Bacteriophages

J Bacteriol, 2002 Oct, 184(20), 5599 - 608
Induction of a DNA nickase in the presence of its target site stimulates adaptive mutation in Escherichia coli; Rodriguez C et al.; Adaptive mutation to Lac(+) in Escherichia coli strain FC40 depends on recombination functions and is enhanced by the expression of conjugal functions . To test the hypothesis that the conjugal function that is important for adaptive mutation is the production of a single-strand nick at the conjugal origin, we supplied an exogenous nicking enzyme, the gene II protein (gIIp) of bacteriophage f1, and placed its target sequence near the lac allele . When both gIIp and its target site were present, adaptive mutation was stimulated three- to fourfold . Like normal adaptive mutations, gIIp-induced mutations were recA(+) and ruvC(+) dependent and were mainly single-base deletions in runs of iterated bases . In addition, gIIp with its target site could substitute for conjugal functions in adaptive mutation . These results support the hypothesis that nicking at the conjugal origin initiates the recombination that produces adaptive mutations in this strain of E . coli, and they suggest that nicking may be the only conjugal function required for adaptive mutation.

J Mol Biol, 2002 Sep 27, 322(4), 697 - 706
Searching for DNA-protein interactions by lambda phage display; Cicchini C et al.; We applied phage display technology to DNA-protein interaction studies . A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line . Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence . One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated . Specific binding of the selected protein domain was confirmed in a phage-independent context . By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified . The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.

Nat Med, 2002 Oct, 8(10), 1166 - 70 Epub 2002 Sep 16.
Stable nonviral genetic correction of inherited human skin disease; Ortiz-Urda S et al.; Current gene-transfer technologies display limitations in achieving effective gene delivery . Among these limitations are difficulties in stably integrating large corrective sequences into the genomes of long-lived progenitor-cell populations . Current larger-capacity viral vectors suffer from biosafety concerns, whereas plasmid-based approaches have poor efficiency of stable gene transfer . These barriers hinder genetic correction of many severe inherited human diseases, such as the blistering skin disorder recessive dystrophic epidermolysis bullosa (RDEB), caused by mutations in the large COL7A1 gene . To circumvent these barriers, we used the phi C31 bacteriophage integrase, which stably integrates large DNA sequences containing a specific 285-base-pair attB sequence into genomic 'pseudo-attP sites' . phi C31 integrase-based gene transfer stably integrated the COL7A1 cDNA into genomes of primary epidermal progenitor cells from four unrelated RDEB patients . Skin regenerated using these cells displayed stable correction of hallmark RDEB disease features, including Type VII collagen protein expression, anchoring fibril formation and dermal-epidermal cohesion . These findings establish a practical approach to nonviral genetic correction of severe human genetic disorders requiring stable genomic integration of large DNA sequences.

Science, 2002 Nov 15, 298(5597), 1387 - 95 Epub 2002 Sep 19.
Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase; Yin YW et al.; To make messenger RNA transcripts, bacteriophage T7 RNA polymerase (T7 RNAP) undergoes a transition from an initiation phase, which only makes short RNA fragments, to a stable elongation phase . We have determined at 2.1 angstrom resolution the crystal structure of a T7 RNAP elongation complex with 30 base pairs of duplex DNA containing a "transcription bubble" interacting with a 17-nucleotide RNA transcript . The transition from an initiation to an elongation complex is accompanied by a major refolding of the amino-terminal 300 residues . This results in loss of the promoter binding site, facilitating promoter clearance, and creates a tunnel that surrounds the RNA transcript after it peels off a seven-base pair heteroduplex . Formation of the exit tunnel explains the enhanced processivity of the elongation complex . Downstream duplex DNA binds to the fingers domain, and its orientation relative to upstream DNA in the initiation complex implies an unwinding that could facilitate formation of the open promoter complex.

Gene, 2002 Jul 24, 295(1), 125 - 34
Mutational analysis of bacteriophage T4 AsiA: involvement of N- and C-terminal regions in binding to sigma(70) of Escherichia coli in vivo; Sharma UK et al.; The T4 AsiA is an anti-sigma factor encoded by an early gene of bacteriophage T4 . AsiA has been shown to inhibit T4 early promoters in vitro and expression of this protein from a plasmid causes transcriptional shut off in the host cells leading to cell death . By reasoning that mutant AsiA expression in Escherichia coli will not inhibit the host transcription and hence lead to healthy colony formation, a strategy was developed wherein inactive or partially active mutants of AsiA could be isolated . These mutants were tested for their ability to bind to sigma(70) in vivo in E . coli, monitored as a relative toxicity assay, co-purification of sigma(70), inhibition of {3H-uridine} incorporation and also in the yeast two hybrid system . A good correlation was found between the loss of toxicity of AsiA to E . coli cells and the inability of mutant AsiAs to bind to sigma(70) It was observed that deletion of C-terminal 17 amino acid residues of AsiA did not affect the activity whereas a mutant asiA lacking C-terminal 28 amino acid residues had the toxicity reduced to a large extent, suggesting that amino acid residues between 64 and 73 played a role in binding to AsiA . A mutant with a deletion of 34 amino acids in the C-terminus did not show any toxicity to E . coli cells . In the N-terminal region, deletion of five amino acid residues was tolerated but extending the deletion to ten amino acids abolished the AsiA activity completely . The conversion of glutamic acid (E10) to either leucine, serine, glutamine, tyrosine or alanine did not affect the toxicity to a great extent suggesting that a negative charge at E10 is not critical for interaction with sigma(70) . The results of our in vivo studies suggest that the primary sigma(70) binding site of AsiA is in N-terminus, but, it requires the presence of C-terminal 64-73 amino acid residues for effective binding in vivo.

J Virol, 2002 Oct, 76(20), 10245 - 55
A P22 scaffold protein mutation increases the robustness of head assembly in the presence of excess portal protein; Moore SD et al.; Bacteriophage with linear, double-stranded DNA genomes package DNA into preassembled protein shells called procapsids . Located at one vertex in the procapsid is a portal complex composed of a ring of 12 subunits of portal protein . The portal complex serves as a docking site for the DNA packaging enzymes, a conduit for the passage of DNA, and a binding site for the phage tail . An excess of the P22 portal protein alters the assembly pathway of the procapsid, giving rise to defective procapsid-like particles and aberrant heads . In the present study, we report the isolation of escape mutant phage that are able to replicate more efficiently than wild-type phage in the presence of excess portal protein . The escape mutations all mapped to the same phage genome segment spanning the portal, scaffold, coat, and open reading frame 69 genes . The mutations present in five of the escape mutants were determined by DNA sequencing . Interestingly, each mutant contained the same mutation in the scaffold gene, which changes the glycine at position 287 to glutamate . This mutation alone conferred an escape phenotype, and the heads assembled by phage harboring only this mutation had reduced levels of portal protein and exhibited increased head assembly fidelity in the presence of excess portal protein . Because this mutation resides in a region of scaffold protein necessary for coat protein binding, these findings suggest that the P22 scaffold protein may define the portal vertices in an indirect manner, possibly by regulating the fidelity of coat protein polymerization.

