Bacteriophages

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 scaffolding subunit resides in the N-terminal portion of the polypeptide chain . Examination of both fragments by circular dichroism, Raman and NMR spectroscopies indicates a highly alpha-helical fold in each case . Nonetheless, pronounced differences are observed between spectral signatures of the two fragments . Notably, Raman spectra of fragments 141-292 and 141-303 indicate that elimination of residues 293 to 303 results in unfolding of an alpha-helical coat protein "recognition" domain encompassing about 20 to 30 residues . The thermostability of fragment 141-303, monitored over a wide concentration range by circular dichroism and Raman spectroscopy, indicates a broad denaturation transition for the monomeric (low concentration) form, while more cooperative unfolding is observed for the dimeric (high concentration) form . A lesser increase in cooperativity upon dimerization is obtained for fragment 141-292 . Additionally, the C-terminal recognition domain constitutes the most stable and cooperative unit in the 141-303 fragment . Measurement of hydrogen-isotope exchange kinetics in scaffolding fragments by time-resolved Raman spectroscopy shows that the C terminus is the only protected segment of the polypeptide chain . On the basis of the measured hydrodynamic and spectroscopic properties, a domain structure is proposed for the scaffolding subunit . The roles of these domains in P22 procapsid assembly are discussed .

J Mol Biol, 1998 Aug 7, 281(1), 69 - 79
Functional domains of bacteriophage P22 scaffolding protein; Parker MH et al.; Assembly of the bacteriophage P22 requires a 303 amino acid residue scaffolding protein . Two scaffolding protein deletion mutants, consisting of residues 141 to 303 and 141 to 292, have been described . We report here that the 141-303 fragment, but not the 141-292 fragment, promoted procapsid assembly in vitro, bound to preformed shells of coat protein, and bound to a coat protein affinity column . These findings suggest that the carboxyl-terminal half of the scaffolding protein is sufficient for promoting assembly, and that the 11 amino acid residues at the extreme carboxyl terminus are required for binding to the coat protein . Analysis of the products of in vitro assembly reactions suggests that the maximum amount of scaffolding protein that can pack into a procapsid is dictated by the internal volume of the procapsid rather than by a finite number of binding sites . However, when the amount of scaffolding protein was reduced to limiting values, both the wild-type protein and the 141-303 fragment assembled procapsids with the same number, rather than the same mass, of scaffolding protein molecules . When the 141-292 fragment was added to a mixture of coat and scaffolding proteins, the initial phase of procapsid assembly was inhibited, but the final yield and composition of the procapsids were not affected . Assembly by a covalent dimeric mutant scaffolding protein (R74C/L177I) was not inhibited by the 141-292 fragment, which suggests that the inhibition is due to the formation of inactive heterodimers between the 141-292 fragment and the monomeric scaffolding protein . The 141-303 fragment, which has less tendency to self-associate than the wild-type protein, formed aberrant species as well as normal procapsid-like particles when the rate of assembly was high, suggesting that scaffolding protein dimerization may play a role in ensuring fidelity of assembly . Alternatively, residues 1 to 140 may play a direct structural role in preventing inappropriate scaffolding/coat protein interactions .

Mol Divers, 1997-1998, 3(3), 149 - 59
Selection of a cyclic nonapeptide inhibitor to alpha-chymotrypsin using a phage display peptide library; Krook M et al.; A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against alpha-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5) . Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with alpha-chymotrypsin than the original library and a wild-type phage did . DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions . The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of alpha-chymotrypsin . The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions . The inhibition constant for alpha-chymotrypsin was estimated to be 10(-6) M . Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified alpha-chymotrypsin than for natural alpha-chymotrypsin as determined by an enzyme immunosorbent assay . This peptide phage clone was also competitively prevented from binding to alpha-chymotrypsin by the corresponding synthetic oxidized peptide . Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe- Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of alpha-chymotrypsin and acts as a specific inhibitor to the enzyme . To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature.

Protein Eng, 1998 Apr, 11(4), 329 - 32
Engineering trimeric fibrous proteins based on bacteriophage T4 adhesins; Miroshnikov KA et al.; The adsorption specificity of bacteriophage T4 is determined by genes 12 and 37, encoding the short tail-fibers (STF) and the distal part of the long tail-fibers (LTF), respectively . Both are trimeric proteins with rod domains made up of similar tandem quasi-repeats, approximately 40 amino acids long . Their assembly requires the viral chaperones gp57A and gp38 . Here we report that fusing fragments of gp12 and gp37 to another trimeric T4 fibrous protein, fibritin, facilitates correct assembly, thereby by-passing the chaperone requirement . Fibritin is an alpha-helical coiled coil protein whose C-terminal part (fibritin E, comprising the last 120 residues) has recently been solved to atomic resolution . Gp12 fragments of 109 and 70 amino acids, corresponding to three and two quasi-repeats respectively, were fused to the C-terminus of fibritin E . A similar chimera was designed for the last 63 residues of gp37, which contain four copies of the pentapeptide Gly-X-His-X-His and assume a narrow rigid structure in the LTF distal tip . Expressed from plasmids, all three chimeras form soluble trimers that are resistant to dissociation by SDS and digestion by trypsin, indicative of correct folding and oligomerization.

Chem Biol Interact, 1998 Apr 24, 111-112, 51 - 67
Regulation of rat glutathione S-transferase A5 by cancer chemopreventive agents: mechanisms of inducible resistance to aflatoxin B1; Hayes JD et al.; The rat can be protected against aflatoxin B1 (AFB1) hepatocarcinogenesis by being fed on a diet containing the synthetic antioxidant ethoxyquin . Evidence suggests that chemoprotection against AFB1 is due to increased detoxification of the mycotoxin by one or more inducible drug-metabolising enzymes . The glutathione S-transferase (GST) isoenzymes in rat liver that contribute to ethoxyquin-induced chemoprotection against AFB1 have been identified by protein purification . This approach resulted in the isolation of several heterodimeric class alpha GST, all of which contained the A5 subunit and possessed at least 50-fold greater activity towards AFB1-8,9-epoxide than previously studied transferases . Molecular cloning and heterologous expression of rat GSTA5-5 has led to the demonstration that it exhibits substantially greater activity for AFB1-8,9-epoxide than other rat transferases . The A5 homodimer can also catalyse the conjugation of glutathione with other epoxides, such as trans-stilbene oxide and 1,2-epoxy-3-(4'-nitrophenoxy)propane, and possesses high catalytic activity for the reactive aldehyde 4-hydroxynonenal . Western blotting has shown that the A5 subunit is not only induced by ethoxyquin but that it is also induced by other cancer chemopreventive agents, such as butylated hydroxyanisole, oltipraz, benzyl isothiocyanate, indole-3-carbinol and coumarin . In addition to GSTA5, we have identified a novel aflatoxin-aldehyde reductase (AFAR) that is similarly induced by ethoxyquin . However, immunoblotting has shown that GSTA5 and AFAR are not always co-ordinately regulated by chemoprotectors . In order to gain a better understanding of the mechanisms responsible for the induction of GSTA5 protein, the GSTA5 gene has been cloned . It was isolated on two overlapping bacteriophage lambda clones and found to be approximately 12 kb in length . The transcriptional start site of GSTA5 has been identified 228 bp upstream from the ATG translational initiation codon . Computer-assisted analysis of the upstream sequence has indicated the presence of a putative antioxidant responsive element (located between -421 and -429 bp) which may be responsible for the induction of GSTA5 by chemopreventive agents.

DNA Res, 1998 Apr 30, 5(2), 77 - 85
Molecular characterization of mouse Pneumocystis carinii surface glycoprotein A; Haidaris CG et al.; Since the mouse offers an easily manipulated experimental animal model for the study of the immunopathogenesis of pneumonia caused by the opportunist Pneumocystis carinii, we cloned and characterized cDNAs encoding an abundant, immunogenic surface antigen termed glycoprotein A (gpA) from mouse P . carinii . A cDNA library was constructed in bacteriophage lambda gt11 from P . carinii-infected mouse lung poly(A+) RNA . Using a nucleic acid probe derived from a conserved region of the mouse P . carinii gpA structural gene, cDNAs encoding gpA were identified . A composite full-length gpA coding sequence was assembled from two overlapping cDNA clones . A DNA element homologous to the rat P . carinii upstream conserved sequence (UCS) was identified at the 5' end of several of the mouse P . carinii gpA cDNA clones, just upstream of the sequences encoding gpA structural gene isoforms . Using primer extension analysis, two neighboring putative transcriptional start sites were located on UCS-gpA mRNAs approximately 25 and 30 nt, respectively, upstream of the most 5' gpA cDNA clone isolated, suggesting a 5' UCS of 489 or 494 nucleotides in mouse P . carinii gpA . A comparative alignment of the composite mouse P . carinii gpA deduced amino acid sequence with gpA homologs from rat, human and ferret P . carinii demonstrated 156 identical residues, including 46 cysteines, further supporting the hypothesis for conserved secondary structure, as well as function, for gpA from all P . carinii.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 159 - 67
Characterization of Escherichia coli lysis using a family of chimeric E-L genes; Witte A et al.; Gene E-L, a chimeric lysis construct from bacteriophages phi X174 and MS2 lysis proteins E and L, respectively, was subjected to internal deletions to create a series of new E-L clones with altered lysis or killing properties . The lytic activities of the parental genes E . L . E-L and the internal truncated forms of E-L were investigated in this study to characterize the different lysis mechanisms, based on differences in the architecture of the different membrane spanning domains . Electron microscopy and release of marker enzymes for the cytoplasmic and periplasmic spaces revealed that two different lysis mechanisms can be distinguished depending on penetrating of the proteins either the inner membrane or the inner and outer membranes of Escherichia coli . Several candidates, which share efficient lysis properties, have biotechnological applications in terms of cell disruption.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 149 - 57
Effect of phi X174 protein E-mediated lysis on murein composition of Escherichia coli; Witte A et al.; Lysis of Escherichia coli by bacteriophage phi X174 is caused by the phage protein E . As protein E is devoid of enzymatic activities it has been postulated that lysis is the result of an induction of the autolytic enzymes of the host . This hypothesis was investigated by comparing the murein composition before and during lysis of either phi X174 infected cells or protein E induced lysis of E . coli . Additionally, protein E-mediated lysis was compared with induction of the autolytic system by EDTA . The analysis showed that the overall composition of murein is not changed after induction of protein E-mediated lysis . Nevertheless, murein degradation seems to be stimulated by the action of protein E as shown by an increase in the total amount of murein turnover products by about 10% . It could be shown that an intact murein sacculus prevents the phages from being released.

Cell, 1998 Jul 10, 94(1), 55 - 60
Protein chainmail: catenated protein in viral capsids; Duda RL; The capsid shells of bacteriophage HK97 and several other phages contain polypeptides that are covalently linked into complexes so large that they do not enter polyacrylamide gels after denaturation . The enormous apparent size of these protein complexes in HK97 derives from a novel protein topology . HK97 subunits cross-link via isopeptide bonds into oligomers that are closed rings of five or six members . However, polypeptides from neighboring pentamer and hexamer rings intertwine before the covalent cross-links form . As a result, adjacent protein rings catenate into a network similar to chainmail armor . In vitro linking and unlinking experiments provide strong support for the chainmail model, which explains the unusual properties of these bacteriophages and may apply to other macromolecular structures.

J Immunol Methods, 1998 Mar 15, 212(2), 131 - 8
Multifunctional g3p-peptide tag for current phage display systems; Beckmann C et al.; We have previously described a monoclonal antibody (mAb), 10C3, directed against the gene-3 protein (g3p) of filamentous phage M13, which was produced to study g3p fusion protein expression in Escherichia coli and its incorporation in the phage capsid {Tesar, M., Beckmann, C., Rottgen, P., Haase, B., Faude, U., Timmis, K., 1995 . Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems . Immunology 1, 53-54} . In this study we report mapping of the antigenic epitope of the mAb 10C3, by means of short overlapping peptide-sequences {Frank, R., Overwin, H., 1996 . Spot synthesis . In: Morris, G.E . (Ed.), Methods in Molecular Biology, Vol . 66: Epitope Mapping Protocols . Humana Press, Totowa, NJ, pp . 149-169.} comprising the C-terminal half of the g3-protein . A minimal recognizable peptide was found which is represented in the 11 amino acid sequence from positions 292 to 302 of g3p {Wezenbeek van, P.M.G.P., Hulsebos, T.J.M., Schoenmakers, J.G.G., 1980 . Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd . Gene 11, 129-148} . In order to use the antibody also for detection and purification of recombinant proteins, such as single chain antibodies, the epitope was introduced as a tag sequence into the phagemid pHEN1 {Hoogenboom, H.R., Griffith, A.D., Johnson, K., Chiswell, D.J., Hudson, P., Winter, G., 1991 . Multi-subunit proteins on the surface of the filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains . Nucleic Acid Res . 19, 4133-4137; Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D., Winter, G., 1994 . Antibody fragments from a single pot phage display library as immunochemical reagents . EMBO J . 13 (3) 692-698} . Purified single chain antibodies containing this tag were detectable down to a concentration of 2 ng ml(-1) under non-denaturing conditions (ELISA) or 4 ng per lane on immunoblots . The high sensitivity of the antibody for the peptide tag was reflected in the antibody affinity constant K(D) of 6.80 x 10(-10) M, which was determined by real time biomolecular interaction analysis (BIA) based on surface plasmon resonance (SPR) {Karlsson, R., Falt, A., 1997 . Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors . J . Immunol . Methods 200, 121-133} . Finally, recombinant proteins in E . coli periplasmic extracts could be purified in a single step by affinity purification using immobilized mAb 10C3 . These studies demonstrated that the new peptide-tag and its corresponding mAb represents a versatile tool for the detection of recombinant proteins selected by phage display technology.

Nucleic Acids Res, 1998 Aug 1, 26(15), 3611 - 3
Transfer of P1 inserts into a yeast-bacteria shuttle vector by co-transformation mediated homologous recombination; Criswell TL et al.; Manipulation of genomic inserts cloned into the bacteriophage P1 vector is hindered by the large size of the inserts . We have used co-transformation mediated recombination between the yeast-bacteria shuttle vector, pClasper, and various P1 clones to transfer the entire insert from the P1 into pClasper . This results in the insert being stably maintained in yeast, facilitating mutagenesis by homologous recombination . The recombinant plasmid can subsequently be transferred to and stably maintained in bacteria for efficient plasmid preparation . This method can also be applied to inserts from P1 artificial chromosome or bacterial artificial chromosome vectors.

J Mol Biol, 1998 Jul 31, 280(5), 799 - 810
Lambda repressor N-terminal DNA-binding domain as an assay for protein transmembrane segment interactions in vivo; Leeds JA et al.; To understand the determinants of membrane protein interactions, we have developed an in vivo genetic assay system for detecting homodimerization of transmembrane (TM) segments from integral membrane proteins . Our approach is to generate gene fusions between potentially dimerizing TM segments and a cytoplasmic DNA-binding protein that lacks its intrinsic dimerization domain . This genetic approach allows us to screen and distinguish among known dimerizing domains and weakly dimerizing mutants, as well as non-dimerizing TM segments . We replaced the bacteriophage lambda cI repressor C-terminal dimerization domain with the human erythrocyte glycophorin A transmembrane segment (GpA TM) . GpA TM forms SDS-resistant homodimers in vitro . Expression of this membrane-associated fusion in Escherichia coli conferred the same degree of immunity to lambda cI phages as the wild-type, intact lambda repressor . Single amino acid substitutions that disrupt the GpA TM dimer interface were introduced into the lambda-GpA TM fusion proteins . These mutations dramatically reduced immunity of E . coli to lambda cI, such that the efficiency of plating these phages increased by greater than 10,000-fold over that conferred by the wild-type lambda-GpA TM fusion . Introduction of the putatively non-dimerizing first TM from E . coli MalF into the lambda-TM fusion vector resulted in no immunity to lambda cI phages . Fusion of the homodimeric, periplasmically localized, mature alkaline phosphatase domain to the C terminus of the lambda-TM fusion proteins containing weakly to non-dimerizing TM segments restored immunity to lambda cI phages . Results from this in vivo genetic assay system demonstrate that (1) dimerization of the lambda cI DNA-binding domain can be promoted by dimerizing TM segments, (2) strongly, weakly, and non-dimerizing TM segments can be distinguished on the basis of their ability to confer immunity to lambda cI phages, and (3) introduction of a dimerizing periplasmic domain can provide functionality to lambda-TM fusions containing weakly to non-dimerizing TM segments .

EMBO J, 1998 Jul 15, 17(14), 4092 - 100
Solution structure of the antitermination protein NusB of Escherichia coli: a novel all-helical fold for an RNA-binding protein; Huenges M et al.; The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination . In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA . The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage lambda, where they play an important role in switching from the lysogenic to the lytic cycle . Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13C, 15N and 2H . The three-dimensional structure of the protein was solved from 1926 NMR-derived distances and 80 torsion angle restraints . The protein folds into an alpha/alpha-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered . Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals . The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM).

Mol Gen Genet, 1998 Jun, 258(5), 494 - 502
Regulation of replication of lambda phage and lambda plasmid DNAs at low temperature; Gabig M et al.; It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein . This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis . Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C . Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature . Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C . Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication . Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII . We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature.

Biochemistry (Mosc), 1998 Jun, 63(6), 702 - 9
Properties of recombinant bacteriophage T4 tail sheath protein and its deletion fragments; Kuznetsova TA et al.; A vector for expression of recombinant bacteriophage T4 tail sheath protein (gp18) under control of phage T7 promoter in Escherichia coli cells has been constructed . The entire length recombinant gp18 (659 amino acids) polymerizes in vivo into extended polysheaths . To study gp18 folding mechanisms, six vectors for expression of deletion mutants have been constructed . Three proteins--1N, 2N, and 3N--contain, respectively, 268, 316, and 372 amino acids of the gp18 N-tail region . The other three fragments--1C, 2C, and 3C--contain, respectively, 455, 356, and 288 amino acids of the gp18 C-tail . The fragments 1N, 2N, 1C, 2C, and 3C form insoluble aggregates during expression . However, fragment 3N accumulates in soluble form in the cellular cytoplasm and does not form polymeric structures; this has allowed an effective purification method to be developed for it . The interaction of monoclonal antibodies against recombinant gp18 with protein fragments and with phage sheath before and after contraction has been studied . The fragment 3N seems to be a stable domain of native phage sheath gp18.

Curr Opin Chem Biol, 1997 Oct, 1(3), 316 - 22
Assembly and disassembly of DNA polymerase holoenzyme; Sexton DJ et al.; The complex task of genomic replication requires a large collection of proteins properly assembled within the close confines of the replication fork . The mechanism and dynamics of holoenzyme assembly and disassembly have been investigated using steady state and pre-steady state methods as opposed to structural studies, primarily due to the intrinsic transient nature of these protein complexes during DNA replication . The key step in bacteriophage T4 holoenzyme assembly involves ATP hydrolysis, whereas disassembly is mediated by subunit dissociation of the clamp protein in an ATP-independent manner.

J Neurol Neurosurg Psychiatry, 1998 Jul, 65(1), 48 - 55
Molecular approach to find target(s) for oligoclonal bands in multiple sclerosis; Rand KH et al.; OBJECTIVES: Oligoclonal bands are a characteristic finding in the CSF of patients with multiple sclerosis, yet their target antigen(s) remain unknown . The objective was to determine whether a filamentous phage peptide library could be employed to allow the oligoclonal bands to select their own target epitopes . METHODS: CSF IgG antibody from 14 patients with multiple sclerosis and 14 controls was used to select individual phage clones from a bacteriophage library containing approximately 4 x 10(7) different hexamers expressed on its surface pIII protein . The amino acid sequence selected was deduced by sequencing the DNA of the genetically engineered insert . RESULTS: In general, after three rounds of selection, CSF from both patients with multiple sclerosis and controls selected one to two consistent peptide motifs . Five out of 14 patients with multiple sclerosis, and one control, selected the amino acid sequence motif, RRPFF . Given 20 possible amino acids per position, the likelihood of five patients selecting the same linear five amino acid sequence is at most 1.6 x 10(-3), corrected for the number of clones sequenced . A GenBank computer search showed that this sequence is found in the Epstein-Barr Virus nuclear antigen (EBNA-1), and a heat shock protein alphaB crystallin . Human serum antibodies to a synthetic peptide containing RRPFF were virtually exclusively found in patients with prior infection by Epstein-Barr virus . Other studies have suggested a relation between Epstein-Barr virus infection and multiple sclerosis, including nearly 100% Epstein-Barr virus seropositivity among patients with multiple sclerosis and increased concentrations of antibody to EBNA in CSF of patients with multiple sclerosis . By antigen specific immunoblotting, antibodies to the RRPFF motif in the CSF were shown to correspond to a subset of oligoclonal bands in the CSF from the same patient . CONCLUSION: This study shows that phage epitope display libraries may be used to select amino acid motifs which are potentially relevant to the pathogenesis of multiple sclerosis.

Biochemistry, 1998 Jul 14, 37(28), 10181 - 7
Mimicking initial interactions of bacteriophage M13 coat protein disassembly in model membrane systems; Stopar D et al.; The structure and changes in environment of the M13 major coat protein were studied in model systems, mimicking the initial molecular process of the phage disassembly . For this purpose we have systematically studied protein associations with various detergents and lipids in two different coat protein assemblies: phage particles and S-forms . It is remarkable that the major coat protein can change its conformation to accommodate three distinctly different environments: phage filament, S-form, and membrane-bound form . The structural and environmental changes during this protein transformations were studied by site-directed spin labeling, fluorescence labeling, and CD spectroscopy in different membrane model systems . The phage particles were disrupted only by strong ionic detergents {sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide and (CTAB)} but were not affected by sodium cholate and sodium deoxycholate, nonionic detergents, and dilauroyl-l-alpha-phosphatidylcholine (DLPC) lipid bilayers . Conversion of the phage particles into S-forms by addition of chloroform rendered the coat protein accessible for the association with different ionic and nonionic detergents, as well as DLPC lipids . The disruption of the S-form by all detergents studied was instantaneous but was slower with DLPC vesicles . Only small unilamellar vesicles effectively solubilized the S-form . The data suggest that the viral protein coat is inherently unstable when the major coat protein is exposed to amphiphilic molecules . During conversion from the phage to the S-form, and subsequently to the membrane-bound form, the coat protein undergoes pronounced changes in environment, and in response the alpha-helix content decreases and the local protein structure changes dramatically . This adaptation of the protein conformation enables a stable association of the protein with the membrane.

Biochemistry, 1998 Jul 14, 37(28), 10156 - 63
Stopped-flow fluorescence study of precatalytic primer strand base-unstacking transitions in the exonuclease cleft of bacteriophage T4 DNA polymerase; Otto MR et al.; DNA polymerases are complex enzymes which bind primer-template DNA and subsequently either extend or excise the terminal nucleotide on the primer strand . In this study, a stopped-flow fluorescence anisotropy binding assay is combined with real-time measurements of a fluorescent adenine analogue (2-aminopurine) located at the 3'-primer terminus . Using this combined approach, the exact time course associated with protein binding, primer terminus unstacking, and base excision by the 3' --> 5' exonuclease of bacteriophage T4 (T4 pol) was examined . T4 pol binding and dissociation kinetics were found to obey simple kinetics, with identical on rates (kon = 4.6 x 10(8) M-1 s-1) and off rates (koff = 9.3 s-1) for both single-stranded primers and double-stranded primer-templates (at 100 microM Mg2+) . Although the time course for T4 pol-DNA association and dissociation obeyed simple kinetics, at suboptimal Mg2+ concentrations (e.g., 100 microM), non-first-order sigmoidal kinetics were observed for the base-unstacking reaction of the primer terminus in double-stranded primer-templates . The observed sigmoidal kinetics for base unstacking demonstrate that T4 pol is a hysteretic enzyme {Frieden, C . (1970) J . Biol . Chem . 245, 5788-5799} and must exist in two DNA bound conformations which differ greatly in base-unstacking properties . A Mg2+-dependent time lag of 10 ms is observed between primer-template binding and the beginning of the unstacking transition, which is 50% complete at 22 +/- 1 ms after addition of 100 microM Mg2+ . Following the hysteretic lag, a simple first-order primer terminus unstacking rate of 130 s-1 is resolved, which is protein and Mg2+ concentration-independent . For the processing of single-stranded primers, all kinetic complexity is lost, and T4 pol binding and primer end base-unstacking kinetics can be superimposed . These data reveal that the kinetic processing of double-stranded primer-template DNA by T4 pol is much more complex than that of single-stranded primers, and suggest that the intrinsic "switching rate" between the polymerase and exonuclease sites may be much faster than previously proposed.

