G-to-C) alone, as predicted by an effect attributable to mRNA secondary structure . We introduced the mutations into the bacteriophage T4 by plasmid-phage recombination . Changes in the plaque and burst sizes of T4 mutants, carrying single and double mutations in the translational initiation region of gene 25, strongly suggest that the predicted mRNA secondary structure controls (enhances) the level of gene 25 expression in vivo . Hybridization of total cellular RNA with a gene 25 specific probe indicated that secondary structure or mutations in the translational initiation region do not notably affect the 25 mRNA stability . Immunoblot analysis of gp25 in Escherichia coli cells infected by T4 mutants showed that mRNA secondary structure increases the level of gp25 synthesis by three- to fourfold . Since the secondary structure increases the level of gp25 synthesis and does not affect mRNA stability, we conclude that this structure enhances translation initiation . We discuss some features of two secondary structures in the translational initiation regions of T4 genes 25 and 38 . J Mol Biol, 1999 Apr 23, 288(1), 21 - 8 Epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (Inoviridae); Kneissel S et al.; To map the accessible surface of filamentous bacteriophage fd particles, the epitope structures of polyclonal rabbit serum and three mouse monoclonal antibodies raised against complete phage were analysed . Western blot analysis confirmed the major coat protein, gene VIII product (g8p or pVIII), to be the antigen . Overlapping peptides were synthesised by spot synthesis on cellulose membranes, covering the whole sequence of g8p . Each of the three tested monoclonal antibodies, B62-FE2, B62-GF3/G12 and B62-EA11, reacted with a core epitope covering ten amino acid residues at or near the amino terminus of g8p . The epitope recognised by B62-FE2 consists of the ten N-terminal amino acid residues of g8p . Extension of the amino terminus by various sequences did not inhibit binding, indicating that a terminal amino group is not essential for the interaction . Both B62-GF3/G12 and B62-EA11 recognise internal epitopes covering amino acid residues 3 to 12 of g8p . The epitopes of the polyclonal rabbit serum were also confined to the 12 N-terminal amino acid residues . The contribution of individual amino acid residues to the binding was analysed by a set of peptides containing individual amino acids exchanged by glycine . Accessible residues were Glu2, Asp4, Asp5, Pro6, Lys8, Phe11 and Asp12 . The positions of the essential amino acid residues within the epitope are in accordance with a helical conformation of the amino-terminal region of g8p . Further, the results suggest new designs of phage display screening vectors to improve their performance in analysing non-linear epitopes . J Virol Methods, 1999 Apr, 79(1), 65 - 74 A functional measles virus replication and transcription machinery encoded by the vaccinia virus genome; Howley PM et al.; Measles virus encodes three proteins required for the encapsidation, transcription and replication of viral genomes . The genes for these proteins have been inserted into the vaccinia virus genome together with the gene for the bacteriophage T7 RNA polymerase . Cells infected with this recombinant virus were able to encapsidate, transcribe and replicate a CAT gene positioned in the negative polarity behind a T7 promoter and flanked by measles virus genomic termini . Inhibition of the accumulation of the nucleocapsid proteins by actinomycin D led to an increase in CAT expression . Thus the measles virus polymerase activity, encoded by the vaccinia genome, was regulated by the level of measles proteins just as the authentic polymerase . The recombinant vaccinia described in this study could be useful for the production of measles virus-like particles encoding foreign genes and employed in vaccination or gene therapy strategies. Mol Gen Genet, 1999 Apr, 261(3), 508 - 13 Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita; Bois F et al.; An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita . The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments . Aa-polB is located between the SSU rDNA (5' region) and a gene for tRNA(Asn) (3' region), and is separated from these genes by two A + T-rich intergenic regions of 1048 (5' region) and 3864 (3' region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin . The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons) . The domains involved in the 3'-->5' exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs . The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA . The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe . As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level . The likelihood of the coexistence of Aa-POLB and Pol gamma in the A . aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol gamma from yeast to human. J Bacteriol, 1999 May, 181(10), 3123 - 8 Bacteriophage T4 rnh (RNase H) null mutations: effects on spontaneous mutation and epistatic interaction with rII mutations; Bebenek A et al.; The bacteriophage T4 rnh gene encodes T4 RNase H, a relative of a family of flap endonucleases . T4 rnh null mutations reduce burst