Bioseparation, 1998, 7(2), 79 - 88 Virus removal from bioproducts using ultrafiltration membranes modified with latex particle pretreatment; Bellara SR et al.; Ultrafiltration is an attractive process for virus removal from bioproducts owing to its high throughput as well as the fact that the operation is carried out under ambient conditions (damage to proteins is highly limited) . The principal concern regarding the adoption of conventional ultrafiltration membranes for virus removal is the possibility of the virus passing through abnormally large pores or surface imperfections on the membrane surface . The chief principle behind the present work is to pretreat the membrane by blocking the abnormally large pores using latex particles . Experimental work was conducted to validate this pretreatment using the bacteriophage phi x 174 as a model virus . The results attained were highly encouraging . Different sizes of latex particles were tested by treating a 100 KD molecular weight cut-off membrane, and the transmission of phage (suspended in buffer) through this membrane assessed . In the absence of any particle pretreatment, a virus clearance of 4.78 log reduction value was observed for this membrane . The transmission of phage through the membrane could be reduced by an order of magnitude using 0.11 micron latex particles, or two orders of magnitude using a combination of 0.11 and 0.50 micron particles . The application of latex particles did not hinder the transport of protein through the 100 KD membrane . Protein sieving coefficients obtained using this membrane were 91%, 16% and 2%, for lysozyme, HSA and IgG, respectively. J Cell Biol, 1998 Oct 5, 143(1), 253 - 66 Cre-loxP-mediated inactivation of the alpha6A integrin splice variant in vivo: evidence for a specific functional role of alpha6A in lymphocyte migration but not in heart development; Gimond C et al.; Two splice variants of the alpha6 integrin subunit, alpha6A and alpha6B, with different cytoplasmic domains, have previously been described . While alpha6B is expressed throughout the development of the mouse, the expression of alpha6A begins at 8.5 days post coitum and is initially restricted to the myocardium . Later in ontogeny, alpha6A is found in various epithelia and in certain cells of the immune system . In this study, we have investigated the function of alpha6A in vivo by generating knockout mice deficient for this splice variant . The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of alpha6A in embryonic stem cells, and the deletion resulted in the expression of alpha6B in all tissues that normally express alpha6A . We show that alpha6A-/- mice develop normally and are fertile . The substitution of alpha6A by alpha6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration . Furthermore, T cells differentiated normally in alpha6A-/- mice . However, the substitution of alpha6A by alpha6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes . Lymphocyte homing to the lymph nodes, which involves various types of integrin-ligand interactions, was not affected in the alpha6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking . Nevertheless, the expression of alpha6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions. J Virol Methods, 1998 Sep, 74(1), 99 - 108 A new and simple method for concentration of enteric viruses from water; Li JW et al.; A new type of electropositive filter media particle was tested to adsorb bacteriophage f2 and enteric viruses from tap water . 3 x nutrient broth (pH 7.2) was used to elute the adsorbed viruses, and the eluate was reconcentrated by polyethylene glycol (Mw 6000) precipitation with a final concentration of 10% (wt./vol.) . The adsorption of bacteriophage was reliable and efficient, and not affected by the pH value, temperature, turbidity and organic materials in water . This method gave a recovery of Polio 1 virus 96.0% for small-volume tap water; 88.7% for large-volume water; and gave a comparable recovery of HAV, Coxsackie B3 and Echo 7 from tap water . The concentration method need not acidify virus-containing water, add exogenous multivalent cation salts, or require expensive equipment. Anal Biochem, 1978 Dec, 91(2), 432 - 40 Isolation of circular viral and complementary strand DNA from bacteriophage f1 duplex replicative-form DNA; Bayne ML et al.; A general method has been developed for the large scale isolation of intact, circular, single-stranded DNA molecules of each strand from supercoiled duplex DNA . The method involves the conversion of the supercoiled duplex DNA to singly nicked, relaxed duplex DNA; denaturation of the duplex DNA; separation of circular DNA molecules from linear DNA molecules; and separation of circular plus and minus strands . All separations involve zone sedimentation . No isopycnic gradient centrifugation is required . The last step in the purification, the separation of plus and minus strands, can be easily adapted for small scale analytical measurements of the amounts of plus and minus strand DNA. Anal Biochem, 1978 Nov, 91(1), 343 - 9 Characterization of density gradients prepared by freezing and thawing a sucrose solution; Davis PB et al.; Density gradients of sucrose can be prepared in large numbers by successive freezing and thawing of sucrose solutions . Gradients of other solute molecules, such as salt and detergents, also form and this could affect subsequent sedimentation behavior of some molecules . However, the sedimentation behavior of native and denatured DNA of bacteriophage lambda was essentially isokinetic under the conditions used thus making these gradients comparable with ones prepared manually, at least for preparative sedimentation work with nucleic acids. J Mol Biol, 1998, 283(1), 155 - 77 Structure of the capsid of Pf3 filamentous phage determined from X-ray fibre diffraction data at 3.1 A resolution; Welsh LC et al.; We have recorded X-ray diffraction patterns at 3.1 A resolution from magnetically aligned fibres of the Pf3 strain of filamentous bacteriophage (Inovirus) . The patterns are similar to patterns from the higher-temperature form of the Pf1 strain, indicating that the Pf3 and Pf1 virions have the same helix symmetry and similar protein subunit shape . This is of particular interest, given that the primary structures of the two protein subunits are quite different; and the nucleotide/protein subunit ratio in the Pf3 virion is more than twice that in Pf1, indicating important differences in DNA packaging . We have built a molecular model of the Pf3 protein capsid based on the model of Pf1, and refined it against the diffraction data using simulated annealing . The refinement confirms that the two structures are similar, which may reflect a fundamental motif of alpha-helix packing . However, there are some differences between the structures: the Pf3 subunit appears to be completely alpha-helical, beginning at the N terminus, whereas the first few residues of the Pf1 subunit are not helical; and the structure of the C-terminal region of the Pf3 subunit at the inner surface of the tubular capsid indicates that DNA/protein interactions in this virion may involve both aromatic side-chains and positively charged side-chains, whereas those in the Pf1 virion involve predominantly only the latter . In the course of this work, we have developed new approaches to refinement and validation of helical structures with respect to continuous transform fibre diffraction data . C R Acad Sci III, 1998 Jan, 321(1), 1 - 4 Accelerated cyclization of lambda DNA; Jary D et al.; In the presence of spermidine, the DNA molecule of the bacteriophage lambda undergoes a coil-globule transition . We report here that the cyclization of this molecule in its globular state is greatly accelerated (by more than 10(4)-fold) in comparison with the cyclization reaction taking place in the coil conformation. Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1008 - 11 Crystallization and preliminary X-ray crystallographic studies of the 13-fold symmetric portal protein of bacteriophage SPP1; Jekow P et al.; Portal proteins are cyclical oligomers which play essential roles in bacteriophage pro-capsid formation, DNA packaging, and in connector formation . Bacteriophage SPP1 portal protein (gp6) is a turbine-like molecule with 13-fold symmetry {Dube et al . (1993) EMBO J . 12, 1303-1309} . The purified protein was crystallized with polyethylene glycol 400 as the precipitating agent using the vapor-diffusion method . Salt conditions were selected based on the properties of gp6 in different ionic environments . X-ray diffraction data up to a resolution of 7.85 A were measured from frozen crystals with orthorhombic space group C2221 and cell dimensions a = 180.5 (5), b = 223.5 (5), c = 417 (1) A . The asymmetric unit contains one tridecameric portal protein with 57.3 kDa subunits . The self-rotation searches confirm the 13-fold symmetry of the crystallized protein. Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 878 - 90 Structure determination of the phiX174 closed procapsid; Dokland T et al.; The structure of a procapsid of the single-stranded DNA bacteriophage ++phiX174 was determined to 3.5 A resolution . The crystal space group was I213 with a unit-cell length of 774 A . The unit cell contained 16 icosahedral virus particles, each situated on a crystallographic threefold axis . Thus, there are two independent one-thirds of a particle per asymmetric unit, and a total of 40-fold non-crystallographic redundancy . To aid in the interpretation of the packing arrangement, crystals were prepared for thin sectioning and analyzed by electron microscopy . Oscillation X-ray diffraction data was collected on image plates using synchrotron radiation and oscillation angles of either 0.25 or 0.30 degrees . A low-resolution 6.5 A data set collected from a single frozen crystal was particularly helpful in the structure determination, because of its completeness and internal consistency . The initial particle orientations were determined using self-rotation functions, while the initial position of one particle was determined from a Patterson map . The structure was solved by molecular replacement real-space averaging using a model based on a cryo-electron microscopy reconstruction as a starting point for the phase determination . The initial structure determination used the data between 20 and 13 A resolution, which was then extended one reciprocal lattice point at a time to 6.5 A resolution . At this point, a 3.5 A resolution data set compiled from a number of crystals collected at 277 K was introduced . Phase extension and averaging continued to 3.5 A resolution after re-determining the particle positions and orientations . The amino-acid sequences of most of the D, F and G proteins and part of the B protein could be unambiguously built into the 3.5 A electron-density map . Partial crystallographic refinement yielded an R factor of 31.6%, consistent with the relatively low resolution and lack of completeness of the data. Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 805 - 16 Structure of bacteriophage T4 fibritin M: a troublesome packing arrangement; Strelkov SV et al.; Fibritin, a 52 kDa product of bacteriophage T4 gene wac, forms 530 A long fibers, named whiskers, that attach to the phage neck and perform a helper function during phage assembly . Fibritin is a homotrimer, with its predominant central domain consisting of 12 consecutive alpha-helical coiled-coil segments linked together by loops . The central domain is flanked by small globular domains at both ends . Fibritin M is a genetically engineered fragment of the wild type and contains 74 amino-acid residues corresponding to the last coiled-coil segment and the complete carboxy-terminal domain . The crystals of fibritin M belong to the rare space group P3 with three crystallographically independent trimers in the unit cell . The structure has been established at 1.85 A resolution by combining molecular and isomorphous replacement techniques . One of the two heavy-atom derivatives used was gaseous xenon . A substantial fraction of residues in each independent trimer is disordered to various extents in proportion to the lack of restraints on the molecules provided by the lattice contacts . Accurate modeling of the solvent present in the crystals was crucial for achieving good agreement with experimental data. Insect Biochem Mol Biol, 1998 Sep, 28(9), 659 - 69 A cuticular protein from the moulting stages of an insect; Marcu O et al.; A 22 kDa peptide was purified from prepupal cuticles of 5th instar Calpodes ethlius caterpillars . It was absent earlier in the stadium and from the egg and adult, i.e . it is related to cuticle turnover rather than cuticle structure . It was present at larval and metamorphic moults, showing that it is related to moulting not just metamorphosis . The cDNA corresponding to the 22 kDa peptide was isolated by antibody screening of an epidermal cDNA expression library . Hybridization to Calpodes genomic DNA showed that the gene was present as a single copy . The deduced amino acid sequence is not like any of the sequences of cuticular structural proteins that have been published, but has a 47 amino acid sequence similar to bacteriophage T7 N-acetylmuramoyl-L-alanine amidase (34% identical, 51% similar) . The amino acid sequence, the timing of expression in development, and the similarity between the substrate of the bacteriophage amidase and components of insect cuticle, all suggest that the 22 kDa protein may have a role in cleaving chitin-peptide bonds as a prerequisite for digestion of the cuticle by chitinases and proteases. J Mol Biol, 1998 Oct 9, 282(5), 947 - 58 Cooperative non-specific DNA binding by octamerizing lambda cI repressors: a site-specific thermodynamic analysis; Pray TR et al.; Relationships between dimerization and site-specific binding have been characterized previously for wild-type and mutant cI repressors at the right operator (OR) of bacteriophage lambda DNA . However, the roles of higher-order oligomers (tetramers and octamers) that are also formed from these cI molecules have remained elusive . In this study, a clear correlation has been established between repressor oligomerization and non-specific DNA-binding activity . A modification of the quantitative DNase I footprint titration technique has been used to evaluate the degree of saturation of non-specific, OR-flanking lambda DNA by cI repressor oligomers . With the exception of one mutant, only those repressors capable of octamerizing were found to exhibit non-specific DNA-binding activity . The non-specific interaction was accurately modeled using either a one-dimensional, univalent, site-specific Ising lattice approximation, or a more traditional, multivalent lattice approach . It was found that non-specific DNA-binding by repressor oligomers is highly cooperative and energetically independent from site-specific binding at OR . Furthermore, the coupling free energy resolved for non-specific binding was similar to that of site-specific binding for each repressor, suggesting that similar structural elements may mediate the cooperative component of both binding processes . It is proposed that the state of assembly of the repressor molecule modulates its relative affinity for specific and non-specific DNA sequences . These specificities are allosterically regulated by the transmission of assembly-state information from the C-terminal domain, which mediates self-association and cooperativity, to the N-terminal domain, which primarily mediates DNA-binding . While dimers have a high affinity for their cognate sites within OR, tetramers and octamers may preferentially recognize non-specific DNA sequences . The concepts and findings developed in this study may facilitate quantitative characterization of the relationships between specific, and non-specific binding in other systems that utilize multiple modes of DNA-binding cooperativity . Gene, 1998 Sep 18, 218(1-2), 1 - 7 Cold-sensitive E-lysis systems; Jechlinger W et al.; The release of recombinant bacteria into the environment is undesirable because of possible risks associated with the genetically modified organisms . The aim of this study was to establish a cold-sensitive killing system with a lethal gene, activated when bacteria encounter lower environmental temperatures . To obtain cold-sensitive lysis vectors, the lambdacI857 repressor/pR promoter expression system was combined with either the lacI/lacZpo or the phage 434 cI/pR system that control the expression of the lysis gene E of bacteriophage phiX174 . Escherichia coli strains harbouring such suicide vectors are able to grow at 37 degrees C, but cell lysis takes place at temperatures below 30 degrees C . By replacing gene E with a beta-galactosidase reporter gene we also showed that the onset of beta-galactosidase activity corresponds with the onset of lysis at 28 degrees C . Results indicate that these newly combined promoter/repressor systems can also be used to confer cold-sensitive expression to any gene of interest. Anal Chem, 1998 Sep 15, 70(18), 3863 - 7 Viral characterization by direct analysis of capsid proteins; Thomas JJ et al.; Matrix-assisted laser desorption/ionization mass spectrometry has enabled viral coat proteins to be characterized directly from the virus . This analysis, demonstrated here with tobacco mosaic virus U2, a bacteriophage MS2, and equine encephalitis TRD, is achieved with a combination of organic acid, UV-absorbing matrix, and high-energy desorption with a nitrogen laser . The molecular weights of these proteins are determined with sufficient accuracy to allow differentiation among viral species and strains . The abundant hydrophobic MS2 coat protein was analyzed in aliquots of culture medium and of the tobacco mosaic virus coat protein in infected leaves . This method provides rapid detection of coat protein in the low-femtomole range, as estimated by titering plaque-forming units of MS2. J Bacteriol, 1998 Oct, 180(19), 5227 - 30 Ndd, the bacteriophage T4 protein that disrupts the Escherichia coli nucleoid, has a DNA binding activity; Bouet JY et al.; Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid . Even at very low levels, the Ndd protein is extremely toxic to cells . In uninfected E . coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection . A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA . The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid. J Bacteriol, 1998 Oct, 180(19), 5151 - 8 A complex control system for transcriptional activation from the sid promoter of bacteriophage P4; Reiter K et al.; The sid gene promoter (Psid), which controls expression of the late genes from satellite phage P4, is activated by a unique class of small DNA-binding proteins . The activators from both satellite and helper phages stimulate transcription from Psid . These activators bind to sites centered at position -55 in all the helper and satellite phage late promoters . P4 Psid is unique in that it has an additional activator binding site centered at position -18 (site II) . We have constructed a mutant of site II that no longer binds activators . Transcription under the control of satellite phage activators is increased by the site II mutation . In contrast, helper phage activators do not show this increase in transcription from Psid mutated at site II . Competition gel shift analysis reveals that the P4 satellite phage activator, Delta, binds eightfold better to site II than to site I . The products of the sid transcription unit are needed only when a helper phage is present; thus, the satellite phage activators repress transcription until the helper is present to supply a nonrepressing activator. Biochemistry, 1998 Sep 22, 37(38), 13300 - 12 Steady-state and pre-steady-state kinetic analysis of 8-oxo-7,8-dihydroguanosine triphosphate incorporation and extension by replicative and repair DNA polymerases; Einolf HJ et al.; The kinetics of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) incorporation into DNA by Escherichia coli polymerases I exo- (KF-) and II exo- (Pol II-), HIV-1 RT reverse transcriptase (HIV-1 RT), and bacteriophage T7 exo- (T7(-)) were examined to determine the misincorporation potential for 8-oxo-dGTP and to investigate the role of base pairing symmetry in DNA polymerase fidelity . 8-Oxo-dGTP was found to be a poor substrate for the four polymerases, with insertion efficiencies >10(4)-fold lower than for dGTP incorporation . Insertion efficiencies of 8-oxo-dGTP were also consistently lower than for incorporation of dNTPs opposite template 8-oxo-G, previously studied in this laboratory . In steady-state reactions, T7(-) had a high preference for 8-oxo-dGTP insertion opposite A (97%) and HIV-1 RT, KF-, and Pol II- preferred to insert 8-oxo-dGTP opposite C . Misinsertion frequencies for 8-oxo-dGTP also varied considerably from frequencies of misinsertion at template 8-oxo-G adducts for Pol II-, HIV-1 RT, and T7(-) . Pre-steady-state incorporation of 8-oxo-dGTP opposite C (but not opposite A) by HIV-1 RT, KF-, and Pol II- displayed biphasic curves, with rates of initial incorporation 2- to 11-fold lower than normal dGTP incorporation . Although extension past template 8-oxo-G adducts had previously been shown to occur preferentially for the mispair, extension past primer 8-oxo-G:template A or C pairs was variable . The low and comparable estimated Kd values for dGTP and 8-oxo-dGTP binding to HIV-1 RT alone or HIV-1 RT.DNA complexes indicated that the initial binding was nonselective and had high affinity . The large difference (>3 orders of magnitude) in kinetic Kdapp values for 8-oxo-dGTP and dGTP binding to HIV-1 RT.DNA indicates that there are contributions to the kinetically determined Kdapp (such as conformational change and/or phosphodiester bond formation) which may be involved in the selection against 8-oxo-dGTP . The differences in binding (Kdapp), incorporation, and extension kinetics of 8-oxo-dGTP compared to normal dNTP incorporation at template 8-oxo-G adducts indicate that polymerase fidelity does not depend solely upon the overall geometry of Watson-Crick base pairs and reflects the asymmetry of the enzyme active site. Virology, 1998 Sep 15, 249(1), 98 - 107 DnaA-mediated regulation of phage lambda-derived replicons in the absence of pR and Cro function; Herman-Antosiewicz A et al.; Bacteriophage lambda-derived replicons can replicate in Escherichia coli cells as plasmids . In the control of replication of these plasmids, an important role was ascribed to the lambda Cro repressor autoregulatory loop . However, the oR/pR-cro-tR-cII' region could be replaced by the ptetA promoter under the control of the TetR repressor, producing plasmid pTClambda . Here, we demonstrate that stable maintenance of pTClambda depends on the host DnaA function because deletion of one of DnaA-binding sequences present in pTClambda resulted in a decrease in the plasmid (pTClambda) copy number and poor maintenance of pTClambda in E . coli . Moreover, in contrast to the replication of the wild-type lambda plasmid, previously found to be positively regulated by DnaA (acting on a relaxed DnaA box situated immediately downstream of the pR promoter), the replication of pTC plasmids (devoid of pR) was found to be negatively regulated by DnaA . Contrary to wild-type lambda plasmids, in cells harboring lambda cro{temperature-sensitive (ts)} or pTClambda (but not pTClambda) plasmid, the lambda replication complex was heat shock resistant; this complex, however, disassembled after inactivation of DnaA function . This disassembly was blocked by DNA gyrase inhibitors . According to our model outlined previously, we propose that the heat shock resistance of the replication complex of lambdacro- plasmids depends on the interaction of the DNA-bound DnaA protein with the DNA-bound lambda replication complex . The replication complex-DnaA-lambda DNA structure may be directly related to the role of DnaA as the Cro-replacing negative regulator of lambdacro- plasmid replication . Virology, 1998 Sep 15, 249(1), 80 - 8 Structure of phage fr capsids with a deletion in the FG loop: implications for viral assembly; Axblom C et al.; The loop between beta-strands F and G in the coat protein of small RNA bacteriophages forms the interactions at the fivefold and threefold (quasi-sixfold) icosahedral axes . In many cases, mutations in this region renders the coat protein unable to form capsids . This FG loop has therefore been suggested to be of major importance for the virus assembly process by guiding the assembly and helping to define the correct curvature of the virus shell . We have determined the crystal structure of a phage fr capsid where the coat protein has a four-residue deletion in the FG loop . This mutant retains the ability to form virus capsids of normal size but has a significantly lower temperature stability than the wild type . The structure reveals that the mutated loops are flexible and too short to interact with each other . This seems incompatible with a role of the FG loop in the regulation of capsid size . Mol Gen Genet, 1998 Jul, 259(1), 39 - 45 The P22 Erf protein and host RecA provide alternative functions for transductional segregation of plasmid-borne duplications; Garzon A et al.; A tandem DNA duplication carried on a ColE1-derived plasmid segregates at high frequency upon generalized transduction by phage P22 HT . Transductional segregation of the plasmid-borne duplication can be promoted either by RecA or by the Erf function of P22, indicating that transductional segregation is a consequence of the recombination