Biochemistry, 1998 Jul 14, 37(28), 10144 - 55
Exonuclease-polymerase active site partitioning of primer-template DNA strands and equilibrium Mg2+ binding properties of bacteriophage T4 DNA polymerase; Beechem JM et al.; The binding of bacteriophage T4 DNA polymerase (T4 pol) to primer-template DNA with 2-aminopurine (2AP) located at the primer terminus results in the formation of a hyperfluorescent 2AP state . Changes in this hyperfluorescent state were utilized to investigate the fractional concentration of primer-templates bound at the exonuclease and statically quenched polymerase sites . In the absence of Mg2+, a hydrophobic exonuclease site dominates over the polymerase site for possession of the primer terminus . The fractional concentration of primer termini in the exonuclease site was found to be 64 and 84% for correct (AP-T) and mismatched (AP-C) primer-templates, respectively . Exonuclease-deficient mutants, polymerase-switching mutants, and nucleoside triphosphates all shift this equilibrium toward the polymerase site . Synthesis of stereospecific hydrolysis resistant phosphorothioate 2AP-labeled DNA allowed Mg2+ ion binding titrations to be performed in the presence of bound DNA without the complication of the excision reaction . High- and low-affinity Mg2+ binding sites were observed in the presence of bound double-stranded (ds) DNA, with dissociation constants in the micromolar (WT Kd = 5.1 microM) and millimolar (WT Kd = 2.5 mM) concentration ranges . Mg2+ binding was found to be a key "conformational switch" for T4 pol . As the high-affinity Mg2+ binding sites are filled, the primer terminus migrates from the exonuclease site to a highly based stacked polymerase active site . Filling the low-affinity Mg2+ sites further shifts the primer terminus into the polymerase site . As the low-affinity Mg2+ sites are filled, T4 pol "loosens its grip" on the primer terminus, as shown by a large amplitude increase in the nanosecond rotational mobility of 2AP within the bound T4 complex . The hyperfluorescent exonuclease site is spatially localized to 2AP positioned on the primer end . The penultimate (n - 1) position, as well as n - 2 and n - 5 positions, reveals no detectable fluorescent enhancement upon binding . The observed position-dependent fluorescence data, when combined with time-resolved total-intensity and anisotropy data, suggest that the creation of the hyperfluorescent state is caused by phenylalanine 120 (F120) of T4 pol intercalating into 2AP primers much like that observed for phenylalanine 123 of RB69 DNA polymerase intercalating into deoxythymidine primers {Wang, J., et al . (1997) Cell 89, 1087-1099} . As Mg2+ binds in the exonuclease site of T4 pol, the primer terminus appears to be "pulled backward" into the active site, decreasing the concentration of F120-intercalated primer termini, and bringing the exonuclease active site residues closer to the primer terminus scissile phosphate bond.

Nat Struct Biol, 1998 Jul, 5(7), 543 - 6
Specific RNA binding proteins constructed from zinc fingers; Friesen WJ et al.; A zinc finger library with degenerate alpha-helices was displayed on the surface of bacteriophage and proteins that bind human immunodeficiency virus type-1 (HIV-1) Rev response element stem loop IIB (RRE-IIB) RNA or 5S rRNA were isolated . DNA encoding affinity selected zinc fingers was shuffled by recombination in vitro to isolate proteins with higher RNA binding affinity . Proteins constructed in this way bind RNA specifically both in vitro and in vivo . These results demonstrate that RNA substrate specificity of zinc fingers can be changed through mutation of alpha-helices to construct novel RNA binding proteins.

J Clin Invest, 1998 Jul 15, 102(2), 430 - 7
Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display; Rajotte D et al.; Vascular beds are known to differ in structure and metabolic function, but less is known about their molecular diversity . We have studied organ-specific molecular differences of the endothelium in various tissues by using in vivo screening of peptide libraries expressed on the surface of a bacteriophage . We report here that targeting of a large number of tissues with this method yielded, in each case, phage that homed selectively to the targeted organ . Different peptide motifs were recovered from each of these tissues . The enrichment in homing to the target organs relative to an unselected phage was 3-35-fold . Peptide sequences that conferred selective phage homing to the vasculature of lung, skin, and pancreas were characterized in detail . Immunohistochemistry showed that the phage localized in the blood vessels of their target organ . When tested, the phage homing was blocked in the presence of the cognate peptide . By targeting several tissues and by showing that specific homing could be achieved in each case, we provide evidence that organ- and tissue-specific molecular heterogeneity of the vasculature is a general, perhaps even universal, phenomenon . Our results also show that these molecular differences can serve as molecular addresses.

FEBS Lett, 1998 Jun 16, 429(3), 269 - 73
A novel peptide, PLAEIDGIELTY, for the targeting of alpha9beta1-integrins; Schneider H et al.; Targeting gene therapy vectors to abundant receptors on airway epithelia may allow a significant enhancement of gene delivery and thereby be of particular importance for the gene therapy of cystic fibrosis . Alpha9beta1-integrins are highly expressed throughout the human airway epithelia in vivo, irrespective of any particular clinical status . Aiming to improve the targeting of our non-viral integrin-mediated gene transfer systems to airway epithelia, we searched for a short tenascin C-derived peptide which would bind to these integrins . By utilizing recombinant bacteriophages that display overlapping regions of the third fibronectin type III repeat of tenascin C (TNfn3), we were able to localize its alpha9beta1-integrin binding site to the B-C loop of TNfn3 . A synthetic Pro-Leu-Ala-Glu-Ile-Asp-Gly-Ile-Glu-Leu-Thr-Tyr peptide (PLAEIDGIELTY) was shown to displace alpha9beta1-integrin-expressing cells completely from binding to TNfn3 . This peptide, therefore, may prove useful both for the examination of the functional importance of alpha9beta1-integrins in vivo and the development of gene therapy vectors or drugs targeting these integrins.

Mol Cell, 1998 Jan, 1(2), 265 - 75
Independent ligand-induced folding of the RNA-binding domain and two functionally distinct antitermination regions in the phage lambda N protein; Mogridge J et al.; The transcriptional antitermination protein N of bacteriophage lambda binds the boxB component of the RNA enhancer nut (boxA + boxB) and the E . coli elongation factor NusA . Efficient antitermination by N requires an RNA-binding domain (amino acids 1-22) and two activating regions for antitermination: a newly identified NusA-binding region (amino acids 34-47) that suppresses NusA's enhancement of termination, and a carboxy-terminal region (amino acids 73-107) that interacts directly with RNA polymerase . Heteronuclear magnetic resonance experiments demonstrate that N is a disordered protein . Interaction with boxB RNA induces only the RNA-binding domain of N to adopt a folded conformation, while the activating regions of the protein remain disordered in the absence of their target proteins.

Biochim Biophys Acta, 1998 May 19, 1384(2), 243 - 52
Mapping of functional sites on the primary structure of the tail lysozyme of bacteriophage T4 by mutational analysis; Takeda S et al.; Tail lysozyme of bacteriophage T4, product of gene 5 (gp5), functions upon infection by locally digging a hole in the peptidoglycan layer, so that the tail tube, through which the phage DNA is injected, can penetrate to the inner membrane . It has been inferred from DNA sequence and expression of the tail lysozyme on a plasmid in Escherichia coli that the tail lysozyme is synthesized as a precursor of 62 K and is later cleaved to form a mature tail lysozyme of 42 K . Furthermore, gp5 has a region that is highly homologous to T4 lysozyme, gpe, that is the product of gene e and functions for 'lysis from within' . As an approach to elucidation of structure-function relationship of gp5, we determined mutational sites of gene 5 mutants that have heat sensitive virions, are temperature sensitive for growth, or require an amber suppressor . All the mutational sites were mapped in the region corresponding to the mature tail lysozyme . Among the ts mutants, 5ts1 was a pseudo-revertant of an amber mutant which bypasses gene e . It was mapped in the region which had a high homology to gpe, which is well known as T4 lysozyme . The other mutational sites will be also discussed in relation to the phenotypes of the mutants.

Biol Pharm Bull, 1998 Jun, 21(6), 621 - 3
Virus inactivation in superoxide dismutase preparations by ultraviolet light irradiation; Hirayama J et al.; Viral inactivation in superoxide dismutase (SOD) derived from human red cells was carried out by ultraviolet light C (UVC) irradiation . With 400 J/m2 UVC irradiation, the titer of canine parvovirus (CPV, a nonenveloped virus), M13 bacteriophage (M13, a nonenveloped phage) and vesicular stomatitis virus (VSV, an enveloped virus), which were spiked into SOD solution, were reduced by > 4.6 log10 (detection limit), 7.0 log10 and 6.2 log10, respectively . The SOD activity was maintained and the band pattern of SOD on an electrophoresis gel was not changed even by 1000 J/m2 UVC irradiation . These results indicate that UVC irradiation is a promising method for the inactivation of both enveloped and nonenveloped viruses in SOD preparations while maintaining its activity.

Virology, 1998 Jun 20, 246(1), 158 - 69
Evolutionary relationships among putative RNA-dependent RNA polymerases encoded by a mitochondrial virus-like RNA in the Dutch elm disease fungus, Ophiostoma novo-ulmi, by other viruses and virus-like RNAs and by the Arabidopsis mitochondrial genome; Hong Y et al.; The nucleotide sequence (2617 nucleotides) of virus-like double-stranded (ds) RNA 3a in a diseased isolate, Log1/3-8d2 (Ld), of the ascomycete fungus Ophiostoma novo-ulmi has been determined . One strand of the dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 718 amino acids, and the complementary strand contains two smaller ORFs with the potential to encode proteins of 178 and 182 amino acids, respectively . The large ORF contains 12 UGA codons which code for tryptophan in ascomycete mitochondria and has a codon bias typical of mitochondrial genes, consistent with the localization of Ld dsRNAs within the mitochondria . The amino acid sequence contains motifs characteristic of RNA-dependent RNA polymerases (RdRps) . This putative RdRp was shown to be related to putative RdRps of mitochondrial dsRNAs of another ascomycete and a basidiomycete fungus and also to a putative RdRp encoded by the mitochondrial genome of Arabidopsis thaliana . In multiple sequence alignments, the fungal mitochondrial dsRNA-encoded RdRp-like proteins formed a cluster, ancestrally related to the RdRps of the yeast 20S and 23S RNA replicons and of the positive-stranded RNA bacteriophages of the Leviviridae family, but distinct from RdRps of other families and genera of fungal RNA viruses and related plant and animal RNA viruses . Northern blot analysis with RNA 3a strand-specific probes indicated that nucleic acid extracts of Ld contain more single-stranded (positive-stranded) RNA than dsRNA, consistent with an evolutionary relationship between RNA 3a and positive-stranded RNA phages.

J Mol Biol, 1998 Jul 10, 280(2), 201 - 13
Pausing and termination by bacteriophage T7 RNA polymerase; Lyakhov DL et al.; Two types of sites are known to cause pausing and/or termination by bacteriophage T7 RNA polymerase (RNAP) . Termination at class I sites (typified by the signal found in the late region of T7 DNA, TPhi) involves the formation of a stable stem-loop structure in the nascent RNA ahead of the point of termination, and results in termination near runs of U . Class II sites, typified by a signal first identified in the cloned human preproparathyroid hormone (PTH) gene, generate no evident structure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U . Termination at class I and class II sites may involve non-equivalent mechanisms, as mutants of T7 RNA polymerase have been identified that fail to recognize class II sites yet continue to recognize class I sites . In this work, we have analyzed pausing and termination at several class II sites, and variants of them . We conclude that the 7 bp sequence ATCTGTT (5' to 3' in the non-template strand) causes transcribing T7 or T3 RNA polymerase to pause . Termination 6 to 8 bp past this sequence is favored by the presence of runs of U, perhaps because they destabilize an RNA:DNA hybrid . The effects of T7 lysozyme on pausing and termination are consistent with the idea that termination involves a reversion of the polymerase from the elongation to the initiation conformation, and that lysozyme inhibits the return to the elongation conformation . A kinetic model of pausing and termination is presented that provides a consistent interpretation of our results .

Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7957 - 62
An N-terminal fragment of the gene 4 helicase/primase of bacteriophage T7 retains primase activity in the absence of helicase activity; Frick DN et al.; Primase and helicase activities of bacteriophage T7 are present in a single polypeptide coded by gene 4 . Because the amino terminal region of the gene 4 protein contributes to primase activity, we constructed a truncated gene 4 encoding the N-terminal 271-aa residues . The truncated protein, purified from cells overexpressing the protein, is a dimer in solution; the full-length protein is a hexamer . Although the fragment is devoid of dTTPase and helicase activities, it catalyzes template-directed synthesis of di-, tri-, and tetranucleotides . The rates for tetraribonucleotide synthesis and for dinucleotide extension on a 20-nucleotide template are similar for the full-length and truncated proteins . However, the activity of the primase fragment is unaffected by dTTP whereas the primase activity of the full-length protein is stimulated >14-fold . The primase fragment is defective in the interaction with T7 DNA polymerase in that primer synthesis cannot be coupled to DNA synthesis.

J Mol Biol, 1998 Jul 3, 280(1), 137 - 51
Crystal structure of lambda-Cro bound to a consensus operator at 3.0 A resolution; Albright RA et al.; The structure of the Cro protein from bacteriophage lambda in complex with a 19 base-pair DNA duplex that includes the 17 base-pair consensus operator has been determined at 3.0 A resolution . The structure confirms the large changes in the protein and DNA seen previously in a crystallographically distinct low-resolution structure of the complex and, for the first time, reveals the detailed interactions between the side-chains of the protein and the base-pairs of the operator . Relative to the crystal structure of the free protein, the subunits of Cro rotate 53 degrees with respect to each other on binding DNA . At the same time the DNA is bent by 40 degrees through the 19 base-pairs . The intersubunit connection includes a region within the protein core that is structurally reminiscent of the "ball and socket" motif seen in the immunoglobulins and T-cell receptors.The crystal structure of the Cro complex is consistent with virtually all available biochemical and related data . Some of the interactions between Cro and DNA proposed on the basis of model-building are now seen to be correct, but many are different . Tests of the original model by mutagenesis and biochemical analysis corrected some but not all of the errors . Within the limitations of the crystallographic resolution it appears that operator recognition is achieved almost entirely by direct hydrogen-bonding and van der Waals contacts between the protein and the exposed bases within the major groove of the DNA . The discrimination of Cro between the operators OR3 and OR1, which differ in sequence at just three positions, is inferred to result from a combination of small differences, both favorable and unfavorable . A van der Waals contact at one of the positions is of primary importance, while the other two provide smaller, indirect effects . Direct hydrogen bonding is not utilized in this distinction .

J Mol Biol, 1998 Jul 3, 280(1), 129 - 36
Refined structure of Cro repressor protein from bacteriophage lambda suggests both flexibility and plasticity; Ohlendorf DH et al.; The structure of the Cro repressor protein from phage lambda has been refined to a crystallographic R-value of 19.3% at 2.3 A resolution . The re fined model supports the structure as originally described in 1981 and provides a basis for comparison with the Cro-operator complex described in the accompanying paper . Changes in structure seen in different crystal forms and modifications of Cro suggest that the individual subunits are somewhat plastic in nature . In addition, the dimer of Cro suggests a high degree of flexibility, which may be important in forming the Cro-DNA complex.The structure of the Cro subunit as determined by NMR agrees reasonably well with that in the crystals (root-mean-square discrepancy of about 2 A for all atoms) . There are, however, only a limited number of intersubunit distance constraints and, presumably for this reason, the different NMR models for the dimer vary substantially among themselves (discrepancies of 1.3 to 5.5 A) . Because of this variation it is not possible to say whether the range of discrepancies between the X-ray and NMR Cro dimers (2.9 to 7.5 A) represent a significant difference between the X-ray and solution structures.It has previously been proposed that substitutions of Tyr26 in Cro increase thermal stability by the "reverse hydrophobic effect", i.e . by exposing 40% more hydrophobic surface to solvent in the folded form than in the unfolded state . The refined structure, however, suggests that Tyr26 is equally solvent exposed in the folded and unfolded states . The most stabilizing substitution is Tyr26-->Asp and in this case it appears that interaction with an alpha-helix dipole is at least partly responsible for the enhanced stability .

J Mol Biol, 1998 Jul 3, 280(1), 73 - 83
Localization and characterization of the dimerization domain of holliday structure resolving endonuclease VII of phage T4; Birkenbihl RP et al.; Endonuclease VII (Endo VII) is a Holliday structure resolving enzyme of bacteriophage T4 . Its nucleolytic activity depends on subactivities, which in order of execution are: (i) dimerization, (ii) binding to DNA, (iii) and cleavage of DNA . In an effort to assign these subfunctions to the primary sequence of the protein, a series of spontaneous point mutations deficient in DNA cleavage was isolated . Some of these mutations affected the dimerization of Endo VII . Compared with wild-type protein, which dimerizes completely in solution, more than 95% of one of the mutant proteins (W87R) remained in the monomeric state . Only the dimeric fraction of this protein bound to DNA . The dimerization domain of Endo VII was mapped by truncating the gene from both ends and analysing the dimerization ability of the purified peptides by crosslinking with glutaraldehyde . The dimerization domain was thus determined to reside between amino acid residues 55 and 105 . Computer analyses predicted two alpha-helices (H2 and H3) in this section of the protein . As demonstrated by heterodimer formation, two copies of helix H3, but only one copy of helix H2, are required for dimerization . Helical wheel analyses revealed that both helices expose a hydrophobic face along their axes, suggesting that hydrophobic interaction between helices H3 mediate formation of Endo VII dimers, while helices H2 stabilize them .

J Mol Biol, 1998 Jul 3, 280(1), 31 - 9
Sequence specificity of bacteriophage 434 repressor-operator complexation; Duong TH et al.; The binding affinity of the bacteriophage 434 repressor for its DNA operator depends strongly on the nature of two central base-pairs that are not in contact with the dimeric protein . In order to investigate the origin of this sequence specificity, we carried out molecular modelling of five model operators with central TA, AT, CG, GC and IC sequences . The five oligomers were studied both before and after complexation with the N-terminal binding domain of the 434 repressor . The relative importance of nucleic acid flexibility on operator-repressor binding was studied via a low frequency normal mode analysis using an internal coordinate method that we developed recently . The results suggest a higher twisting flexibility for TA and AT central steps than for CG, GC or IC steps, but the differences appear to be too small to account for the strength of repressor binding . An energetic analysis of the model operator-repressor complexes reveals rather that the preference for A.T and T.A base-pairs is electrostatic in origin and is linked to the presence of cationic Arg43 side-chains of repressor . This conclusion is supported by comparison with an R43A mutant and correlates with the sequence dependence of the electrostatic potential in the central minor groove of the operators .

J Mol Biol, 1998 Jul 3, 280(1), 11 - 29
Termination of packaging of the bacteriophage lambda chromosome: cosQ is required for nicking the bottom strand of cosN; Cue D et al.; Termination of packaging of the lambda chromosome involves completion of translocation of the DNA into the head shell, and conversion of the translocation complex into a cleavage complex . The cleavage reaction introduces staggered nicks into the downstream cosN to generate the right cohesive end of the chromosome . cosQ, a site adjacent to cosN, was found to be required for nicking the bottom strand of cosN; bottom strand nicking was also sequence-specific for bps at the nick site . Nicking of the top strand of cosN (cosNL) was stimulated by cosQ, but fidelity and efficiency of cosNL nicking were largely dictated by other cos subsites (i.e . cosB and I2) . Aberrant top-strand cleavage within cosQ was observed in the absence of I2, and nicking at a site 8 nt 5' to the normal cosNL nick site occurred in the absence of cosB . The presence of cosQ was found to be insufficient to arrest DNA translocation in vivo, indicating that cosQ, per se, is not a packaging stop signal . A model is presented in which the role of cosQ is to depolarize the asymmetric arrangement of terminase protomers in the translocation complex so that protomers are configured to match the 2-fold rotational symmetry of cosN .

Mol Cell, 1998 Jun, 1(7), 1001 - 10
Coordinated leading and lagging strand DNA synthesis on a minicircular template; Lee J et al.; The coordinated synthesis of both leading and lagging DNA strands is thought to involve a dimeric DNA polymerase and a looping of the lagging strand so that both strands can be synthesized in the same direction . We have constructed a minicircle with a replication fork that permits an assessment of the stoichiometry of the proteins and a measurement of the synthesis of each strand . The replisome consisting of bacteriophage T7 DNA polymerase, helicase, primase, and single-stranded DNA-binding protein mediates coordinated replication . The criteria for coordination are fulfilled: (1) a replication loop is formed, (2) leading and lagging strand synthesis are coupled, (3) the lagging strand polymerase recycles from one Okazaki fragment to another, and (4) the length of Okazaki fragments is regulated . T7 single-stranded DNA-binding protein is essential for coordination.

Appl Microbiol Biotechnol, 1998 May, 49(5), 560 - 7
Production of a new DNA vehicle for gene transfer using site-specific recombination; Kreiss P et al.; Supercoiled DNA molecules, minicircles, were produced by in vivo site-specific recombination . They contained exclusively the desired excisable fragment . Recombination was driven by bacteriophage lambda integrase from a plasmid substrate containing the attP and attB recombination sites in the same orientation . Conditions for minicircle production within the lysogen Escherichia coli D1210HP were optimised . Up to 1.5 mg minicircles could be produced per litre bacterial culture, and the remaining, unrecombined plasmid comprised less than about 15% of the minicircle produced . However minicircle multimers were also produced, and comprised up to 30% of all minicircles synthesised . The par ABCDE' locus from plasmid RK2 was introduced into the minicircle fragment, resulting in minicircle dimers being reduced to less than 2% of all minicircles . The par A gene encodes a resolvase that catalyses recombination at the multimer resolution site in the parABCDE' locus . Minicircle multimers were also resolved when par A was introduced downstream from the integrase gene of the lambda pL transcript in D1210HP together with a multimer resolution site carried by the minicircle fragment.

EMBO J, 1998 Jul 1, 17(13), 3758 - 65
lambda bar minigene-mediated inhibition of protein synthesis involves accumulation of peptidyl-tRNA and starvation for tRNA; Hernandez-Sanchez J et al.; Expression of the bacteriophage lambda two-codon, AUG AUA, barI minigene (bar+) leads to the arrest of protein synthesis in cells defective in peptidyl-tRNA hydrolase (Pth) . It has been hypothesized that translation of the bar+ transcript provokes premature release and accumulation of peptidyl-tRNA (p-tRNA) . Inhibition of protein synthesis would then result from either starvation of sequestered tRNA or from toxicity of accumulated p-tRNA . To test this hypothesis and to investigate the cause of arrest, we used a coupled in vitro transcription-translation system primed with DNA containing bar+ and the beta-lactamase-encoding gene of the vector as a reporter . The results show that expression of bar+ minigene severely inhibits beta-lactamase polypeptide synthesis by Pth-defective extracts and partially inhibits synthesis by wild-type extracts . Fractions enriched for Pth, or a homogeneous preparation of Pth, prevented and reversed bar+-mediated inhibition . A mutant minigene, barA702, which changes the second codon AUA (Ile) to AAA (Lys), was also toxic for Pth-defective cells . Expression of barA702 inhibited in vitro polypeptide synthesis by Pth-defective extracts and, as with bar+, exogenous Pth prevented inhibition . Addition of pure tRNALys prevented inhibition by barA702 but not by bar+ . Expression of bar+ and barA702 led to release and accumulation of p-tRNAIle and p-tRNALys respectively but bar+ also induced accumulation of p-tRNALys . Finally, bar+ stimulated association of methionine with ribosomes probably as fMet-tRNAfMet and the accumulation of methionine and isoleucine in solution as peptidyl-tRNA (p-tRNA) . These results indicate that minigene-mediated inhibition of protein synthesis involves premature release of p-tRNA, misincorporation of amino acyl-tRNA, accumulation of p-tRNAs and possibly sequestration of tRNAs.

EMBO J, 1998 Jul 1, 17(13), 3521 - 33
Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants; Puntoriero G et al.; The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus . It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response . Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task . We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage . This library was affinity selected using many different sera from infected patients . Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants . When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants . In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity . These data may hold the key for future development of a prophylactic vaccine against HCV.

Biophys J, 1998 Jul, 75(1), 513 - 20
Scanning force microscopy of DNA molecules elongated by convective fluid flow in an evaporating droplet; Wang W et al.; Scanning force microscopy (SFM) was used to image intact, nearly fully elongated lambda bacteriophage DNA molecules, fixed onto freshly cleaved mica surfaces . Molecular elongation and fixation were accomplished using a newly characterized fixation technique, termed "fluid fixation." Here convective fluid flows generated within an evaporating droplet of DNA solution efficiently elongate DNA molecules for fixation onto suitably charged surfaces . SFM images of a very large bacteriophage genome, G, showed the presence of double-stranded bubbles . We speculate that these structures may contain putative replication forks . Overall, the experiments presented here demonstrate the viability of using fluid fixation for the preparation of DNA molecules for SFM imaging . The combination of largely automatable optically based techniques with the high-resolution SFM imaging presented here will likely produce a high-throughput system for detailed physical mapping of genomic DNA or clones.

Appl Environ Microbiol, 1998 Jul, 64(7), 2443 - 8
Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7 and that carry the Shiga toxin 2 gene; Muniesa M et al.; Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available . An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage . The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage . Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E . coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml . Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells . By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E . coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins . These values were approximately 1% of all phages infecting E . coli O157:H7.

FEBS Lett, 1998 May 22, 428(1-2), 68 - 70
Efficient introduction of alkene functionality into proteins in vivo; van Hest JC et al.; The methionine analogue 2-amino-5-hexenoic acid (homoallylglycine, Hag) can be utilized by Escherichia coli in the initiation and elongation steps of protein biosynthesis . Use of an E . coli methionine auxotroph and Hag-supplemented medium resulted in replacement of ca . 85% of the methionine residues in mouse dihydrofolate reductase expressed under control of a bacteriophage T5 promoter . N-terminal sequencing indicated 92+/-5% occupancy of the initiator site by Hag . The vinyl function of Hag remains intact in the purified protein and suggests new chemistries for modification of natural and artificial proteins prepared in bacterial hosts.

Arch Virol, 1998, 143(5), 903 - 14
Ceratobium mosaic potyvirus: another virus from orchids; Mackenzie AM et al.; A potyvirus, which we call ceratobium mosaic virus, has been detected in about one third of more than 100 plants representing c . 33 orchid genera in two collections in Australia . It was detected using RT-PCR with redundant primers that are Potyviridae-specific and have additional sequences corresponding to either the SP6 or T7 bacteriophage promoters at their 5'-termini . Thus the nucleotide sequence of the resulting PCR fragments, consisting of about 1.7 kb of the 3' portion of the viral genome, could be determined directly . Viral sequences obtained from five infected orchids indicate that they contained different isolates of a single potyvirus species most closely related to the bean common mosaic group of potyviruses, but clearly distinct from all whose virion protein genes have been reported to the international gene sequence databases.