sizes, increase sensitivity to DNA damage, and increase the frequency of acriflavin resistance (Acr) mutations . Because mutations in the related Saccharomyces cerevisiae RAD27 gene display a remarkable duplication mutator phenotype, we further explored the impact of rnh mutations upon the mutation process . We observed that most Acr mutants in an rnh+ strain contain ac mutations, whereas only roughly half of the Acr mutants detected in an rnhDelta strain bear ac mutations . In contrast to the mutational specificity displayed by most mutators, the DNA alterations of ac mutations arising in rnhDelta and rnh+ backgrounds are indistinguishable . Thus, the increase in Acr mutants in an rnhDelta background is probably not due to a mutator effect . This conclusion is supported by the lack of increase in the frequency of rI mutations in an rnhDelta background . In a screen that detects mutations at both the rI locus and the much larger rII locus, the r frequency was severalfold lower in an rnhDelta background . This decrease was due to the phenotype of rnh rII double mutants, which display an r+ plaque morphology but retain the characteristic inability of rII mutants to grow on lambda lysogens . Finally, we summarize those aspects of T4 forward-mutation systems which are relevant to optimal choices for investigating quantitative and qualitative aspects of the mutation process. Biochemistry, 1999 May 11, 38(19), 6035 - 42 The solution structure and dynamics of an Arc repressor mutant reveal premelting conformational changes related to DNA binding; Nooren IM et al.; The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Arc repressor . A backbone rmsd versus the average of 0.37 A was obtained for the well-defined core region . For both Arc-MYL and the wild-type Arc repressor, evidence for a fast equilibrium between a packed ("in") conformation and an extended ("out") conformation of the side chain of Phe 10 was found . In the MYL mutant, the "out" conformation is more highly populated than in the wild-type Arc repressor . The Phe 10 is situated in the DNA-binding beta-sheet of the Arc dimer . While its "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Arc, where the Phe 10 ring contacts the phosphate backbone {Raumann, B . E., et al . (1994) Nature 367, 754-757} . As well as DNA binding, denaturation by urea and high temperatures induces the functionally active "out" conformation . With a repacking of the hydrophobic core, this characterizes a premelting transition of the Arc repressor . The dynamical properties of the Arc-MYL and the wild-type Arc repressor were further characterized by 15N relaxation and hydrogen-deuterium exchange experiments . The increased main chain mobility at the DNA binding site compared to that of the core of the protein as well as the reorientation of the side chain of Phe 10 is suggested to play an important role in specific DNA binding. J Nucl Med, 1999 May, 40(5), 883 - 8 Identification of receptor ligands with phage display peptide libraries; Koivunen E et al.; With the development and maturation of the technology of displaying peptides on bacteriophage, it has become possible to isolate peptide ligands to various targets . In the phage display strategy, up to 10(9) peptides of different permutations are expressed on the surface of filamentous phage . Thus, peptides capable of binding target molecules in vitro and even target tissues in vivo can be identified . In recent years, a series of libraries that display degenerate peptides of different lengths have been constructed, and specific ligands to cell surface receptors, such as integrins, have been isolated . In the in vivo biopanning, peptides targeting distinct organs or tumors have been rescued after intravenous administration of phage libraries into mice . In one application, the isolated peptide ligands have been used to direct a cytotoxic drug to tumor vasculature in mice . Further applications in radioimaging and radiotherapy are being investigated. J Biol Chem, 1999 May 14, 274(20), 13999 - 4005 Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease; Gonciarz-Swiatek M et al.; It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease . We report for the bacteriophage lambda O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX . The N-terminal portion of lambda O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly . Consistent with these results, deletion of the first 18 amino acids of lambda O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids . We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O . However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease . Based on our results with lambda O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX. Protein Eng, 1999 Mar, 12(3), 225 - 33 Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor; Jana NK et al.; The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid . The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing . The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively . Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions . Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied . Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C . Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures. Biochemistry (Mosc), 1999 Apr, 64(4), 379 - 83 Co-expression of gene 31 and 23 products of bacteriophage T4; Kurochkina LP et al.; Folding of the major capsid protein of bacteriophage T4 encoded by gene 23 is aided by Escherichia coli GroEL chaperonin and phage co-chaperonin gp31 . In the absence of gene product (gp) 31, aggregates of recombinant gp23 accumulate in the cell similar to inclusion bodies . These aggregates can be solubilized with 6 M urea . However, the protein cannot form regular structures in solution . A system of co-expression of gp31 and gp23 under the control of phage T7 promoter in E . coli cells has been constructed . Folding of entire-length gp23 (534 amino acid residues) in this system results in the correctly folded recombinant gp23, which forms long regular structures (polyheads) in the cell. Immunotechnology, 1999 Mar, 4(3-4), 175 - 84 Cross-reactive epitope mimics in a fragmented-genome phage display library derived from the rickettsia, Cowdria ruminantium; Fehrsen J et al.; BACKGROUND: Epitopes can be mapped by comparing immunoaffinity-selected peptides from fragmented-gene display libraries with the target gene . With larger libraries derived from unsequenced genomes, this is not possible . Spurious epitope mimics may be created by expressing DNA in a variety of meaningless reading frames and orientations . OBJECTIVES: To determine empirically whether panning a large fragmented-genome phage display library with antibodies to MAP1, the major antigenic protein of the rickettsial parasite Cowdria ruminantium, would result in the selection of irrelevant, cross-reactive mimotopes . STUDY DESIGN: A gene III phage library displaying peptides derived from C . ruminantium was constructed using cloned DNA from a bacteriophage lambda genomic library . After in vivo excision, plasmids were cleaved with PvuII followed by PCR . Genes with a PvuII site, including MAP1 were therefore not amplified . DNA was sonicated, partially digested with DNase and cloned into the display vector fUSE2 . Affinity-purified MAP1 antibodies were used for panning . Peptides expressed by panned phages were tested for recognition in Western blot and ELISA . Oligonucleotides representing antigenic sequences were used to locate their encoding DNA sequences in the original lambda library . The phage display library was also panned with two monoclonal antibodies (Mabs) against bluetongue virus (BTV) . RESULTS: Five different peptide sequences were selected from the MAP1-deficient phage display library . None was identical to MAP1, but four peptides had regions that were similar, both to each other, and to the parasite protein . They produced strong signals in ELISA and Western blot . None could be located to any C . ruminantium open reading frame . Two BTV Mabs recognised a sequence similar to their authentic epitope . CONCLUSION: Large genome-targeted phage display libraries may be sufficiently diverse to allow the selection of peptides that mimic actual antigenic determinants . This diversity may be exploited in the search for useful epitopes. Curr Biol, 1999 Apr 22, 9(8), R296 - 300 Virus assembly: Imaging a molecular machine; Wikoff WR et al.; A recent structural study of a double-stranded DNA bacteriophage has provided remarkable new insights into the assembly of a complex virus particle and ushers in a new era in the imaging of non-icosahedral viruses. Int Arch Allergy Immunol, 1999 Feb-Apr, 118(2-4), 119 - 21 Mimotope and anti-idiotypic vaccines to induce an anti-IgE response; Stadler BM et al.; We have defined epitopes on human IgE by screening different phage display random peptide libraries with a monoclonal anti-IgE antibody termed BSW17 . The selected mimotopes and epitopes within the Cepsilon3 and Cepsilon4 region of IgE induced antibodies that were nonanaphylactogenic and had biological activity similar to BSW17 . The chemically synthesized and KLH-coupled IgE epitopes or mimotopes were used to induce an anti-IgE response in rhesus monkeys . The immunized rhesus monkeys were subsequently protected in a PCA test when sensitized with human IgE and triggered with the corresponding allergen . Furthermore, using the same monoclonal anti-IgE antibody, we also generated an anti-idiotypic antibody that showed sequence homology with the IgE epitope in the Cepsilon3 domain . This anti-idiotypic antibody as well as the mimotopes were then used in a mouse model to induce orally an anti-IgE immune response . For this purpose mice were fed by intragastric gavages with bacteriophages displaying the small IgE-homologous structures . Orally immunized mice produced serum anti-IgE antibodies that were inhibited by BSW17 suggesting that it may be possible to induce a systemic anti-IgE response orally. J Clin Invest, 1999 May, 103(9), 1243 - 52 CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris; Abrams JR et al.; Engagement of the B7 family of molecules on antigen-presenting cells with their T cell-associated ligands, CD28 and CD152 (cytotoxic T lymphocyte-associated antigen-4 {CTLA-4}), provides a pivotal costimulatory signal in T-cell activation . We investigated the role of the CD28/CD152 pathway in psoriasis in a 26-week, phase