events that re-circularize the plasmid in the recipient cell . RecA-mediated and Erf-mediated transduction give similar frequencies of duplication segregation and yield the same types of segregation products, indicating that two distinct recombination machineries (RecA + RecBCD and Erf + RecBCD) perform similar or identical recombination reactions on plasmid DNA substrates transduced by bacteriophage P22 HT. J Mol Biol, 1998 Sep 25, 282(3), 543 - 56 Genome plasticity in the distal tail fiber locus of the T-even bacteriophage: recombination between conserved motifs swaps adhesin specificity; Tetart F et al.; The adsorption specificity of the T-even phages is determined by the protein sequence near the tip of the long tail fibers . These adhesin sequences are highly variable in both their sequence and specificity for bacterial receptors . The tail fiber adhesin domains are located in different genes in closely related phages of the T-even type . In phage T4, the adhesin sequence is encoded by the C-terminal domain of the large tail fiber gene (gene 37), but in T2, the adhesin is a separate gene product (gene 38) that binds to the tip of T2 tail fibers . Analysis of phage T6 and Ac3 sequences reveals additional variant forms of this locus . The tail fiber host specificity determinants can be exchanged, although the different loci have only limited homology . Chimeric fibers can be created by crossovers either between small homologies within the structural part of the fiber gene or in conserved motifs of the adhesin domain . For example, the T2 adhesin determinants are flanked by G-rich DNA motifs and exchanges involving these sequences can replace the specificity determinants . These features of the distal tail fiber loci genetically link their different forms and can mediate acquisition of diverse host range determinants, including those that allow it to cross species boundaries and infect taxonomically distant hosts . Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11128 - 33 Simultaneous formation of functional leading and lagging strand holoenzyme complexes on a small, defined DNA substrate; Berdis AJ et al.; The biochemical characterization of leading and lagging strand DNA synthesis by bacteriophage T4 replication proteins has been addressed utilizing a small, defined primer/template . The ATP hydrolysis activity of 44/62, the clamp loading complex responsible for holoenzyme assembly, was monitored during assembly of both the leading and lagging strand holoenzyme complex . The ATPase activity of 44/62 diminishes once a functional holoenzyme is assembled on both the leading and lagging strand . The assembly of the lagging strand holoenzyme is facilitated by several factors including biotinylated streptavidin blocks at the end of the fork strands, preassembly of the leading strand holoenzyme, and by the presence of the DNA primase with ribonucleoside triphosphates . The resultant minimal replicative complex consists of two holoenzymes and a primase nested on a model replication fork derived from a 62-mer template/34-mer primer/36-mer lagging strand in an apparent 2:2:1:1 ratio of 45 protein:polymerase:primase:forked DNA . The 44/62 protein complex does not remain associated with the complex . The primase alone slowly synthesizes pentaribonucleotides on the forked DNA when the lagging strand contains a nonannealed TTG initiation site with the rate of synthesis greatly stimulated by the addition of the 41 helicase . The addition of deoxy-NTPs to this complex results in leading strand synthesis, but extension of the synthesized RNA primer does not occur . DNA synthesis in both the leading and lagging strand directions is achieved, however, when a 6-mer DNA primer is annealed to the primase recognition site of the forked DNA substrate . A model is presented that describes how leading and lagging strand DNA synthesis might be coordinated as well as the associated molecular interactions of the replicative proteins. Plasmid, 1998 Sep, 40(2), 113 - 25 Replication and maintenance of lambda plasmids devoid of the Cro repressor autoregulatory loop in Escherichia coli; Herman-Antosiewicz A et al.; Plasmids derived from bacteriophage lambda are known as lambda plasmids . These plasmids contain the ori lambda region and lambda replication genes O and P . Typical lambda plasmids also contain the cro gene, the product of which is a repressor of the pR promoter when present at relatively high concentrations . These genes stably maintain the plasmid in Escherichia coli at copy numbers of 20 to 50 per cell . According to a generally accepted model, stable maintenance of lambda plasmids is possible due to the Cro repressor autoregulatory loop (the cro gene is under control of pR) . Here we demonstrate that lambda plasmids devoid of the Cro autoregulatory loop can also be stably maintained in E . coli strains . We present data for two such plasmids: pTC lambda 1 in which the pR-cro region has been replaced by the ptetA promoter and the tetR gene (coding for the TetR repressor), and a standard lambda plasmid with inactivated cro gene (lambda cro-null plasmid) . Thus, the presence of the Cro repressor autoregulatory loop does not appear to be essential to the maintenance of lambda plasmids in vivo. J Mol Biol, 1998 Sep 18, 282(2), 401 - 19 Solution structure of the M13 major coat protein in detergent micelles: a basis for a model of phage assembly involving specific residues; Papavoine CH et al.; The three-dimensional structure of the major coat protein of bacteriophage M13, solubilized in detergent micelles, has been determined using heteronuclear multidimensional NMR and restrained molecular dynamics . The protein consists of two alpha-helices, running from residues 8 to 16 and 25 to 45, respectively . These two helices are connected by a flexible and distorted helical hinge region . The structural properties of the coat protein make it resemble a flail, in which the hydrophobic helix (residues 25 to 45) is the handle and the other, amphipathic, helix the swingle . In this metaphor, the hinge region is the connecting piece of leather . The mobility of the residues in the hinge region is likely to enable a smooth transformation from the membrane-bound form, mimicked by the structure in detergent micelles, into the structure in the mature phage . A specific distribution of the residues over the surface of the two helices was observed in the presented high-resolution structure of the membrane-bound form of the major coat protein as well as in the structure in the mature phage . All data suggest that this arrangement of residues is important for the interactions of the protein with the membrane, for correct protein-DNA and protein-protein interactions in the phage and for a proper growth of the phage during the assembly process . By combining our findings with earlier NMR results on the major coat protein in detergent micelles, we were able to construct a model that addresses the role of specific residues in the assembly process . Anal Biochem, 1998 Aug 15, 262(1), 67 - 76 Probing RegA/RNA interactions using electrospray ionization-fourier transform ion cyclotron resonance-mass spectrometry; Liu C et al.; The interactions of bacteriophage T4 regA protein, a unique translational regulator, with RNAs of various size and sequence were studied using electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry . Using very gentle interface conditions, regA/RNA complexes with a 1:1 binding stoichiometry were observed for all four target RNAs studied, consistent with solution binding studies . Competitive binding of target RNAs and their degradation products with regA demonstrated that the loss of a single nucleotide resulted in a dramatic change in binding affinity in some cases . Competitive binding of regA with four target RNAs revealed similar relative binding affinity order to that suggested by previous in vitro repression experiments . The use of sustained off-resonance irradiation for collisionally induced dissociation of a regA/RNA complex suggested the potential for directly obtaining information regarding the regA binding domain . J Biol Chem, 1998 Sep 18, 273(38), 24853 - 60 Localization and characterization of two nucleotide-binding sites on the anaerobic ribonucleotide reductase from bacteriophage T4; Olcott MC et al.; We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP) to investigate the nucleotide-binding sites on the NrdD subunit of the anaerobic ribonucleotide reductase from T4 phage . Saturation studies revealed two saturable sites for this photoaffinity analog of ATP . One site exhibited half-maximal saturation at approximately 5 microM {gamma-32P}8-N3ATP, whereas the other site required 45 microM . To localize the sites of photoinsertion, photolabeled peptides from tryptic and chymotryptic digests were isolated by immobilized Al3+ affinity chromatography and high performance liquid chromatography and subjected to amino acid sequence and mass spectrometric analyses . The molecular masses of the photolabeled products of cyanogen bromide cleavage were estimated using tricine-SDS-polyacrylamide gel electrophoresis . Overlapping sequence analysis localized the higher affinity site to the region corresponding to residues 289-291 and the other site to the region corresponding to residues 147-160 . Site-directed mutagenesis of Cys290, a residue conserved in all known class III reductases, resulted in a protein that exhibited less than 10% of wild type enzymatic activity . These observations indicate that Cys290 may reside in or near the active site . High performance liquid chromatography analysis revealed that photoinsertion of {gamma-32P}8-N3ATP into the site corresponding to residues 147-160 was almost completely abolished when 100 microM dATP, dGTP, or dTTP was included in the photolabeling reaction mixture, whereas 100 microM ATP, GTP, CTP, or dCTP had virtually no effect . Based on these nucleotide binding properties, we conclude that this site is an allosteric site analogous to the one that has been shown to regulate substrate specificity of other ribonucleotide reductases . There was no evidence for a second allosteric nucleotide-binding site as observed in the anaerobic ribonucleotide reductase from Escherichia coli. J Biol Chem, 1998 Sep 18, 273(38), 24822 - 31 Fidelity and mutational specificity of uracil-initiated base excision DNA repair synthesis in human glioblastoma cell extracts; Sanderson RJ et al.; The fidelity of DNA synthesis associated with uracil-initiated base excision repair was measured in human whole cell extracts . An M13mp2 lacZalpha DNA-based reversion assay was developed to assess the error frequency of DNA repair synthesis at a site-specific uracil residue . All three possible base substitution errors were detected at the uracil target causing reversion of opal codon 14 in the Escherichia coli lacZalpha gene . Using human glioblastoma U251 whole cell extracts, approximately 50% of the heteroduplex uracil-containing DNA substrate was completely repaired, as determined by the insensitivity of form I DNA reaction products to cleavage by a combined treatment of E . coli uracil-DNA glycosylase and endonuclease IV . The majority of repair occurred by the uracil-initiated base excision repair pathway, since the addition of the bacteriophage PBS2 uracil-DNA glycosylase inhibitor protein to extracts significantly blocked this process . In addition, the formation of repaired form I DNA molecules occurred concurrently with limited DNA synthesis, which was largely restricted to the HinfI DNA fragment initially containing the uracil residue and specific to the uracil-containing DNA strand . Based on the reversion frequency of repaired M13mp2 DNA, the fidelity of DNA repair synthesis at the target was determined to be about one misincorporated nucleotide per 1900 repaired uracil residues . The major class of base substitutions propagated transversion mutations, which were distributed almost equally between T to G and T to A changes in the template . A similar mutation frequency was also observed using whole cell extracts from human colon adenocarcinoma LoVo cells, suggesting that mismatch repair did not interfere with the fidelity measurements. J Mol Biol, 1998 Sep 11, 282(1), 125 - 35 Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda; Santini C et al.; We describe the construction and characterization of a hepatitis C virus (HCV) cDNA expression library displayed as a fusion to the carboxy terminus of the capsid protein D of bacteriophage lambda . cDNA inserts were obtained by tagged random-priming of the HCV genome and cloned into a lambda vector from which chimeric phage bearing both wild-type D protein and D fusion products on the capsid surface were produced . The resulting library was affinity-selected with anti-HCV human