Int J Biol Macromol, 1998 Jul, 23(1), 37 - 48
The phage lambda terminase enzyme: 2 . Refolding of the gpNu1 subunit from the detergent-denatured and guanidinium hydrochloride-denatured state yields different oligomerization states and altered protein stabilities; Hanagan A et al.; The terminase enzyme from bacteriophage lambda is responsible for packaging a single genome within the viral capsid . Gold and co-workers have developed a scheme for the solubilization of the small terminase subunit (gpNu1) from inclusion bodies using the strong detergent sarkosyl and purification of the protein to homogeneity (gpNu1SRK) (Parris et al., J Biol Chem 1994;269:13564-13574) . We have developed a similar purification scheme except that guanidinium hydrochloride was used to denature the insoluble protein (gpNu1GDN) . The circular dichroism (CD) spectra of both protein preparations suggest that they are predominantly alpha-helical when purified and stored in Tris buffers . Moreover, thermal denaturation of the proteins thus purified yielded similar thermodynamic parameters for unfolding (T(m), delta Hm and delta Sm of unfolding of approximately 306 K, approximately 22 kcal/mol and approximately 70 cal/mol.K, respectively) . Interestingly, however, when the proteins were purified and stored in imidazole buffers, the gpNu1SRK preparation lost a significant amount of secondary structure and was more stable to both thermally-induced and guanidinium HCl-induced denaturation than was gpNu1GDN . The purified gpNu1 monomers oligomerize into apparent tetramers and hexamers in solution and the distribution between these two oligomeric states and into higher order aggregates depends upon buffer composition, salt concentration and protein concentration . Moreover, differences in the oligomerization state of gpNu1SRK and gpNu1GDN under identical buffer conditions were observed . The significance of these results with respect to the biological role of the phage lambda gpNu1 protein are discussed.

Int J Biol Macromol, 1998 Jul, 23(1), 27 - 36
The phage lambda terminase enzyme: 1 . Reconstitution of the holoenzyme from the individual subunits enhances the thermal stability of the small subunit; Meyer JD et al.; The terminase enzyme from bacteriophage lambda is a hetero-trimeric complex composed of the viral gpA and gpNu1 proteins (gpA1.gpNu1(2)) and is responsible for packaging a single genome within the viral capsid . Current expression systems for these proteins require thermal induction which may be responsible for the formation of insoluble aggregates observed in E . coli . We report the re-cloning of the terminase subunits into vectors which allow low temperature induction . While this has resulted in increased solubility of the large gpA subunit of the enzyme, the small gpNu1 subunit remains insoluble under all conditions examined . This paper describes the solublization of gpNu1 with guanidinium hydrochloride and purification of the protein to homogeneity . Reconstitution of the enzyme from the individually purified subunits yields a catalytically-competent complex which exhibits activity identical to wild-type enzyme . Thermal denaturation of the proteins was monitored by circular dichroism (CD) spectroscopy and demonstrates that while unfolding of gpA is irreversible, the gpNu1 subunit refolds into a conformation which is essentially identical to the pre-heated protein . Moreover, while denaturation of gpA is highly cooperative, the small subunit unfolds over a wide temperature range and with thermodynamic parameters lower than expected for a small globular protein . Thermally-induced denaturation of the enzyme reconstituted from the individual subunits is highly cooperative with no evidence of multiple transitions . Our data demonstrate that the terminase subunits directly interact in solution, and that this interaction alters the thermal stability of the smaller gpNu1 subunit . The implication of these results with respect to assembly of a catalytically competent enzyme complex are discussed.

J Mol Biol, 1998 Jun 5, 279(2), 347 - 59
Structure and NTPase activity of the RNA-translocating protein (P4) of bacteriophage phi 6; Juuti JT et al.; The RNA polymerase complex of bacteriophage phi 6 comprises four proteins, P1, P2, P4 and P7, and forms the core of the virion . Protein P4 is a non-specific NTPase that provides the energy required for RNA translocation (packaging) . Characterization of purified recombinant P4 shows that the protein assembles into stable hexamers in the presence of ADP and divalent cations . Image averaging of electron micrographs reveals this hexamer as a slightly skewed ring with outer and inner diameters of 12 and 2 nm, respectively . NTPase activity of P4 is associated only with the hexameric form . Ca2+ and Zn2+ and non-specific single-stranded RNA stimulate the NTPase activity, while Mg2+ acts as a non-competitive inhibitor, presumably via a separate Mg2+ binding site . Binding affinities of different nucleotide mono-, di- and triphosphates and non-hydrolyzable analogs indicate that the beta-phosphate moiety is required for substrate binding . A slight preference for binding of purine nucleotides is also observed . Analysis of P4 by CD and Raman spectroscopy indicates an alpha/beta subunit fold that is altered only slightly by hexamer assembly . Raman markers of P4 secondary and tertiary structures are also largely invariant to nucleotide exchange and hydrolysis, suggesting that the mechanisms of RNA translocation involves movement of subunits relative to one another rather than large scale changes in the alpha/beta subunit fold . The stoichiometry of P4 in the mature phi 6 virion is estimated as 120 copies . Because the recombinant P4 hexamers exhibit hydrodynamic and enzymatic properties that are identical to those of P4 oligomers released from native phi 6, we propose that P4 occurs as hexamers in the native viral core particle.

Mol Microbiol, 1998 May, 28(4), 719 - 27
Replication strand preference for deletions associated with DNA palindromes; Pinder DJ et al.; We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli . All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats . This is consistent with deletion by strand slippage during DNA replication . The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event . It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known . In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork . The data include replication slippage in three situations: on the chromosome of E . coli, in bacteriophage lambda and in high-copy-number pUC-based plasmids.

J Mol Biol, 1998 Jun 12, 279(3), 545 - 64
Non-nearest neighbor effects on the thermodynamics of unfolding of a model mRNA pseudoknot; Theimer CA et al.; The upstream autoregulatory mRNA leader sequence of gene 32 of 17 T-even and related bacteriophages folds into a simple tertiary structural motif, a hairpin-type RNA pseudoknot . In phage T4, the pseudoknot is contained within 28 contiguous nucleotides which adopt a pseudocontinuous helical structure derived from two coaxially stacked helical stems of four (stem 1) and seven (stem 2) base-pairs connected by two inequivalent single-stranded loops of five and one nucleotide(s) . These two loops cross the minor and major grooves of stems 1 and 2, respectively . In this study, the equilibrium unfolding pathway of a 35-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T4 gene 32 mRNA has been determined through analysis of dual-wave-length, equilibrium thermal melting profiles via application of a van't Hoff model based on multiple sequential, two-state transitions . The melting profile of the wild-type RNA is well-described by two sequential melting transitions over a wide range of magnesium concentration . Compensatory base-pair substitutions incorporated into helical stems 1 and 2 were used to assign the first low enthalpy, moderate tm melting transition to the denaturation of the short three to four base-pair stem 1, followed by unfolding of the larger seven base-pair stem 2 . We find that loop 1 substitution mutants (A10 to G10, C10, U10 or GA10) strikingly uncouple the melting of stems 1 and 2, with the U10 substitution and the GA10 loop expansion more destabilizing than the G10 and C10 substitutions . A significant increase in the extent of cleavage by RNase T1 following the conserved G26 (the 3' nucleotide in loop 2) in the U10, G10, and GA10 mutants suggests that an altered helix-helix junction region in this mutant may be responsible, at least in part, for this uncoupling . In addition to a modest destabilization of stem 2, the major effect of deletion or nucleotide substitution in the 3' single-stranded tail is a destabilization of stem 1, a non-nearest neighbor tertiary structural effect, which may well be transmitted through an altered loop 1-core helix interaction . In contrast, truncation of the 5' tail has no effect on the stability of the molecule.

J Immunol, 1998 Jun 15, 160(12), 5990 - 7
Structural requirements for a specificity switch and for maintenance of affinity using mutational analysis of a phage-displayed anti-arsonate antibody of Fab heavy chain first complementarity-determining region; Wong YW et al.; We previously showed that a single mutation at heavy (H) position 35 of Abs specific for p-azophenylarsonate (Ars) resulted in acquisition of binding to the structurally related hapten p-azophenylsulfonate (Sulf) . To explore the sequence and structural diversity of the H chain first complementarity-determining region (HCDR1) in modulating affinity and specificity, positions 30-36 in Ab 36-65 were randomly mutated and expressed as Fab in a bacteriophage display vector . Ab 36-65 is germline encoded, lacking somatic mutations . Following affinity selection on Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCDR1 sequences containing different amino acids at position H:35 . All Fab bound Sulf, but not Ars . Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten specificity, independent of the sequence of the remainder of HCDR1 . At H:35, Asn is required for Ars specificity, consistent with the x-ray crystal structure of the somatically mutated anti-Ars Ab 36-71, while Sulf binding occurs with at least seven different H:35 residues . All Sulf-binding clones selected following phage display contained H:Gly33, observed previously for Ars-binding Abs that use the same germline V(H) sequence . Site-directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Abs.

Mutat Res, 1998 Mar, 407(2), 157 - 68
Altered UV resistance and UV mutational spectrum in repair-proficient murine fibroblasts expressing endonuclease V; Kusewitt DF et al.; In previously reported studies, we transfected repair-proficient murine fibroblasts with the denV gene of bacteriophage T4 and showed that expression of encoded endonuclease V markedly enhanced cyclobutane pyrimidine dimer (CPD) repair and reduced the frequency of ultraviolet radiation (UV)-induced mutations . In the present studies, we compared the spectra of UV-induced mutations at the hprt locus in denV-transfected and control cells . A significant difference in mutation types was observed . While multiple base deletions and single base insertions were found in denV-transfected but not control cells, multiple tandem and non-tandem point mutations identified in control cells were absent in denV-transfected cells . When we compared colony survival following UV exposure in the two cell lines, it appeared that endonuclease V expression did not enhance UV resistance, instead denV-transfected cells had increased susceptibility to low fluences of UV . The effects of endonuclease V expression on UV resistance and on UV mutational spectrum are likely to be due both to the removal of CPDs and to the novel enzymatic activity of endonuclease V.

J Mol Biol, 1998 May 29, 279(1), 175 - 88
Different pathways for protein degradation by the FtsH/HflKC membrane-embedded protease complex: an implication from the interference by a mutant form of a new substrate protein, YccA; Kihara A et al.; Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent zinc-metalloproteinase, which forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC . It is the protease that degrades uncomplexed forms of the SecY subunit of protein translocase . Here, we characterized a new class of SecY-stabilizing mutation on the E . coli chromosome . The mutation (yccA11) is an internal deletion within a gene (yccA) known as an open reading frame for a hydrophobic protein with putative seven transmembrane segments . The YccA protein was found to be degraded in an FtsH-dependent manner in vivo and in vitro, whereas the YccA11 mutant protein, lacking eight amino acid residues within the amino-terminal cytoplasmic domain, was refractory to the degradation . The yccA11 mutation exhibited partial dominance when overexpressed . Cross-linking, co-immunoprecipitation, and histidine tagging experiments showed that YccA11 as well as YccA can associate with both the FtsH and the HflKC proteins . Thus, the mutant YccA protein appeared to compete with SecY for recognition by the FtsH proteolytic system and the residues deleted by the yccA mutation are required for the initiation of proteolysis by FtsH . Interestingly, the inhibitory action of YccA11 was mediated by HflKC, since the deletion of hflK-hflC suppressed the yccA11 phenotype . The yccA11 mutation stabilized subunit a of the proton ATPase F0 segment as well, but not the CII protein of bacteriophage lambda or the sigma 32 protein . From these results we suggest that there are at least two pathways for FtsH-dependent protein degradation, only one of which (probably for membrane proteins) is subject to the HflKC-dependent interference by the YccA11 mutant substrate.

J Mol Biol, 1998 May 29, 279(1), 19 - 29
The major coat protein of filamentous bacteriophage f1 specifically pairs in the bacterial cytoplasmic membrane; Haigh NG et al.; Filamentous bacteriophage are long, thin single-stranded DNA viruses that infect male strains of Escherichia coli without killing the host . Each phage contains approximately 2700 copies of the major coat protein, pVIII, which exists as a 5.2 kDa cytoplasmic membrane protein prior to incorporation into phage . Studies from a number of groups analyzing the behavior of wild-type and mutant pVIII in detergents suggested that pVIII might pair under these conditions . In order to test whether pVIII molecules pair in vivo in the cytoplasmic membrane, four plasmidencoded pVIII variants were constructed in which specific residues in the transmembrane region were mutated to cysteine in an attempt to stabilize any pair via disulfide bridges . Variants A35C and I39C were unable to complement phage with an amber mutation in gene VIII . The I39C variant was unable to be packaged into phage particles even though it was inserted into the membrane . In the case of A35C, the inability to complement was not due to a packaging defect because the variant protein could be packaged into phage in the presence of wild-type pVIII . Western blot analysis of cytoplasmic membrane samples revealed that the A35C variant formed stable disulfide dimers in vivo . Expression of A35C interfered with wild-type phage infection, indicating that the assembly machinery may recognize the disulfide dimers in some non-productive way . The results indicate that pVIII may specifically pair along a particular face in the cytoplasmic membrane prior to assembly; however, these pairs must be able to be separated in order for normal assembly to occur.

J Mol Biol, 1998 May 29, 279(1), 9 - 18
Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA; Severinova E et al.; The 10 kDa bacteriophage T4 antisigma protein AsiA binds the Escherichia coli RNA polymerase promoter specificity subunit, sigma 70, with high affinity and inhibits its transcription activity . AsiA binds to sigma 70 primarily through an interaction with sigma 70 conserved region 4.2, which has also been implicated in sequence-specific recognition of the -35 consensus promoter element . Here we show that AsiA forms a stable ternary complex with core RNA polymerase (RNAP) and sigma 70 and thus does not inhibit sigma 70 activity by preventing its binding to core RNAP . We investigated the effect of AsiA on open promoter complex formation and abortive initiation at two -10/-35 type promoters and two "extended -10" promoters . Our results indicate that the binding of AsiA to sigma 70 and the interaction of sigma 70 region 4.2 with the -35 consensus promoter element of -10/-35 promoters is mutually exclusive . In contrast, AsiA has much less effect on open promoter complex formation and abortive initiation from extended -10 promoters, which lack a -35 consensus element and do not require sigma 70 conserved region 4.2 . From these results we conclude that T4 AsiA inhibits E . coli RNAP sigma 70 holoenzyme transcription at -10/-35 promoters by interfering with the required interaction between sigma 70 conserved region 4.2 and the -35 consensus promoter element.

Biotechnology (N Y), 1995 Oct, 13(10), 1105 - 9
Human stress protein hsp70: overexpression in E coli, purification and characterization; Jindal S et al.; The gene encoding the stress-inducible member of human heat shock protein hsp70, was expressed in E . coli using the bacteriophage T7 RNA polymerase-based gene expression system . Recombinant hsp70 (R-hsp70) was purified from inclusion bodies after solubilization and refolding, using a combination of ATP-agarose affinity chromatography and ion-exchange chromatography . R-hsp70 was shown to be monomeric and free of its structurally similar E . coli counterpart, DnaK . In addition, R-hsp70 is functional as demonstrated by its ability to bind to peptides and to ATP . The availability of pure, correctly folded R-hsp70 in sufficient quantity will assist in the structural and functional characterization of hsp70 . Furthermore, an understanding of the cytoprotective function of hsp70 and its role in immune responses during infections will be facilitated by the availability of pure R-hsp70.

Biochemistry, 1998 Jun 23, 37(25), 9127 - 37
Sequence context profoundly influences the mutagenic potency of trans-opened benzo{a}pyrene 7,8-diol 9,10-epoxide-purine nucleoside adducts in site-specific mutation studies; Page JE et al.; The postoligomerization method was used to prepare oligonucleotide 16-mers that contained dAdo or dGuo adducts, derived from trans opening of each enantiomer of the two diastereomeric benzo{a}pyrene 7,8-diol 9,10-epoxides, in two sequence contexts . These 16 oligonucleotides, along with the four corresponding oligonucleotides containing unsubstituted purines, were ligated into single-stranded DNA from bacteriophage M13mp7L2 and transfected into Escherichia coli SMH77 . The mutagenic effects of replication past these adducts were then evaluated . The various adduct isomers induced point mutations at different frequencies and with different distributions of mutation types, as was anticipated . However, sequence context had the most substantial effects on mutation frequency . A high frequency of deletions of a single guanine was found in a context where the dGuo adduct was at the 3'-end of a run of five guanines, whereas no single base deletion was found in the other context studied, 5'-CGA-3' . Mutation frequencies in constructs containing dAdo adducts were much higher in a 5'-TAG-3' context (37-58%, depending on the individual isomer) than in a 5'-GAT-3' context (5-20%), and for a given adduct, mutation frequency was up to 10-fold higher in the former sequence than in the latter . These findings indicate that sequence context effects need more thorough evaluation if the goal of understanding the mechanism through which DNA adducts lead to mutation is to be achieved.

Biophys J, 1998 Jun, 74(6), 3217 - 25
Orientations of tyrosines 21 and 24 in coat subunits of Ff filamentous virus: determination by Raman linear intensity difference spectroscopy and implications for subunit packing; Matsuno M et al.; Virions of the Ff group of bacteriophages (fd, f1, M13) are morphologically identical filaments (approximately 6-nm diameter x approximately 880-nm length) in which a covalently closed, single-stranded DNA genome is sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit (pVIII) . Orientations of pVIII tyrosines (Tyr21 and Tyr24) with respect to the filament axis have been determined by Raman linear intensity difference (RLID) spectroscopy of flow-oriented mutant virions in which the tyrosines were independently mutated to methionine . The results show that the twofold axis of the phenolic ring (C1-C4 line) of Tyr21 is inclined at 39.5 +/- 1.4 degrees from the virion axis, and that of Tyr24 is inclined at 43.7 +/- 0.6 degrees . The orientation determined for the Tyr21 phenol ring is close to that of a structural model previously proposed on the basis of fiber x-ray diffraction results (Protein Data Bank, identification code 1IFJ) . On the other hand, the orientation determined for the Tyr24 phenol ring differs from the diffraction-based model by a 40 degrees rotation about the Calpha-Cbeta bond . The RLID results also indicate that each tyrosine mutation does not greatly affect the orientation of either the remaining tyrosine or single tryptophan (Trp26) of pVIII . On the basis of these results, a refined model is proposed for the coat protein structure in Ff.

Biotechnology (N Y), 1995 Jun, 13(6), 583 - 6
Whole-virus vaccine development by continuous culture on a complementing host; Kong D et al.; We have evaluated an adaptive strategy for generating whole-virus vaccines using a bacteriophage model . Wildtype phage T7 was cultivated in a two-stage continuous stirred-tank reactor (CSTR) utilizing a recombinant E . coli host that constitutively expressed T7 RNA polymerase, an essential enzyme of the early viral metabolism . Over the course of 180 generations a diversity of phage variants emerged, outgrew the wildtype, and were subsequently eclipsed by yet fitter variants, based on host-ranges, restriction patterns, and one-step growth responses of isolated clones . The fittest variant, which required complementation by the recombinant host in order to grow, deleted at least 12 percent of its genome and replicated twice as fast as the wildtype . Moreover, this variant was immunogenically indistinguishable from the wildtype, based on cross-reactivities of antisera raised against both . These results suggest the feasibility of the proposed strategy for the development of safe whole-virus vaccines.

Biotechnology (N Y), 1995 Mar, 13(3), 261 - 4
A hybrid baculovirus-bacteriophage T7 transient expression system; van Poelwijk F et al.; A hybrid recombinant baculovirus-bacteriophage T7 expression system was developed for transient expression in insect cells of plasmids with foreign genes provided with a T7 promoter . The coding sequence for T7 RNA polymerase, with or without a nuclear localization signal, was inserted into the genome of Autographa californica nuclear polyhedrosis virus . Recombinant viruses stably expressed T7 RNA polymerase in insect cells . Upon transfection of infected insect cells with plasmids containing the genes for chloramphenicol acetyltransferase (CAT), the hepatitis B virus precore-, core- or e- antigens under control of the T7 promoter, transient expression of these genes was detected by ELISA . The results obtained indicate that this baculovirus/T7 system provides a simple and widely applicable tool for transient gene expression studies.

Biotechnology (N Y), 1995 Feb, 13(2), 165 - 9
Binding epitope of somatostatin defined by phage-displayed peptide libraries; Wright RM et al.; We have developed a versatile phagemid system to display peptides on the surface of M13 bacteriophage at a copy number which approaches monovalency . In this system, a phagemid encodes a peptide fused to the amino-terminus of the second domain (dII) of the minor coat protein pIII under control of the inducible lac promoter . The fusion protein is displayed in combination with several copies of wild-type pIII on the surface of phage . Two diverse random octapeptide libraries, one linear and one which contained flanking cysteines capable of forming disulfide bridges, were were generated using an in vitro mutagenesis approach and affinity selected on an anti-somatostatin mAb . Peptides with high affinity for the mAb were enriched only from the cyclic library and the tetrapeptide, FWKT, was identified by consensus as the binding epitope . The selected peptides exhibited not only the primary amino acid sequence but also shared structural features with somatostatin . One peptide, CRFWKTWC, also exhibited nanomolar affinities for the five known somatostatin receptor subtypes . This system can easily be adapted to display individual peptides or a wide range of custom peptide libraries.

J Biol Chem, 1998 Jun 26, 273(26), 16205 - 9
Expressed protein ligation, a novel method for studying protein-protein interactions in transcription; Severinov K et al.; Expressed protein ligation is a novel protein semi-synthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element . In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription . Here we describe the successful application of expressed protein ligation to the approximately 600 amino acid sigma70 subunit of Escherichia coli RNA polymerase . The resulting semi-synthetic sigma70 constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of sigma70 . The success of these semi-synthesis studies sets the stage for the future generation of semi-synthetic sigma70 molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.

Biochemistry, 1998 May 26, 37(21), 7749 - 56
Dissecting the order of bacteriophage T4 DNA polymerase holoenzyme assembly; Sexton DJ et al.; Most biological organisms rely upon a DNA polymerase holoenzyme for processive DNA replication . The bacteriophage T4 DNA polymerase holoenzyme is composed of the polymerase enzyme and a clamp protein (the 45 protein), which functions as a processivity factor by strengthening the interaction between DNA and the holoenzyme . The 45 protein must be loaded onto DNA by a clamp loader ATPase complex (the 44/62 complex) . In this paper, the order of events leading to holoenzyme formation is investigated using a combination of rapid-quench and stopped-flow fluorescence spectroscopy kinetic methods . A rapid-quench strand displacement assay in which the order of holoenzyme component addition is varied provided data indicating that the rate-limiting step in holoenzyme assembly is associated with the clamp loading process . Pre-steady-state analysis of the clamp loader ATPase activity demonstrated that the four bound ATP molecules are hydrolyzed stepwise during the clamp loading process in groups of two . Clamp loading was examined with stopped-flow fluorescence spectroscopy from the perspective of the clamp itself, using a site-specific, fluorescently labeled 45 protein . A mechanism for T4 DNA polymerase holoenzyme assembly is proposed in which the 45 protein interacts with the 44/62 complex leading to the hydrolysis of 2 equiv of ATP, and upon contacting DNA, the remaining two ATP molecules bound to the 44/62 complex are hydrolyzed . Once all four ATP molecules are hydrolyzed, the 45 protein is poised on DNA for association with the polymerase to form the holoenzyme.

Nat Biotechnol, 1996 Aug, 14(8), 986 - 91
In vitro selection of peptides acting at a new site of NMDA glutamate receptors; Li M et al.; Oligomeric N-methyl D-aspartate receptor (NMDAR) in brain is a ligand-gated ion channel that becomes selectively permeable to ions upon binding to ligands . For NMDAR channel, the binding of glutamate and glycine results in opening of the calcium permeable channel . Because the calcium influx mediated by NMDAR is important for synaptic plasticity and excitotoxicity, the function of NMDA receptors has been implicated in both health and disease . Native NMDA receptors are thought to be heteromeric pentamers with a central ion conduction pathway . There are five genes (NR1, 2A, 2B, 2C, and 2D) encoding various subunits that have been cloned, and NR1 is thought to be the essential subunit since it forms a functional channel by itself . To study NMDAR structure and function, we have searched for peptide modulators of NR1 using random peptide bacteriophage libraries . The peptides were identified based on their specific association with a purified receptor fusion protein that contains the putative ligand binding domain . We report the identification of one group of cyclic peptides (Mag-1) with a consensus sequence of CDGLRHMWFC . Using biochemical binding analysis and patch clamp electrophysiological recording, we show that the synthetic Mag-1 peptides cause noncompetitive inhibition of the receptor channel activity.

Nat Biotechnol, 1996 Apr, 14(4), 491 - 3
Detection of evolving viruses; Lee Y et al.; The spread of viruses on a homogeneous lawn of receptive hosts provides an opportunity to detect the dynamics of their evolution . We have previously found that when repeated virus passages are confined to the expanding perimeter of a growing plaque, the appearance and outgrowth of genetically diverse strains (all descended from the same parent strain) can be traced along different radii of the plaque . As a plaque grows, the random mutation and selection of new fast-growing strains reduce the roundness or circularity of the growing plaque . Here we have quantified such changes in growing plaques of bacteriophage T7 using a digital imaging system . We find that T7 populations not adapted for fast growth exhibit a broader diversity of growth rates than populations adapted for fast growth . These results provide a foundation for understanding how viruses exploit mutation and selection processes to persist in nature.