I, open-label dose-escalation study . The importance of this pathway in the generation of humoral immune responses to T cell-dependent neoantigens, bacteriophage phiX174 and keyhole limpet hemocyanin, was also evaluated . Forty-three patients with stable psoriasis vulgaris received 4 infusions of the soluble chimeric protein CTLA4Ig (BMS-188667) . Forty-six percent of all study patients achieved a 50% or greater sustained improvement in clinical disease activity, with progressively greater effects observed in the highest-dosing cohorts . Improvement in these patients was associated with quantitative reduction in epidermal hyperplasia, which correlated with quantitative reduction in skin-infiltrating T cells . No markedly increased rate of intralesional T-cell apoptosis was identified, suggesting that the decreased number of lesional T cells was probably likely attributable to an inhibition of T-cell proliferation, T-cell recruitment, and/or apoptosis of antigen-specific T cells at extralesional sites . Altered antibody responses to T cell-dependent neoantigens were observed, but immunologic tolerance to these antigens was not demonstrated . This study illustrates the importance of the CD28/CD152 pathway in the pathogenesis of psoriasis and suggests a potential therapeutic use for this novel immunomodulatory approach in an array of T cell-mediated diseases. Mutat Res, 1999 Apr 26, 441(1), 59 - 72 Ethylnitrosourea-induced mutation and molecular analysis of transgenic mice containing the gpt shuttle vector; Yamada T et al.; Novel transgenic mice were developed in order to study the in vivo mutagenesis . The transgenic mice carried pCGK shuttle vector, which contained the Escherichia coli gpt gene as a mutational target, the kanamycin-resistant gene (Kanr) and cos region derived from bacteriophage lambda . The shuttle vector can be recovered from the transgenic mouse genome into the gpt-deficient E . coli by an in vitro packaging method and is selectable as a Kanr phenotype . Mutations induced at the gpt gene can be easily detected with a selective agent, 6-thioguanine (6-TG) . In the previous study, the pCGK shuttle vector was incorporated into Chinese hamster CHL/IU cells and the resultant transgenic cell line was shown to be a useful system to study in vitro mutagenesis at the gpt gene . Therefore, an advantage of the shuttle vector is that in vivo mutational data obtained from the transgenic mouse can be compared with those of transgenic cell line in vitro . A transgenic CD-1 mouse line, designated as #128, that carried approximately 50 copies of pCGK shuttle vectors, was selected among 4 transgenic mouse lines . To investigate the sensitivity of the #128 line, the transgenic mice were treated with a single intraperitoneal injection of 250 mg/kg of N-ethyl-N-nitrosourea (ENU) or with 50 mg kg-1 day-1 of ENU for 5 consecutive days, and bone marrow, spleen and liver were dissected to investigate their mutational responses . The background mutant frequency was between 18x10(-6) and 75x10(-6) among all tissues tested . ENU induced significant increases in the mutant frequency above the background level in all three tissues at 14 days after single or 5-day treatment with the chemical . The increases in the mutant frequencies in bone marrow, spleen and liver were 6.4- to 6.8-fold, 3.0- to 5.6-fold and 3.0- to 3.3-fold, respectively . The shuttle vector DNA was recovered from the bone marrow of both spontaneous and ENU-treated mice and the gpt gene was amplified by polymerase chain reaction . The amplified DNA was subject to DNA sequence analysis . Out of 79 spontaneous and 52 ENU-induced mutants, the gpt gene could be amplified from 28 spontaneous and 46 ENU-induced mutants . DNA sequence analysis showed that predominant mutations were identified as A:T to T:A transversions (22 out of 46 sequenced mutants) and G:C to A:T transitions (9/46) in ENU-induced mutants, whereas G:C to T:A transversions (7 out of 28 sequenced mutants) were predominant in spontaneous mutants . These results demonstrate that this transgenic mouse, in combination with the transgenic CHL/IU cell line, is a useful system to study in vivo and in vitro mutational events at the same target gene . J Mol Biol, 1999 Apr 16, 287(5), 867 - 76 The primary immunity determinant in modulating the lysogenic immunity of the filamentous bacteriophage cf; Cheng CM et al.; Bacteriophage cf is the first single-stranded DNA phage that has been shown to set up a stable lysogenic state with its genome integrated into the host chromosome . From the isolation and characterization of a virulent mutant, cf-tv2, we report the first investigation into the mechanisms of the immunity established by the filamentous bacteriophage . The mutation in cf-tv2 enables the phage to produce plaques on lawns of cf lysogenic cells . The mutation was defined as a 49-nucleotide deletion located in a 0.59 kb NcoI/KpnI fragment of cf replicative form DNA . Two messages, cM1 and cM2, transcribed from the immunity region of wild-type cf but in opposite directions, were detected . In cf-tv2, the 49-nucleotide deletion abolishes cM2 transcription . The primer extension assay suggests a possible RNA-RNA interaction directed by base-pairing