monoclonal antibodies recognizing linear or conformational epitopes, and human sera from HCV-infected patients . Selection was monitored by immuno-screening experiments, ELISA, and sequence analysis of positive clones . The performance of this library was compared with two additional HCV cDNA display libraries generated as N-terminal fusions to the III and VIII capsid proteins of filamentous phage M13 . The results obtained demonstrate the great potential of the lambda display system for constructing complex cDNA libraries for natural ligand discovery . Genes Dev, 1998 Sep 1, 12(17), 2791 - 802 Amino acid-amino acid contacts at the cooperativity interface of the bacteriophage lambda and P22 repressors; Whipple FW et al.; The bacteriophage lambda repressor and its relatives bind cooperatively to adjacent as well as artificially separated operator sites . This cooperativity is mediated by a protein-protein interaction between the DNA-bound dimers . Here we use a genetic approach to identify two pairs of amino acids that interact at the dimer-dimer interface . One of these pairs is nonconserved in the aligned sequences of the lambda and P22 repressors; we show that a lambda repressor variant bearing the P22 residues at these two positions interacts specifically with the P22 repressor . The other pair consists of a conserved ion pair; we reverse the charges at these two positions and demonstrate that, whereas the individual substitutions abolish the interaction of the DNA-bound dimers, these changes in combination restore the interaction of both lambdacI and P22c2 dimers. Mol Endocrinol, 1998 Sep, 12(9), 1452 - 62 Insulin-like growth factor-I affects perinatal lethality and postnatal development in a gene dosage-dependent manner: manipulation using the Cre/loxP system in transgenic mice; Liu JL et al.; Insulin-like growth factor-I (IGF-I) is essential for cell growth, differentiation and postnatal development . A null mutation in igf-1 causes intrauterine growth retardation and perinatal lethality . The present study was designed to test the lower limit of igf-1 gene dosage that ensures survival and postnatal growth by using the Cre/loxP system . Mice with variable reductions in IGF-I levels were generated by crossing EIIa-cre transgenic mice and mice with loxP-flanked igf-1 locus (igf-1/flox) . EIIa-cre mice express bacteriophage P1 Cre (causes recombination) recombinase under the adenovirus promoter EIIa, during early embryonic development before implantation, and cause genomic recombination of the igf-1/flox locus . Mice with the most extensive recombination die immediately after birth, while the survivors have significant growth retardation in proportion to the reduction in their igf-1 gene . Interestingly, this gene dosage effect on body weight was not very significant before weaning . However, when the young animals were weaned at 3 weeks, the igf-1 gene dosage was the only independent predictor of the weight gain between 3 and 6 weeks among the parameters tested . Although growth retarded, mice with Cre-induced partial igf-1 deficiency were fertile and gave birth to null mice . Thus Cre-induced genomic recombination using the EIIa promoter occurs during development and creates distinct phenotypes compared with the conventional null mutation . This variability allows for postnatal survival and will enable one to begin to explore the role of the endocrine vs . paracrine effects of IGF-I. AIDS, 1998 Aug 20, 12(12), 1413 - 8 Structure-based design of peptides that recognize the CD4 binding domain of HIV-1 gp120; Fontenot JD et al.; DESIGN: Envelope protein-specific antiviral peptides, called mucibodies, that can specifically recognize and bind to the surface unit protein gp120 of HIV-1 were designed . The initial mucibody binding target was the V3 loop of HIV-1 gp120 . Here, the gp120-CD4 binding domain was chosen as the site of mucibody binding . The CD4 binding domain of gp120 is known to be a conformational epitope and is involved in the earliest events of viral entry into many cells . METHODS: The design of the mucibody antivirals was based on previous observations that antibody complementarity determining regions (CDR) are generally similar to the repeating loops or knob structures found in the 20-residue tandem repeat domain of human mucin MUC1 . The heavy chain CDR3 from the bacteriophage display antibody b12 was used to construct two mucibodies, b12-CDR1 and b12-26 . RESULTS: Peptides corresponding to three tandem repeats were shown to bind directly to the CD4 binding domain of HIV-1 gp120 in a solid-phase enzyme-linked immunosorbent assay . These mucibody peptides also disrupted the gp120-CD4 interaction in a solution-phase inhibition assay . Finally, mucibodies neutralized primary and laboratory macrophage-tropic isolates of HIV-1 . CONCLUSIONS: There is a potential for medical use of these peptides as topical vaginal microbicides in preventing HIV-1 transmission during sexual contact . These results also suggest that multivalent, non-immunogenic binding proteins of virtually any specificity could be constructed for use in therapeutic applications involving infectious diseases and immune system dysfunction. J Biol Chem, 1998 Sep 11, 273(37), 24258 - 65 Analysis of higher order intermediates and synapsis in the bent-L pathway of bacteriophage lambda site-specific recombination; Segall AM; The integrase protein of bacteriophage lambda mediates recombination via four distinct pathways . The recent in vitro reconstitution of the efficient bidirectional reaction between two attLtenP'1 target sites now allows comparisons of this pathway, known as the bent-L pathway, with the inefficient bidirectional straight-L pathway and with the efficient but unidirectional pathways of integration and excision . To this end, a series of higher order intermediates of the bent-L pathway was characterized using gel mobility shift assays, two-dimensional gel analysis, and footprinting . The analysis spans the initial binding of proteins to individual DNA target sites, synapsis of two partner DNA targets, and strand exchange . This study identifies a presynaptic "checkpoint" of recombination . It shows that synapsis is a slow step in the recombination reaction, while subsequent strand exchange is comparatively fast . Synaptic complexes contain a preponderance of recombinant products, suggesting that an energetically favorable but somewhat subtle conformational change drives strand exchange . In addition, comparison of wild-type integrase with a catalytically defective mutant of integrase, IntF, showed that, in addition to the catalysis defect, this mutant has different DNA-binding properties than the wild-type protein. Biophys J, 1998 Sep, 75(3), 1223 - 7 Hexagonally packed DNA within bacteriophage T7 stabilized by curvature stress; Odijk T; A continuum computation is proposed for the bending stress stabilizing DNA that is hexagonally packed within bacteriophage T7 . Because the inner radius of the DNA spool is rather small, the stress of the curved DNA genome is strong enough to balance its electrostatic self-repulsion so as to form a stable hexagonal phase . The theory is in accord with the microscopically determined structure of bacteriophage T7 filled with DNA within the experimental margin of error. Virus Res, 1998 Jun, 55(2), 129 - 41 Characterization of a recombinant human calicivirus capsid protein expressed in mammalian cells; Pletneva MA et al.; The capsid protein of the Hawaii strain of human calicivirus was expressed in the transient MVA/bacteriophage T7 polymerase hybrid expression system in order to examine its processing in mammalian cells . Selected amino acid modifications (an insertion, deletion, and substitution) at the predicted amino terminus of the capsid protein as well as the presence or absence of the ORF3 gene were examined for their effect on capsid expression . The protein was expressed efficiently in cell lines derived from three different species, with most of the expressed protein remaining localized within the cells . There was no evidence for N-linked glycosylation or myristylation of the 57 kDa capsid protein . Hawaii virus-like particles (HV VLPs), efficiently produced in the baculovirus expression system, were not observed in this expression system under the conditions in this study. Mol Microbiol, 1998 Aug, 29(3), 859 - 69 An Escherichia coli gene (yaeO) suppresses temperature-sensitive mutations in essential genes by modulating Rho-dependent transcription termination; Pichoff S et al.; An extragenic multicopy suppressor of the cell division inhibition caused by a MalE-MinE fusion protein in Escherichia coli has been mapped and identified as yaeO, one of the two short open reading frames (ORFs) of an operon located at 4.6 min . Overexpressed yaeO also suppressed some temperature-sensitive mutations in division genes ftsA and ftsQ, in chaperone gene groEL and in co-chaperone gene grpE . Gene yaeO, whose expression is regulated by growth rate, codes for a 9 kDa acidic protein with no obvious resemblance to other proteins . Transcription termination protein Rho co-purified with a histidine-tagged derivative of YaeO protein on Ni2+-NTA agarose columns in a manner that suggested direct YaeO-Rho interaction . In vivo, yaeO expression reduced termination at rho-dependent bacteriophage terminator tL1 and at the terminator of autogenously regulated gene rho . The suppression of temperature-sensitive phenotypes was a consequence of anti-termination, as it could be mimicked by a Prho::Tn10 mutation that reduces the expression and activity of gene rho . Our data indicate that the suppression is not caused by overexpression of the mutated genes, but presumably by indirect stabilization of the mutated proteins. J Biol Chem, 1998 Sep 4, 273(36), 22969 - 76 The proofreading pathway of bacteriophage T4 DNA polymerase; Reha-Krantz LJ et al.; The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase . "Mutator" DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps . The G255S- and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction . The hydrolysis-defective D112A/E114A-DNA polymerase was also examined . Fluorescent enzyme-DNA complexes were preformed in the absence of Mg2+, and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg2+ . Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway . These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex . Residue Asp131 acts at a later step in formation of the preexonuclease complex. J Biol Chem, 1998 Sep 4, 273(36), 22884 - 91 Cre mutants with altered DNA binding properties; Hartung M et al.; The recombinase Cre of bacteriophage P1 is a member of the family of site-specific recombinases and integrases that catalyze inter- and intramolecular DNA rearrangements . To understand how this protein specifically recognizes its target sequence, we constructed Cre mutants with amino acid substitutions in different positions of the presumptive DNA binding region . Here we present the results of in vitro DNA binding and in vivo recombination experiments with these Cre mutants . Most substitutions of presumptive DNA-binding amino acids in in vitro tests resulted either in the loss of target binding or in a broadening of target recognition specificity . Of the mutations resulting in a broadening of target specificity, one, N317A, results in a reduced recombination efficiency with the wild-type loxP target but recombines, in contrast to wild-type Cre, in in vivo experiments, with a symmetric variant of the wild-type target sequence . This target variant differs from wild-type loxP by the symmetric C to A replacement in position 6 of the inverted repeats . We propose a common multihelical DNA binding motif for the family of integrases and recombinases . This model implies a major structural rearrangement for the DNA binding region of lambda integrase, analogous to the structural rearrangements of the DNA binding motifs of other proteins when contacting their target DNA. J Bacteriol, 1998 Sep, 180(17), 4339 - 43 Efficiency and frequency of translational coupling between the bacteriophage T4 clamp loader genes; Torgov MY et al.; The bacteriophage T4 DNA polymerase holoenzyme is composed of the core polymerase, gene product 43 (gp43), in association with the "sliding clamp" of the T4 system, gp45 . Sliding clamps are the processivity factors of DNA replication systems . The T4 sliding clamp comes to encircle DNA via the "clamp loader" activity inherent in two other T4 proteins: 44 and 62 . These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins 44 and 62 . Previous work established that T4 genes 44 and 62, which are directly adjacent on polycistronic mRNA molecules, are-to some degree-translationally coupled . In the present study, measurement of the levels (monomers/cell) of the clamp loader