J Biochem (Tokyo), 1998 May, 123(5), 821 - 6
Effects of cis-diamminedichloroplatinum(II) on Escherichia coli and bacteriophage systems; Tanaka N et al.; The effects of cis-diamminedichloroplatinum(II) (cisplatin) on Escherichia coli cells and bacteriophages were investigated . The bacteriocidal effect of cisplatin was stronger on uvrA or recA mutants than on wild type cells . The drug, like UV, induced prophage development in lysogenic bacteria . Host cell reactivation of alpha3 replicative form (RF) I DNA treated with cisplatin in vitro was more efficient in wild type or recA cells than in uvrA host . When wild type cells were exposed to cisplatin, decay of the host's capacity to sustain the viral multiplication proceeded nearly in parallel with the loss of colony-forming ability, whereas the capacity of uvrA mutant was much more resistant to the drug, as compared with the viability . In the DNA preparation from cisplatin-treated alpha3-infected wild type cells, RF II was deficient, but the RF I molecules extracted from the cells were moderately infective . The microvirid gene A protein, required for RF I-->RF II conversion, was hardly detectable in wild type cells exposed to cisplatin . The possible relationship between uvr+-dependent repair and synthesis of the viral protein is discussed.

Gene, 1998 May 12, 211(2), 277 - 85
Amplification of target-specific, ligation-dependent circular probe; Zhang DY et al.; We describe a novel polymerase chain reaction (PCR)-based gene amplification method utilizing a circularizable oligodeoxyribonucleotide probe (C-probe) . The C-probe contains two target complementary regions located at each terminus and an interposed generic PCR primer binding region . The hybridization of C-probe to a target brings two termini in direct apposition as the complementary regions of C-probe wind around the target to form a double helix . Subsequent ligation of the two termini results in a covalently linked C-probe that becomes 'locked on to' the target . The circular nature of the C-probe allows for the generation of a multimeric single-stranded DNA (ssDNA) via extension of the antisense primer by Taq DNA polymerase along the C-probe and displacement of downstream strand, analogous to 'rolling circle' replication of bacteriophage in vivo . This multimeric ssDNA then serves as a template for multiple sense primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex . Subsequent thermocycling denatures the dsDNA and initiates the next round of primer extension and ramification . This model results in significantly improved amplification kinetics (super-exponential) as compared to conventional PCR . Our results show that the C-probe was 1000 times more sensitive than the corresponding linear hemiprobes for detecting Epstein-Barr virus early RNA . The C-probe not only increases the power of amplification but also offers a means for decontaminating carryover amplicons . As the ligated C-probes possess no free termini, they are resistant to exonuclease digestion, whereas contaminated linear amplicons are susceptible to digestion . Treatment of the ligation reaction mixture with exonuclease prior to amplification eliminated the amplicon contaminant, which could also have been co-amplified with the same PCR primers; only the ligated C-probes were amplified . The combined advantages of the C-probe and thermocycling have a broad applicability for the detection of both DNA and RNA . Finally, we described a novel isothermal amplification method, ramification extension amplification, utilizing circular nature of C-probe and displacement activity of DNA polymerase.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3242 - 6
Long-range translational coupling in single-stranded RNA bacteriophages: an evolutionary analysis; Licis N et al.; In coliphage MS2 RNA a long-distance interaction (LDI) between an internal segment of the upstream coat gene and the start region of the replicase gene prevents initiation of replicase synthesis in the absence of coat gene translation . Elongating ribosomes break up the repressor LDI and thus activate the hidden initiation site . Expression studies on partial MS2 cDNA clones identified base pairing between 1427-1433 and 1738-1744, the so-called Min Jou (MJ) interaction, as the molecular basis for the long-range coupling mechanism . Here, we examine the biological significance of this interaction for the control of replicase gene translation . The LDI was disrupted by mutations in the 3'-side and the evolutionary adaptation was monitored upon phage passaging . Two categories of pseudorevertants emerged . The first type had restored the MJ interaction but not necessarily the native sequence . The pseudorevertants of the second type acquired a compensatory substitution some 80 nt downstream of the MJ interaction that stabilizes an adjacent LDI . In one examined case we confirmed that the second site mutations had restored coat-replicase translational coupling . Our results show the importance of translational control for fitness of the phage . They also reveal that the structure that buries the replicase start extends to structure elements bordering the MJ interaction.

Biol Chem, 1998 Apr-May, 379(4-5), 621 - 3
Escherichia coli bacteriophage T1 DNA methyltransferase appears to interact with Escherichia coli enolase; Gassner C et al.; Infection of Escherichia coli cells with bacteriophage T1 induces synthesis of a bacteriophage-specific DNA methyltransferase (M.EcoT1, EC No: 2.1.1.72) with a specificity for adenine residues in the sequence 5'-GATC-3' . Purification of M.EcoT1 allowed the determination of the coding sequence of the gene (Schneider-Scherzer et al., 1990) . The peptide of the entire coding sequence was over-expressed as a histidine-hexapeptide tagged protein in E . coli . Affinity purification using a Ni2+ chelating (Ni-NTA) resin yielded a recombinant enzyme with almost the same enzymatic properties as the protein purified from T1 infected E . coli cells . Interestingly, in both purification procedures, a protein with a molecular weight of 50000 was found to copurify with M.EcoT1 . The N-terminal amino acid sequence identified these proteins in both cases as E . coli enolase (EC No: 4.2.1.11).

Biol Chem, 1998 Apr-May, 379(4-5), 579 - 82
Design of a novel regulatory circuit for expression of restriction endonucleases; Chandrashekharan S et al.; We have developed a new strategy with a very tight control for the expression of cloned genes . The system employed here is the T7 promoter-based expression system in which transcription activator protein C of bacteriophage Mu (Mu C) has been cloned to serve as a repressor in the regulatory circuit . The system also includes pLysE, which encodes T7 lysozyme, an inhibitor of T7 RNA polymerase . This ensures tight regulation of cloned genes in the uninduced state . Upon induction, the expressed Mu C protein binds to its cognate site thereby repressing lys transcription driven by the tet promoter . In order to evaluate the tight control achieved in the system, and to check leaky expression, if any, we have cloned the gene for the SmaI restriction endonuclease without its cognate methylase . For this purpose, a dicistronic unit was constructed by cloning the smaIR gene downstream of the Mu C gene . SmaI expression was observed only in the induced cell extracts, demonstrating a tight control . The system could be used to express the genes of other cloned restriction enzymes and has the potential for general applications.

Hybridoma, 1998 Apr, 17(2), 151 - 6
Generation of a recombinant bacteriophage antibody library to mycobacterium tuberculosis; Cummings PJ et al.; Monoclonal antibodies to Mycobacterium tuberculosis (Mtb) may be useful for laboratory diagnosis, antigenic characterization, and studying the immune response to infection and vaccination . To investigate the potential of the recombinant phage antibody technique for the isolation of single-chain fragments (ScFv) reactive with Mtb culture proteins, we generated a bacteriophage antibody library from splenic tissue of mice immunized with heat-killed Mtb . Heavy- and light-chain immunoglobulin genes were isolated and amplified by the polymerase chain reaction (PCR), and the products were assembled into ScFv gene fragments and cloned into the pCANTAB5-E phagemid . Phagemids were introduced into E . coli TG1 by electrotransformation, followed by rescue of antibody-expressing phage using M13K07 helper-phage superinfection . Immunoselection of the Mtb phage library against Mtb cell wall extract or culture filtrate proteins selected antigen-specific and cross-reactive phage antibodies, none of which demonstrated reactivity to the immunodominant 65-kDa Mtb antigen . These results suggest that the phage antibody system has the potential to generate a diverse population of the reactive phage that may prove useful for investigating the immune response to Mtb infection and vaccination.

Biotechnol Prog, 1998 May-Jun, 14(3), 496 - 9
Artificial antibodies for affinity chromatography of homologous proteins: application to blood clotting proteins; Wu H et al.; A method to readily isolate antibodies that bind to only one member of a family of homologous proteins is described . A library of different single chain antibody fragments can be displayed on the surface of a bacteriophage vector . Individual antibodies from this library recognizing a particular protein from a family of homologous proteins can be readily isolated by a two-step affinity screening process . In the first step antibodies which bind specifically to the undesired proteins or to homologous regions of the proteins are removed . In the second step, those antibodies specifically recognizing the desired protein are then isolated . Using this procedure and starting with a naive antibody library, a single chain antibody fragment specific to the blood clotting protein, Protein C, which did not recognize either of the homologous proteins, Factor IX or Factor X, was isolated . Similarly an antibody specific to Factor IX, but not Factor X or Protein C, was also isolated . The isolated antibodies can be readily produced, purified, and affixed to sepharose beads for affinity chromatography of the blood clotting factors . One of the key advantages to this procedure over conventional monoclonal antibody isolation is that the antibodies are isolated and produced in vitro so a broad range of related proteins, toxins, viruses, or other products can be targeted.

Nat Biotechnol, 1998 Jun, 16(6), 581 - 6
Cloning of cell-specific secreted and surface proteins by subtractive antibody screening; Scherer PE et al.; To identify and clone genes that encode cell- or tissue-specific secreted and surface proteins, a polyclonal antiserum was raised against a complex mixture of surface or secreted proteins from the target cell, followed by immunodepletion of antibodies that recognize proteins from a nontarget cell or tissue . The depleted antiserum is used to screen bacteriophage cDNA expression libraries . Because of our interest in how adipocytes communicate with other cells, we have used this method to clone cDNAs encoding secreted and plasma membrane proteins that are induced during adipocyte differentiation . We describe several of these, including a novel plasma membrane-associated protein, S3-12.

Biochemistry, 1998 May 5, 37(18), 6446 - 55
Single-chain lambda Cro repressors confirm high intrinsic dimer-DNA affinity; Jana R et al.; The overall affinity of the bacteriophage lambda Cro repressor for its operator DNA site is limited by dimer dissociation at submicromolar concentrations . Since Cro dimer-operator complexes form at nanomolar concentrations of Cro subunits where free dimers are rare, these dimers must bind with compensating high affinities . Previous studies of the covalent dimer Cro V55C suggest little change in DNA binding affinity even though the dimeric species is quantitatively populated; this is an apparent contradiction to the expectation of high intrinsic dimer-DNA affinity . In contrast to the disulfide linkage at the center of the dimer interface in Cro V55C, polypeptide linkers that join the two subunits allow single-chain Cro repressors to bind operator DNA with picomolar affinities . A series of five single-chain Cro repressors have been expressed from fused tandem cro genes . Each contains a peptide linker of 8-16 hydrophilic residues that connects the C-terminus of one subunit to the N-terminus of the next . All bind to operator DNA with at least 100-fold higher affinity than Cro V55C . Proteins containing the longest and shortest linkers have been purified and characterized in detail . Both exhibit similar CD spectra to wild-type Cro and enhanced thermal stability . Sedimentation equilibrium experiments show that single-chain Cro repressors do not associate at concentrations up to 30 microM . The rate of dissociation of Cro-DNA complexes is almost unchanged by covalent linkage . Biophysical characterization of Cro variants such as these, where DNA binding is uncoupled from subunit assembly, is necessary for a quantitative understanding of the structural and energetic determinants of DNA recognition in this simple model system.

J Bacteriol, 1998 Jun, 180(12), 3257 - 9
Activation of bacteriophage Mu mom transcription by C protein does not require specific interaction with the carboxyl-terminal region of the alpha or sigma 70 subunit of Escherichia coli RNA polymerase; Sun W et al.; Late in its growth cycle, transcription of the phage Mu mom Promoter (Pmom) is activated by the phage gene product, C, a site-specific DNA binding protein . In vitro transcription analyses showed that this activation does not require specific contacts between C and the carboxyl-terminal region of the alpha or sigma 70 subunit of Escherichia coli RNA polymerase . Unexpectedly, these results are in contrast to those known for another Mu-encoded transcriptional activator, Mor, which has a high degree of sequence identity with C and appears to interact with the carboxyl termini of both alpha and sigma 70.

Plant Mol Biol, 1998 May, 37(1), 77 - 85
Characterization of rbcL group IA introns from two colonial volvocalean species (Chlorophyceae); Nozaki H et al.; Group I introns were reported for the first time in the large subunit of Rubisco (rbcL) genes, using two colonial green algae, Pleodorina californica and Gonium multicoccum (Volvocales) . The rbcL gene of P . californica contained an intron (PIC intron) of 1320 bp harboring an open reading frame (ORF) . The G . multicoccum rbcL gene had two ORF-lacking introns of 549 (GM1 intron) and 295 (GM2 intron) base pairs . Based on the conserved nucleotide sequences of the secondary structure, the PIC and GM1 introns were assigned to group IA2 whereas the GM2 intron belonged to group IA1 . Southern hybridization analyses of nuclear and chloroplast DNAs indicated that such intron-containing rbcL genes are located in the chloroplast genome . Sequencing RNAs from the two algae revealed that these introns are spliced out during mRNA maturation . In addition, the PIC and GM1 introns were inserted in the same position of the rbcL exons, and phylogenetic analysis of group IA introns indicated a close phylogenetic relationship between the PIC and GM1 introns within the lineage of bacteriophage group IA2 introns . However, P . californica and G . multicoccum occupy distinct clades in the phylogenetic trees of the colonial Volvocales, and the majority of other colonial volvocalean species do not have such introns in the rbcL genes . Therefore, these introns might have been recently inserted in the rbcL genes independently by horizontal transmission by viruses or bacteriophage.

Biochem Biophys Res Commun, 1998 May 29, 246(3), 634 - 9
Random inheritance of the replication complex by one of two daughter lambda plasmid copies after a replication round in Escherichia coli; Wegrzyn A et al.; There are two pathways for replication of plasmids derived from bacteriophage lambda (so-called lambda plasmids) in Escherichia coli . One pathway is based on the assembly of the new replication complex at ori lambda, and the second requires activity of the replication complex inherited by one of two daughter plasmid copies after each replication round . Although these two replication pathways proceed at the same time in the host cell, we previously found conditions for specific elimination of the pathway based on the assembly of the new replication complex; thus, replication is restricted to that carried out by the heritable replication complex . These conditions are (i) the relaxed response to amino acid starvation and (ii) temperature upshift of the culture of cells harboring the lambda crotsPts1 plasmid . Here we asked whether the replication complex is inherited randomly by one of two daughter plasmid copies or whether the inheritance is preferred by one particular copy, that containing the parental DNA r strand or that bearing the l strand . We performed density shift experiments which allowed us to separate plasmid DNA molecules replicated by the heritable replication complex from those devoid of the replication complex and therefore not able to replicate . Then, {3H}thymidine-labelled plasmid DNA strands were separated and hybridized to membrane-bound ssDNA containing a fragment of either the r or l strand of lambda DNA . We found roughly equal efficiency of hybridization to both r and l strands in all experimental systems used . Therefore, we conclude that the lambda replication complex is randomly inherited by one of two daughter plasmid copies rather than preferentially inherited by either the copy carrying the parental r strand or that containing the l strand.

Anal Biochem, 1998 Jun 1, 259(2), 226 - 34
Quantitative reverse transcription strand displacement amplification: quantitation of nucleic acids using an isothermal amplification technique; Nycz CM et al.; Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens . The most prevalent testing is carried out for HIV viral load . Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties . SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences . We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication . In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence . QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting .

Nucleic Acids Res, 1998 May 15, 26(10), 2457 - 63
Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phiC31; Wilson SE et al.; Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage phiC31 . Because expression of the two smaller isoforms, 54 and 42 kDa, is sufficient for superinfection immunity, the interaction between these isoforms was studied . The native 42 kDa repressor (Nat42) and an N-terminally 6x histidine-tagged 54 kDa isoform (His54) were shown by co-purification on a Ni-NTA column to interact in Streptomyces lividans . In vitro three repressor preparations, containing Nat42, His54 and the native 54 and 42 kDa isoforms expressed together (Nat54&42), were subjected to chemical crosslinking and gel filtration analysis . Homo- and hetero-tetramers were observed . Previous work showed that the smallest isoform bound to 17 bp operators containing aconservedinvertedrepeat (CIR) and that the CIRs were located at 16 loci throughout the phiC31 genome . One of the CIRs (CIR6) is believed to be critical for regulating the lytic pathway . The DNA binding activities of the three repressor preparations were studied using fragments containing CIRs (CIR3-CIR6) from the essential early region as templates for DNase I footprinting . Whereas Nat42 bound to CIR6, poorly to CIR5 but undetectably to CIR3 or CIR4, the Nat54&42 preparation could bind to all CIRs tested, albeit poorly to CIR3 and CIR4 . The His54 isoform bound all CIRs tested . Isoforms expressed from the phiC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

Virology, 1998 May 25, 245(1), 11 - 7
Bacteriophage P2 and P4 morphogenesis: structure and function of the connector; Rishovd S et al.; The connector, the structure located between the bacteriophage capsid and tail, is interesting from several points of view . The connector is in many cases involved in the initiation of the capsid assembly process, functions as a gate for DNA transport in and out of the capsid, and is, as implied by the name, the structure connecting a tail to the capsid . Occupying a position on a 5-fold axis in the capsid and connected to a coaxial 6-fold tail, it mediates a symmetry mismatch between the two . To understand how the connector is capable of all these interactions its structure needs to be worked out . We have focused on the bacteriophage P2/P4 connector, and here we report an image reconstruction based on 2D crystalline layers of connector protein expressed from a plasmid in the absence of other phage proteins . The overall design of the connector complies well with that of other phage connectors, being a toroid structure having a conspicuous central channel . Our data suggests a 12-fold symmetry, i.e., 12 protrusions emerge from the more compact central part of the structure . However, rotational analysis of single particles suggests that there are both 12- and 13-mers present in the crude sample . The connectors used in this image reconstruction work differ from connectors in virions by having retained the amino-terminal 26 amino acids normally cleaved off during the morphogenetic process . We have used different late gene mutants to demonstrate that this processing occurs during DNA packaging, since only mutants in gene P, coding for the large terminase subunit, accumulate uncleaved connector protein . The suggestion that the cleavage might be intimately involved in the DNA packaging process is substantiated by the fact that the fragment cleaved off is highly basic and is homologous to known DNA binding sequences.

J Mol Biol, 1998 Mar 27, 277(2), 317 - 32
Small binding proteins selected from a combinatorial repertoire of knottins displayed on phage; Smith GP et al.; Knottins are a group of small, disulphide-bonded proteins that bind with high specificity to their target molecules . These proteins appear to use different faces of the protein for their interactions with different targets . Here, we attempted to create knottins with novel binding activities based on the cellulose-binding domain of the fungal enzyme cellobiohydrolase I . Variation was introduced to the face of the protein that binds cellulose . Seven residues, which are located in two regions of the polypeptide chain and form a patch of about 400 A2 on the protein surface, were simultaneously varied by random mutation of the gene . The repertoire was cloned for display on filamentous bacteriophage (5.5 x 10(8) clones), and selected for binding to cellulose or to one of three enzymes (alpha-amylase, alkaline phosphatase and beta-glucuronidase) . We thereby isolated variant knottins against cellulose (differing in sequence from the parent knottin) and also against alkaline phosphatase . The binding to (glycosylated) alkaline phosphatase was highly specific with an affinity of about 10 microM, required the presence of disulphide bonds and was mediated through protein (rather than carbohydrate) contacts . Knottin scaffolds therefore appear to be a promising architecture for the creation of small folded proteins with binding activities, with the potential for improvement of binding affinities by mutation, or of using other faces of the protein to provide greater structural diversity in the primary repertoire .

Nucleic Acids Res, 1998 Jun 15, 26(12), 2891 - 8
Auxiliary downstream elements are required for efficient polyadenylation of mammalian pre-mRNAs; Chen F et al.; We have previously identified a G-rich sequence (GRS) as an auxiliary downstream element (AUX DSE) which influences the processing efficiency of the SV40 late polyadenylation signal . We have now determined that sequences downstream of the core U-rich element (URE) form a fundamental part of mammalian polyadenylation signals . These novel AUX DSEs all influenced the efficiency of 3'-end processing in vitro by stabilizing the assembly of CstF on the core downstream URE . Three possible mechanisms by which AUX DSEs mediate efficient in vitro 3'-end processing have been explored . First, AUX DSEs can promote processing efficiency by maintaining the core elements in an unstructured domain which allows the general polyadenylation factors to efficiently assemble on the RNA substrate . Second, AUX DSEs can enhance processing by forming a stable structure which helps focus binding of CstF to the core downstream URE . Finally, the GRS element, but not the binding site for the bacteriophage R17 coat protein, can substitute for the auxiliary downstream region of the adenovirus L3 polyadenylation signal . This suggests that AUX DSE binding proteins may play an active role in stimulating 3'-end processing by stabilizing the association of CstF with the RNA substrate . AUX DSEs, therefore, serve as a integral part of the polyadenylation signal and can affect signal strength and possibly regulation.

Nucleic Acids Res, 1998 Jun 15, 26(12), 2879 - 85
Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by replication protein A; Shen JC et al.; Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer . The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro . Here we report further biochemical characterization of the WRN helicase . The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand . Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement . K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively . Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs) . Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN . A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.

Math Biosci, 1998 Apr, 149(1), 57 - 76
Modeling and analysis of a marine bacteriophage infection; Beretta E et al.; A mathematical model for the marine bacteriophage infection is proposed and its essential mathematical features are analyzed . Since bacteriophage infection induces bacterial lysis which releases into the marine environment, on the average, 'b' viruses per cell, the parameter b epsilon (1, t infinity) or 'virus replication factor' is chosen as the main parameter on which the dynamics of the infection depends . We proved that a threshold b* exists beyond which the endemic equilibrium bifurcates from the free disease one . Still, for increasing b values the endemic equilibrium bifurcates toward a periodic solution . We proved that a compact attractor set omega within the positive cone exists and within omega the free disease equilibrium is globally stable whenever b < or = b*, whereas it becomes a strong uniform repeller for b > b* . A concluding discussion with numerical simulation is then presented.

Biochemistry, 1998 May 19, 37(20), 7251 - 9
Repressive effect on oligonucleosome transcription of the core histone tail domains; Chirinos M et al.; Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from histone octamers and three different DNA species, two circular (pGEMEX-1 and pT207-18) and one linear (T7-207-18) . pGEMEX is devoid of nucleosome positioning sequences, while in pT207-18 and T7-207-18 the region downstream of the promoter contains 18 tandem repeats of a 207 bp positioning sequence derived from the 5S RNA gene of the sea urchin Lytechinus variegatus . Elimination of the histone tails in the assembled oligonucleosomes by trypsin digestion is accompanied, in all three DNA species, by substantial increases in transcription efficiency, assayed at different KCl and MgCl2 concentrations, after allowing for the aggregation observed under certain conditions . In the absence of KCl and at low MgCl2 concentration, the presence of 2 mM spermidine causes substantial aggregation of the intact oligonucleosomes but has a much smaller effect on those trypsin digested . The untreated histone-DNA templates, assembled on pGEMEX-1 and T7-207-18, give transcription products significantly shorter than those obtained with the corresponding free DNA . With oligonucleosome templates lacking histone tails, the transcripts have an average length intermediate between those corresponding to free DNA and intact histone-DNA, which indicates a partial elimination of the elongation restrictions imposed by intact histone octamers . The absence of histone terminal domains facilitates both transcriptional initiation and elongation . Apparently, the interaction of the histone tails with DNA at the nucleosomal level is responsible, at least in part, for their repressive effect on transcription.

Methods, 1998 Apr, 14(4), 381 - 92
Inducible gene targeting in mice using the Cre/lox system; Sauer B; Molecular techniques now allow the design of precise genetic modifications in the mouse . Not only can defined nucleotide changes be engineered into the genome of the mouse, but genetic switches can be designed to target expression or ablation of any gene (for which basic molecular information is available) to any tissue at any defined time . These strategies promise to contribute substantially to an increased understanding of individual gene function in development and pathogenesis . A powerful tool, both for the design of such genetic switches and for speeding the creation of gene-modified animals, is the Cre site-specific DNA recombinase of bacteriophage P1 . Precise DNA rearrangements and genetic switches can be efficiently generated in a straightforward manner using Cre recombinase . In conjunction with inducible systems for controlling Cre expression and function, these recombination-based strategies are likely to have a profound impact on developmental biology and the generation of useful animal models of human disease.

Hematopathol Mol Hematol, 1998, 11(2), 63 - 71
Structure and linkage relationships of the region containing the human L-type pyruvate kinase (PKLR) and glucocerebrosidase (GBA) genes; Demina A et al.; Both the L-type pyruvate kinase gene (PKLR) and glucocerebrosidase (GBA) gene are on band q21 of chromosome 1 in humans . Two overlapping P1 bacteriophage clones containing PKLR and GBA were identified and mapped, defining the locations of these two genes as well as those of the GBA pseudogene (psi GBA) metaxin (MTX), the MTX pseudogene (psi MTX), and thrombospondin 3 (THBS3) . The distance between the 5' ends of GBA and PKLR was determined to be 71 kb . The direction of transcription PKLR gene was convergent to that of the GBA gene . All 195 Gaucher disease patients homozygous for the 1226G mutation, representing 390 chromosomes with the 1226G mutation, had a PvuII -/- GBA haplotype and a C/C at nt 1705 of the PKLR gene (-/- haplotype) . All 56 Gaucher disease patients who were 1226G/84GG compound heterozygotes manifested a -/+ GBA haplotype and 55 of 56 patients were -/+ at PKLR nt 1705 . Only 1 patient with 1226G/84GG genotype showed a crossover with the PKLR polymorphism, with a -/- haplotype at nt 1705 . Similarly, 9 patients deficient in pyruvate kinase with the PKLR 1529A/1529A genotype were all found to have the same -/- GBA haplotype.