of the cM1 and cM2 RNAs . A frameshift mutation of the open reading frame ORF 165, encoded by cM2, resulted in a 10(5) plating efficiency on the cf lysogen . These observations suggest that both RNA-RNA interaction and repressor protein inhibition are involved in the mechanism of cf immunity . A model is proposed for the regulation of cf immunity . J Comput Biol, 1999 Spring, 6(1), 1 - 12 On the structure of the scaffolding core of bacteriophage T4; Berger B et al.; The scaffolding core in bacteriophages is a temporary structure that plays a major role in determining the shape of the protein shell that encapsulates the viral DNA . In the currently accepted structure for the scaffolding core in bacteriophage T4, there is a symmetry mismatch between the protein shell, which has fivefold symmetry, and the scaffolding core, which is believed to consist of six helical chains . The analysis of T4 giant prohead data that was used to determine this structure made an implicit assumption about the manner in which giant proheads flatten during preparation for electron microscopy . Namely, it was assumed that techniques for analysis of Fourier transforms of flattened single-layer cylinders could be applied independently to the shell and the core . This analysis makes the implicit assumption that connections between the core and the shell do not affect the flattening process, and thus are stretched or broken during the flattening process . Reexamination of the experimental data shows that this assumption is likely to be incorrect . A reanalysis shows that the data could be consistent with six, eight, or 10 helical chains, and is a better match for eight or 10 helical chains . Ten helical chains would match the fivefold symmetry of the shell . The 10-helix core model is particularly attractive because it suggests a Vernier mechanism, which is able to explain the process of length determination in giant head mutants of T4 . It is possible that the same assumption has been made for structural analysis of other biological systems . If this is the case, any results obtained should also be reexamined. J Struct Biol, 1999 Apr-May, 125(2-3), 216 - 22 A data mining approach for analyzing density maps representing macromolecular structures; Ravantti JJ et al.; Results of electron microscopy-based three-dimensional reconstructions of macromolecules or their complexes are usually stored as density maps . Each point ("voxel") in the map represents a density value and one approach for studying details of the map is to display an isosurface enclosing areas of interest . We have taken a data mining approach not only focusing on the areas of immediate interest but determining all possible separate entities ("blobs") from a density map . After the entire density map is analyzed with our mining program BLOBBER, properties of all detected blobs can be browsed and sets of blobs can be visualized using our VIZBLOB program . Since BLOBBER analyzes density maps using only density information and relates it to spatial relationships, BLOBBER can be used to analyze symmetrical or asymmetrical density maps from any source . To test our program we have analyzed published bacteriophage PRD1 reconstructions . We identified various structural details ranging from individual proteins to major complexes such as the whole capsid shell and more elaborate details of possible connections between membrane interfaces . This approach can also be a useful preprocessing tool for visualizing reconstructions . Biospectroscopy, 1999, 5(1), 3 - 8 A novel Raman spectrophotometric method for quantitative measurement of nucleoside triphosphate hydrolysis; Jenkins RH et al.; A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity . The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm(-1)) and diphosphate (1085 cm(-1)) moieties of the NTPase substrate and product, respectively . The reliability of the method is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4) of bacteriophage phi6, for which comparative NTPase activities have been estimated independently by radiolabeling assays . The Raman-determined rate for adenosine triphosphate substrate (8.6 +/- 1.3 micromol x mg(-1) x min(-1) at 40 degrees C) is in good agreement with previous estimates . The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP) . Advantages of the present protocol include conservative sample requirements (approximately 10(-6) g enzyme/protocol) and relative ease of data collection and analysis . The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity. Transfusion, 1999 Apr, 39(4), 364 - 71 A molecular approach for isolating high-affinity Fab fragments that are useful in blood group serology; Czerwinski M et al.; BACKGROUND: Multiple mouse hybridoma antibodies recognize the antigens of the MNS blood group system . The Fab fragments of several of these antibodies were expressed on bacteriophage and as soluble proteins . The parental N92 anti-N IgG monoclonal antibody (parental N92 MoAb), but not its monovalent, soluble Fab fragment (N92 Fab fragment), agglutinated antigen-positive red cells by an antiglobulin method . Light-chain shuffling was used to isolate mutant N92 Fab fragments with higher affinity that would functio