subunits during the course of various T4 infections in different host cell backgrounds was accomplished by quantitative immunoblotting . The efficiency of translational coupling was obtained by determining the in vivo levels of gp62 that were synthesized when its translation was either coupled to or uncoupled from the upstream translation of gene 44 . Levels of gp44 were also measured to determine the relative stoichiometry of synthesis and the percentage of gp44 translation that was transmitted across the intercistronic junction (coupling frequency) . The results indicated a coupling efficiency of approximately 85% and a coupling frequency of approximately 25% between the 44-62 gene pair during the course of infection . Thus, translational coupling is the major factor in maintaining the 4:1 stoichiometry of synthesis of the clamp loader subunits . However, coupling does not appear to be an absolute requirement for the synthesis of gp62. Microbiology, 1998 Aug, 144 ( Pt 8), 2217 - 24 Excess production of phage lambda delayed early proteins under conditions supporting high Escherichia coli growth rates; Gabig M et al.; Bacteriophage lambda is unable to lysogenize Escherichia coli hosts harbouring the rpoA341 mutation due to a drastic reduction in transcription from CII-activated lysogenic promoters (pE, pI and paQ) . In addition, the level of early transcripts involved in the lytic pathway of lambda development is also decreased in this genetic background due to impaired N-dependent antitermination . Here, it is demonstrated that despite the reduced level of early lytic pL- and pR-derived transcripts, lytic growth of bacteriophage lambda is not affected in rich media . The level of the late lytic, pR-derived transcripts also remains unaffected by the rpoA341 mutation under these conditions . However, it was found that whilst there is no significant difference in the phage burst size in rpoA+ and rpoA341 hosts growing in rich media, phage lambda is not able to produce progeny in the rpoA341 mutant growing in minimal medium, in contrast to otherwise isogenic rpoA+ bacteria . Provision of an excess of the phage replication proteins O and P in trans or overproduction of the antitermination protein N restore the ability of phage lambda to produce progeny in the rpoA341 mutant under the latter conditions . These results suggest that in rich media phage lambda produces some early proteins in excess of that needed for its effective propagation and indicate that replication proteins may be limiting factors for phage lytic growth in poor media. J Mol Biol, 1998 Sep 4, 281(5), 803 - 14 Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4; Kuebler D et al.; In bacteriophage T4, the terminase complex constituted by the large subunit gp17 (69 kDa) and the small subunit gp16 (18 kDa) is a critical component of the ATP-driven DNA-packaging pump that translocates DNA into an empty capsid shell . Evidence suggests that the large subunit gp17 is the critical component and consists of a number of the functional sites required for DNA-packaging . It exhibits a terminase activity that introduces non-specific cuts into DNA, a portal vertex binding site that allows linkage of cleaved DNA to an empty prohead, an in vitro DNA-packaging activity, and an ATPase activity . In addition, a consensus metal-binding motif and two consensus ATP-binding sites have been identified by sequence analysis . In order to understand the mechanism of action of the multifunctional gp17, we developed an expression-based selection strategy to select for mutants that are defective in terminase function . Characterization of one of the mutants revealed a unique phenotype in which a single H436R mutation resulted in a dramatic loss of both the terminase and the DNA-packaging functions . Indeed, in vivo substitution of H436 with any of the 12 amino acids for which a suppressor is available was lethal to T4 development . According to one hypothesis, H436 is part of a metal-binding motif that is essential for gp17 function . This hypothesis was tested by introducing mutations at each of the three histidine pairs, the H382-X2-H385 pair, the H411-X2-H414 pair and the H430-X5-H436 pair, which constitute the histidine-rich region near the C terminus of gp17 . A mutation at either the H411 pair or the H430 pair resulted in a loss of gp17 function, whereas a mutation at the H382 pair had no effect . In addition to the putative metal-binding motif, substitutions at residue K166 within the putative N terminus-proximal ATP-binding site also resulted in a loss of gp17 function . We propose that a metal-binding motif involving the histidine residues within the sequence H411-X2-H414-X15-H430-X5-H436 is essential for gp17 function . Metal-terminase interactions may be required for structural alignment and stabilization of functional sites in phage T4 terminase and other double-stranded DNA phage terminases . Microbiol Immunol, 1998, 42(7), 515 - 9 Combined use of bacteriophage typing and pulsed-field gel electrophoresis in the epidemiological analysis of Japanese isolates of enterohemorrhagic Escherichia coli O157:H7; Izumiya H et al.; A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed-field gel electrophoresis (PFGE) . Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks . All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July-August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types . These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys. Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 9885 - 90 Mechanism of capsid maturation in a double-stranded DNA virus; Tuma R et al.; Folding mechanisms of proteins incorporated within supramolecular assemblies, including viruses, are little understood and may differ fundamentally from folding mechanisms of small globular proteins . We describe a novel Raman dynamic probe of hydrogen-isotope exchange to investigate directly these protein folding/assembly pathways . The method is applied to subunit folding in assembly intermediates of the double-stranded DNA bacteriophage P22 . The icosahedral procapsid-to-capsid maturation (shell expansion) of P22 is shown to be accompanied by a large increase in exchange protection of peptide beta-strands . The molecular mechanism of shell expansion involves unfolding of metastable tertiary structure to form more stable quaternary contacts and is governed by a surprisingly high activation energy . The results demonstrate that coat subunit folding and capsid expansion are strongly coupled processes . Subunit structure in the procapsid represents a late intermediate along the folding/assembly pathway to the mature capsid . Coupling of folding and assembly is proposed as a general pathway for the construction of supramolecular complexes. Eur J Biochem, 1998 Jul 15, 255(2), 409 - 13 Flexibility of the nascent polypeptide chain within the ribosome--contacts from the peptide N-terminus to a specific region of the 30S subunit; Choi KM et al.; The ribosomal environment of the N-terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes; the peptides each carry a photoreactive diazirine moiety at their N-terminus, so as to generate cross-links to neighbouring ribosomal components . Our previous studies {Choi, K . M . & Brimacombe, R . (1998) Nucleic Acids Res . 26, 887-895} with three independent families of peptides, derived from the E . coli ompA protein gene, the tetracycline-resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross-linked . The distribution of these cross-links indicated that the nascent peptide is very flexible within the 50S subunit . Here, we demonstrate that the N-termini of the ompA and gene-60 peptides can, in addition, even become concomitantly cross-linked to the 30S subunit . The cross-linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region . This result is discussed in terms of the flexibility of the nascent peptide during the co-translational folding process, and in terms of the 'ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA. Infect Immun, 1998 Sep, 66(9), 4100 - 7 Identification and characterization of a newly isolated shiga toxin 2-converting phage from shiga toxin-producing Escherichia coli; Watarai M et al.; Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded by toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1- and one Stx2-converting phages have been isolated from two STEC strains (A . D . O'Brien, J . W . Newlands, S . F . Miller, R . K . Holmes, H . W . Smith, and S . B . Formal, Science 226:694-696, 1984) . In this study, we isolated two Stx2-converting phages, designated Stx2Phi-I and Stx2Phi-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Phi-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E . coli K-12 strain C600 while Stx2Phi-II was distinct from them . The sizes of the plaques of Stx2Phi-I and Stx2Phi-II in C600 were different; the former was larger than the latter . The restriction maps of Stx2Phi-I and Stx2Phi-II were not identical; rather, Stx2Phi-II DNA was approximately 3 kb larger than Stx2Phi-I DNA . Furthermore, Stx2Phi-I and Stx2Phi-II showed different phage immunity, with Stx2Phi-I and 933W belonging to the same group . Infection of C600 by Stx2Phi-I or 933W was affected by environmental osmolarity differently from that by Stx2Phi-II . When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Phi-I and 933W was greatly decreased compared with that of Stx2Phi-II . Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Phi-I and 933W for the fadL mutant decreased to less than 10(-7) compared with that for C600 whereas the efficiency of Stx2Phi-II decreased to 0.1% of that for C600 . In contrast, while the plating efficiency of Stx2Phi-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Phi-I and 933W were not changed . This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600, fadL mutant, or lamB mutant or the purified His6-tagged FadL and LamB proteins . Based on the data, we concluded that FadL acts as the receptor for Stx2Phi-I and Stx2Phi-II whereas LamB acts as the receptor only for Stx2Phi-II. J Mol Biol, 1998 Aug 28, 281(4), 651 - 61 Defining the structural and functional roles of the carboxyl region of the bacteriophage lambda excisionase (Xis) protein; Wu Z et al.; The bacteriophage lambda excisionase (Xis) protein is required for excisive site-specific recombination . Xis is composed of 72 amino acids and binds cooperatively to two DNA sites (X1 and X2) that are arranged as direct repeats . Alternatively, Xis binds cooperatively with the host-encoded factor for inversion stimulation (FIS) protein at the X1 and F sites, respectively . Here we analyzed the effects of missense substitutions from codon 57 to the carboxyl end of the protein and nonsense mutations that truncate the protein at various positions from residues 60 to 69 . We find that all of the mutant proteins promote excision to some extent and interact cooperatively with FIS . Some mutants have no detectible phenotype while others are altered in their abilities to promote excision or to interact cooperatively with integrase (Int) . Computer modeling predicts that amino acids from residues 59 to 65 are in an alpha-helix conformation . Mutants with substitutions on one side of the helix at residues 57, 60, 63 and 64 as well as truncated mutants containing 60, 61 or 63 amino acids, fail to interact cooperatively with Int suggesting that this region of the protein forms the interface with Int . Mutants with substitutions at other positions in the putative helix have no detectible phenotype . Residues 66 to 68 may form a reverse turn and the last four amino acids (69 to 72) may not be crucial for the structure or function of the protein . FEBS Lett, 1998 Jul 31, 432(1-2), 70 - 2 Rapid degradation of polyadenylated oop RNA; Szalewska-Palasz A et al.; The oop RNA is a short (77 nucleotides (nt)) transcript encoded by bacteriophage lambda which acts as an antisense RNA for lambda cII gene expression . Recently we demonstrated that oop RNA is specifically polyadenylated at its 3' end by poly(A) polymerase I (PAP I), the pcnB gene product . Here we demonstrate that the half life of oop RNA is 3 times longer in the pcnB mutant relative to the pcnB+ host, indicating that polyadenylation of this transcript causes its accelerated degradation . Although it was proposed that polyadenylation of RNAs in bacteria leads to their enhanced degradation, in most cases stabilization of these molecules was observed only when other mutations (pnp, rnb and rne) were present in the pcnB- strain . Therefore it seems that oop RNA may serve as a very useful model in further studies on molecular mechanisms of RNA polyadenylation and degradation in bacteria . Analysis of oop RNA and its degradation product isolated from Escherichia coli cells suggests that both polyadenylated and