Plant Cell, 1998 May, 10(5), 801 - 12
Comparative mapping of the Brassica S locus region and its homeolog in Arabidopsis . Implications for the evolution of mating systems in the Brassicaceae; Conner JA et al.; The crucifer family includes self-incompatible genera, such as Brassica, and self-fertile genera, such as Arabidopsis . To gain insight into mechanisms underlying the evolution of mating systems in this family, we used a selective comparative mapping approach between Brassica campestris plants homozygous for the S8 haplotype and Arabidopsis . Starting with markers flanking the self-incompatibility genes in Brassica, we identified the homeologous region in Arabidopsis as a previously uncharacterized segment of chromosome 1 in the immediate vicinity of the ethylene response gene ETR1 . A total of 26 genomic and 21 cDNA markers derived from Arabidopsis yeast artificial and bacterial artificial chromosome clones were used to analyze this region in the two genomes . Approximately half of the cDNAs isolated from the region represent novel expressed sequence tags that do not match entries in the DNA and protein databases . The physical maps that we derived by using these markers as well as markers isolated from bacteriophage clones spanning the S8 haplotype revealed a high degree of synteny at the submegabase scale between the two homeologous regions . However, no sequences similar to the Brassica S locus genes that are known to be required for the self-incompatibility response were detected within this interval or other regions of the Arabidopsis genome . This observation is consistent with deletion of self-recognition genes as a mechanism for the evolution of autogamy in the Arabidopsis lineage.

Cytogenet Cell Genet, 1997, 79(3-4), 188 - 92
Rapid isolation of genomic clones for individual members of human multigene families: identification and localisation of UBE2L4, a novel member of a ubiquitin conjugating enzyme dispersed gene family; Ardley HC et al.; We describe a rapid, PCR-based, screening procedure for the isolation of human genomic clones in lambda bacteriophage, containing sequences coding for individual homologous members of a multigene family . The approach is based upon the identification, by dilution, of sub-pools of the genomic library that contain members of the gene family, prior to phage isolation . The presence of specific genes is established by PCR of aliquots of individually amplified library pools, using consensus primers and subsequent sequencing . We have used the approach to isolate a fourth member of the UBE2L gene family, UBE2L4, and located it on chromosome 19q13.1-->q13.2 . This PCR-based approach to library screening has wider applicability in that it could be used to isolate alternate-spliced products from cDNA libraries.

Gene, 1998 May 28, 212(1), 21 - 9
Host vector system for high-level expression and purification of recombinant, enzymatically active alanine dehydrogenase of Mycobacterium tuberculosis; Hutter B et al.; The 40-kDa antigen of M . tuberculosis, which is an alanine dehydrogenase, is a species-specific antigen that is potentially useful for strain identification . Large quantities of the purified protein are required for immunological, as well as for detailed biochemical and structural, characterization . The AlaDH gene was cloned by PCR from H37Rv (virulent) and H37Ra (partially attenuated) strains of M . tuberculosis, and their DNA sequence was determined . A host-vector system suitable for the production of sufficient quantities of the recombinant AlaDH antigen was developed . The AlaDH gene was expressed under the control of strong, transcriptional (bacteriophage pLpR) and translational (atpE) signals . High-level expression of soluble AlaDH was obtained using the recombinant E . coli K-12 strain CAG629 {pMSK12}, which is deficient in Lon protease and the heat-shock response . A simple two-step procedure for the rapid purification of the recombinant protein was developed . The protein was purified to near homogeneity, and the purified AlaDH showed a specific enzyme activity comparable to the native protein isolated from M . tuberculosis . In addition, the product showed an expected amino acid sequence and reacted strongly to the 40-kDa (AlaDH)-specific mAb HBT-10 . Furthermore, the epitope of the mAb HBT-10 was mapped to a 12-amino-acid region . Contrary to the published results, we show that the AlaDH and the PNT (pyridine nucleotide transhydrogenase) of M . tuberculosis do not share common epitopes reacting to the species-specific mAb HBT-10 . The availability of highly purified AlaDH should now enable a detailed biochemical and structural characterization of this important enzyme of M . tuberculosis.

Biochim Biophys Acta, 1998 May 29, 1398(1), 9 - 17
Genomic structure of Unp, a murine gene encoding a ubiquitin-specific protease; Di Fruscio M et al.; The murine Unp gene encodes a ubiquitin-specific protease, a member of a family of enzymes that includes the product of the human tre-2 oncogene . The Unp gene has previously been mapped to chromosome 9 . We have cloned in bacteriophage a 50 kilobase region of chromosome 9 containing the Unp gene, and have determined the nucleotide sequence of the gene . The gene has 22 exons, distributed over 47.4 kb . A processed ribosomal S2 pseudogene was identified in the third intron of the Unp gene . Expression of Unp is driven by a GC-rich, 'housekeeping' type promoter .

Gene, 1998 Apr 28, 211(1), 125 - 31
Structure and chromosomal localization of the mouse bombesin receptor subtype 3 gene; Weber HC et al.; Bombesin (BN)-like peptides/neurotransmitters mediate a broad range of physiological funtions in the gastrointestinal tract and the central nervous system through binding to their specific, high-affinity mammalian bombesin receptors . This family of heptahelical, G-protein coupled receptors includes the gastrin-releasing peptide receptor (GRP-R, or bb2), neuromedin B receptor (NMB-R, or bb1), and the bombesin receptor subtype 3 (BRS-3, or bb3) . The tissue distribution of BRS-3 is quite dissimilar compared to the other two BN receptors, GRP-R and NMB-R, and a natural ligand for BRS-3 is currently unknown . Nothing is known about mechanisms regulating BRS-3 gene expression and possible association with disease . To gain insight into the underlying structure and chromosomal localization of the BRS-3 genes, bacteriophage P1 genomic clones, harboring the genes for the human and mouse BRS-3, respectively, were isolated and their structure and chromosomal localizations determined . The protein-coding region of both genes is divided into three exons and spans approximately 5kb . The loci of the BRS-3 genes were mapped to a syntenic region of the human (Xq25) and mouse (XA7.1-7.2) X-chromosome, respectively . The structural data of the BRS-3 genes derived from this study will permit future investigations of the mechanisms regulating their expression.

J Biol Chem, 1998 May 22, 273(21), 13136 - 42
Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1; McCullough AK et al.; Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific glycosylase . Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1) . Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG . Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer . This is the first trans-syn-II-specific glycosylase identified to date . Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies . Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond . cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA . The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.

Biochem Cell Biol, 1997, 75(6), 687 - 96
Chitosanase from Streptomyces sp . strain N174: a comparative review of its structure and function; Fukamizo T et al.; Novel information on the structure and function of chitosanase, which hydrolyzes the beta-1,4-glycosidic linkage of chitosan, has accumulated in recent years . The cloning of the chitosanase gene from Streptomyces sp . strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances . Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module . From energy minimization based on the X-ray crystal structure of Streptomyces sp . strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites . The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism . Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues . The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues . Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities . This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation.

J Mol Biol, 1998 Apr 3, 277(3), 541 - 57
The largest (70 kDa) product of the bacteriophage T4 DNA terminase gene 17 binds to single-stranded DNA segments and digests them towards junctions with double-stranded DNA; Franklin JL et al.; Bacteriophage terminases are oligomeric multifunctional proteins that bind to vegetative DNA, cut it and, together with portal proteins, translocate the DNA into preformed heads . Most terminases are encoded by two partially overlapping genes . In phage T4 they are genes 16 and 17 . We have shown before that the larger of these, gene 17, can yield, in addition to a full-length 70 kDa product, several shorter peptides . At least two of these, gene product (gp) 17' and gp17", are initiated in the same reading frame as the 70 kDa gp17 from internal ribosome binding sites . Most of the shorter gp17 s contain predicted ATPase motifs, but only the largest (70 kDa) peptide has a predicted single-stranded DNA binding domain.Here we describe the DNA binding and cutting properties of the purified 70 kDa protein, expressed from two different clones containing gene 17 but no other T4 gene . Epitope-specific antibodies, which recognize several different gene 17 products in extracts of induced clones or of T4-infected cells, precipitate the purified 70 kDa gp17 . When Mg2+ is chelated by EDTA this 70 kDa protein binds to single-stranded DNA, preferentially to junctions of single- and double-stranded DNA segments . It does not bind to blunt-ended double-stranded DNA . When Mg2+ is present the purified 70 kDa gp17 digests single-stranded segments preferentially up to junctions with double-stranded DNA . A 70 kDa gp17 from a P379L temperature sensitive (ts) mutant, which has lost the nuclease and ATPase activities, retains the single-stranded DNA binding activity . Taken together with earlier findings these results support a model for packaging of T4 DNA from single-stranded regions in recombinational or replicative intermediates, which occur at nearly random positions of the genome . This mechanism may be an alternative to site-specific initiation of packaging proposed by other investigators .

J Mol Biol, 1998 Apr 3, 277(3), 529 - 40
Epitope mapping of T4 endonuclease VII with monoclonal antibodies reveals importance of both ends of the protein for target binding; Christoph A et al.; Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure . The protein has a molecular mass of 18 kDa and exists as a dimer in solution . Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme . From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established . The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays . Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII . The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization . Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs .

Gene, 1998 Mar 27, 210(1), 135 - 42
Translationally repressive RNA structures monitored in vivo using temperate DNA bacteriophages; Fouts DE et al.; The RNA challenge phage system enables genetic selection of proteins with RNA-binding activity in bacteria . These phages are modified versions of the temperate DNA bacteriophage P22 in which post-transcriptional regulatory events control the developmental fate of the phage . The system was originally developed to identify novel RNA ligands that display reduced affinity for the R17/MS2 coat protein, as well as to select for suppressor coat proteins that recognize mutant RNA ligands . During the course of evaluating whether the HIV-1 Rev protein could direct lysogen development for bacteriophage derivatives that encode Rev response element (RRE) RNA sequences, two examples of RRE RNA ligands that interfere with challenge phage development were identified . In the phage examples described, RRE RNA secondary structure prevents Ant protein biosynthesis and lytic development . Phage lysogen formation occurs efficiently in recipient cells, independent of the expression status of the Rev protein or trans-acting competitor RRE RNA ligands . These studies provide the first example whereby RNA challenge phages may be applied to study RNA folding events and RNA structural interactions in an in vivo context.

J Mol Biol, 1998 Apr 17, 277(5), 1059 - 70
Recognition of core binding sites by bacteriophage integrases; Dorgai L et al.; Bacteriophage integrases promote recombination between DNA molecules that carry attachment sites . They are members of a large and widely distributed family of site-specific recombinases with diverse biological roles . The integrases of phages lambda and HK022 are closely related members of this family, but neither protein efficiently recombines the attachment sites of the other phage . The nucleotides responsible for this specificity difference are located close to the points of recombinational strand exchange, within an integrase binding motif called the extended core binding site . There are four imperfectly repeated copies of this motif in each set of phage attachment sites, but only two, B' and C, contain major specificity determinants . When these specificity determinants were replaced by the corresponding nucleotides from a site with the alternative specificity, the resulting mutant was recombined by both integrases . Thus, the determinants act by impeding recombination promoted by the non-cognate integrase . We found that identical nucleotide substitutions within different core site copies had different effects on recombination, suggesting that integrase does not recognize each of the extended core binding sites in the same way . Finally, substitution at several positions in lambda integrase with the corresponding HK022-specific amino acids prevents recombination of lambda attachment sites, and this defect can be suppressed in an allele-specific manner by appropriate substitutions of HK022-specific nucleotides in the extended core binding sites .

Proteins, 1998 May 1, 31(2), 116 - 27
Domain motions in bacteriophage T4 lysozyme: a comparison between molecular dynamics and crystallographic data; de Groot BL et al.; A comparison of a series of extended molecular dynamics (MD) simulations of bacteriophage T4 lysozyme in solvent with X-ray data is presented . Essential dynamics analyses were used to derive collective fluctuations from both the simulated trajectories and a distribution of crystallographic conformations . In both cases the main collective fluctuations describe domain motions . The protein consists of an N- and C-terminal domain connected by a long helix . The analysis of the distribution of crystallographic conformations reveals that the N-terminal helix rotates together with either of these two domains . The main domain fluctuation describes a closure mode of the two domains in which the N-terminal helix rotates concertedly with the C-terminal domain, while the domain fluctuation with second largest amplitude corresponds to a twisting mode of the two domains, with the N-terminal helix rotating concertedly with the N-terminal domain . For the closure mode, the difference in hinge-bending angle between the most open and most closed X-ray structure along this mode is 49 degrees . In the MD simulation that shows the largest fluctuation along this mode, a rotation of 45 degrees was observed . Although the twisting mode has much less freedom than the closure mode in the distribution of crystallographic conformations, experimental results suggest that it might be functionally important . Interestingly, the twisting mode is sampled more extensively in all MD simulations than it is in the distribution of X-ray conformations.

Nucleic Acids Symp Ser, 1997, (37), 217 - 8
Study on site specific cleavage of RNA; Shirakura H et al.; The precursor of an RNA molecule from bacteriophage T4 infected Escherichia coli cell (p2Sp1 RNA) has the ability to cleave itself . It has been found that the site specific RNA cleavage reaction occurred at the pyridine-adenosine sequence in the presence of a monovalent cation and a non-ionic detergent . In order to investigate the mechanism of this cleavage reaction, we designed a RNA oligonucleotide (UUUAUU) and this RNA was cleavage activity at the U-A sequence.

Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5505 - 10
In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family; Thorpe HM et al.; The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases . The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase . The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S . ambofaciens . In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required . Recombination occurred irrespective of whether the substrates were supercoiled or linear . A mutant integrase containing an S12F mutation was completely defective in recombination both in E . coli and in vitro . No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor . Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites . The results show that directionality in phiC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.

Curr Microbiol, 1998 May, 36(5), 298 - 301
Low-frequency electromagnetic fields alter the replication cycle of MS2 bacteriophage; Staczek J et al.; The effect of exposure to 60-Hz electromagnetic fields (EMFs) on RNA coliphage MS2 replication was studied . EMF exposure commenced when the bacterial cultures were inoculated with the phage (t = 0) . In 12 experiments in which the strength of the field was 5 G, a significant delay in phage yield was found in the EMF-exposed cultures 45-65 min after inoculation, compared with control cultures . However, the EMF did not alter the final phage concentration . Experiments at 25 G (N = 5) suggested that the stronger field resulted in both impeded phage replication and increased phage yield . No differences between test groups were found in experiments involving sham-EMF exposure, thereby indicating that the results obtained with the EMFs were not due to systematic error . It appears that MS2, which codes for only four proteins, is the simplest biological system in which an EMF-induced effect has been demonstrated . The MS2 system is, therefore, conducive to follow-up studies aimed at understanding the level and nature of the underlying interaction process, and perhaps to biophysical modeling of the interaction process.

Biochemistry, 1998 Apr 21, 37(16), 5673 - 81
The uvsY recombination protein of bacteriophage T4 forms hexamers in the presence and absence of single-stranded DNA; Beernink HT et al.; A prerequisite to genetic recombination in the T4 bacteriophage is the formation of the presynaptic filament-a helical nucleoprotein filament containing stoichiometric amounts of the uvsX recombinase in complex with single-stranded DNA (ssDNA) . Once formed, the filament is competent to catalyze homologous pairing and DNA strand exchange reactions . An important component in the formation of the presynaptic filament is the uvsY protein, which is required for optimal uvsX-ssDNA assembly in vitro, and essential for phage recombination in vivo . uvsY enhances uvsX activities by promoting filament formation and stabilizing filaments under conditions of low uvsX, high salt, and/or high gp32 (ssDNA-binding protein) concentrations . The molecular properties of uvsY include noncooperative binding to ssDNA and specific protein-protein interactions with both uvsX and gp32 . Evidence suggests that all of these hetero-associations of the uvsY protein are important for presynaptic filament formation . However, there is currently no structural information available on the uvsY protein itself . In this study, we present the first characterization of the self-association of uvsY . Using hydrodynamic methods, we demonstrate that uvsY associates into a stable hexamer (s020,w = 6.0, M = 95 kDa) in solution and that this structure is competent to bind ssDNA . We further demonstrate that uvsY hexamers are capable of reversible association into higher aggregates in a manner dependent on both salt and protein concentration . The implications for presynaptic filament formation are discussed.

Biochemistry, 1998 Apr 14, 37(15), 5184 - 93
Misincorporation of nucleotides opposite five-membered exocyclic ring guanine derivatives by escherichia coli polymerases in vitro and in vivo: 1,N2-ethenoguanine, 5,6,7,9-tetrahydro-9-oxoimidazo{1, 2-a}purine, and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo{1, 2-a}purine; Langouet S et al.; A variety of exocyclic modified bases have been shown to be formed in DNA from various procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan) and are also found in untreated animals and humans, presumably arising as a result of lipid peroxidation . 1, N2-Ethenoguanine (1,N2-epsilon-Gua), a product known to be formed from several 2-carbon electrophiles, was placed in a known site (6256) in bacteriophage M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G-->A, 0.74% G-->T, and 0.09% G-->C changes found in uvrA- bacteria . 5,6,7, 9-Tetrahydro-7-hydroxy-9-oxoimidazo{1,2-a}purine (HO-ethanoGua), formally the hydrated derivative of 1,N2-epsilon-Gua, is a stable DNA product also derived from vinyl halides . When this base was placed in the same context, the mutation rate was 0.007-0.19% for G-->A, C, or T changes . The saturated etheno ring derivative of 1, N2-epsilon-Gua, 5,6,7,9-tetrahydro-9-oxoimidazo{1,2-a}purine (ethanoGua) produced G-->A and G-->T mutations (0.71% each) . All mutants were SOS-dependent and were attenuated by uvrA activity in E . coli . In vitro studies with four polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N2-epsilon-Gua . Both E . coli polymerases (pol) I exo- and II exo- and bacteriophage pol T7 exo- showed extensive misincorporation opposite ethanoGua in vitro, with pol I exo- incorporating G and T, pol II exo- incorporating A, and pol T7 exo- incorporating A and G . All modified bases reduced the use of the minus strand bearing the modified guanine in E . coli cells . It is of interest that even though the normal base pairing site of guanine is completely blocked, all of the five-membered ring derivatives incorporate the normal base (C) in >80% of the replication events in E . coli . Major differences in blockage and misincorporation are seen due to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.

Biochemistry, 1998 Apr 7, 37(14), 4977 - 84
The MotA transcriptional activator of bacteriophage T4 binds to its specific DNA site as a monomer; Cicero MP et al.; During bacteriophage T4 middle mode gene expression, the MotA transcription factor binds to T4 middle promoters at a -30 mot box consensus sequence to allow activation . Previous binding studies showed that MotA forms multiple gel-shifted complexes with DNA, and structural evidence suggested that MotA dimerizes upon DNA binding . We have shown that a short (13 bp) mot box DNA substrate binds MotA protein but fails to form slower migrating complexes . Therefore, the slower migrating complexes in gel shift assays are caused by DNA-mediated binding events . Competition experiments indicate that the slower migrating complexes are formed by nonspecific binding events, while the first-shifted complex is caused by specific binding to the mot box . Saturation binding experiments revealed that the stoichiometry of MotA binding to DNA is 1:1 in the first-shifted complex, while the slower complexes apparently contain MotA multimers . Gel shift assays using mixtures of MotA and a GST-MotA fusion protein supported the conclusion that the first-shifted complex contains one protein molecule bound to DNA . Furthermore, MotA monomers were cross-linked by glutaraldehyde under conditions where slower complexes exist, but not under conditions that lead to only the first-shifted complex . We conclude that MotA binds specifically to the mot box as a monomer and that additional nonspecific binding events require flanking DNA.

Arch Virol, 1998, 143(3), 523 - 36
Genome diversity in temperate bacteriophages of Oenococcus oeni; Santos R et al.; The genome structure of six bacteriophages of Oenococcus oeni was compared . Two distinct groups with no apparent restriction site conservation were defined . In members of the alpha group (fOgML34, fOg4029, fOg30 and fOg218) a 7.5 kb region containing the origin of DNA packaging (cos) was highly conserved . Stretches of DNA heterogeneity could also be assigned to particular regions and were mostly evident in the right area of the genomes . fOg44 and fOgPSU1 (beta group) were indistinguishable in the left half of their genomes, including cos, but were markedly dissimilar in other regions . Strong labelling signals detected in cross-hybridizations involving members of different groups were confined to fragments centrally located in their physical maps . The attachment site (attP) of fOg44 was assigned to this conserved region . It is suggested that recombination events at this location may have been important in generating the observed diversity of oenophage genomes.

Oral Microbiol Immunol, 1998 Apr, 13(2), 124 - 8
Functional analysis of pVT745, a plasmid from Actinobacillus actinomycetemcomitans; Novak KF et al.; Plasmid pVT745 is a 25.1-kb replicon isolated from Actinobacillus actinomycetemcomitans strain VT745 . A previous report described the hybridization of pVT745 in 5 strain-specific patterns to chromosomal DNA from 15 other A . actinomycetemcomitans strains . However, pVT745 does not share homology with the chromosome of the strain from which it was isolated, VT745 . It was hypothesized that the shared areas of homology might represent insertion sequence elements and/or transposons possibly encoding resistance to one or more antibiotics . An antibiogram of strain VT745 demonstrated that this strain was uniformly susceptible to all antibiotics examined . Because insertion sequence elements and transposons are mobile genetic elements, a series of cell passaging experiments, followed by Southern hybridization was conducted in a attempt to detect transposition of pVT745 homologous DNA within the chromosomes of several A . actinomycetemcomitans strains . The results of these experiments suggested stability of the homologous DNA both within the chromosome and on the plasmid . It was also possible that pVT745 represented a lysogenic bacteriophage . Phage induction experiments were conducted under conditions that induced a previously described A . actinomycetemcomitans lysogenic phage, but no phage could be induced from strain VT745 . Attempts to obtain isolates of VT745 cured of pVT745 were also unsuccessful.

J Biol Chem, 1998 Apr 3, 273(14), 7880 - 7
Increased DNA unwinding efficiency of bacteriophage T7 DNA helicase mutant protein 4A'/E348K; Washington MT et al.; Bacteriophage T7 4A' protein is a DNA helicase that unwinds DNA in a reaction coupled to dTTP hydrolysis . To understand better its mechanism of DNA unwinding, we characterized a set of 4A' mutant proteins (Washington, M . T., Rosenberg, A . H., Griffin, K., Studier, F . W., and Patel, S . S . (1996) J . Biol . Chem . 271, 26825-26834) . We showed here, using single turnover DNA unwinding assays, that the 4A'/E348K mutant protein had the unusual property of unwinding DNA (with a 5-6-fold slower rate) despite a significant defect in its dTTPase activity (a 25-30-fold slower rate) . Comparing the DNA unwinding rates to the dTTPase rates, we estimated the DNA unwinding efficiencies of both wild-type (about 1 base pair unwound per dTTP hydrolysis) and mutant (4 to 6 base pairs unwound per dTTP hydrolysis) . Thus the mutant had a 4-6-fold improvement in its DNA unwinding efficiency over that of the wild-type . We believe that this mutant undergoes less slippage (uncoupled dTTP hydrolysis) than the wild-type . We speculate that nature has selected for a high rate of DNA unwinding rather than a high efficiency of DNA unwinding . Thus even though the mutant is more efficient at DNA unwinding, the wild-type probably was selected because it unwinds DNA faster.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3920 - 4
Directed changes in the number of double-stranded RNA genomic segments in bacteriophage phi6; Onodera S et al.; Bacteriophage Phi6 has a genome of three segments of double-stranded RNA . The segments are designated S, M, and L . Each segment has a unique packaging site, pac, near the 5' end of the plus strand . The plus strands of the segments are normally packaged in the order S, M, L . Chimeras of segment M and S in which segment M is at the 5' end of the plus strand can be stably incorporated into the virion; however, an independent segment S must be included along with normal segment L, even if it contains no active genes . A chimera of segment M and S in which segment S is at the 5' end of the plus strand can be stably incorporated into the virion along with normal segment L to form a two-segment genome . A chimera of segments S, M, and L in which the packaging sequence is that of S can also form a stable nonsegmented genome . These findings are consistent with a model that we have proposed for the packaging of the Phi6 genome.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3507 - 12
Identification of a transient excision intermediate at the crossroads between DNA polymerase extension and proofreading pathways; Baker RP et al.; DNA polymerases achieve accurate DNA replication through a delicate balance between primer elongation and proofreading . While insufficient proofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may prevent completion of DNA synthesis . The transition between polymerization and proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is significantly reduced by the presence of an incorrect base pair at the primer-terminus . We have used mutational analysis, coupled with a sensitive, fluorescence-based assay to characterize intermediate steps in the proofreading pathway . A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer-templates labeled at the 3' terminus with the base analog 2-aminopurine . Formation of the fluorescent complex appears to be a rate-determining step in the proofreading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain . Although these mutant DNA polymerases are proficient in hydrolysis, their reduced ability to form the fluorescent complex imposes a higher kinetic barrier . As a consequence, the mutant DNA polymerases proofread less frequently, resulting in more DNA replication errors.

J Mol Biol, 1998 Mar 6, 276(4), 733 - 44
The beta protein of phage lambda promotes strand exchange; Li Z et al.; Bacteriophage lambda encodes a 28 kDa protein called beta that binds to single-stranded DNA and promotes the renaturation of complementary single strands . beta Protein fails to bind directly to duplex DNA but remains bound to the DNA product of renaturation that beta itself catalyzes . These observations led to an examination of the ability of beta protein to promote strand exchange . beta Protein caused the replacement of a 43-mer oligonucleotide annealed to M13 circular single-stranded DNA by a homologous 63-mer whose 20 extra nucleotide residues were complementary to the adjacent 3' region of M13 DNA . The role of beta protein in this reaction was manifested in several ways: beta protein pushed the exchange through four to eight mismatches, which blocked exchange mediated by spontaneous renaturation and branch migration; beta imposed a polarity on the strand exchange that was lacking in the spontaneous reaction; and beta remained bound to the heteroduplex product of strand exchange . These observations reveal a mechanism by which a protein can drive strand exchange in one direction without using ATP or any other exogenous source of energy .