J Virol, 2002 Oct, 76(20), 10122 - 7
Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage phi6 is required for single-stranded RNA packaging and transcription; Pirttimaa MJ et al.; Bacteriophage phi6 has a segmented double-stranded RNA genome . The genomic single-stranded RNA (ssRNA) precursors are packaged into a preformed protein capsid, the polymerase complex, composed of viral proteins P1, P2, P4, and P7 . Packaging of the genomic precursors is an energy-dependent process requiring nucleoside triphosphates . Protein P4, a nonspecific nucleoside triphosphatase, has previously been suggested to be the prime candidate for the viral packaging engine, based on its location at the vertices of the viral capsid and its biochemical characteristics . In this study we were able to obtain stable polymerase complex particles that are completely devoid of P4 . Such particles were not able to package ssRNA segments and did not display RNA polymerase (either minus- or plus-strand synthesis) activity . Surprisingly, a mutation in P4, S250Q, which reduced the level of P4 in the particles to about 10% of the wild-type level, did not affect RNA packaging activity or change the kinetics of packaging . Moreover, such particles displayed minus-strand synthesis activity . However, no plus-strand synthesis was observed, suggesting that P4 has a role in the plus-strand synthesis reaction also.

J Gen Virol, 2002 Oct, 83(Pt 10), 2601 - 6
Various morphological aspects of Escherichia coli lysis by two distinct RNA bacteriophages; Nishihara T; Transmission electron micrographs of Escherichia coli cells induced by cloned lysis genes from RNA bacteriophages GA (group A-II) and SP (group B-IV) revealed various morphological aspects of intermediates of lysing cells . Cells induced by the SP lysis gene became stretched and also tapered in shape and fragmentation of parts of the cells had also occurred . Cells induced by the GA lysis gene showed many ballooning structures on the cell surfaces and others leaked material through the cell wall . Some balloon-like structures also appeared on the surfaces of cells induced by the cloned lysis gene of RNA phage SP and material also appeared to be leaking through the cell wall in the photographs . The lysing cells observed by transmission electron microscopy showed various morphological aspects of intermediates of the lysing process.

J Gen Virol, 2002 Oct, 83(Pt 10), 2593 - 600
Molecular structures of viruses from Raman optical activity; Blanch EW et al.; A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level . Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced . Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle chi(2,1) for tryptophan in fd, may also sometimes be obtained . By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar . This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

J Biol Chem, 2002 Nov 22, 277(47), 44778 - 83 Epub 2002 Sep 16.
Interaction of the DnaK and DnaJ chaperone system with a native substrate, P1 RepA; Kim SY et al.; DnaK, the Hsp70 chaperone of Escherichia coli interacts with protein substrates in an ATP-dependent manner, in conjunction with DnaJ and GrpE co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes . To understand how DnaJ targets specific proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with a known natural substrate, bacteriophage P1 RepA protein . By characterizing RepA deletion derivatives, we found that DnaJ interacts with a region of RepA located between amino acids 180 and 200 of the 286-amino acid protein . A peptide corresponding to amino acids 180-195 inhibited the interaction of RepA and DnaJ . Two site-directed RepA mutants with alanine substitutions in this region were about 4-fold less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA . We also identified by deletion analysis a site in RepA, in the region of amino acids 35-49, which interacts with DnaK . An alanine substitution mutant in amino acids 36-39 was constructed and found defective in activation by DnaJ and DnaK . Taken together the results suggest that DnaJ and DnaK interact with separate sites on RepA.

J Am Chem Soc, 2002 Sep 25, 124(38), 11324 - 33
Secondary structure of DNA is recognized by slightly cross-linked cationic hydrogel; Sergeyev VG et al.; Interaction of salmon sperm DNA (300-500 bp) and ultrahigh molecular mass DNA (166 kbp) from bacteriophage T4dC with linear poly(N-diallyl-N-dimethylammonium chloride) (PDADMAC) and slightly cross-linked (#) PDADMAC (#PDADMAC) hydrogel in water has been studied by means of UV-spectroscopy, ultracentrifugation, atomic force, and fluorescence microscopy (FM) . It is found that the linear polycation induced compaction of either native (double-stranded) or denatured (single-stranded) DNA by forming PDADMAC-DNA interpolyelectrolyte complexes (IPEC)s . At the same time, #PDADMAC hydrogel is able to distinguish between native and denatured DNA . Native DNA is adsorbed and captured in the hydrogel surface layer, while denatured DNA diffuses to the hydrogel interior until the whole hydrogel sample is transformed into the cross-linked IPEC . Both native and denatured DNA can be completely released from the hydrogel in appropriate conditions with no degradation by adding a low molecular salt . The data observed using conventional physicochemical methods with respect to DNA of a moderate molecular mass remarkably correlate with the pictures directly observed for ultrahigh molecular mass DNA in dynamics by using FM.

Nucleic Acids Res, 2002 Sep 15, 30(18), 3936 - 44
Interaction of the ocr gene 0.3 protein of bacteriophage T7 with EcoKI restriction/modification enzyme; Atanasiu C et al.; The ocr protein, the product of gene 0.3 of bacteriophage T7, is a structural mimic of the phosphate backbone of B-form DNA . In total it mimics 22 phosphate groups over approximately 24 bp of DNA . This mimicry allows it to block DNA binding by type I DNA restriction enzymes and to inhibit these enzymes . We have determined that multiple ocr dimers can bind stoichiometrically to the archetypal type I enzyme, EcoKI . One dimer binds to the core methyltransferase and two to the complete bifunctional restriction and modification enzyme . Ocr can also bind to the component subunits of EcoKI . Binding affinity to the methyltransferase core is extremely strong with a large favourable enthalpy change and an unfavourable entropy change . This strong interaction prevents the dissociation of the methyltransferase which occurs upon dilution of the enzyme . This stabilisation arises because the interaction appears to involve virtually the entire surface area of ocr and leads to the enzyme completely wrapping around ocr.

Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12703 - 8 Epub 2002 Sep 12.
Interaction of adjacent primase domains within the hexameric gene 4 helicase-primase of bacteriophage T7; Lee SJ et al.; The interaction of primase monomers within the hexameric gene 4 helicase-primase of bacteriophage T7 has been examined by using two genetically distinct gene 4 proteins . The T7 56-kDa gene 4 protein differs from the full-length 63-kDa protein in that it lacks the N-terminal zinc motif essential for the recognition of primase recognition sites . A second gene 4 protein, gp4-K122A, is unable to catalyze the synthesis of phosphodiester bonds as the result of an amino acid change in the catalytic site . Although each protein alone is inactive, the two together catalyze the synthesis of RNA primers . Reconstitution of activity depends on hexamer formation . We propose that the zinc motif of one subunit in the hexamer interacts with the catalytic sites of adjacent subunits.

Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12709 - 14 Epub 2002 Sep 12.
Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains; Ho CK et al.; RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure . Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases . Here, we identified another RNA ligase from the bacterial domain--a second RNA ligase (Rnl2) encoded by phage T4 . Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates . Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction . The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA capping enzymes . Rnl2 has scant amino acid similarity to T4 gp63 . Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea . These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.