non-modified oop transcripts can act as antisense RNA. Transfusion, 1998 Aug, 38(8), 729 - 37 Preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue; Wagner SJ et al.; BACKGROUND: All published reports have described methods for virus photoinactivation which significantly alter red cell (RBC) properties during storage . In order to improve virucidal activity and reduce damage to RBCs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux . One compound, 1,9-dimethylmethylene blue (dimethyl-methylene blue), had superior screening results and was chosen for further characterization . STUDY DESIGN AND METHODS: White cell reduced RBC suspensions (30% hematocrit) were deliberately inoculated with extracellular virus or virus-infected VERO cells, incubated with 4 microM dimethyl-methylene blue and illuminated with cool-white fluorescent light . Control and treated samples were titered for virus inactivation . In parallel studies, RBC suspensions were exposed to dimethylmethylene blue and light under identical conditions and assayed for in vitro RBC storage properties . RESULTS: Phototreatment of RBC suspensions inactivated > 4.4 log10 of extracellular vesicular stomatitis virus (VSV), > 3.0 log10 of intracellular VSV, > 5.0 log10 of extracellular pseudorabies virus (PRV), > 4.8 log10 of intracellular PRV, > 4.7 log10 of extra-cellular bovine virus diarrhea virus, 5.8 log10 of bacterio-phage phi 6 and > 7 log10 of bacteriophage R17 . Encephalo-myocarditis virus, a nonenveloped picornavirus, was resistant to photoinactivation . Virucidal conditions resulted in no detectable IgG binding in 11 of 13 samples, unchanged RBC morphology, normal banding patterns of RBC membrane proteins on SDS PAGE, and unaltered characteristics of 12 of 13 RBC antigens during storage as measured by antibody titrations . In addition, minimal changes were observed in RBC osmotic fragility, lysis, potassium efflux, ATP and 2,3-DPG levels, and the strength of one RBC antigen during storage of phototreated samples compared with controls . CONCLUSION: Dimethylmethylene blue photo-treatment can inactivate several intracellular and extracellular model viruses under conditions which minimally alter RBC properties during 42 days storage at 1-6 degrees C. Exp Parasitol, 1998 Sep, 90(1), 65 - 76 Testing promoter activity in the trypanosome genome: isolation of a metacyclic-type VSG promoter, and unexpected insights into RNA polymerase II transcription; McAndrew M et al.; In trypanosomes, most genes are arranged in polycistronic transcription units . Individual mRNAs are generated by 5'-trans splicing and 3' polyadenylation . Remarkably, no regulation of RNA polymerase II transcription has been detected although many RNAs are differentially expressed during kinetoplastid life cycles . Demonstration of specific class II promoters is complicated by the difficulty in distinguishing between genuine promoter activity and stimulation of trans splicing . Using vectors that were designed to allow the detection of low promoter activities in a transcriptionally silent chromosomal context, we isolated a novel trypanosome RNA polymerase I promoter . We were however unable to detect class II promoter activity in any tested DNA fragment . We also integrated genes which were preceded by a T3 promoter into the genome of cells expressing bacteriophage T3 polymerase: surprisingly, transcription was alpha-amanitin sensitive . One possible interpretation of these results is that in trypanosomes, RNA polymerase II initiation is favored by genomic accessibility and double-strand melting . Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 9861 - 6 In vivo activities of GroEL minichaperones; Chatellier J et al.; Fragments encompassing the apical domain of GroEL, called minichaperones, facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL . We have identified the smallest minichaperone that is active in vitro in chaperoning the refolding of rhodanese and cyclophilin A: GroEL(193-335) . This finding raises the question of whether the minichaperones are active under more stringent conditions in vivo . The smallest minichaperones complement two temperature-sensitive Escherichia coli groEL alleles, EL44 and EL673, at 43 degreesC . Although they cannot replace GroEL in cells in which the chromosomal groEL gene has been deleted by P1 transduction, GroEL(193-335) enhances the colony-forming ability of such cells when limiting amounts of GroEL are expressed from a tightly regulated plasmid . Surprisingly, we found that overexpression of GroEL prevents plaque formation by bacteriophage lambda and inhibits replication of the lambda origin-dependent plasmid, Lorist6 . The minichaperones also inhibit Lorist6 replication, but less markedly . The complex quaternary structure of GroEL, its central cavity, and the structural allosteric changes that take place on the binding of nucleotides and GroES are not essential for all of its functions in vivo. Virology, 1998 Aug 1, 247(2), 251 - 64 ATP-reactive sites in the bacteriophage lambda packaging protein terminase lie in the N-termini of its subunits, gpA and gpNu1; Babbar BK et al.; ATP-reactive sites in terminase and its subunits have been successfully identified using three different affinity analogs of ATP (2-and 8-azidoATP and FITC) GpA, the larger subunit of terminase, was shown to have a higher affinity for these analogs than gpNu1, the smaller subunit . The suitability of these reagents as affinity analogs of ATP was demonstrated by ATP protection experiments and in vitro assays done with the modified proteins . These analogs were thus shown to modify the ATP-reactive sites . The results obtained from these experiments also indicate the importance of subunit-subunit interactions in the holoenzyme . Terminase, gpA, and gpNu1 were modified with these analogs and the ATP-reactive sites were identified by isolating the modified peptide by reverse-phase chromatography . The sequence analysis of the modified peptides indicates a region including amino acids 18-35 in the N-terminus of gpNu1 and a region including amino acids 59-85 in the N-terminus of gpA as being the ATP-reactive sites. J Struct Biol, 1998, 121(3), 285 - 94 On the Structure of the Scaffolding Core of Bacteriophage T4 and Its Role in Head Length Determination; 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 . Alternate structures for the scaffolding core in T4 are investigated . Starting with the hypothesis that the shell and a 10-helix core would have matching symmetry, a Vernier mechanism is proposed that explains the previously unexplained behavior of the length determination process in giant head mutants of T4 . Other possible Vernier mechanisms for core structures containing six and eight helices are also explored . J Biol Chem, 1998 Aug 21, 273(34), 21980 - 7 Cyclic peptides as non-carboxyl-terminal ligands of syntrophin PDZ domains; Gee SH et al.; Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain . Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC . Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13 . Affinity selection from a library of X10C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-Psi-C, where Psi represents any hydrophobic amino acid . These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains . PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity . Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding . Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains. Virology, 1998 Aug 15, 248(1), 148 - 55 Involvement of other bacteriophage T4 genes in the blockade of protein synthesis and mRNA destabilization by a mutation of gene 61 . 5; Kai T et al.; Gene 61.5 of bacteriophage T4 is required for gene expression at late stages of infection; a mutant of gene 61.5 shows a reduced rate of synthesis of late proteins and accumulates short transcripts upon infection of nonpermissive host cells at 30 degreesC (T . Kai, H . E . Sellick, and T . Yonesaki, Genetics 144, 7-14, 1996) . Here we describe that, although the defects are only apparent at late stages, gene 61.5 is expressed early after infection and that the requirement of a functional gene 61.5 is partially compensated by the passage of early and middle stages of infection at high temperatures, suggesting that T4 genes expressed at early and middle stages bypass the requirement for the gene 61.5 at high temperatures . Furthermore, we isolated five pseudorevertants of a gene 61.5 mutant, designated as ssf1 through ssf5, which partially restored the growth ability at low temperatures, stabilized mRNAs, and stimulated protein synthesis at late stages . These results strongly suggest that the products of other T4 genes interact with the product of gene 61.5 in stabilizing mRNAs late in T4 infection . Virology, 1998 Aug 15, 248(1), 117 - 30 The late-expressed region of the temperate coliphage 186 genome; Portelli R et al.; The late-lytic region of the genome of bacteriophage 186 encodes the phage proteins that synthesize the complex viral particle and lyse the bacterial host . We report the completion of the DNA sequence of the late region and the assignment of 18 previously identified genes to open reading frames in the sequence . The 186 late region is similar to the late region of phage P2, sharing 26 genes of known function: the single gene for activation of late gene transcription, 6 genes for construction of DNA-containing heads, 16 for tail morphogenesis, and 3 for cell lysis . We identified two 186 late genes with unknown function; one is homologous to previously unrecognised genes in P2, HP1, and phiCTX, and the other may modulate DNA packaging . The 186 late region, like the rest of the genome, lacks the lysogenic conversion genes that are carried by P2, allowing the 186 late region to be transcribed from only three late promoters rather than four . The relative absence of lysogenic conversion genes in 186 suggests that the two phages have evolved to use the lytic and lysogenic reproductive modes to different extents . Biotechnol Annu Rev, 1995, 1, 149 - 83 Peptide and protein display on the surface of filamentous bacteriophage; Felici F et al.; The isolation of ligands that bind biologically relevant molecules is fundamental to the understanding of biological processes and to the search for therapeutics . Filamentous phage can be used to display foreign peptides and proteins in physical association with their DNA coding sequences . Repertoires larger than 10(8) phage clones expressing different peptide sequences can be prepared using molecular genetic techniques . The strategies utilizing this technology promise to provide not only new binding and possibly catalytic activities, but also lead structures for the development of new drugs and vaccines. Mol Cell, 1998 Jul, 2(1), 149 - 55 Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation; Guo P et al.; Ds-DNA viruses package their DNA into a preformed protein shell (procapsid) during maturation . Bacteriophage phi29 requires an RNA (pRNA) to package its genomic DNA into the procapsid . We report here that the pRNA upper and lower loops are involved in RNA/RNA interactions . Mutation in only one loop results in inactive pRNAs . However, mixing of two, three and six inactive mutant pRNAs restores DNA packaging activity as long as an interlocking hexameric ring can be predicted to form by base pairing of the mutated loops in separate RNA molecules . The stoichiometry of pRNA for the packaging of one viral DNA genome is six . Homogeneous pRNA purified from a single band in denaturing gels showed six bands when rerun in native gels . These results suggest that six pRNAs form a hexameric ring by the intermolecular interaction of two RNA loops to serve as part of the DNA transportation machinery. Mol Cell, 1998 Jul, 2(1), 141 - 7 Function of hexameric RNA in packaging of bacteriophage phi 29 DNA in vitro; Zhang F et al.; A cyclic hexamer of the 120-base prohead RNA (pRNA) is needed for efficient in vitro packaging of the B . subtilis bacteriophage phi 29 genome . This capacity of pRNA to form higher multimers by intermolecular base pairing of identical subunits represents a new RNA structural motif . Dimers of pRNA are likely intermediates in formation of the cyclic hexamer . A three-dimensional model of the pRNA hexamer is presented. Acta Biochim Pol, 1998, 45(1), 271 - 80 The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein; Gabig M et al.; Bacteriophage lambda is not able to lysogenise the Escherichia coli rpoA341 mutant . This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) . Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for alphaCTD in this process . Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the cII gene product . Our results indicate that amino-acid residues 265, 268 and 271 in the a subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between alphaCTD and an UP-like element near pE (most probably residues 265 and 268) . Measurement of the activity of pE-lacZ, pI-lacZ and p(aQ)-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different. Acta Biochim Pol, 1998, 45(1), 251 - 9 Biochemical and genetic analysis of lambdaW, the newly isolated lambdoid phage; Wrobel B et al.; Otherwise isogenic Escherichia coli CP78 (relA+) and CP79 (relA-) strains are commonly used in studies on the stringent control, the bacterial response to amino acid starvation . We found that these strains are lysogenic for a phage which is spontaneously induced with a low frequency, producing virions able to infect other E . coli strains . Genetic studies, restriction analysis of the phage DNA genome, and electron microscopy revealed that this phage is very similar to, but not identical with, bacteriophage lambda . We called the newly isolated phage lambdaW, and found that most of CP78/CP79 ancestor strains are lysogenic for this phage. RNA, 1998 Aug, 4(8), 948 - 57 A long-range interaction in Qbeta RNA that bridges the thousand nucleotides between the M-site and the 3' end is required for replication; Klovins J et al.; The genome of the positive strand RNA bacteriophage Qbeta folds into a number of structural domains, defined by long-distance interactions . The RNA within each domain is ordered in arrays of three- and four-way junctions that confer rigidity to the chain . One such domain, RD2, is about 1,000-nt long and covers most of the replicase gene . Its downstream border is the 3' untranslated region, whereas upstream the major binding site for Qbeta replicase, the M-site, is located . Replication of Qbeta RNA has always been puzzling because the binding site for the enzyme lies some 1,500-nt away from the 3' terminus . We present evidence that the long-range interaction defining RD2 exists and positions the 3' terminus in the vicinity of the replicase binding site . The model is based on several observations . First, mutations destabilizing the long-range interaction are virtually lethal to the phage, whereas base pair substitutions have little effect . Secondly, in vitro analysis shows that destabilizing the long-range pairing abolishes replication of the plus strand . Thirdly, passaging of nearly inactive mutant phages results in the selection of second-site suppressor mutations that restore both long-range base pairing and replication . The data are interpreted to mean that the 3D organization of this part of Qbeta RNA is essential to its replication . We propose that, when replicase is bound to the internal recognition site, the 3' terminus of the template is juxtaposed to the enzyme's active site. J Chromatogr B Biomed Sci Appl, 1998 Jun 26, 711(1-2), 127 - 38 Separation of proteins and viruses using two-phase aqueous micellar systems; Liu CL et al.; We discuss the utilization of a novel two-phase aqueous nonionic micellar system for the purification and concentration of biomolecules, such as proteins and viruses, by liquid-liquid extraction . The nonionic surfactant n-decyl tetra(ethylene oxide), C10E4, phase separates in water into two coexisting aqueous micellar phases by increasing temperature . The mild interactions of the C10E4 nonionic surfactant with biomolecules, combined with the high water content of the two coexisting micellar phases, suggest the potential utility of two-phase aqueous C10E4 micellar systems for the purification and concentration of biomolecules . In this paper, we review our recent experimental and theoretical studies involving the partitioning of several water-soluble proteins, including cytochrome c . soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and catalase, in the two-phase aqueous C10E4 micellar system . In addition, we present results of our preliminary experimental investigation on the partitioning of bacteriophages, including phiX174, P22, and T4. J Mol Biol, 1998 Aug 14, 281(2), 219 - 25 Crystallographic analysis reveals the 12-fold symmetry of the bacteriophage phi29 connector particle; Guasch A et al.; The internal symmetry of the connector or portal particle from the double-stranded DNA bacteriophage phi29 has been examined by X-ray crystallography . This large multimeric structure (420 kDa) is built up by a number of identical subunits of the p10 protein . It connects the head of the virus with the tail and plays a central role in the prohead assembly and DNA packaging . For the first time a bacteriophage connector has been crystallized and X-ray data have been collected up to a resolution of 3.2 A . A self-rotation function has been calculated, unambigously revealing the 12-fold symmetry of the particle and its orientation in the crystal lattice . The orientation has been confirmed by calculating a cross-rotation function using a low resolution model based on electron microscopy reconstructions . J Infect Dis, 1998 Aug, 178(2), 318 - 24 Parvovirus B19-induced anemia as the presenting manifestation of X-linked hyper-IgM syndrome; Seyama K et al.; Parvovirus B19 (B19) can cause chronic anemia due to persistent infection in immunocompromised hosts who cannot produce neutralizing antibody necessary for clearing B19 . Three patients with X-linked hyper-IgM syndrome (XHIM), who were all asymptomatic until they developed B19-induced chronic anemia at the ages of 8, 14, and 17 years, respectively, were found to have mutations of the CD40L gene, including a missense mutation (T254M), a nonsense mutation resulting in a new initiation codon and loss of the intracellular domain (R11X), and a splice site mutation (nt 309+2t-->a) . Antibody responses to the T cell-dependent antigen, bacteriophage phiX174, were impaired, but neutralizing antibody titers were higher than in XHIM patients with classic phenotype . All 3 patients responded to intravenous immune globulin (IVIG) treatment . Certain mutations of the CD40L gene result in a mild XHIM phenotype that may become apparent following B19 infection in patients not on IVIG therapy and therefore not protected from B19 infection. J Virol, 1998 Sep, 72(9), 7428 - 39 Herpes simplex virus type 1 cleavage and packaging proteins UL15 and UL28 are associated with B but not C capsids during packaging; Yu D et al.; At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32, and UL33) that are required for DNA cleavage and packaging of herpes simplex virus type 1 (HSV-1) DNA . Sequence analysis reveals that UL15 shares homology with gp17, the large catalytic subunit of the bacteriophage T4 terminase . Thus, UL15 may play a direct role in the cleavage of viral DNA replication intermediates into monomers . In this study, we asked whether UL15 and other cleavage and packaging proteins could be detected in capsids isolated from infected cells . Consistent with previous studies showing that UL6 and UL25 are minor protein constituents of the capsids, we detected these proteins in both B and C capsids . In contrast, the previously identified full-length version (81 kDa) of UL15 was found predominantly in B capsids and in much smaller amounts in C capsids . In addition, the UL28 protein was found predominantly in B but not C capsids in a distribution similar to that of the 81-kDa version of UL15 . These results suggest that UL28 and the 81-kDa form of UL15 are transiently associated with capsid intermediates during the packaging process . Surprisingly, however, a previously unidentified 87-kDa form of UL15 was found in the B and C capsids and in virions . Analysis of cells infected with mutants individually lacking UL6, UL15, UL25, UL28, or UL32 demonstrates that the lack of one cleavage and packaging protein does not affect the expression of the others . Furthermore, this analysis, together with guanidine HCl extraction analysis of purified capsids, indicates that UL6, UL25, and UL28 are able to associate with B capsids in the absence of other DNA cleavage and packaging proteins . On the other hand, the two UL15-related proteins (81 and 87 kDa) do not associate efficiently with B capsids in cells infected with UL6 and UL28 mutants . These results suggest that the ability of the UL15-related proteins to bind to B capsids may be mediated through interactions with UL6 and UL28. J Bacteriol, 1998 Aug, 180(16), 4199 - 211 Oligohistidine tag mutagenesis of the lambda holin gene; Smith DL et al.; Holins are a diverse group of small integral membrane proteins elaborated by bacteriophages to lyse bacterial hosts and effect release of progeny phages in a precisely timed manner . Recently, the holin S gene of phage lambda was overexpressed and the holin protein was purified to homogeneity by means of an oligohistidine tag procedure and immobilized metal affinity chromatography (D . L . Smith, D . K . Struck, J . M . Scholtz, and R . Young, J . Bacteriol . 180:2531-2540, 1998) . Numerous locations within the S gene were tested as sites for an oligohistidine-tag-encoding insertion which preserves holin function . The lysis phenotypes of these alleles, expressed from moderate-copy-number transactivation plasmids, were characterized . A striking class of mutants, previously referred to as early-dominant, have been found to have severe lysis defects but are fully functional in the presence of wild-type protein . Results presented here reveal that the early-dominance phenotype is independent of S107 inhibitor function . The results provide insight into the mechanism of hole formation and indicate that, while oligomerization is required in the pathway to hole formation, a nucleation event may also be required. J Autoimmun, 1998 Jun, 11(3), 205 - 11 Mimotopes identified by phage display for the monoclonal antibody CII-C1 to type II collagen; Cook AD et al.; The characterization of B cell epitopes has been advanced by the use of random peptide libraries displayed within the coat protein of bacteriophage . This technique was applied to the monoclonal antibody (mAb) C1 to type II collagen (CII-C1) . CII-C1 is known to react with a conformational epitope on type II collagen that includes residues 359-363 . Three rounds of selection were used to screen two random nonameric phage libraries and 18 phagotopes were isolated . CII-C1 reacted by ELISA with 17 of the 18 phagotopes: one phagotope contained a stop codon . Of the eight most reactive phage, seven inhibited the reactivity by ELISA of CII-C1 with type II collagen . Of the 18 phage isolated, 11 encoded the motif F-G-x-Q with the sequence F-G-S-Q in 6, 2 encoded F-G-Q, and one the reverse motif Q-x-y-F . Most phagotopes that inhibited the reactivity of CII-C1 encoded two particular motifs consisting of two basic amino acid residues and a hydrophobic residue in the first part of the insert and the F-G-x-Q or F-G-Q motif in the second part; phagotopes which contained only one basic residue in the first part of the sequence were less reactive . These motifs are not represented in the linear sequence of type II collagen and thus represent mimotopes of the epitope for CII-C1 on type II collagen . There were five phagotopes with peptide inserts containing the sequence RLPFG occurring in the Epstein-Barr virus nuclear antigen, EBNA-1 . This is of interest because EBV has been implicated in the initiation of rheumatoid arthritis (RA) by reason of increased reactivity to EBNA-1 in RA sera . In conclusion, the phage display technique disclosed mimotopes for a conformational epitope of type II collagen, and revealed an interesting homology with a sequence of the EBNA-1 antigen from Epstein Barr virus. Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1041 - 7 Antibody fragments as inhibitors of Japanese radish acid phosphatase; Takata R et al.; VH (heavy-chain variable region) and VL (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase . Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VH and identical VL domains . Initially, the VH and VL genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments . Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs . Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources . ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine . The PCR-amplified VH and VL genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase . The fusion proteins had little effect on Japanese radish acid phosphatase . Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library . These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none . Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18. Microb Comp Genomics, 1997, 2(4), 287 - 98 Clustering of chi sequence in Escherichia coli genome; Handa N et al.; An 8-mer DNA sequence called chi (5'-GCTGGTGG) is present on the Escherichia coli chromosome at a high frequency . It is responsible for both the attenuation of RecBCD exonuclease activity and the promotion of RecABCD-mediated homologous recombination . chi was first identified as a site that increased plaque size of bacteriophage lambda . lambda containing chi makes very small plaques on a recC* (recC1004) mutant because chi is poorly recognized by the RecBC*D mutant enzyme . We cloned E . coli chromosomal fragments in lambda that allowed lambda to form larger plaques on this recC* mutant as well as on the rec+ parent . One identified fragment contained a cluster of two copies of chi and several chi-like sequences with the same orientation . It increased recombination in the rec+ strain more than a fragment with one chi did . This fragment was within the rep gene, whose helicase product is known to be required for growth in the absence of functional RecBCD enzyme . The possibility that RecBCD enzyme might interact both with the rep gene and its product is discussed . Many of the other chi clusters identified in the E . coli genome database lie within genes for membrane proteins . The possible significance of these findings is discussed. Proc Natl Acad Sci U S A, 1998 Aug 4, 95(16), 9244 - 9 A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein; Johansson HE et al.; Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect . However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold . Guided by the structure of the protein-RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to (i) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and (ii) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue . The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at the thermodynamically important sites. Food Chem Toxicol, 1998 Jul, 36(7), 543 - 53 Modulation of metal-induced genotoxicity by Maillard reaction products isolated from coffee; Wijewickreme AN et al.; PM2 bacteriophage DNA was exposed to non-dialysable Maillard reaction products (MRPs) isolated from brewed (Br), boiled (Bo) and instant (I) coffee brew extracts in a Fe2+ catalysed Fenton reaction at four pH conditions (i.e . 7.5, 4.0, 3.2, 2.6) . MRPs were incubated with DNA either directly with Fe2+, or following a short preincubation period conducted with Fe2+ in an atmosphere of oxygen or argon . Damage to supercoiled DNA resulting in strand scissions as characterized by both nicked circular and linear forms were found to occur either with coffee MRPs or Fe2+ alone, in a dose-dependent manner at all pH conditions tested . At low MRP concentrations, damage to DNA with respect to Fe2+ was lowered only when MRPs were preincubated with Fe2+ in argon or oxygen before incubating with DNA . The addition of MRPs and Fe2+ to DNA without preincubation, had no effects in protecting DNA damage . This finding showed that a preincubation step is necessary for MRPs to chelate Fe2+ in order to mitigate the Fenton reaction . In contrast, the protective effects against Fe2+-induced DNA breakage by MRPs were lost at high coffee MRP concentrations, irrespective of the incubation method used . Increasingly higher concentrations of MRPs in combination with Fe2+ actually enhanced the breakage of DNA with respect to the control . These results indicate that MRPs at high concentrations do not improve Fe2+ ion chelation, but rather accelerate the DNA breakage by possibly changing the redox state of the transition element. EMBO J, 1998 Aug 3, 17(15), 4527 - 34 Endonuclease VII has two DNA-binding sites each composed from one N- and one C-terminus provided by different subunits of the protein dimer; Birkenbihl RP et al.; Endonuclease VII (endo VII) is a Holliday structure-resolving enzyme of bacteriophage T4 . Its activity depends on dimerization, DNA binding and hydrolysis of two phosphodiester bonds flanking the Holliday junction . We analysed the DNA-binding activity of truncated monomeric and covalently linked dimeric endo VII proteins . We show that both ends of endo VII are involved in DNA binding . In particular, the C-terminus of one subunit interacts with the N-terminus of the other subunit, constituting one DNA-binding site; the other two termini form the second binding site of the dimer . One binding site is sufficient to bind cruciform DNA . The concerted mechanism involving termini from different subunits ensures that only dimers bind to Holliday structures, thus providing two catalytic centres which introduce two cleavages in opposite strands . This is a precondition for precise resolution of Holliday structures. Appl Environ Microbiol, 1998 Aug, 64(8), 2780 - 7 Gene transfer by transduction in the marine environment; Jiang SC et al.; To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates . Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays . Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies . Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5 . The transduction frequencies ranged from 1.33 x 10(-7) to 5.13 x 10(-9) transductants/PFU in studies performed with the bacterial isolates . With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed . These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed with two sets of primers specific for pQSR50 . The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 x 10(-8) to 3.7 x 10(-8) transductants/PFU . Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 x 10(14) transduction events per year could occur in the Tampa Bay Estuary . The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment. Structure, 1998 Jul 15, 6(7), 849 - 61 A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution; Shepard W et al.; BACKGROUND: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC . This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA . ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function . Nuclear magnetic resonance (NMR) studies indicate that the GCGAAAGC sequence forms a hairpin loop in solution, while centromere-like repeats dimerise into unusual duplexes . The factors stabilising these unusual secondary structure elements in ssDNA, however, are poorly understood . RESULTS: The nonamer d(GCGAAAGCT) was crystallised as a bromocytosine derivative in the presence of cobalt hexammine . The crystal structure, solved by the multiple wavelength anomalous dispersion (MAD) method at the bromine K-edge, reveals an unexpected zipper-like motif in the middle of a standard B-DNA duplex . Four central adenines, flanked by two sheared G.A mismatches, are intercalated and stacked on top of each other without any interstrand Watson-Crick base pairing . The cobalt hexammine cation appears to participate only in crystal cohesion . CONCLUSIONS: The GAAA consensus sequence can dimerise into a stable zipper-like duplex as well as forming a hairpin loop . The arrangement closes the minor groove and exposes the intercalated, unpaired, adenines to the solvent and DNA-binding proteins . Such a motif, which can transform into a hairpin, should be considered as a structural option in modelling DNA and as a potential binding site, where it could have a role in DNA replication, nuclease resistance, ssDNA genome packaging and centromere function. Mutat Res, 1998 May 25, 400(1-2), 59 - 68 Endogenous lesions, S-phase-independent spontaneous mutations, and evolutionary strategies for base excision repair; Holmquist GP; We calculate from published levels of endogenous base lesions that our cells constantly generate and excise during base excision repair (BER) about one million lesions per day . Repair glycosylases may also non-specifically excise an additional number of undamaged bases . The resulting abasic sites are repaired daily by BER . The fidelity of polymerase-beta is 2.4x10(-5) and one must postulate additional fidelity mechanisms in the BER complex to explain the low mutation rate of resting cells . Any strategy which constitutively increases glycosylase activity to prevent endogenous lesions from entering S-phase and becoming mutations will also serve to increase the number of mutations per day caused by non-specific excision of normal undamaged bases . The best break-even strategy for reducing endogenous lesion-induced mutations is clearly not one of avid repair . Lower organisms from bacteriophage to fungi have adopted strategies to generate 0.0033 consequential mutations per cell division, no more and no less . Strategies such as down regulating glycosylase activity outside of S-phase to reduce time-dependent mutation frequency while leaving lesion replication-induced mutation frequency unchanged are discussed . Micron, 1998 Apr-Jun, 29(2-3), 145 - 60 Cryo-negative staining; Adrian M et al.; A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we term cryo-negative staining . The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thinner than the aqueous film produced by conventional negative staining on a continuous carbon layer . Because of this, a higher than usual concentration of negative stain (ca . 16% rather than 2%) is required for cryo-negative staining in order to produce an optimal image contrast . The maintenance of the hydrated state, the absence of adsorption to a carbon film and associated sample flattening, together with reduced stain granularity, generates high contrast cryo-images of superior quality to conventional air-dry negative staining . Image features characteristic of unstained vitrified cryo-electron microscopic specimens are present, but with reverse contrast . Examples of cryo-negative staining of several particulate biological samples are shown, including bacteriophage T2, tobacco mosaic virus (TMV), bovine liver catalase crystals, tomato bushy stunt virus (TBSV), turnip yellow mosaic virus (TYMV), keyhole limpet hemocyanin (KLH) types 1 and 2, the 20S proteasome from moss and the E . coli chaperone GroEL . Densitometric quantitation of the mass-density of cryo-negatively stained bacteriophage T2 specimens before and after freeze-drying within the TEM indicates a water content of 30% in the vitreous specimen . Determination of the image resolution from cryo-negatively stained TMV rods and catalase crystals shows the presence of optical diffraction data to ca . 10 A and 11.5 A, respectively . For cryo-negatively stained vitrified catalase crystals, electron diffraction shows that atomic resolution is preserved (to better than 20 diffraction orders and less than 3 A) . The electron diffraction resolution is reduced to ca . 10 A when catalase crystal specimens are prepared without freezing or when they are freeze-dried in the electron microscope . Thin vitrified films of TMV, TBSV and TYMV in the presence of 16% ammonium molybdate show a clear indication of two-dimensional (2-D) order, confirmed by single particle orientational analysis of TBSV and 2-D crystallographic analysis of TYMV . These observations are in accord with earlier claims that ammonium molybdate induces 2-D array and crystal formation from viruses and macromolecules during drying onto mica . Three-dimensional analysis of the TBSV sample using the tools of icosahedral reconstruction revealed that a significant fraction of the particles were distorted . A reconstruction from a subset of undistorted particles produced the characteristic T = 3 dimer clustered structure of TBSV, although the spikes are shortened relative to the structure defined by X-ray crystallography . The 20S proteasome, GroEL, catalase, bacteriophage T2, TMV, TBSV and TYMV all show no indication of sample instability during cryo-negative staining . However, detectable dissociation of the KLH2 oligomers in the presence of the high concentration of ammonium molybdate conforms with existing knowledge on the molybdate-induced dissociation of this molecule . This indicates that the possibility of sample-stain interaction in solution, prior to vitrification, must always be carefully assessed. J Mol Biol, 1998 Aug 7, 281(1), 81 - 94 A helical coat protein recognition domain of the bacteriophage P22 scaffolding protein; Tuma R et al.; The scaffolding protein of bacteriophage P22 directs the assembly of an icosahedral procapsid, a metastable shell that is the precursor for DNA packaging . The full-length protein has been shown previously to exist in a monomer-dimer-tetramer equilibrium of elongated and predominantly alpha-helical molecules . Two deletion-mutant fragments of the scaffolding protein, comprising amino acid residues 141 to 303 and 141 to 292, respectively, have been constructed, overexpressed in Escherichia coli, and purified . Removal of residues 1 to 140 yields a protein that is assembly-active both in vitro and in vivo, while the removal of the C-terminal 11 residues (293 to 303) leads to complete loss of scaffolding activity . Sedimentation analysis reveals that both scaffolding fragments exist in a monomer-dimer equilibrium governed by apparent dissociation constants Kd(141-303)=640 microM and Kd(141-292)=880 microM . Tetramer formation is not observed for either fragment; thus, the tetramerization domain of the scaffoldi