Gene Expr, 1998, 7(1), 39 - 52
The lambda holin accumulates beyond the lethal triggering concentration under hyperexpression conditions; Smith DL et al.; Most bacteriophages terminate infection by creating lesions in the cytoplasmic membrane, which not only cause immediate cell death but also allow escape of a phage-encoded endolysin . Destruction of the peptidoglycan and cell lysis follows very rapidly, allowing efficient release of the progeny virions . These membrane lesions are formed by a small integral membrane protein called a holin . Holins have highly charged carboxyl-termini that are thought to have two transmembrane alpha-helical domains . Holins are believed to oligomerize and form large holes in the inner membrane . The prototype holin is the S protein from bacteriophage lambda . Scheduling of the lytic event is determined in part by the "structure directed initiation" or sdi translational control region . Inductions of S, cloned under a variety of native and nonnative promoters but with native translational control, resulted in cell lysis at about 1000 molecules of holin per cell, and thus do not produce biochemically useful amounts of S protein . By utilizing a plasmid-based system with the T7 RNA polymerase promoter in tandem with a consensus ribosome binding site, Coomassie blue-detectable quantities of S protein were obtained upon induction, corresponding to an approximately 100-fold increase over the normal lethal level of holin . Characterization of this expression system is presented and discussed with respect to the current model of holin function.

Cell, 1998 Apr 17, 93(2), 289 - 99
NMR structure of the bacteriophage lambda N peptide/boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif; Legault P et al.; The structure of the complex formed by the arginine-rich motif of the transcriptional antitermination protein N of phage lambda and boxB RNA was determined by heteronuclear magnetic resonance spectroscopy . A bent alpha helix in N recognizes primarily the shape and negatively charged surface of the boxB hairpin through multiple hydrophobic and ionic interactions . The GAAGA boxB loop forms a GNRA fold, previously described for tetraloops, which is essential for N binding . The fourth nucleotide of the loop extrudes from the GNRA fold to enable the E . coli elongation factor NusA to recognize the N protein/RNA complex . This structure reveals a new mode of RNA-protein recognition and shows how a small RNA element can facilitate a protein-protein interaction and thereby nucleate formation of a large ribonucleoprotein complex.

Mutagenesis, 1998 Mar, 13(2), 173 - 9
Induction of mutations by 2-acetylaminofluorene in lacI transgenic B6C3F1 mouse liver; Ross JA et al.; Mutations induced in liver cells by the hepatocarcinogen 2-acetylaminofluorene (2-AAF) were characterized after i.p . administration on 4 consecutive days at 100 mg/kg per injection in male B6C3F1 Big Blue transgenic mice that harbored the Escherichia coli lacI reporter gene . Animals were sacrificed at 5, 10 or 60 weeks following the last injection, livers removed and DNA packaged in vitro into bacteriophage lambda particles . The bacteriophage were assayed for lacI function by plating on E . coli in the presence of X-gal . Approximately 3 x 10(5) plaques were assayed per animal . Solvent-treated control mice exhibited a slight increase in mutant frequency over time, from 3.93 x 10(-5) at 5 weeks to 5.02 x 10(-5) at 60 weeks . In contrast, treatment with 2-AAF yielded an approximately 2-fold increase in mutant frequency at 5 and 10 weeks after treatment relative to controls, with frequencies of 8.13 x 10(-5) and 7.43 x 10(-5) respectively . However, by 60 weeks post-treatment the mutant frequency was not significantly increased over concurrent controls . Similar to results in other systems, 2-AAF induced predominantly single base changes targeted to G:C base pairs, primarily G:C-->T:A transversions (27%) . In contrast to results in other bacterial and eukaryotic systems, no deletions were observed among the 2-AAF-induced mutations and the 4 base hot spot deletion that is frequently observed in lacI in E . coli was not observed in this system, suggesting that the lacI transgene may be relatively refractory to frameshift mutations in vivo in the mouse.

Biofizika, 1998 Jan-Feb, 43(1), 53 - 6
{DNA inside bacteriophage lambda forms a Z-form}; Ulanov BP et al.; The site of the transition regions between the B- and Z-forms of the DNA duplex (B-Z junction) may serve as marker of the existence of Z-DNA in situ . The structure of (dC-dG)10 insert in the bacteriophage lambda gt10 has been studied in situ by modification of B-Z junction with O-beta-diethylaminoethylhydroxylamine (OHA) . The latter is an analogue of hydroxylamine possessing specificity with respect to unpaired cytidine . This modification inhibited the process of restriction at BamHI site adjacent to the Z-insert . Judging by the extent of the inhibition about 5% of all inserts has been converted in Z-form . The certain role of Z-form at process of the packaging DNA into bacteriophage's capsid suggest.

Chem Res Toxicol, 1998 Apr, 11(4), 311 - 6
Polymerase blockage and misincorporation of dNTPs opposite the ethylene dibromide-derived DNA adducts S-{2-(N7-guanyl)ethyl}glutathione, S-{2-(N2-guanyl)ethyl}glutathione, and S-{2-(O6-guanyl)ethyl}glutathione; Kim MS et al.; The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems . The known DNA adducts S-{2-(N7-guanyl)ethyl}GSH, S-{2-(N2-guanyl)ethyl}GSH, and S-{2-(O6-guanyl)ethyl}GSH were individually placed at a site in a single oligonucleotide . Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events . Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-{2-(N7-guanyl)ethyl}GSH using all of these polymerases . dCTP was the most preferred nucleotide incorporated opposite S-{2-(N7-guanyl)ethyl}GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-{2-(N7-guanyl)ethyl}GSH with pol II- . Both S-{2-(N2-guanyl)ethyl}GSH and S-{2-(O6-guanyl)ethyl}GSH strongly blocked replication by all polymerases . Only dATP and dGTP were incorporated opposite S-{2-(N2-guanyl)ethyl}GSH by both Kf- and pol II- . S-{2-(O6-Guanyl)ethyl}GSH was shown to strongly code for dATP incorporation by Kf- . With pol II-, dTTP was incorporated opposite S-{2-(O6-guanyl)ethyl}GSH . In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.

Virus Genes, 1998, 16(1), 47 - 58
Evolution of viral DNA-dependent DNA polymerases; Knopf CW; DNA viruses as their host cells require a DNA-dependent DNA polymerase (Pol) to faithfully replicate their genomic information . Large eukaryotic DNA viruses as well as bacterial viruses encode a specific Pol equipped with a proofreading 3'-5'-exonuclease, and other replication proteins . All known viral Pol belong to family A and family B Pol . Common to all viral Pol is the conservation of the 3'-5'-exonuclease domain manifested by the three sequence motifs Exo I, Exo II, and Exo III . The polymerase domain of family A and B Pol is clearly distinguishable . Family A Pol share 9 distinct consensus sequences, only two of them are convincingly homologous to sequence motif B of family B Pol . The putative sequence motifs A, B, and C of the polymerase domain are located near the C-terminus in family A Pol and more central in family B Pol . Thus, family A Pol show a significant greater spacing between the Exo III motif and the Pol motif A that is especially extended in the case of the mitochondrial Pol gamma . From each host and virus family whenever possible the consensus sequences of two distantly related polymerase species were aligned for assessment of phylogenetic trees, using both maximum parsimony and distance methods, and evaluated by bootstrap analysis . Three alternative methods yielded trees with identical major groupings . A subdivision of viral family B Pol was achieved resulting in a branch with Pol carrying out a protein-primed mechanism of DNA replication, including adenoviruses, bacteriophages and linear plasmids of plant and fungal origin . Archaebacterial Pol and cellular Pol epsilon were consistently found at the base of this branch . Another major branch comprised alpha- and delta-like viral Pol from mammalian herpesviruses, fish lymphocystis disease virus, insect ascovirus, and chlorella virus . Due to a lower branch integrity Pol of T-even bacteriophages, poxviruses, African swine fever virus, fish herpesvirus, and baculoviruses were not clearly resolved and placed in alternate groupings . A composite and rooted tree of family A and B Pol shows that viral Pol with a protein-priming requirement represent the oldest viral Pol species suggesting that the protein-primed mechanism is one of the earliest modes of viral DNA replication.

Genetics, 1998 Apr, 148(4), 1655 - 65
The spectrum of acridine resistant mutants of bacteriophage T4 reveals cryptic effects of the tsL141 DNA polymerase allele on spontaneous mutagenesis; Wang FJ et al.; Mutations in the ac gene of bacteriophage T4 confer resistance to acridine-inhibition of phage development . Previous studies had localized the ac gene region; we show that inactivation of T4 Open Reading Frame 52.2 confers the Acr phenotype . Thus, 52.2 is ac . The resistance mechanism is unknown . The ac gene provides a convenient forward mutagenesis assay . Its compact size (156 bp) simplifies mutant sequencing and diverse mutant types are found: base substitutions leading to missense or nonsense codons, in-frame deletions or duplications within the coding sequence, deletion or duplication frameshifts, insertions, complex mutations, and large deletions extending into neighboring sequences . Comparisons of spontaneous mutagenesis between phages bearing the wild-type or tsL141 alleles of DNA polymerase demonstrate that the impact of the mutant polymerase is cryptic when total spontaneous mutant frequencies are compared, but the DNA sequences of the ac mutants reveal a substantial alteration of fidelity by the mutant polymerase . The patterns of base substitution mutagenesis suggest that some site-specific mutation rate effects may reflect hotspots for mutagenesis arising by different mechanisms . A new class of spontaneous duplication mutations, having sequences inconsistent with misaligned pairing models, but consistent with nick-processing errors, has been identified at a hotspot in ac.

Genetics, 1998 Apr, 148(4), 1611 - 8
Aspects of the ultraviolet photobiology of some T-even bacteriophages; Smith LA et al.; Bacteriophage T4 DNA metabolism is largely insulated from that of its host, although some host functions assist in the repair of T4 DNA damage . Environmental factors sometimes affect survival and mutagenesis after ultraviolet (UV) irradiation of T4, and can affect mutagenesis in many organisms . We therefore tested the effect of certain environmental factors and host genetic defects upon spontaneous and UV-induced mutagenesis and survival in T4 and some related T-even phages . Plating at pH 9 enhances UV resistance in T4 by about 14% compared to pH 7 . The host cAMP regulatory system affects host survival after UV irradiation but does not affect T4 survival . Thermal rescue, the increasing survival of irradiated T4 with increasing plating temperature, occurs also in phage T6, but only weakly in phages T2 and RB69; this temperature effect is not altered by supplementing infected cells with additional Holliday resolvase (gp49) early in infection . Phage RB69 turns out to have almost 50% greater UV resistance than T4, but has a genome of about the same size; RB69 is UV-mutable but does not produce r mutants, which are easily seen in T2, T4, and T6 . Spontaneous mutagenesis in T4 shows no dependence on medium and little dependence on temperature overall, but mutation rates can increase and probably decrease with temperature at specific sites . UV mutagenesis is not affected by incubating irradiated particles under various conditions before plating, in contrast to phage S13.

Genetics, 1998 Apr, 148(4), 1539 - 50
The roles of the bacteriophage T4 r genes in lysis inhibition and fine-structure genetics: a new perspective; Paddison P et al.; Seldom has the study of a set of genes contributed more to our understanding of molecular genetics than has the characterization of the rapid-lysis genes of bacteriophage T4 . For example, T4 rII mutants were used to define gene structure and mutagen effects at the molecular level and to help unravel the genetic code . The large-plaque morphology of these mutants reflects a block in expressing lysis inhibition (LIN), the ability to delay lysis for several hours in response to sensing external related phages attacking the cell, which is a unique and highly adaptive attribute of the T4 family of phages . However, surprisingly little is known about the mechanism of LIN, or how the various r genes affect its expression . Here, we review the extensive old literature about the r genes and the lysis process and try to sort out the major players affecting lysis inhibition . We confirm that superinfection can induce lysis inhibition even while infected cells are lysing, suggesting that the signal response is virtually instantaneous and thus probably the result of post-translational regulation . We identify the rI gene as ORF tk.-2, based on sequence analysis of canonical rI mutants . The rI gene encodes a peptide of 97 amino acids (Mr = 11.1 kD; pI = 4.8) that probably is secreted into the periplasmic space . This gene is widely conserved among T-even phage . We then present a model for LIN, postulating that rI is largely responsible for regulating the gpt holin protein in response to superinfection . The evidence suggests that the rIIA and B genes are not directly involved in lysis inhibition; rather, when they are absent, an alternate pathway for lysis develops which depends on the presence of genes from any of several possible prophages and is not sensitive to lysis inhibition.

Genetics, 1998 Apr, 148(4), 1475 - 82
DNA polymerase fidelity: from genetics toward a biochemical understanding; Goodman MF et al.; This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and antimutator strains . Early genetic studies on T4 identified mutator and antimutator variants of DNA polymerase that, in turn, stimulated the development of model systems for the study of DNA polymerase fidelity in vitro . Later enzymatic studies using purified T4 mutator and antimutator polymerases were essential in elucidating mechanisms of base selection and exonuclease proofreading . In both cases, the base analogue 2-aminopurine (2AP) proved tremendously useful-first as a mutagen in vivo and then as a probe of DNA polymerase fidelity in vitro . Investigations into mechanisms of DNA polymerase fidelity inspired theoretical models that, in turn, called for kinetic and thermodynamic analyses . Thus, the field of DNA synthesis fidelity has grown from many directions: genetics, enzymology, kinetics, physical biochemistry, and thermodynamics, and today the interplay continues . The relative contributions of hydrogen bonding and base stacking to the accuracy of DNA synthesis are beginning to be deciphered . For the future, the main challenges lie in understanding the origins of mutational hot and cold spots.

Genetics, 1998 Apr, 148(4), 1461 - 73
A species barrier between bacteriophages T2 and T4: exclusion, join-copy and join-cut-copy recombination and mutagenesis in the dCTPase genes; Gary TP et al.; Bacteriophage T2 alleles are excluded in crosses between T2 and T4 because of genetic isolation between these two virus species . The severity of exclusion varies in different genes, with gene 56, encoding an essential dCT(D)Pase/dUT(D)Pase of these phages, being most strongly affected . To investigate reasons for such strong exclusion, we have (1) sequenced the T2 gene 56 and an adjacent region, (2) compared the sequence with the corresponding T4 DNA, (3) constructed chimeric phages in which T2 and T4 sequences of this region are recombined, and (4) tested complementation, recombination, and exclusion with gene 56 cloned in a plasmid and in the chimeric phages in Escherichia coli CR63, in which growth of wild-type T2 is not restricted by T4 . Our results argue against a role of the dCTPase protein in this exclusion and implicate instead DNA sequence differences as major contributors to the apparent species barrier . This sequence divergence exhibits a remarkable pattern: a major heterologous sequence counter-clockwise from gene 56 (and downstream of the gene 56 transcripts) replaces in T2 DNA the T4 gene 69 . Gene 56 base sequences bordering this substituted region are significantly different, whereas sequences of the dam genes, adjacent in the clockwise direction, are similar in T2 and in T4 . The gene 56 sequence differences can best be explained by multiple compensating frameshifts and base substitutions, which result in T2 and T4 dCTPases whose amino acid sequences and functions remain similar . Based on these findings we propose a model for the evolution of multiple sequence differences concomitant with the substitution of an adjacent gene by foreign DNA: invasion by the single-stranded segments of foreign DNA, nucleated from a short DNA sequence that was complementary by chance, has triggered recombination-dependent replication by "join-copy" and "join-cut-copy" pathways that are known to operate in the T-even phages and are implicated in other organisms as well . This invasion, accompanied by heteroduplex formation between partially similar sequences, and perhaps subsequent partial heteroduplex repair, simultaneously substituted T4 gene 69 for foreign sequences and scrambled the sequence of the dCTPase gene 56 . We suggest that similar mechanisms can mobilize DNA segments for horizontal transfer without necessarily requiring transposase or site-specific recombination functions.

Clin Chem, 1998 Apr, 44(4), 731 - 9
Automated fluorescent analysis procedure for enzymatic mutation detection; Del Tito BJ Jr et al.; The Enzymatic Mutation Detection (EMD) assay detects mutations or polymorphisms in DNA . The assay procedure takes <1 h and is followed by electrophoretic detection . We report an automated procedure, using fluorescently labeled probe and quantitative analysis on the ABI Prism 377 DNA Sequencer, that improves on earlier methods (1, 2) by eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio . The EMD assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves the heteroduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis . Unlike existing mutation techniques, the EMD method uses a single protocol to identify point mutations, deletions, and insertions for all DNA fragments . Test DNA samples are assayed directly from PCR reactions, and fragments up to 4 kb in size have been assayed successfully . A independent analysis on the p53 tumor suppressor gene from clinical samples has shown 100% sensitivity and 94% specificity . Because the fluorescent EMD assay has been optimized for high signal-to-noise ratios, mutations can be identified in mixed samples containing up to a 20-fold excess of normal DNA.

Biochemistry (Mosc), 1998 Apr, 63(4), 399 - 406
Chaperones in bacteriophage T4 assembly; Marusich EI et al.; Protein folding in the cell is controlled at the levels of translation and post-translational modification, depends on a number of conserved proteins known as chaperones, and is catalyzed by specific enzymes, such as protein disulfide isomerase and peptidyl prolyl cis-trans isomerase . The chaperones stabilize folding intermediates and participate in assembly and disaggregation of supramolecular structures . Bacteriophage T4 is an especially convenient system for studying of protein folding mechanisms, since its genome encodes several virus-specific chaperones . In this review, the chaperones of phage T4 that take part in capsid formation (gp31 and gp40) and in folding and assembly of virion tail fibers (gp38, gp57A) have been considered . Protein encoded by gene 31 completely substitutes co-chaperonin GroES of the host cell in folding of the major capsid protein, gp23, aided by chaperonin GroEL . The product of gene 40, which is homologous to analogs of eukaryotic GroEL and peptidyl prolyl cis-trans isomerase, participates in assembly of gp20 while the formation of procapsid connector . The chaperone encoded by gene 57A is essential for folding and oligomerization of both long and short phage tail fibers . gp38, together with gp57A, participates in the formation of the distal part of the long fibers . This protein seems to represent a principally new group of chaperones that change steric structure of folded polypeptide . One phage chaperone, fibritin, encoded by gene wac (whiskers antigen control) and taking part in assembly the subunits of the long tail fibers is a constituent of the virion . Fibritin is a convenient model for studying mechanisms of folding and oligomerization of fibrous proteins due to its labile triple-stranded alpha-helical coiled-coil structure.

J Bacteriol, 1998 Apr, 180(8), 2248 - 52
Specific binding of Escherichia coli ribosomal protein S1 to boxA transcriptional antiterminator RNA; Mogridge J et al.; We show that ribosomal protein S1 specifically binds the boxA transcriptional antiterminator RNAs of bacteriophage lambda and the Escherichia coli ribosomal RNA operons . Although S1 competes with the NusB-S10 antitermination complex for binding to boxA, it does not affect antitermination by the lambda N protein in vitro, and its role, if any, in rRNA synthesis is still unknown.

J Bacteriol, 1998 Apr, 180(8), 2063 - 71
Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli; Murphy KC; Replacement of Escherichia coli's RecBCD function with phage lambda's Red function generates a strain whose chromosome recombines with short linear DNA fragments at a greatly elevated rate . The rate is at least 70-fold higher than that exhibited by a recBC sbcBC or recD strain . The value of the system is highlighted by gene replacement with a PCR-generated DNA fragment . The deltarecBCD::Plac-red kan replacement allele can be P1 transduced to other E . coli strains, making the hyper-Rec phenotype easily transferable.

J Bacteriol, 1998 Apr, 180(8), 2005 - 13
Divergence of a DNA replication gene cluster in the T4-related bacteriophage RB69; Yeh LS et al.; The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo . We describe here the biological implications of divergence in a genomic segment that encodes four essential DNA replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein) . We have cloned, sequenced, and expressed several overlapping segments of the RB69 gene 46-45.2-(rpbA)-45-44-62 cluster and compared its features to those of the homologous gene cluster from T4 . The deduced primary structures of all four RB69 replication proteins and gp45.2 from this cluster are very similar (80 to 95% similarity) to those of their respective T4 homologs . In contrast, the rpbA region (which encodes a nonessential protein in T4) is highly diverged (approximately 49% similarity) between the two phage genomes and does not encode protein in RB69 . Expression studies and patterns of high divergence of intercistronic nucleotide sequences of this cluster suggest that T4 and RB69 evolved similar transcriptional and translational control strategies for the cistrons contained therein, but with different specificities . In plasmid-phage complementation assays, we show that posttranslationally, RB69 and T4 homologs of gp45 and the gp44/62 complex can be effectively exchanged between the two phage replicase assemblies; however, we also show results which suggest that mixed clamp loader complexes consisting of T4 gp62 and RB69 gp44 subunits are not active for phage DNA replication . Thus, specificity of the gp44-gp62 interaction in the clamp loader marks a point of departure between the T4 and RB69 replication systems.

Mutat Res, 1998 Feb 2, 397(2), 161 - 8
DNA breakage by L-DOPA and Cu(II): breakage by melanin and bacteriophage inactivation; Husain S et al.; We have previously shown that L-DOPA in the presence of Cu(II) caused DNA cleavage through the generation of reactive oxygen species such as the hydroxyl radical . Since L-DOPA is the precursor for the synthesis of melanin, we have studied the action of melanin on DNA in a similar reaction . In this paper, we show that melanin in the presence of Cu(II) also causes DNA strand breakage . However, the rate of such strand breakage is considerably less than l-DOPA . Melanin and L-DOPA are both capable of generating superoxide anion . Furthermore, the action of L-DOPA and Cu(II) on bacteriophage lambda reduces its viability . The results are discussed in relation to the putative role of L-DOPA-Cu(II) system as a source of endogenous generation of reactive oxygen species.

Eur J Biochem, 1998 Mar 15, 252(3), 408 - 15
Oligomeric structure of the repressor of the bacteriophage Mu early operon; Alazard R et al.; The regulation of the lytic and lysogenic development in the life cycle of bacteriophage Mu is regulated in part by its repressor, c, which binds to three operator sites, O1, O2 and O3, overlapping two divergent promoters . The oligomeric structure of this repressor protein was investigated by hydrodynamic and biochemical methods . Size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering, crosslinking and direct electron microscopy observations suggest that c exists primarily as a hexamer with a molecular mass of 120-140 kDa at low concentrations, i.e . in the 10-microM range . This molecule undergoes a self-assembly process leading to dodecamers and higher order species as the concentration is further increased in a manner depending on the nature of the solvent . Our results also suggest that these species have an elongated structure, and a possible arrangement of the subunits within the hexamer is proposed . The implication of this unusual quaternary structure for a repressor in its interaction with the operator sites O1 and O2 remains to be elucidated.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2828 - 33
A genetic screen for the isolation and characterization of site-specific proteases; Sices HJ et al.; Site-specific proteolysis is an important regulatory mechanism in basic cellular and viral processes . Using the protease of the HIV as a model, a genetic system has been developed for the isolation and characterization of site-specific proteases . The system utilizes the well defined bacteriophage lambda regulatory circuit where the viral repressor, cI, is specifically cleaved to initiate the lysogenic-to-lytic switch . The model system is rapid, highly specific, and demonstrates the ability to isolate and characterize enzymes of limited expression or activity . In addition, the system has a significant potential for the selection of clinically relevant mutant enzymes and in the development of anti-protease therapeutics.

EMBO J, 1998 Mar 2, 17(5), 1542 - 52
Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site; Kusakabe T et al.; The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities . The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis . In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound . Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA . When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound . Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand . The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.

EMBO J, 1998 Mar 2, 17(5), 1467 - 75
Core-sigma interaction: probing the interaction of the bacteriophage T4 gene 55 promoter recognition protein with E.coli RNA polymerase core; Leonetti JP et al.; The bacterial RNA polymerase sigma subunits are key participants in the early steps of RNA synthesis, conferring specificity of promoter recognition, facilitating promoter opening and promoter clearance, and responding to diverse transcriptional regulators . The T4 gene 55 protein (gp55), the sigma protein of the bacteriophage T4 late genes, is one of the smallest and most divergent members of this family . Protein footprinting was used to identify segments of gp55 that become buried upon binding to RNA polymerase core, and are therefore likely to constitute its interface with the core enzyme . Site-directed mutagenesis in two parts of this contact surface generated gene 55 proteins that are defective in polymerase-binding to different degrees . Alignment with the sequences of the sigma proteins and with a recently determined structure of a large segment of sigma70 suggests that the gp55 counterpart of sigma70 regions 2.1 and 2.2 is involved in RNA polymerase core binding, and that sigma70 and gp55 may be structurally similar in this region . The diverse phenotypes of the mutants implicate this region of gp55 in multiple aspects of sigma function.

J Bacteriol, 1998 Apr, 180(7), 1822 - 30
Efficiency of T4 gene 60 translational bypassing; Maldonado R et al.; Ribosomes translating bacteriophage T4 gene 60 mRNA bypass 50 noncoding nucleotides from a takeoff site at codon 46 to a landing site just upstream of codon 47 . A key signal for efficient bypassing is contained within the nascent peptide synthesized prior to takeoff . Here we show that this signal is insensitive to the addition of coding information at its N terminus . In addition, analysis of amino-terminal fusions, which allow detection of all major products synthesized from the gene 60 mRNA, show that 50% of ribosomes bypass the coding gap while the rest either terminate at a UAG stop codon immediately following codon 46 or fail to resume coding . Bypassing efficiency estimates significantly lower than 50% were obtained with enzymatic reporter systems that relied on comparing test constructs to constructs with a precise excision of the gap (gap deletion) . Further analysis showed that these estimates are distorted by differences between test and gap deletion functional mRNA levels . An internal translation initiation site at Met12 of gene 60 (which eliminates part of the essential nascent peptide) also distorts these estimates . Together, these results support an efficiency estimate of approximately 50%, less than previously reported . This estimate suggests that bypassing efficiency is determined by the competition between reading signals and release factors and gives new insight into the kinetics of bypassing signal action.