J Biol Chem, 2002 Nov 15, 277(46), 43785 - 91 Epub 2002 Sep 10.
Multiple mechanisms of transcription inhibition by ppGpp at the lambdap(R) promoter; Potrykus K et al.; General stress conditions in bacterial cells cause a global cellular response called the stringent response . The first event in this control is production of large amounts of a regulatory nucleotide, guanosine-3',5'-(bis)pyrophospahte (ppGpp) . It was proposed recently that ppGpp acts by decreasing stability of open complexes at promoters that make short-lived open complexes, e.g . the rRNA promoters . However, here we report that the bacteriophage lambdap(R) promoter, which forms long-lived open complexes, is inhibited by ppGpp in vitro as observed in vivo . We performed a systematic investigation of the ppGpp-specific inhibition of transcription initiation at lambdap(R) and found that ppGpp does decrease stability of open complexes at lambdap(R), but only slightly . Likewise the equilbrium binding constant and rate of open complex formation by RNA polymerase at lambdap(R) are only slightly affected by ppGpp . The major effect of ppGpp-mediated inhibition is to decrease the rate of promoter escape . We conclude that ppGpp-mediated inhibition of transcription initiation is not restricted to promoters that make short-lived open complexes . Rather we conclude that the initial catalytic step of transcript formation is affected by ppGpp, specifically formation of the first phosphodiester bond is inhibited by ppGpp at lambdap(R).

J Biol Chem, 2002 Nov 15, 277(46), 43778 - 84 Epub 2002 Sep 10.
Kinetic pathway of dTTP hydrolysis by hexameric T7 helicase-primase in the absence of DNA; Jeong YJ et al.; Bacteriophage T7 gp4A' protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis . Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA . Pre-steady state dTTP hydrolysis kinetics showed a distinct burst whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer . The pre-steady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs . The medium {(18)O}P(i) exchange experiments failed to detect dTTP synthesis, indicating that the less than six-site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site . The P(i)-release rate was measured directly using a stopped-flow fluorescence assay, and it was found that the rate of dTTP hydrolysis on the helicase active site is eight times faster than the P(i)-release rate, which in turn is three times faster than the dTDP release rate . Thus, the rate-limiting step in the pathway of helicase-catalyzed deoxythymidine triphosphatase (dTTPase) reaction is the release of dTDP . Chase-time dTTPase kinetics in the steady state phase provided evidence for two to three slowly hydrolyzing dTTPase sites on the hexamer . The results of this study are therefore consistent with those reported earlier (Hingorani, M . M., Washington, M . T., Moore, K . C., and Patel, S . S . (1997) Proc . Natl . Acad . Sci . U.S.A . 94, 5012-5017), and they support a model of dTTP hydrolysis by T7 helicase hexamer that is similar to the binding change mechanism of F(1)-ATPase with dTTP hydrolysis occurring sequentially at the catalytic sites.

J Am Chem Soc, 2002 Sep 18, 124(37), 10966 - 7
Designed arginine-rich RNA-binding peptides with picomolar affinity; Austin RJ et al.; Arginine-rich peptide motifs (ARMs) capable of binding unique RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging . Bacteriophage ARMs necessary for transcription antitermination bind to distinct boxB RNA hairpin sequences with a characteristic induced alpha-helical structure . Characterization of ARMs from lambdoid phages reveals that the dissociation constant of the P22 bacteriophage model-antitermination complex (P22(N21)-P22boxB) is 200 +/- 56 pM in free solution at physiologic concentrations of monovalent cation, significantly stronger than previously determined by gel mobility shift and polyacrylamide gel coelectophoresis, and 2 orders of magnitude stronger than the tightest known native ARM-RNA interaction at physiological salt . Here, we use a reciprocal design approach to enhance the binding affinity of two separate alpha-helical ARM-RNA interactions; one derived from the native lambda phage antitermination complex and a second isolated using mRNA display selection experiments targeting boxB RNA.

J Mol Biol, 2002 Aug 30, 321(5), 839 - 49
Flexibility of the rings: structural asymmetry in the DnaB hexameric helicase; Yang S et al.; DnaB is the primary replicative helicase in Escherichia coli and the hexameric DnaB ring has previously been shown to exist in two states in the presence of nucleotides . In one, all subunits are equivalent, while in the other, there are two different subunit conformations resulting in a trimer of dimers . Under all conditions that we have used for electron microscopy, including the absence of nucleotide, some rings exist as trimers of dimers, showing that the symmetry of the DnaB hexamer can be broken prior to nucleotide binding . Three-dimensional reconstructions reveal that the N-terminal domain of DnaB makes two very different contacts with neighboring subunits in the trimer of dimers, but does not form a predicted dimer with a neighboring N-terminal domain . Within the trimer of dimers, the helicase domain exists in two alternate conformations, each of which can form symmetrical hexamers depending upon the nucleotide cofactor used . These results provide new information about the modular architecture and domain dynamics of helicases, and suggest, by comparison with the hexameric bacteriophage T7 gp4 and SV40 large T-antigen helicases, that a great structural and mechanistic diversity may exist among the hexameric helicases.

J Mol Biol, 2002 Aug 30, 321(5), 807 - 19
T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA; Kim DE et al.; DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation . Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers . Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA . Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C . While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA . One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating . We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.

J Mol Biol, 2002 Aug 30, 321(5), 767 - 84
The mechanism of transcriptional activation by the topologically DNA-linked sliding clamp of bacteriophage T4; Kolesky SE et al.; Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the sigma-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45 . Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer-template junctions by its clamp loader . The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes . The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly . Activation ( approximately 320-fold in this work) increases affinity in the closed complex and accelerates promoter opening . Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 degrees C, but once opened at 30 degrees C remains open upon shift to the lower temperature . At a hybrid promoter for sigma(70) and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening . Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33.

Nat Struct Biol, 2002 Oct, 9(10), 756 - 63
Minor proteins, mobile arms and membrane-capsid interactions in the bacteriophage PRD1 capsid; San Martin C et al.; Bacteriophage PRD1 shares many structural and functional similarities with adenovirus . A major difference is the PRD1 internal membrane, which acts in concert with vertex proteins to translocate the phage genome into the host . Multiresolution models of the PRD1 capsid, together with genetic analyses, provide fine details of the molecular interactions associated with particle stability and membrane dynamics . The N- and C-termini of the major coat protein (P3), which are required for capsid assembly, act as conformational switches bridging capsid to membrane and linking P3 trimers . Electrostatic P3-membrane interactions increase virion stability upon DNA packaging . Newly revealed proteins suggest how the metastable vertex works and how the capsid edges are stabilized.

J Mol Biol, 2002 Sep 13, 322(2), 357 - 67
A minimized M13 coat protein defines the requirements for assembly into the bacteriophage particle; Roth TA et al.; The M13 filamentous bacteriophage coat is a symmetric array of several thousand alpha-helical major coat proteins (P8) that surround the DNA core . P8 molecules initially reside in the host membrane and subsequently transition into their role as coat proteins during the phage assembly process . A comprehensive mutational analysis of the 50-residue P8 sequence revealed that only a small subset of the side-chains were necessary for efficient incorporation into a wild-type (wt) coat . In the three-dimensional structure of P8, these side-chains cluster into three functional epitopes: a hydrophobic epitope located near the N terminus and two epitopes (one hydrophobic and the other basic) located near the C terminus on opposite faces of the helix . The results support a model for assembly in which the incorporation of P8 is mediated by intermolecular interactions involving these functional epitopes . In this model, the N-terminal hydrophobic epitope docks with P8 molecules already assembled into the phage particle in the periplasm, and the basic epitope interacts with the acidic DNA backbone in the cytoplasm . These interactions could facilitate the transition of P8 from the membrane into the assembling phage, and the incorporation of a single P8 would be completed by the docking of additional P8 molecules with the second hydrophobic epitope at the C terminus . We constructed a minimized P8 that contained only nine non-Ala side-chains yet retained all three functional epitopes . The minimized P8 assembled into the wt coat almost as efficiently as wt P8, thus defining the minimum requirements for protein incorporation into the filamentous phage coat . The results suggest possible mechanisms of natural viral evolution and establish guidelines for the artificial evolution of improved coat proteins for phage display technology.

J Mol Biol, 2002 Sep 13, 322(2), 311 - 24
Derepression of bacteriophage mu transposition functions by truncated forms of the immunity repressor; O'Handley D et al.; To trigger bacteriophage Mu transposition and replication in response to physiological signals, its immunity repressor must be synchronously inactivated . Two repressor mutants (Vir), which have an altered C-terminal domain and are highly susceptible to degradation by ClpXP protease, confer a dominant negative phenotype by promoting degradation of the wild-type repressor . To search for other modified repressors that can induce Mu derepression in vivo and to determine what part of the inducing repressor molecules are needed to target the unmodified repressor population, repressor peptides with nested deletions starting at the C-terminal end were constructed . Such peptides with a C-terminal ssrA degradation tag promoted a sharp reduction in cellular levels of full-length unmodified repressor, a process largely dependent upon the clpP protease function . Only the repressor DNA-binding domain, located at the N-terminal end, was required in tagged peptides to target unmodified repressor . In addition, some repressor peptides containing the DNA-binding domain promoted derepression without the clpP function, being able to promote repressor inactivation without promoting its degradation . None of the modified repressors could promote derepression if immunity was established by a mutant repressor lacking 18 residues at its C-terminal end . The results indicate that inducing repressor peptides influence the function of the C-terminal domain of the intact repressor, a domain that regulates its degradation and DNA binding . They suggest the possibility that tagged repressor molecules, produced by stalled ribosomes, can be inducers of transposition under starvation conditions.

J Mol Biol, 2002 Sep 13, 322(2), 291 - 309
Toward understanding the mutagenicity of an environmental carcinogen: structural insights into nucleotide incorporation preferences; Perlow RA et al.; Bulky carcinogen-DNA adducts, including (+)-trans-anti-{BP}-N(2)-dG derived from the reaction of (+)-anti-benzo{a}pyrene diol epoxide with guanine, often block the progression of DNA polymerases . However, when rare bypass of the lesions does occur, they may be misreplicated . Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-{BP}-N(2)-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A>T>or=G>C . To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure . We modeled the (+)-trans-anti-{BP}-N(2)-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct . The modeling required that the (+)-trans-anti-{BP}-N(2)-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions . While the dATP: (+)-trans-anti-{BP}-N(2)-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation . Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-{BP}-N(2)-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond . The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-{BP}-N(2)-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively . In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide . The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation . The simulations of dTTP and dGTP opposite (+)-trans-anti-{BP}-N(2)-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system . However, the active-site pocket size of the dTTP and dGTP simulations remained stable . The dCTP: (+)-trans-anti-{BP}-N(2)-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct . These simulations elucidated why A is inserted opposite (+)-trans-anti-{BP}-N(2)-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated . Structural rationalization of the incorporation preference opposite (+)-trans-anti-{BP}-N(2)-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.

Nature, 2002 Sep 5, 419(6902), 43 - 8 Epub 2002 Jul 31.
Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase; Di Noia J et al.; A functional immune system depends on the production of a wide range of immunoglobulin molecules . Immunoglobulin variable region (IgV) genes are diversified after gene rearrangement by hypermutation . In the DNA deamination model, we have proposed that deamination of dC residues to dU by activation-induced deaminase (AID) triggers this diversification . In hypermutating chicken DT40 B cells, most IgV mutations are dC --> dG/dA or dG --> dC/dT transversions, which are proposed to result from replication over sites of base loss produced by the excision activity of uracil-DNA glycosylase . Blocking the activity of uracil-DNA glycosylase should instead lead to replication over the dU lesion, resulting in dC --> dT (and dG --> dA) transitions . Here we show that expression in DT40 cells of a bacteriophage-encoded protein that inhibits uracil-DNA glycosylase shifts the pattern of IgV gene mutations from transversion dominance to transition dominance . This is good evidence that antibody diversification involves dC --> dU deamination within the immunoglobulin locus itself.

RNA, 2002 Aug, 8(8), 981 - 96
Comparative studies of frameshifting and nonframeshifting RNA pseudoknots: a mutational and NMR investigation of pseudoknots derived from the bacteriophage T2 gene 32 mRNA and the retroviral gag-pro frameshift site; Wang Y et al.; Mutational and NMR methods were used to investigate features of sequence, structure, and dynamics that are associated with the ability of a pseudoknot to stimulate a -1 frameshift . In vitro frameshift assays were performed on retroviral gag-pro frameshift-stimulating pseudoknots and their derivatives, a pseudoknot from the gene 32 mRNA of bacteriophage T2 that is not naturally associated with frameshifting, and hybrids of these pseudoknots . Results show that the gag-pro pseudoknot from human endogenous retrovirus-K10 (HERV) stimulates a -1 frameshift with an efficiency similar to that of the closely related retrovirus MMTV . The bacteriophage T2 mRNA pseudoknot was found to be a poor stimulator of frameshifting, supporting a hypothesis that the retroviral pseudoknots have distinctive properties that make them efficient frameshift stimulators . A hybrid, designed by combining features of the bacteriophage and retroviral pseudoknots, was found to stimulate frameshifting while retaining significant structural similarity to the nonframeshifting bacteriophage pseudoknot . Mutational analyses of the retroviral and hybrid pseudoknots were used to evaluate the effects of an unpaired (wedged) adenosine at the junction of the pseudoknot stems, changing the base pairs near the junction of the two stems, and changing the identity of the loop 2 nucleotide nearest the junction of the stems . Pseudoknots both with and without the wedged adenosine can stimulate frameshifting, though the identities of the nucleotides near the stem1/stem2 junction do influence efficiency . NMR data showed that the bacteriophage and hybrid pseudoknots are similar in their local structure at the junction of the stems, indicating that pseudoknots that are similar in this structural feature can differ radically in their ability to stimulate frameshifting . NMR methods were used to compare the internal motions of the bacteriophage T2 pseudoknot and representative frameshifting pseudoknots . The stems of the investigated pseudoknots are similarly well ordered on the time scales to which nitrogen-15 relaxation data are sensitive; however, solvent exchange rates for protons at the junction of the two stems of the nonframeshifting bacteriophage pseudoknot are significantly slower than the analogous protons in the representative frameshifting pseudoknots.