J Bacteriol, 1998 Apr, 180(7), 1723 - 8
The TolQRA proteins are required for membrane insertion of the major capsid protein of the filamentous phage f1 during infection; Click EM et al.; Infection of Escherichia coli by the filamentous bacteriophage f1 is initiated by interaction of the end of the phage particle containing the gene III protein with the tip of the F conjugative pilus . This is followed by the translocation of the phage DNA into the cytoplasm and the insertion of the major phage capsid protein, pVIII, into the cytoplasmic membrane . DNA transfer requires the chromosomally encoded TolA, TolQ, and TolR cytoplasmic membrane proteins . By using radiolabeled phages, it can be shown that no pVIII is inserted into the cytoplasmic membrane when the bacteria contain null mutations in tolQ, -R and -A . The rate of infection can be varied by using bacteria expressing various mutant TolA proteins . Analysis of the infection process in these strains demonstrates a direct correlation between the rate of infection and the incorporation of infecting bacteriophage pVIII into the cytoplasmic membrane.

J Biol Chem, 1998 Mar 13, 273(11), 6556 - 64
Role of the acidic carboxyl-terminal domain of the single-stranded DNA-binding protein of bacteriophage T7 in specific protein-protein interactions; Kong D et al.; The gene 2.5 single-stranded DNA (ssDNA) binding protein of bacteriophage T7 is essential for T7 DNA replication and recombination . Earlier studies have shown that the COOH-terminal 21 amino acids of the gene 2.5 protein are essential for specific protein-protein interaction with T7 DNA polymerase and T7 DNA helicase/primase . A truncated gene 2.5 protein, in which the acidic COOH-terminal 21 amino acid residues are deleted no longer supports T7 growth, forms dimers, or interacts with either T7 DNA polymerase or T7 helicase/primase in vitro . The single-stranded DNA-binding protein encoded by Escherichia coli (SSB protein) and phage T4 (gene 32 protein) also have acidic COOH-terminal domains, but neither protein can substitute for T7 gene 2.5 protein in vivo . To determine if the specificity for the protein-protein interaction involving gene 2.5 protein resides in its COOH terminus, we replaced the COOH-terminal region of the gene 2.5 protein with the COOH-terminal region from either E . coli SSB protein or T4 gene 32 protein . Both of the two chimeric proteins can substitute for T7 gene 2.5 protein to support the growth of phage T7 . The two chimeric proteins, like gene 2.5 protein, form dimers and interact with T7 DNA polymerase and helicase/primase to stimulate their activities . In contrast, chimeric proteins in which the COOH terminus of T7 gene 2.5 protein replaced the COOH terminus of E . coli SSB protein or T4 gene 32 protein cannot support the growth of phage T7 . We conclude that an acidic COOH terminus of the gene 2.5 protein is essential for protein-protein interaction, but it alone cannot account for the specificity of the interaction.

Genetika, 1998 Jan, 34(1), 38 - 44
{Cloning of large imperfect palindromes in circular and linear vectors}; Ravin NV et al.; Data on comparative analysis of cloning of large imperfect artificial palindromes and one natural palindrome in the circular pUC19 and linear pN15L vectors, constructed on the basis of temperate N15 bacteriophage minireplicon are presented . The artificial palindromes consisted of the two head-to-head oriented 5:5-kb lambda bacteriophage DNA fragments interrupted by a short sequence of varied size . Natural palindrome was represented by the 12.5-kb BamHI fragment of Tetrahymena pyriformis ribosomal genes cluster . Integration of some artificial palindromes and a natural palindrome into a circular vector resulted in a considerable decrease of its copy number . This was assumed to result from cruciform formation mediated by supercoiling of a circular vector DNA . Thus, a linear is vector preferable in cloning of inverted repeated DNA sequences.

Wien Klin Wochenschr, 1998 Jan 30, 110(2), 40 - 4
Uptake of foreign DNA from the environment: the gastrointestinal tract and the placenta as portals of entry; Doerfler W et al.; Foreign DNA (deoxyribonucleic acid) is part of our environment . Considerable amounts of foreign DNA of very different origin are ingested daily with food . In a series of experiments we fed the DNA of bacteriophage M13 as test DNA to mice and showed that fragments of this DNA survive the passage through the gastrointestinal (GI) tract in small amounts (1-2%) . Food-ingested M13 DNA reaches peripheral white blood cells, the spleen and liver via the intestinal epithelia and cells in the Peyer's patches of the intestinal wall . There is evidence to assume that food-ingested foreign DNA can become covalently linked to mouse-like DNA . When M13 DNA is fed to pregnant mice the test DNA can be detected in cells in various organs of the fetuses and of newborn animals, but never in all cells of the mouse fetus . It is likely that the M13 DNA is transferred by the transplacental route and not via the germ line . The consequences of foreign DNA uptake for mutagenesis and oncogenesis have not yet been investigated.

J Pept Res, 1998 Mar, 51(3), 216 - 25
A peptide inhibitor of cholesteryl ester transfer protein identified by screening a bacteriophage display library; Bonin PD et al.; We screened a bacteriophage display library of random decapeptides to identify peptide inhibitors of cholesteryl ester transfer protein (CETP) . After affinity selection against CETP, bacteriophage-infected Escherichia coli were plated at clonal density and 36 random clones were isolated . Analysis of the relevant portion of the bacteriophage DNA from a group of 12 clones that had a relatively high affinity for CETP revealed that the corresponding amino acid sequences of the displayed peptides exhibited an .. . Xaa-Arg-Met-Arg-Tyr-Xaa .. . composite motif . Based on those results, decapeptides from this group were synthesized and one of them, DP1 (NH2-VTWRMWYVPA-COOH), inhibited CETP-catalyzed transfer of cholesteryl esters and triglycerides . Amino- and carboxy-terminal truncations of DP1 demonstrated that the original decapeptide could be reduced to a pentapeptide without loss of either its ability to bind to CETP or its ability to inhibit CETP-mediated lipid transfer . That pentapeptide, NH2-WRMWY-COOH (WRMWY, PNU-107368E), binds directly to CETP and its inhibition is consistent with that of a competitive inhibitor of CETP with a Ki of 164 microM . WRMWY or modified versions of this peptide may be useful in studying the interactions between CETP and plasma lipoproteins.

Mamm Genome, 1998 Apr, 9(4), 327 - 30
Structure of the human biotinidase gene; Knight HC et al.; Biotinidase cleaves biotin from biocytin, thereby recycling the vitamin . We have determined the structure of the human biotinidase gene . A genomic clone, containing three exons that code for the mature enzyme, was obtained by screening a human genomic bacteriophage library with the biotinidase cDNA by plaque hybridization . To obtain a clone containing the most 5' exon of the biotinidase cDNA, a human PAC library by PCR was screened . The human biotinidase gene is organized into four exons and spans at least 23 kb . The 5'-flanking region of exon 1 contains a CCAAT element, three initiator sequences, an octamer sequence, three methylation consensus sites, two GC boxes, and one HNF-5 site, but has no TATA element . The region from nt -600 to +400 has features of a CpG island and resembles a housekeeping gene promoter . The structure and sequence of this gene are useful for identifying and characterizing mutations that cause biotinidase deficiency.

Mol Gen Genet, 1998 Feb, 257(4), 490 - 5
Guanosine tetraphosphate (ppGpp)-mediated inhibition of the activity of the bacteriophage lambda pR promoter in Escherichia coli; Wrobel B et al.; It was previously demonstrated that the activity of bacteriophage lambda promoter pR is decreased in wild-type Escherichia coli cells starved for amino acids (during the stringent response) . Since pR activity is necessary for the transcriptional activation of ori lambda, this leads to inhibition of the replication of plasmids derived from phage lambda . These results led to the proposal that the pR promoter susceptible to control by the stringent response . However, subsequent studies demonstrated that this promoter is activated by the host dnaA gene product and since the dnaA promoter was reported to be controlled by the stringent response, it is possible that the inhibition of pR activity in amino acid-starved cells is indirect, and results from the impairment of DnaA-mediated transcriptional activation . Here we present evidence that pR is negatively regulated by ppGpp, even when DnaA protein is provided in excess as well as in cells devoid of DnaA function . We have checked that the level of ppGpp is increased during prolonged (up to 4 h) starvation for isoleucine in relA+ cells but not in the relA- mutant . At the same time we observed inhibition of lambda plasmid replication during the stringent, but not relaxed, response, even when DnaA was overproduced . Finally, we found that the activity of a pR-lacZ fusion is inhibited after gratuitously induced overproduction of ppGpp in unstarved cells, irrespective of the status of the dnaA gene product . We conclude that the activity of the pR promoter is inhibited directly by ppGpp.

Virology, 1998 Mar 30, 243(1), 113 - 8
Crystallization and preliminary X-ray analysis of the dsDNA bacteriophage HK97 mature empty capsid; Wikoff WR et al.; HK97 is a temperate dsDNA bacteriophage of Escherichia coli that is structurally similar to phage lambda, with an icosahedral head of triangulation (7) number 7 . Although the capsids of several large dsDNA phages have been studied extensively using a variety of biophysical approaches, no high-resolution structure is available . We have grown crystals of mature but empty bacteriophage HK97 capsids that diffract to at least 3.5 A using synchrotron radiation . The HK97 Head II crystals are the first capsid crystals from a dsDNA bacteriophage that diffract X-rays to high resolution . A capsid precursor (prohead) was made in vivo by expressing capsid proteins in E . coli . This prohead was purified, converted to Head II in vitro, and used to grow crystals . The empty Head II has the same form as the mature HK97 capsid, but without DNA . The crystals were grown in a mixture of ammonium sulfate and PEG 8000, directly in an X-ray capillary to minimize crystal handling . The unit cell is monoclinic, with dimensions a = 580 A, b = 625 A, c = 790 A, beta = 90.0 degrees and two particles per unit cell.

Virology, 1998 Mar 30, 243(1), 32 - 44
Genetic analysis of the UL 15 gene locus for the putative terminase of herpes simplex virus type 1; Yu D et al.; The herpes simplex virus (HSV-1) UL15 gene encodes one of the six viral gene products required for viral DNA cleavage and packaging . UL15 is a spliced gene and encodes two separately translated proteins, UL15 and UL15.5 . Sequence analysis reveals that UL15 shares homology with gp 17, the large catalytic subunit of the bacteriophage T4 terminase, a protein which cleaves the polymeric T4 DNA into monomers . Both proteins contain a putative ATP binding motif known as the Walker A and B boxes . In this report, immunofluorescence was used to show that UL15 localizes to the nucleus in the absence of any other viral proteins; this indicates that UL15 contains its own nuclear localization signal . In addition, we found that UL15 colocalizes with replication compartments at early times (6 h postinfection) . Since, at this time, preformed capsids as well as other cleavage and packaging proteins are also recruited to replication compartments, it seems likely that cleavage and packaging occurs in the same compartments in which DNA synthesis occurs . Also in this report, we have investigated UL15.5, the N-terminally truncated gene product of the UL15 open reading frame (ORF) . The start codon has been mapped to Met443 within the UL15 ORF . Furthermore, we have shown that plasmids containing a UL15.5 knockout mutation still complement the growth of UL15 insertion mutant viruses, indicating that UL15.5 is not required for viral growth in cell culture . Last, we constructed a UL15 mutant, UL15C(G263A), in which the invariant Gly263 in the Walker box A of the ATP binding motif (GKT) was substituted with an alanine . We show that the mutant gene fails to support the growth of UL15 insertion mutant viruses, indicating that the putative ATP binding motif of UL15 is indispensable for its function.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1337 - 44
Dissecting the key recognition features of the MS2 bacteriophage translational repression complex; Lago H et al.; The MS2 RNA operator capsid offers an unparalleled opportunity to study sequence-specific protein-protein and RNA-protein interactions in molecular detail . RNA molecules encompassing the minimal translational operator recognition elements can be soaked into crystals of RNA-free coat protein shells, allowing the RNA to access the interior of the capsids and make contact with the operator binding sites . Correct interpretation of these structural studies depends critically on functional analysis in solution to confirm that the interactions seen in the crystal are not an artefact of the unusual approach used to generate the RNA-protein complexes . Here we present a series of in vivo and in vitro functional assays, using coat proteins carrying single amino acid substitutions at residues which either interact with the operator RNA or are involved in stabilizing the conformation of the FG loop, the site of the major quasi-equivalent conformational change . Variant operator RNAs have been assayed for coat protein affinity in vitro . The results reveal the robustness of the operator-coat protein interaction and the requirement for both halves of a protein dimer to contact RNA in order to achieve tight binding . They also suggest that there may be a direct link between the conformation of the FG loop and RNA binding.

Photochem Photobiol, 1998 Mar, 67(3), 350 - 7
Potential mechanisms of photodynamic inactivation of virus by methylene blue . I . RNA-protein crosslinks and other oxidative lesions in Q beta bacteriophage; Schneider JE Jr et al.; A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes . When suspensions of the intact bacteriophage Q beta were exposed to methylene blue plus light (MB + L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8-dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks . Formation of carbonyl groups associated with protein in the Q beta phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal . Strand breaks in the Q beta genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Q beta RNA was observable well beyond the 99% inactivation point in MB dosage . It was shown that the number of 8-oxoGua lesions were unlikely to be sufficient to account for the number of lethal events . Following exposure to MB + L, crosslink formation between Q beta RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions . A significant increase over background of RNA-protein complexes (including full-length Q beta RNA) was observed at the lowest concentration of MB tested (0.5 microM), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival) . Due to its close correlation with Q beta inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Q beta following MB + L exposure.

Photochem Photobiol, 1998 Mar, 67(3), 343 - 9
Factors affecting virus photoinactivation by a series of phenothiazine dyes; Wagner SJ et al.; A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions . One of the dyes, 1,9-dimethyl-3-dimethylamino-7-dimethylaminophenothiazine (1,9-dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties . In order to understand why the virucidal specificity of 1,9-dimethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9-dimethyl-3-diethylamino-7-dibutylaminophenothiazine {compound 4-140}, 1,9-dimethyl-3-dimethylamino-7-diethylaminophenothiazine {compound 6-136}) . All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (> 0.5), but 1,9-dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB . In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2-octanol : water) ranging from 0.11 for MB to 3560 for compound 4-140 . The dyes had the following affinities for DNA: 1,9-dimethylmethylene blue > compound 6-136 > MB approximately compound 4-140 . This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds . Results with the most hydrophobic compound, 4-140, contrasted with those obtained with 1,9-dimethylmethylene blue . Compound 4-140 had a high affinity for protein and a low affinity for DNA . Although compound 4-140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer . However, unlike results with 1,9-dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4-140 was completely inhibited by the presence of red cells and plasma . Thus, the high affinity of 1,9-dimethylmethylene blue for DNA and the dye's efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizer's ability to inactivate viruses without adversely affecting anucleate red cells.

Biochemistry, 1998 Mar 17, 37(11), 3831 - 8
Mg2+ mediated sequence-specific binding of transcriptional activator protein C of bacteriophage Mu to DNA; De A et al.; The contributions from the secondary structure of the transcriptional activator protein C of bacteriophage Mu to its specific DNA binding and the influence of various factors, viz., electrolytes, and minor groove and major groove binders on this protein-DNA interaction have been addressed . Circular dichroism (CD) spectral results suggest that, in the absence of Mg2+, C protein exhibits a beta-pleated sheetlike structure and Mg2+ changes the conformation to a more alpha-helical structure which could provide specific geometrical constraints complementary to those of DNA-helix . Thus, Mg2+ acts as a cofactor for the binding of the C protein to its specific site in DNA by inducing conformational changes in the protein . Competitive binding studies with minor and major groove binding drugs, viz., distamycin A and methyl green, respectively, and the DMS footprinting data indicate that the C protein recognizes the major groove of DNA during complex formation . Further, upon major groove binding, C protein brings about changes in DNA conformation; such conformational changes could have implications in the transcription process.

Biochemistry, 1998 Mar 10, 37(10), 3567 - 74
Pre-steady-state kinetics of nucleotide insertion following 8-oxo-7,8-dihydroguanine base pair mismatches by bacteriophage T7 DNA polymerase exo-; Furge LL et al.; 8-Oxo-7,8-dihydroguanine (8-oxoGua) can base pair with either cytosine (C) or adenine (A) when replicated by DNA polymerases . The 8-oxoGua.A mismatch is extended in preference to the 8-oxoGua.C pair . Using a model 25-mer/36-mer DNA duplex containing either guanine (Gua).C, 8-oxoGua.C, or 8-oxoGua.A base pairs at the primer terminus and A at the standing start position, we found that the pre-steady-state addition of dTTP opposite A following all three base pairs by bacteriophage T7 DNA polymerase exo- showed burst kinetics, suggesting that extension of all three base pairs is controlled by the rate of a step at or before phosphodiester bond formation . Substitution of dTTP alpha S for dTTP yielded modest thio effects of 1-6, suggesting that extension of all three pairs is limited by the rate of the conformational change prior to phosphodiester bond formation . Pre-steady-state values for kpol (maximum polymerization rate) were 120, 12, and 28 s-1, and Kd values were 2, 75, and 22 microM for insertion of dTTP following Gua.C, 8-oxoGua.C, and 8-oxoGua.A base pairs, respectively . Additional analysis of extension was provided by substitution of A in the standing start position by 2-aminopurine (2-AP), a fluorescent base analogue . Comparison of rapid-quench gel-based assays with stopped-flow fluorescence quenching assays suggested that during addition of dTTP opposite 2-AP phosphodiester bond formation was rate-limiting when 8-oxoGua.C or 8-oxoGua.A were the preceding base pairs, while conformational change was rate-limiting when Gua.C was the preceding base pair . Furthermore, the difference in apparent conformational change rates for addition of dTTP opposite 2-AP following the 8-oxoGua base pairs was greater than the differences in their phosphodiester bond formation rates, suggesting that discrimination in extension may be influenced more by conformational change rates than the rates of phosphodiester bond formation in this mispaired system.

Eur J Immunol, 1998 Feb, 28(2), 589 - 98
Diminished expression of CD40 ligand may contribute to the defective humoral immunity in patients with MHC class II deficiency; Nonoyama S et al.; Major histocompatibility complex (MHC) class II deficiency (bare lymphocyte syndrome, BLS) is a rare primary immunodeficiency classified as a subgroup of severe combined immunodeficiency . We studied T and B lymphocyte function by examining the CD40 ligand/CD40 system in three BLS patients from two unrelated families . CD40 ligand expression by maximally activated BLS T cells was diminished . This abnormality may represent immunological naivete rather than a general T cell defect, since expression of activation marker CD69 and proliferative responses to PHA or anti-CD3 were normal, and BLS T cells primed and restimulated in vitro expressed normal amounts of CD40 ligand . BLS B cells proliferated and produced IgE if stimulated with anti-CD40 or soluble CD40 ligand and IL-4 . Activation of BLS B cells with soluble CD40 ligand and IL-4 induced normal expression of activation markers, although MHC class II expression remained absent . Depressed antibody titers, lack of amplification and failure to undergo isotype switching in response to immunization with bacteriophage phi x 174 demonstrated defective T cell help . We conclude that BLS B cells are functionally normal if appropriately stimulated, and that the defective humoral immunity observed may be related to diminished expression of CD40 ligand on BLS T cells.

J Gen Virol, 1998 Mar, 79 ( Pt 3), 629 - 37
The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene; Durantel D et al.; Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase . This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication . It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells . The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon . The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ . This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells . Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins . Shut-down of host protein synthesis was not abolished in recombinant infection . When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution . This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

Structure, 1998 Feb 15, 6(2), 121 - 5
Processivity of DNA polymerases: two mechanisms, one goal; Kelman Z et al.; Replicative DNA polymerases are highly processive enzymes that polymerize thousands of nucleotides without dissociating from the DNA template . The recently determined structure of the Escherichia coli bacteriophage T7 DNA polymerase suggests a unique mechanism that underlies processivity, and this mechanism may generalize to other replicative polymerases.

Curr Opin Struct Biol, 1998 Feb, 8(1), 54 - 63
Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes; Brautigam CA et al.; New levels in the understanding of DNA replication have been achieved from recent crystal structure determinations of several DNA polymerases and their substrate complexes . The structure of an alpha family DNA polymerase from bacteriophage RB69 shows some similarities, but also considerable differences in structure and organization from the pol I family DNA polymerases . Also, the functions of three polymerase domains and their conserved residues have been clarified by studying structures of pol I family DNA polymerases complexed to their substrates . These structures also confirm that an identical two-metal ion catalytic mechanism proposed previously is used by both the nonhomologous pol I and pol beta family DNA polymerases.

J Biol Chem, 1998 Feb 27, 273(9), 5260 - 70
Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins; Park K et al.; Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy . Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail . A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein . Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication . Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork . The loops are fully double-stranded with an average length of approximately 1 kilobase . Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand . A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork . This study provides the first direct demonstration of such loops.

J Biol Chem, 1998 Feb 13, 273(7), 4149 - 54
A novel soluble tissue factor variant with an altered factor VIIa binding interface; Lee GF et al.; Tissue factor (TF) residues Lys20 and Asp58 form part of a binding epitope previously shown by alanine scanning to be critical for high affinity interactions with factor VIIa (FVIIa) . To explore the possibility of enhancing the affinity of a TF-based antagonist for FVIIa, we created libraries in which residues at 20, 58, and adjacent positions were varied in constructs containing the soluble extracellular domain of TF (sTF) fused to the bacteriophage M13 tail coat protein . TF variants monovalently displayed on phage were then sorted on the basis of binding to FVIIa . Sorting of preliminary libraries, in which position 58 and/or 20 and surrounding residues were randomized, led to the selection of TF proteins of essentially wild-type sequence . Therefore, we devised a strategy wherein TF position 20 was held fixed as alanine and 5 specific residues near to, and including, position 58 were randomized to effectively obtain alternative sequences at this interface . The consensus sequence reached with this library included wild-type residues at positions 61, 62, 65, and 66 but exclusively tryptophan at position 58 . Analyses of the soluble K20A,D58W (A20W58) TF protein indicated that it binds FVIIa with an affinity comparable with wild-type sTF but is defective as a cofactor for FVIIa-dependent factor X activation . Further experiments designed to elucidate the mechanism of binding suggest that the new binding interactions involve more than the simple addition of hydrophobic surface area.

J Biol Chem, 1998 Feb 13, 273(7), 4143 - 8
Involvement of boxA nucleotides in the formation of a stable ribonucleoprotein complex containing the bacteriophage lambda N protein; Mogridge J et al.; The association of the transcriptional antitermination protein N of bacteriophage lambda with Escherichia coli RNA polymerase depends on nut site RNA (boxA + boxB) in the nascent transcript and the host protein, NusA . This ribonucleoprotein complex can transcribe through Rho-dependent and intrinsic termination sites located up to several hundred base pairs downstream of nut . For antitermination to occur farther downstream, this core antitermination complex must be stabilized by the host proteins NusB, NusG, and ribosomal protein S10 . Here, we show that the assembly of NusB, NusG, and S10 onto the core complex involves nucleotides 2-7 of lambda boxA (CGCUCUUACACA) and is a fully cooperative process that depends on the presence of all three proteins . This assembly of NusB, NusG, and S10 also requires the carboxyl-terminal region (amino acids 73-107) of N, which interacts directly with RNA polymerase . NusB and S10 assemble in the absence of NusG when lambda boxA is altered at nucleotides 8 and 9 to create a consensus version of boxA (CGCUCUUUAACA) . These experiments suggest that multiple protein-protein and protein-RNA interactions are required to convert a core antitermination complex into a complete complex.

J Bacteriol, 1998 Mar, 180(6), 1573 - 7
Lambda Xis degradation in vivo by Lon and FtsH; Leffers GG Jr et al.; Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R . A . Weisberg and M . E . Gottesman, p . 489-500, in A . D . Hershey, ed., The Bacteriophage Lambda, 1971) . We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB) . Xis was stabilized two- to threefold more than in the wild type in a lon mutant and as much as sixfold more in a lon ftsH double mutant at the nonpermissive temperature for the ftsH mutation . Integration of lambda into the bacterial chromosome was delayed in the lon ftsH background, suggesting that accumulation of Xis in vivo interferes with integration . Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage.

Mol Microbiol, 1998 Feb, 27(4), 819 - 29
The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters; Colland F et al.; The AsiA protein, encoded by bacteriophage T4, inhibits Esigma70-dependent transcription at bacterial and early-phage promoters . We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the -35 consensus promoter sequence by host RNA polymerase . In vitro experiments were performed with a C-terminally labelled sigma factor that is competent for functional holoenzyme reconstitution . By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of sigma70, which recognizes the -35 sequence . Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments . The stationary-phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a -35 region, is insensitive to AsiA inhibition . The recognition of a galP1 promoter by Esigma70 is not affected by the presence of AsiA . Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of sigma70 with the -35 hexamer.