Biotechnol Bioeng, 2002 Oct 20, 80(2), 233 - 6
Separating lysozyme from bacteriophage P22 in two-phase aqueous micellar systems; Kamei DT et al.; This communication demonstrates that two-phase aqueous mixed (nonionic/ionic) micellar systems have the potential for improving the separation of proteins from viruses . Specifically, two separation experiments were performed to show that the addition of the anionic surfactant sodium dodecyl sulfate (SDS) to the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C(10)E(4)) micellar system increases the yield of a model net positively charged protein, lysozyme, in the micelle-rich phase from 75 to 95%, while still maintaining approximately the same yield of a model net negatively charged virus, bacteriophage P22, in the micelle-poor phase (97% vs . 98%) .

Genesis, 2002 Aug, 33(4), 191 - 7
Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements; Forde A et al.; Summary: The versatility of the bacteriophage Cre/LoxP system is dependent on the availability of a spectrum of tissue-specific Cre transgenic mice to address a host of biological questions . In this paper, we report on the generation of an inducible Tie2Cre transgenic mouse line that facilitates gene targeting exclusively in endothelial cells . The temporal manner of recombination is feasible through the use of a Cre-estrogen receptor fusion protein ER(T2) and was, in practical terms, achieved by feeding the animals the estrogen antagonist tamoxifen orally for 5 weeks . High efficiency of recombination was found in the vast majority of endothelial cell populations examined, as monitored by an EGFP reporter mouse line . Critically, no EGFP expression was observed in any uninduced mice . This inducible Cre line will be a very beneficial asset to investigating the role of endothelial specific genes in the adult mouse and to induce transgenes in the endothelium in an extremely efficient manner . genesis 33:191-197, 2002 .

Virology, 2002 Aug 1, 299(2), 182 - 91
Microarray analysis of gene expression during bacteriophage T4 infection; Luke K et al.; Genomic microarrays were used to examine the complex temporal program of gene expression exhibited by bacteriophage T4 during the course of development . The microarray data confirm the existence of distinct early, middle, and late transcriptional classes during the bacteriophage replicative cycle . This approach allows assignment of previously uncharacterized genes to specific temporal classes . The genomic expression data verify many promoter assignments and predict the existence of previously unidentified promoters.

Virology, 2002 Aug 15, 300(1), 71 - 8
Catalytic efficiency and phenotype of HIV-1 proteases encoding single critical resistance substitutions; Cabana M et al.; We have shown that a bacteriophage lambda genetic screen system may be useful in predicting the activity and phenotype of HIV-1 protease in the course of viral infection and antiretroviral therapy . This simple and rapid genetic screening system has been used here to characterize HIV-1 proteases encoding single primary resistance substitutions . Except for proteases with amino acid changes at positions 46 and 84, proteases containing single-resistance substitutions displayed a lower catalytic efficiency than the WT enzyme . Single mutants could be identified by their efficiency, demonstrating that modest differences in protease activity can be monitored with this simple assay . Overall, drug susceptibility could be reduced by introduction of single mutations . However, high-level protease inhibitor (PI) resistance was only achieved by multiple mutated proteases . The small but reproducible increase in resistance displayed by single mutants also demonstrated the ability of this genetic screen system for detecting minor reductions in drug susceptibility . These results show that the bacteriophage lambda genetic screen system used here is a useful tool in the analysis of specific contribution of mutations in the HIV protease-coding region or in specific cleavage sites that affect the process of PI resistance.

J Bacteriol, 2002 Sep, 184(18), 5200 - 3
Interactions between integrase and excisionase in the phage lambda excisive nucleoprotein complex; Cho EH et al.; Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome . Lambda integrase (Int) carries out both reactions . During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex . The carboxyl-terminal alpha-helix of Xis is thought to interact with Int through direct protein-protein interactions . In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis . This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.

J Bacteriol, 2002 Sep, 184(18), 4952 - 61
N4 RNA polymerase II, a heterodimeric RNA polymerase with homology to the single-subunit family of RNA polymerases; Willis SH et al.; Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII) . Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3 . These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases . The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed . Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.

J Immunol Methods, 2002 Jun 1, 264(1-2), 163 - 71
An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries; Al-bukhari TA et al.; Libraries of random peptides displayed by bacteriophage can be screened to select phage expressing peptides that specifically bind antibodies, so that the peptide sequence motifs expressed by the phage can help to define the epitopes of the antibodies . It is often desirable to screen antibody-selected phage for binding of the selecting antibody in an immunoassay in order to verify the specificity of the interaction . Enzyme-linked immunosorbent assays (ELISAs) are commonly used for this purpose . However, for many antibodies, the best techniques for measuring specific, high affinity interactions are immuno-precipitation assays . Immuno-precipitation was therefore investigated as a means of measuring interactions between antibodies and phage clones selected from random peptide display libraries . Three mouse monoclonal antibodies specific for glutamic acid decarboxylase were used to select peptides as 9-mers on T7 phage, linear 12-mers on pIII of M13 phage, or constrained 15-mers on pVIII of M13 phage . Following the cloning and sequencing of selected phage, mixtures of antibody and phage were incubated in solution and the immune complexes were precipitated with Protein G bound to Sepharose beads . In order to detect and quantitate the phage that had formed immune complexes and been precipitated, advantage was taken of the biological properties of the phage by inducing infection of Escherichia coli by the precipitated phage . The aim was to quantitate the phage precipitated by determining the number of plaques produced, which would therefore be proportional to the degree of interaction between the phage and the antibody in solution . The results presented here indicate that this method of measuring monoclonal antibody interactions with phage selected for expression of peptides recognised by the monoclonal antibody is highly specific and sensitive.

EMBO Rep, 2002 Sep, 3(9), 893 - 8 Epub 2002 Aug 16.
Bacteriophage-encoded cochaperonins can substitute for Escherichia coli's essential GroES protein; Keppel F et al.; The Escherichia coli chaperonin machine is composed of two members, GroEL and GroES . The GroEL chaperonin can bind 10-15% of E . coli's unfolded proteins in one of its central cavities and help them fold in cooperation with the GroES cochaperonin . Both proteins are absolutely essential for bacterial growth . Several large, lytic bacteriophages, such as T4 and RB49, use the host-encoded GroEL in conjunction with their own bacteriophage-encoded cochaperonin for the correct assembly of their major capsid protein, suggesting a cochaperonin specificity for the in vivo folding of certain substrates . Here, we demonstrate that, when the cochaperonin of either bacteriophage T4 (Gp31) or RB49 (CocO) is expressed in E . coli, the otherwise essential groES gene can be deleted . Thus, it appears that, despite very little sequence identity with groES, the bacteriophage-encoded Gp31 and CocO proteins are capable of replacing GroES in the folding of E . coli's essential, housekeeping proteins.

J Biol Chem, 2002 Oct 25, 277(43), 40640 - 9 Epub 2002 Aug 19.
Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA polymerases; Fidalgo da Silva E et al.; The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides . Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation . The most significant difference detected was in the K(d) for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (approximately 367 microm) than for incorporation of dTMP opposite template A (approximately 31 microm) . In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s(-1) with template A to about 165 s(-1) for template 2AP . Discrimination is due to the high selectivity in the initial nucleotide-binding step . T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides . If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.

J Biochem Mol Biol Biophys, 2002 Feb, 6(1), 55 - 8
Cloning, expression and display of the PreS domain of hepatitis B virus on filamentous bacteriophage M13; Kok WL et al.; The PreS domain of hepatitis B virus (HBV) is believed to be involved in virion assembly and attachment to a hepatocyte receptor during infection . In order to study the functions of this region, we fused it to the g3p protein of bacteriophage M13 that allows the fusion protein to be displayed at the tip of the filament . The fusion protein was detected by the anti-E tag antibody on a Western blot . The polypeptide in a soluble form was produced by transfecting a non-suppressor E . coli host cell with the recombinant phagemid . The soluble protein was detected in cytoplasm, in the periplasmic space and also in the medium . The functional display of the PreS domain would provide an alternative means to study its interactions with the nuleocapsid and hepatocytes.

Acad Radiol, 2002 Aug, 9(8), 927 - 32
Development of novel tumor imaging agents with phage-display combinatorial peptide libraries; Campa MJ et al.; RATIONALE AND OBJECTIVES: Current radiologic methods do not provide sufficient information for unambiguous diagnosis and prognosis of cancer . The present investigation sought to address this deficiency by developing a system for designing novel small molecules targeted against tumor-specific molecules for use as radionuclide imaging agents . MATERIALS AND METHODS: Part of a tumor-specific receptor, purified recombinant epidermal growth factor receptor (EGFR), variant III, extracellular domain (rEGFRvIII-ecd), was used as the target in the selection of EGFRvIII-specific peptide ligands from random peptide bacteriophage (phage) display libraries . After three rounds of screening, phage isolates were tested for binding affinity with an enzyme-linked immunosorbent assay . Positive phage were sequenced, and the peptides were synthesized and tested for binding affinity with a surface plasmon resonance assay . RESULTS: Affinity screening identified 49 peptide-expressing phage that showed enhanced binding to the variant receptor compared with wild-type EGFR . Free peptides from the two phage isolates exhibiting the most favorable binding were tested for target binding . One of these demonstrated a binding affinity for rEGFRvIII-ecd in the 30-nmol/L range . CONCLUSION: These data suggest that phage display libraries may be very useful in the design of novel, high-affinity tumor imaging agents.

J Gen Virol, 2002 Sep, 83(Pt 9), 2215 - 23
Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes; Potgieter AC et al.; Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic . Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes . Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Phi12 . Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0.8 to 6.8 kb . Rotavirus dsRNA was extracted directly from stool samples . Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres . This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5' GUU and UAC 3') and coding assignment as AHSV and BTV . To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification . The method is simple and efficient compared to other methods . Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22-30 cycles compared to 30-35 of other methods) . This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.

J Contam Hydrol, 2002 Aug, 57(3-4), 259 - 79
Two-site kinetic modeling of bacteriophages transport through columns of saturated dune sand; Schijven JF et al.; Breakthrough curves, on a semi-log scale, from tests in porous media with block-input of viruses, bacteria, protozoa and colloidal particles often exhibit a typical skewness: a rather slowly rising limb and a smooth transition of a declining limb to a very long tail . One-site kinetic models fail to fit the rising and declining limbs together with the tail satisfactorily . Inclusion of an equilibrium adsorption site does not seem to improve simulation results . This was encountered in the simulation of breakthrough curves from a recent field study on the removal of bacteriophages MS2 and PRD1 by passage through dune sand . In the present study, results of laboratory experiments for the study of this issue are presented . Breakthrough curves of salt and bacteriophages MS2, PRDI, and phiX174 in 1 D column experiments have been measured . One- and two-site kinetic models have been applied to fit and predict breakthrough curves from column experiments . The two-site model fitted all breakthrough curves very satisfactorily, accounting for the skewness of the rising limb as well as for the smooth transition of the declining limb to the tail of the breakthrough curve . The one-site model does not follow the curvature of the breakthrough tail, leading to an overestimation of the inactivation rate coefficient for attached viruses . Interaction with kinetic site 1 is characterized by relatively fast attachment and slow detachment, whereas attachment to and detachment from kinetic site 2 is fast . Inactivation of viruses and interaction with kinetic site 2 provide only a minor contribution to removal . Virus removal is mainly determined by the attachment to site 1 . Bacteriophage phiX174 attached more than MS2 and PRD1, which can be explained by the greater electrostatic repulsion that MS2 and PRD1 experience compared to the less negatively charged phiX174.

Mol Gen Mikrobiol Virusol, 2002, (2), 23 - 6
{RNA transduction by phage MS2 noninfectious virions}; Kniazhev VA et al.; Two fragments (1.9 and 1.8 kb) including, respectively, "structural" genes (A, B, and lysis polypeptide) and "replicase" gene, were obtained by reverse transcription PCR on the template of bacteriophage MS2 genome RNA . The fragments contained a common sequence which allowed the assembly of the entire functional genome sDNA . 1.8 kb fragment was subjected to two independent modifications: 1) so that the replicase gene could be replaced by other sequences no more than 1.5 kb in size and 2) it was inserted (on an artifician Tn-like structure) into E . coli chromosome . A system for replication of MS2-like corpuscles was thus created, containing a "shortened" genome and having extra genes . For extra genes, aminoglycoside-3'-phosphotransferase (kanamycin resistance) or a sequence of antiviral genetic program directed against hepatitis C virus were used.

J Biol Chem, 2002 Nov 1, 277(44), 41954 - 9 Epub 2002 Aug 09.
Myo-inositol monophosphatase is an activated target of calbindin D28k; Berggard T et al.; Calbindin D(28k) (calbindin) is a member of the calmodulin superfamily of Ca(2+)-binding proteins . An intracellular target of calbindin was discovered using bacteriophage display . Human recombinant calbindin was immobilized on magnetic beads and used in affinity purification of phage-displayed peptides from a random 12-mer peptide library . One sequence, SYSSIAKYPSHS, was strongly selected both in the presence of Mg(2+) and in the presence of Ca(2+) . Homology search against the protein sequence data base identified a closely similar sequence, ISSIKEKYPSHS, at residues 55-66 in myo-inositol-1(or 4)-monophosphatase (IMPase, EC ), which constitute a strongly conserved and exposed region in the three-dimensional structure . IMPase is a key enzyme in the regulation of the activity of the phosphatidylinositol-signaling pathway . It catalyzes the hydrolysis of myo-inositol-1(or 4)-monophosphate to form free myo-inositol, maintaining a supply that represents the precursor for inositol phospholipid second messenger signaling systems . Fluorescence spectroscopy showed that isolated calbindin and IMPase interact with an apparent equilibrium dissociation constant, K(D), of 0.9 microm . Both apo and Ca(2+)-bound calbindin was found to activate IMPase up to 250-fold, depending on the pH and substrate concentration . The activation is most pronounced at conditions that otherwise lead to a very low activity of IMPase, i.e . at reduced pH and at low substrate concentration.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(3), 263 - 266
Screening of Cellulose Binding Motif (CBM) from Phage Peptides Library; Han ZZ et al.; Bacteriophages capable of binding cellulose matrix were screened from a 15-mer phage peptides library . In the deduced amino acid sequences of the screened phages, a characteristic region containing a conserved aromatic residue {tyrosine (Y) or phenylalanine (F)} was found, which is similar to the normal cellulose binding domains found in fungal and bacterial cellulose catalysase . Dot-ELISA showed that the phage containing sequence as SWYL has higher affinity to cellulose fibre than other phages, such as those containing sequences as CWYGNC, CWYGEC and XSWYDXXSWFSX . Results indicated that SWYL maybe a good candidate for cellulose binding motif . This work laid basis for further study on cellulose binding motif.