J Mol Biol, 1998 Feb 13, 276(1), 151 - 64
Crystal structure of the lysozyme from bacteriophage lambda and its relationship with V and C-type lysozymes; Evrard C et al.; Like other lysozymes, the bacteriophage lambda lysozyme is involved in the digestion of bacterial walls . This enzyme is remarkable in that its mechanism of action is different from the classical lysozyme's mechanism . From the point of view of protein evolution, it shows features of lysozymes from different classes . The crystal structure of the enzyme in which all tryptophan residues have been replaced by aza-tryptophan has been solved by X-ray crystallography at 2.3 A using a combination of multiple isomorphous replacement, non-crystallographic symmetry averaging and density modification techniques . There are three molecules in the asymmetric unit . The characteristic structural elements of lysozymes are conserved: each molecule is organized in two domains connected by a helix and the essential catalytic residue (Glu19) is located in the depth of a cleft between the two domains . This cleft shows an open conformation in two of the independent molecules, while access to the cavity is much more restricted in the last one . A structural alignment with T4 lysozyme and hen egg white lysozyme allows us to superpose about 60 C alpha atoms with a rms distance close to 2 A . The best alignments concern the helix preceding the catalytic residue, some parts of the beta sheets and the helix joining the two domains . The results of sequence alignments with the V and C lysozymes, in which weak local similarities had been detected, are compared with the structural results.

J Mol Biol, 1998 Feb 13, 276(1), 7 - 17
The natural 6 S RNA found in Q beta-infected cells is derived from host and phage RNA; Avota E et al.; The RNA of Escherichia coli infected with RNA bacteriophage Q beta was isolated and screened for replicable short-chained RNA . In contrast to earlier assumptions we show that (i) short-chained replicable RNA is a very minor part of the RNA synthesized in the infection cycle, and (ii) that the replicable RNA isolated from infected cells is derived from cellular RNA, in particular 23 S rRNA and 10 Sa RNA, and from Q beta RNA itself . None of the many RNA species known from in vitro experiments was found . The RNA species isolated were all inefficient templates . No replicable RNA could be isolated from non-infected cells . Even in cells expressing high amounts of Q beta replicase very few RNA species could be isolated . RNA generated in vitro in template-free synthesis is therefore not derived from RNA species found in vivo, and replicable RNA found in vitro is generated by a mechanism fundamentally different from the one operating in vivo.

Protein Eng, 1997 Nov, 10(11), 1311 - 8
Novel selection method for engineered antibodies using the mechanism of Fv fragment stabilization in the presence of antigen; Tsumoto K et al.; Although the heavy and light chain domains of some antibody variable region fragments (Fvs) readily dissociate under physiological conditions, the Fvs are stable in the presence of antigen . This 'antigen-driven Fv stabilization mechanism' was applied to the selection of clones with specificity toward target antigens . The results can be summarized as follows . (i) Some of the residues in the heavy chain complementarity determining region 2 (HCDR2) of anti-hen egg white lysozyme (HEL) monoclonal antibody HyHEL10 heavy chain variable region (VH) were randomized . (ii) The randomized VH fragments of HyHEL10 were displayed on a filamentous bacteriophage and mixed with the target antigen, before being applied to a light chain variable region (VL) which was immobilized on microtiter plates and subjected to selection by panning . (iii) After four rounds of panning, four clones that showed significant binding to human lysozyme (hL), which HyHEL10 recognized poorly, were selected from the HCDR2 library . (iv) The soluble Fv fragments selected were expressed in Escherichia coli, purified, and subjected to an inhibition assay of lysozyme enzymatic activities and an isothermal titration calorimetry . These Fv fragments had increased affinity toward hL, and thermodynamic analysis suggested that the reduced entropy loss due to binding by the replacement of residues in HCDR2 resulted in the higher hL binding activity.

Biochem Pharmacol, 1998 Feb 15, 55(4), 441 - 6
Nonsteroidal antiinflammatory drug-photosensitized formation of pyrimidine dimer in DNA; Chouini-Lalanne N et al.; Phototoxic nonsteroidal antiinflammatory drugs (NSAIDs) may induce DNA damage in vitro upon irradiation . In this study, we investigated the ability of ketoprofen (KP), tiaprofenic acid (Tia), naproxen (NP) and indomethacin (IND) to photosensitize the formation of pyrimidine dimers and single strand breaks . Both kinds of damage were sought by analyzing DNA-drug mixtures irradiated at 313 nm by agarose gel electrophoresis . The formation of pyrimidine dimers was evidenced by using endonuclease V from bacteriophage T4 and compared to that induced by acetophenone, a well-known photosensitizer of thymine dimerization . Upon irradiation of DNA alone, pyrimidine dimers were observed while single strand breaks were not detected under our conditions . DNA, in the presence of NSAIDs, undergoes single strand breaks, the quantum yield of the DNA cleavage so induced (phiC) varying from 5 x 10(-4) for KP to 10(-5) for IND . The formation of dimers was only increased in the presence of KP or Tia . The quantum yields of pyrimidine dimers formed by photosensitization (phiD) were 2 x 10(-4) for KP and 10(-5) for Tia, respectively . The oxygen and concentration dependence of both processes was analyzed in the case of KP . In aerated solution, KP-photoinduced cleavage of DNA was predominant on the photodimerization process of pyrimidines, whereas in deaerated solution the cleavage was decreased and the dimerization increased . These results reflect competition between a radical process leading to DNA cleavage and a poorly efficient energy transfer between the drug and the pyrimidines at the origin of the dimerization process.

Biochim Biophys Acta, 1998 Feb 11, 1395(3), 293 - 300
Cloning and expression of a cDNA encoding the large subunit of duck replication factor C; Guo JT et al.; A cDNA encoding an avian homologue of the large subunit of replication factor C (RFC-L) has been cloned from a duck liver cDNA expression library prepared in bacteriophage lambda . The full length cDNA encodes a protein with a predicted size of approximately 130 kDa, consistent with the size of the polypeptide detected in duck liver . The duck RFC-L amino acid sequence shares 66.4% and 68.4% identity with mouse and human RFC-L proteins, respectively . We identified a 4kb RFC-L mRNA expressed in most duck tissues.

J Immunol, 1998 Mar 15, 160(6), 2619 - 25
Local synthesis of C3 within the splenic lymphoid compartment can reconstitute the impaired immune response in C3-deficient mice; Fischer MB et al.; Mice bearing a disrupted C3 locus (C3-/-) have an impaired Ab response to T-dependent Ags (bacteriophage phiX 174 and nuclear protein-keyhole limpet hemocyanin) characterized by a reduction in number and size of germinal centers and impaired retention of Ag by follicular dendritic cells . To test the importance of C3 synthesized locally within the lymphoid compartment during an immune response to T-dependent Ag, we reconstituted C3-/- mice with wild-type bone marrow of MHC-identical littermates . Engraftment not only restored local C3 synthesis in the spleen, but also rescued the impaired humoral response . The major source of C3 mRNA was MOMA-2+ macrophages localized within the white pulp areas of the spleen . Interestingly, C3 expression is apparently regulated as C3 mRNA was not detected in splenic sections of nonimmune mice . Furthermore, local C3 synthesis by donor macrophages reversed the impaired Ag trapping by splenic follicular dendritic cells in C3-deficient mice.

Mol Cells, 1997 Dec 31, 7(6), 816 - 9
Cloning and characterization of cDNAs encoding VH and VL of a monoclonal anti-CEA antibody (CEA 79) cross-reactive with NCA-95 and generation of a single-chain Fv molecule (scFv); Chung JH et al.; We cloned complementary DNA (cDNA) encoding the variable regions of heavy chain (VH) and of light chain (VL) of a monoclonal anti-carcinoembryonic antigen (CEA) antibody cross-reactive with nonspecific cross-reacting antigen-95 (NCA-95), which had been previously prepared and designated as CEA 79 (gamma 2a, kappa) . From these cDNAs, a phagemid expression vector for the CEA79 single chain variable fragment (scFv) was generated . Enzyme-linked immunosorbent assay (ELISA), competitive ELISAs, and Western blotting confirmed that the scFv displayed on the surface of the bacteriophage had retained affinity for CEA and NCA-95 . We then determined the nucleotide sequences of the cloned cDNAs for VH and VL . The sequence analysis revealed that VH and VL of the CEA 79 antibody represent new members of the mouse heavy chain subgroup "miscellaneous" and the kappa light chain subgroup "V", respectively.

Electrophoresis, 1997 Dec, 18(15), 2880 - 92
Identification of bacteriophage T4 virion proteins by transverse pore-gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dual amino acid labeling; Ferguson PL et al.; We have developed a horizontal N,N'-methylenebisacrylamide (Bis) acrylamide gradient sodium dodecyl sulfate (SDS) gel system that permits the evaluation of the purity of individual protein bands in complex mixtures . A Bis gradient gel is poured vertically and, after polymerization, reoriented horizontally . A single large sample spanning the top of the gel is then run down at right angles to the gradient . The relative mobility of a few proteins varies considerably from the rest, causing them to merge with and cross other bands as the Bis concentration changes . Band splitting revealed that several bands previously thought to represent a single species are actually comprised of comigrating proteins . Once the Bis/monomer concentration offering the best separation was identified, we sought a simple method for identifying the genetic origin of bands, since many proteins now migrated in new positions on the gel . We reasoned that if infected cells were simultaneously labeled with {35S}methionine and {3H}leucine and the purified virion proteins analyzed to determine their 35S/3H ratio, we could identify most proteins by comparing this ratio with one calculated from the T4 DNA sequence . Our expectations were realized, and we here report the separation and identification of all T4 virion proteins . Finally, we comment on the incorporation of various changes to the original Laemmli SDS-polyacrylamide gel formulations that have been reported in the literature.

Biol Chem, 1998 Jan, 379(1), 51 - 8
Overexpression and structural characterization of the phage T4 protein DsbA; Sieber P et al.; The double strand binding protein A (DsbA) of bacteriophage T4 is one of several viral gene products participating in transcriptional regulation . These proteins interact or associate with the host RNA polymerase core enzyme, enabling the enzyme to successively initiate transcription at different classes of viral promoters: early, middle and late . This leads to a temporally controlled expression of the T4 gene products . The DsbA binding site overlaps the late promoter region, and DsbA binding seems to intensify transcription of late genes in vitro, possibly acting as an enhancer protein (Molecular Biology of Phage T4, Karam, 1994) . To further investigate the function and structure of DsbA, we overexpressed the protein in E . coli and purified it to homogeneity . Physiological functionality of the recombinant protein was shown by gel retardation experiments and by circular dichroism (CD) spectroscopy . DsbA shows strong bands in the near UV-CD spectra . The far UV-CD spectroscopy analysis shows alpha-helices to be the main secondary structure elements . This is in agreement with secondary structure predictions . A possible helix-turn-helix motif in the center of the protein could be identified . Results from crosslinking and sedimentation analyses show that DsbA forms a dimer in solution . The thermal unfolding curve fits a dimer-two-state-folding-model, and the unfolding temperature was concentration dependent . Therefore, dimerization should supply the main portion of the free energy of stabilization of deltaG0 = 42 kJ/mol.

Nat Struct Biol, 1998 Mar, 5(3), 203 - 12
Solution structure of P22 transcriptional antitermination N peptide-boxB RNA complex; Cai Z et al.; We have determined the solution structure of a 15-mer boxB RNA hairpin complexed with a 20-mer basic peptide of the N protein involved in bacteriophage P22 transcriptional antitermination . Complex formation involves adaptive binding with the N peptide adopting a bent alpha-helical conformation that packs tightly through hydrophobic and electrostatic interactions against the major groove face of the boxB RNA hairpin, orienting the open opposite face for potential interactions with host factors and/or RNA polymerase . Four nucleotides in the boxB RNA hairpin pentaloop form a stable GNRA like tetraloop structural scaffold on complex formation, allowing the looped out fifth nucleotide to make extensive hydrophobic contacts with the bound peptide . The guanidinium group of a key arginine is hydrogen-bonded to the guanine in a loop-closing sheared G.A mismatch and to adjacent backbone phosphates . The identified intermolecular contacts account for the consequences of N peptide and boxB RNA mutations on bacteriophage transcriptional antitermination.

Invest Ophthalmol Vis Sci, 1998 Mar, 39(3), 665 - 70
Evaluation of the human arrestin gene in patients with retinitis pigmentosa and stationary night blindness; Sippel KC et al.; PURPOSE: To establish the DNA sequence of the coding regions of the human arrestin locus and to determine whether defects in this sequence are present among patients with retinitis pigmentosa (RP) or types of stationary night blindness in addition to Oguchi disease . METHODS: The human genomic locus encoding arrestin was cloned in bacteriophage and P1 vectors . The sequence of the intron DNA flanking each exon was determined from these clones . Single-strand conformation polymorphism analysis and direct genomic sequencing techniques were used to screen 272 unrelated patients, comprising 177 patients with autosomal dominant RP, 85 with recessive RP, and 10 with stationary night blindness . RESULTS: The arrestin gene is divided into 16 exons ranging in size from 10 bp to 194 bp, with the open reading frame spanning exons 2 through 16 . The authors identified several discrepancies between the genomic sequence the authors obtained and the previously published cDNA and genomic sequences . In the set of patients with dominant RP, the authors found one of three heterozygous missense changes (Arg84Cys, Thr125Met, and Val378Ile) in each of four unrelated patients; none of these changes cosegregated with disease in the respective families . In the set of patients with recessive RP, the authors found one of two heterozygous missense changes in each of two unrelated patients with recessive RP (Pro364Leu and Arg384Cys) . One of the patients was the offspring of a consanguineous marriage; because the Arg384Cys change in him was heterozygous, it is unlikely to have been the cause of his RP . Cosegregation studies could not be performed on the patient with the Pro364Leu change . The authors confirmed the existence of two previously described polymorphisms (Ile76Val and a multiallelic polymorphism at codon 403), and the authors identified several silent polymorphisms and rare sequence variants . No sequence changes, other than polymorphic changes also found in some patients with RP, were identified in the patients with stationary night blindness . CONCLUSIONS: We found no evidence that mutations in arrestin are a cause of RP or stationary night blindness other than Oguchi disease . According to the genomic sequence obtained, a region in exon 8 that has been postulated to represent the site of interaction between arrestin and rhodopsin is 100% conserved between humans and all other mammals studied to date.

Science, 1998 Mar 6, 279(5356), 1504 - 13
Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA; Redinbo MR et al.; Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination . The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA . The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases . A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.

Nucleic Acids Res, 1998 Feb 15, 26(4), 988 - 93
Embryonic stem cell gene targeting using bacteriophage lambda vectors generated by phage-plasmid recombination; Tsuzuki T et al.; Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest . Currently the rate determining step in any gene targeting experiment is construction of the targeting vector (TV) . In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of lambda phage in targeted mutagenesis . The recombination-proficient phage vector lambda2TK permits generation of TVs by conventional restriction-ligation or recombination-mediated methods . The resulting lambdaTV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets . We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination . We describe two methods for eliminating single crossover recombinants, spi selection and size restriction, both of which result in phage TVs bearing double crossover insertions . Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information.

Nucleic Acids Res, 1998 Feb 15, 26(4), 887 - 95
The path of the growing peptide chain through the 23S rRNA in the 50S ribosomal subunit; a comparative cross-linking study with three different peptide families; Choi KM et al.; As part of a programme to investigate the path of the nascent peptide through the large ribosomal subunit, peptides of different lengths (up to 30 amino acids), corresponding to the signal peptide sequence and N-terminal region of the Escherichia coli ompA protein, were synthesized in situ on E.coli ribosomes . The peptides each carried a diazirine moiety attached to their N-terminus which, after peptide synthesis, was photoactivated so as to induce cross-links to the 23S rRNA . The results showed that, with increasing length, the peptides became progressively cross-linked to sites in Domains V, II, III and I of the 23S rRNA, in a similar manner to that previously observed with a family of peptides derived from the tetracycline resistance gene . However, the cross-links to Domain III appeared at a shorter peptide length (12 aa) in the case of the ompA sequence, and an additional cross-link in Domain II (localized to nt 780-835) was also observed from this peptide . As with the tetracycline resistance sequence, peptides of all lengths were still able to form cross-links from their N-termini to the peptidyl transferase centre in Domain V . A further set of peptides (30 or 50 aa long), derived from mutants of the bacteriophage T4 gene 60 sequence, did not show the cross-links to Domain III, but their N-termini were nevertheless cross-linked to Domain I and to the sites in Domains II and V . The ability of relatively long peptides to fold back towards the peptidyl transferase centre thus appears to be a general phenomenon.

Arch Virol, 1998, 143(1), 163 - 71
A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene; Yu HH et al.; A full-length DNA clone encoding the genome of odontoglossum ringspot tobamovirus (ORSV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter . Capped-RNA transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey . A representative clone, designated pOT2, caused a disease phenotype identical to that produced by parental viral RNA . ELISA, Western blot analysis, Northern blot hybridization and electron microscopy verified the infectivity of pOT2 . A coat protein deficient mutant of the clone, pO delta CP1, was produced with the initiation codon of the coat protein cistron of ORSV abolished . Transcripts from pO delta CP1 were infective, able to move in N . benthamiana but produced no coat protein . This demonstrates that the coat protein was dispensable for RNA replication and for movement . This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.

J Biochem (Tokyo), 1997 Dec, 122(6), 1088 - 91
Determination of the DNA-binding sequences of the Zn(II)2Cys6 zinc-cluster-containing PRIB protein, derived from the basidiomycete Lentinus edodes gene; Miyazaki Y et al.; The 565 amino-acid PRIB protein with a Zn(II)2Cys6 zinc-cluster DNA-binding motif is the expression product of the priB gene, which is most actively transcribed in an early stage of fruiting-body formation by the basidiomycete, Lentinus edodes . PRIB produced in Escherichia coli using the bacteriophage T7 expression system was purified by ion-exchange chromatographies and then subjected to random binding-site selection analysis using a pool of random 24-bp oligonucleotides with 13-bp PCR primer sites at each end . The oligonucleotides (50 bp) selected for PRIB binding were cloned into pUC19 . A total of 303 cloned DNA fragments were picked randomly and sequenced . The PRIB binding sites could be grouped into 25 individual sequences, suggesting a consensus sequence of 16 bp, 5' GGGGGGGACAGGANCC 3' . Gel mobility-shift assaying of 10 randomly selected sequences all revealed a reasonable band shift . DNase I footprinting analysis of the 50-bp DNA fragment containing the sequence most similar to the consensus sequence showed that PRIB protects the entire 16-bp sequence from digestion by DNase I.

J Bacteriol, 1998 Mar, 180(5), 1154 - 8
Regulation of proteolysis of the stationary-phase sigma factor RpoS; Zhou Y et al.; RpoS, the stationary-phase sigma factor of Escherichia coli, is responsible for increased transcription of an array of genes when cells enter stationary phase and under certain stress conditions . RpoS is rapidly degraded during exponential phase and much more slowly during stationary phase; the resulting changes in RpoS accumulation play an important role in providing differential expression of RpoS-dependent gene expression . It has previously been shown that rapid degradation of RpoS during exponential growth depends on RssB (also called SprE and MviA), a protein with homology to the family of response regulators, and on the ClpXP protease . We find that RssB regulation of proteolysis does not extend to another ClpXP substrate, bacteriophage lambda O protein, suggesting that RssB acts on the specific substrate RpoS rather than on the protease . In addition, the activity of RpoS is down-regulated by RssB when degradation is blocked . In cells blocked for RpoS degradation by a mutation in clpP, cells devoid of RssB show a four- to fivefold-higher activity of an RpoS-dependent reporter fusion than cells overproducing RssB . Therefore, RssB allows specific environmental regulation of RpoS accumulation and may also modulate activity . The regulation of degradation provides an irreversible switch, while the regulation of activity may provide a second, presumably reversible level of control.

J Bacteriol, 1998 Mar, 180(5), 1148 - 53
Assembly of both the head and tail of bacteriophage Mu is blocked in Escherichia coli groEL and groES mutants; Grimaud R et al.; Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normally in groE hosts (M . Pato, M . Banerjee, L . Desmet, and A . Toussaint, J . Bacteriol . 169:5504-5509, 1987) . We show here that lysates obtained upon induction of groE Mu lysogens contain free inactive tails and empty heads . GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails . Evidence is presented that groE mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.

Biochem Cell Biol, 1997, 75(4), 435 - 43
Genotoxin resistance properties of transgenic tobacco plants expressing bacteriophage T4 DenV and Saccharomyces cerevisiae Apn1 proteins; Lapointe G et al.; We have examined whether the resistance to genotoxic agents can be altered in transgenic plants by introducing heterologous DNA repair enzymes . Transformation of tobacco tissue produced two lines of plants, one expressing bacteriophage T4 UV endonuclease (DenV) and the other expressing Saccharomyces cerevisiae apurinic-apyrimidinic endonuclease and 3'-diesterase (Apn1) . Some of the transformants were subsequently crossed, with the expectation that Apn1 activity might complement DenV activity in hybrid plants . Apn1 transgenotes behaved similarly to control plants upon exposure to UV-C light, oxidizing agents, or alkylating agents, as measured by chlorophyll bleaching . This is in contrast to plants expressing DenV activity, which have been previously shown to exhibit varying degrees of sensitivity to UV-C light and the alkylating agent dimethyl sulfate . APN1/denV hybrid plants were more sensitive to UV-C light than were parental lines, but reproducibly displayed enhanced resistance to dimethyl sulfate . These data indicate that repair processes are an important component of natural protective systems in tobacco, because exogenous repair genes compromised the natural resistance of denV-transformed plants . In the hybrid plants, the two proteins appeared to act in concert, potentiating the effects of UV damage but enhancing the resistance to alkylation damage.

Biochemistry, 1998 Feb 17, 37(7), 1819 - 27
Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly; Soumillion P et al.; Clamp proteins confer processivity to the DNA polymerase during DNA replication . These oligomeric proteins are loaded onto DNA by clamp loader protein complexes in an ATP-dependent manner . The mechanism by which the trimeric bacteriophage T4 clamp protein (the 45 protein) loads and dissociates from DNA was investigated as a function of its intersubunit protein-protein interactions . These interactions were continuously monitored using a fluorescence resonance energy transfer (FRET) based assay . A cysteine mutant of the 45 protein was constructed to facilitate site-specific incorporation of a fluorescent probe at the subunit interface . This site was chosen such that FRET was observed between the introduced fluorescent probe and a tryptophan residue located on the opposing subunit . By use of this fluorescently labeled 45 protein, it was possible to obtain an estimate of an apparent trimer dissociation constant from either a cooperative (0.08 +/- 0.04 microM2 at 25 degrees C) or a noncooperative (0.51 microM and 0.17 microM at 25 degrees C) model . Upon mixing the fluorescently labeled 45 protein with a 45 protein containing 4-fluorotryptophan, a nonfluorescent tryptophan analogue, subunit exchange between the two variants of the 45 protein was observed according to a reduction in intersubunit FRET . Subunit exchange rate constants measured in the presence or absence of the clamp loader (44/62 complex), the polymerase (43 protein), and/or a primer template DNA substrate demonstrate (a) that the 45 protein is not loaded onto DNA by subunit exchange and (b) that the disassembly dissociation of a stalled holoenzyme from DNA is dictated by 45 protein subunit dissociation.

Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 566 - 71
Gene 1, one of the dna genes of bacteriophage phi 29, represses other dna genes through binding to mRNA; Takeuchi A et al.; The dna genes, essential for protein priming DNA replication of bacteriophage phi 29, are transcribed as a long polycistronic mRNA . In the previous study, gene 1 product (gp1) was shown to repress the expression of the upstream dna genes for DNA polymerase and primer protein . To investigate the details of the repression by gp1, we have examined the amount and integrity of polycistronic mRNA encoding DNA polymerase and primer protein by agarose gel electrophoresis and nuclease S1 protection assay . As a result, the amount, size, and integrity of the polycistronic mRNA were not influenced by the presence of gene 1 . Furthermore, the RNA binding ability of gp1 was demonstrated by in vitro system using histidine-tagged gp1 . These results strongly suggested that translation of the dna genes was affected by gp1 through binding to mRNA . Other possible mechanisms of gene regulation by gp1 were discussed.

Immunol Res, 1998, 17(1-2), 239 - 51
The human immune response to red blood cell antigens as revealed by repertoire cloning; Siegel DL; A major goal of current immunologic research is to develop specific therapeutic strategies by which the enormous diversity in immune response can be enhanced, attenuated, or eliminated, depending on the particular disease process . For nearly a century, the human immune response to red blood cell antigens has served as a paradigm for understanding the pathophysiology of autoimmune disorders and alloimmune reactions to foreign cells and tissues . Recent developments in molecular biology have facilitated the expression of immune repertoires in the form of immunoglobulin Fab fragments on the surface of filamentous bacteriophage . Such approaches have provided powerful means for producing monoclonal antibodies for research, clinical, and therapeutic applications . Our laboratory has combined these techniques with novel cell-surface selection methods to isolate extraordinarily large arrays of human antibodies to the clinically relevant red blood cell Rh(D) antigen . Our results have provided a comprehensive genetic and serologic analysis of anit-Rh(D) antibodies within single alloimmunized individuals thereby offering new insights into the development of human immune repertoires.

Virology, 1998 Feb 1, 241(1), 73 - 9
Regulation of the bacteriophage Mu gem operon; Fabozzi G et al.; The gem operon of bacteriophage Mu, responsible for the complex phenomenon of phage conversion, is included in the so called "semiessential early" region of phage DNA . Unlike the other early genes of the phage which are transcribed from the pe promoter, expression of the gem operon is driven by its own promoter, which escapes the control of the repressor . In fact, the transcript corresponding to gem was detected in immune lysogens by using a combined reverse transcription and a subsequent amplification of the resulting cDNA . The transcription initiation site from pgem was determined by primer extension mapping experiments and localized at 8217 bp from the left end of phage DNA . Two elements which could perform the negative control of gem were also identified . The first is a phage product, GemB, which presumably interferes with gem expression at a posttranscriptional level, whereas the second is a structural element, an inverted repeat immediately downstream of pgem, which acts as a terminator for the transcripts starting from pe . These transcripts could regulate gem expression by interfering with the initiation of transcription from pgem .

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