Vopr Virusol, 2002 May-Jun, 47(3), 34 - 7
{Detection of DNA packing density in bacteriophages Phi KZ and T4 by atomic force microscopy}; Klinov DV et al.; Comparative analysis of DNA packing density in Phi KZ and T4 bacteriophages was carried out by atomic force microscopy . Irrespective of the support (mica or highly ordered pyrrolytic graphite), Phi KZ bacteriophage was compressed stronger than T4 . The most probable causes of this difference are analyzed.

J Protein Chem, 2002 May, 21(4), 287 - 96
Identification and characterization of peptides that bind human ErbB-2 selected from a bacteriophage display library; Karasseva NG et al.; The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies . The overexpression of ErbB-2 in cancer cells as well as its extracellular accessibility makes it an attractive target for the development of tumor-specific agents . In this study, random peptide bacteriophage display technology was employed to identify peptides that bound the extracellular domain of human ErbB-2 . The peptide KCCYSL, most frequently occurring in the affinity-selected phage population, was chemically synthesized and characterized for its binding activities to ErbB-2 . The synthetic peptide exhibited high specificity for ErbB-2 and an equilibrium dissociation constant of 30 microM . Peptide binding to ErbB-2 positive human breast and prostate carcinoma cells was visualized in direct cell binding assays . In conclusion, the peptide KCCYSL has the potential to be developed into a cancer imaging or therapeutic agent targeting malignant cells overexpressing the ErbB-2 receptor.

Theor Popul Biol, 2002 Jun, 61(4), 471 - 80
Bacteriophages: evolution of the majority; Hendrix RW; The dsDNA-tailed bacteriophages are probably the largest evolving group in the Biosphere and they are arguably very ancient . Comparative examination of genomes indicates that the hallmark of phage evolution is horizontal exchange of sequences . This is accomplished, first, by rampant non-homologous recombination between different genomes and, second, by reassortment of the variant sequences so created through homologous recombination . The comparative analysis suggests mechanisms by which new genes can be added to phage genomes and by which genes with novel functions may be assembled from parts . Horizontal exchange of sequences occurs most frequently among closely related phages, but it also extends across the entire global population at lower frequency . Bacteriophages also have probable ancestral connections with viruses of eukaryotes and archaea .

RNA, 2002 Jul, 8(7), 924 - 32
Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution; Lima S et al.; The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine . In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix . This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom . The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay . Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com.

EMBO Rep, 2002 Aug, 3(8), 792 - 7 Epub 2002 Jul 15.
The Methanobacterium thermoautotrophicum MCM protein can form heptameric rings; Yu X et al.; Mini-chromosome maintenance (MCM) proteins form a conserved family found in all eukaryotes and are essential for DNA replication . They exist as heteromultimeric complexes containing as many as six different proteins . These complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks . In most archaea a single MCM protein exists . The protein from Methanobacterium thermoautotrophicum (mtMCM) has been reported to assemble into a large complex consistent with a dodecamer . We show that mtMCM can assemble into a heptameric ring . This ring contains a C-terminal helicase domain that can be fit with crystal structures of ring helicases and an N-terminal domain of unknown function . While the structure of the ring is very similar to that of hexameric replicative helicases such as bacteriophage T7 gp4, our results show that such ring structures may not be constrained to have only six subunits.

Plasmid, 2002 May, 47(3), 210 - 5
Lambdap(o), a promoter for oop RNA synthesis, has a role in replication of plasmids derived from bacteriophage lambda; Potrykus K et al.; Transcription initiated at the bacteriophage lambdap(o) promoter gives a short RNA, called oop RNA . Early studies led to a proposal that this transcript plays a role in the initiation of lambda DNA replication . In fact, the p(o) promoter is located in the lambda replication region and it was suggested that oop RNA may be a primer for replication proceeding leftward from orilambda . However, since in vitro experiments demonstrated that primers for lambda DNA replication are produced by the dnaG gene product (DnaG primase) and subsequent in vivo studies indicated that oop RNA is an antisense RNA for the lambda cII gene expression, the above-mentioned hypothesis has fallen into oblivion . Nevertheless, here we demonstrate that the p(o) promoter plays a role in lambda DNA replication, indeed . We found that lambda plasmids bearing a mutation that inactivates p(o) occur in Escherichia coli cells in a copy number significantly lower than wild-type lambda plasmids . Amplification of lambdap(o)(-) plasmids during the relaxed response was less efficient relative to lambdap(o)(+) plasmids suggesting less frequent initiation of replication from orilambda in the absence of transcription from p(o) . This suggestion was confirmed by measurement of incorporation of {(3)H}thymidine into lambda plasmid DNA during pulse-labeling experiments . Therefore, we propose that transcription from the p(o) promoter stimulates replication initiation at orilambda as suggested a long time ago, however, contrary to that suggestion, we assume that the process of p(o)-initiated transcription per se but not the transcription product (oop RNA) might play a role at early steps of lambda DNA replication.

EMBO J, 2002 Aug 1, 21(15), 4154 - 61
The global regulator RNase III modulates translation repression by the transcription elongation factor N; Wilson HR et al.; Efficient expression of most bacteriophage lambda early genes depends upon the formation of an antiterminating transcription complex to overcome transcription terminators in the early operons, p(L) and p(R) . Formation of this complex requires the phage-encoded protein N, the first gene product expressed from the p(L) operon . The N leader RNA contains, in this order: the NUTL site, an RNase III-sensitive hairpin and the N ribosome-binding site . N bound to NUTL RNA is part of both the antitermination complex and an autoregulatory complex that represses the translation of the N gene . In this study, we show that cleavage of the N leader by RNase III does not inhibit antitermination but prevents N-mediated translation repression of N gene expression . In fact, by preventing N autoregulation, RNase III activates N gene translation at least 200-fold . N-mediated translation repression is extremely sensitive to growth rate, reflecting the growth rate regulation of RNase III expression itself . Given N protein's critical role in lambda development, the level of RNase III activity therefore serves as an important sensor of physiological conditions for the bacteriophage.

J Mol Biol, 2002 Aug 9, 321(2), 249 - 63
Blocking transcription through a nucleosome with synthetic DNA ligands; Gottesfeld JM et al.; Pyrrole-imidazole (Py-Im) polyamides are synthetic ligands that bind in the minor groove of DNA . Previous studies have established that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible for molecular recognition by Py-Im polyamides, and that nucleosomes remain fully folded upon ligand binding . Two polyamides that bind within the sea urchin 5S gene nucleosome positioning sequence inhibit both heat-induced nucleosome sliding and transcription by bacteriophage T7 RNA polymerase from the nucleosomal template, but not from histone-free DNA . These polyamides prevent repositioning of the histone octamer by RNA polymerase, and thereby inhibit passage of the elongating polymerase through nucleosomal DNA . These results establish unambiguously the requirement for octamer mobility for transcription of nucleosomal templates by T7 RNA polymerase.

J Bacteriol, 2002 Aug, 184(16), 4626 - 9
Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12; Poteete AR et al.; The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted . Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways . The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell . Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny . The results of these experiments indicated that the lambda rap gene could functionally substitute for the E . coli ruvC gene in Red-mediated recombination.

Annu Rev Genomics Hum Genet, 2002, 3, 341 - 69 Epub 2002 Apr 15.
Genetic "code": representations and dynamical models of genetic components and networks; Gilman A et al.; Dynamical modeling of biological systems is becoming increasingly widespread as people attempt to grasp biological phenomena in their full complexity and make sense of an accelerating stream of experimental data . We review a number of recent modeling studies that focus on systems specifically involving gene expression and regulation . These systems include bacterial metabolic operons and phase-variable piliation, bacteriophages T7 and lambda, and interacting networks of eukaryotic developmental genes . A wide range of conceptual and mathematical representations of genetic components and phenomena appears in these works . We discuss these representations in depth and give an overview of the tools currently available for creating and exploring dynamical models . We argue that for modeling to realize its full potential as a mainstream biological research technique the tools must become more general and flexible, and formal, standardized representations of biological knowledge and data must be developed.

Nucleic Acids Res, 2002 Aug 1, 30(15), 3341 - 8
RNA determinants of translational operator recognition by the DNA polymerases of bacteriophages T4 and RB69; Petrov VM et al.; The DNA polymerases (gp43s) of the two related phages T4 and RB69 are DNA-binding proteins that also function as mRNA-binding autogenous translational repressors . As repressors, T4 gp43 is narrowly specific to its own mRNA whereas RB69 gp43 is equally effective against mRNA for either protein . We used in vitro RNase-sensitivity and RNA footprinting assays to identify features of the non-identical T4 and RB69 mRNA targets (translational operators) that allow for their identical binding affinities and biological responses to RB69 gp43 . We observed that T4 gp43 and RB69 gp43 produce identical footprints on RNA substrates bearing the T4-derived operator, suggesting that the two gp43s make identical contacts with this operator . In contrast, the footprint produced by RB69 gp43 on its autogenous RNA target was shorter than its footprint on operator RNA from T4 . As expected, we also observed only weak protection of RB69-derived operator RNA from RNase by T4 gp43; however, photocross-linking studies suggested that T4 gp43 recognizes structural features of the RB69-derived operator that are not detected by RNase- sensitivity assays . The results suggest that RB69 gp43 and T4 gp43 differ in their abilities to use RNA-sequence-independent interactions to configure potential RNA targets for translational repression.

Nucleic Acids Res, 2002 Aug 1, 30(15), 3323 - 32
Effect of manganese on in vitro replication of damaged DNA catalyzed by the herpes simplex virus type-1 DNA polymerase; Villani G et al.; In vitro bypass of damaged DNA by replicative DNA polymerases is usually blocked by helix-distorting or bulky DNA lesions . In this study, we report that substitution of the divalent metal ion Mg2+ with Mn2+ promotes quantitative replication of model DNA substrates containing the major cisplatin or N-2-acetylaminofluorene adducts by the catalytic subunit (UL30) of the replicative DNA polymerase of herpes simplex virus . The ability of Mn2+ ions to confer bypass of bulky lesions was not observed with other replicative DNA polymerases of the B family, such as bacteriophage T4 or delta polymerases . However, for these enzymes, manganese induced the incorporation of one nucleotide opposite the first (3') guanine of the d(GpG) intrastrand cisplatin lesion . Translesion replication of the cisplatin adduct by UL30 led to the incorporation of mismatched bases, with the preferential incorporation of dAMP opposite the 3' guanine of the lesion . Furthermore, substitution of MgCl2 with MnCl2 greatly inhibited the 3' to 5' exonuclease of UL30 but had a far lesser effect on that of T4 DNA polymerase . Finally, manganese induced a conformational change in the structure of UL30 bound to the platinated substrate . Taken together, the latter findings suggest a mechanism by which manganese might allow UL30 to efficiently promote translesion DNA synthesis in vitro.

Mol Microbiol, 2002 Aug, 45(3), 697 - 710
Action at a distance in CI repressor regulation of the bacteriophage 186 genetic switch; Dodd IB et al.; The non-lambdoid coliphage 186 provides an alternative model to the lytic-lysogenic switch of phage lambda . Like lambda, the key switch regulator, the CI repressor, associates to octamers . Unlike lambda, the lytic promoter (pR) and the lysogenic promoter (pL) are face-to-face, 62 bp apart and are flanked by distal CI binding sites (FL and FR) located approximately 300 bp away . Using reporter and footprinting studies, we show that the outcome, but not the mechanism, of regulation by 186 CI is very similar to lambda . 186 CI stimulates pL transcription indirectly by repressing convergent interfering transcription from pR . However, in the absence of the flanking FL and FR sites, CI bound at pR interacts co-operatively with a weak CI binding site at pL and represses both promoters . FL and FR play a critical role; they assist repression of pR and simultaneously alleviate repression of pL, thus allowing high pL activity . We propose that the 186 switch is regulated by a novel mechanism in which a CI octamer bound at pR forms alternative DNA loops to pL or to a flanking site, depending on CI concentration.

Biochemistry (Mosc), 2002 Jul, 67(7), 815 - 21
Chaperonin-mediated folding of bacteriophage T4 major capsid protein . II . Production of gene product 23 deletion mutants; Aijrich LG et al.; Folding of bacteriophage T4 major capsid protein, gene product 23 (534 a.a.), is aided by two proteins: E . coli GroEL chaperonin and viral gp31 co-chaperonin . In the present work a set of mutants with extensive deletions inside gene 23 using controlled digestion with Bal31 nuclease has been constructed . Proteins with deletions were co-expressed from plasmid vectors with phage gp31 co-chaperonin . Deletions from 8 to 33 a.a . in the N-terminal region of the gp23 molecule covering the protein proteolytic cleavage site during capsid maturation have no influence on the mutants' ability to produce in E . coli cells proteins which form regular structures--polyheads . Deletions in other regions of the polypeptide chain (187-203 and 367-476 a.a.) disturb the correct folding and subsequent assembly of gp23 into polyheads.

J Infect Dis, 2002 Aug 1, 186(3), 432 - 5 Epub 2002 Jul 11.
Role of bacteriophage MAV1 as a mycoplasmal virulence factor for the development of arthritis in mice and rats; Tu AH et al.; The lysogenic bacteriophage MAV1 has been shown to be a virulence factor for the development of arthritis in rats infected with Mycoplasma arthritidis . In the present study, arthritis was evaluated by histopathologic examination to demonstrate that MAV1 is a virulence factor not only in the rat but also in the mouse . Specifically, the MAV1 lysogen 158L3-1 was more virulent than the nonlysogen strain 158 in DBA/2NCr, C3H/HeNCr, C3H/HeJ, and C3Smn.CB17-Prkdc(scid)/J mice, as well as in LEW rats.

Virology, 2002 Jul 5, 298(2), 224 - 31
The structure of P4 procapsids produced by coexpression of capsid and external scaffolding proteins; Dokland T et al.; The double-stranded DNA bacteriophage P4 has a T = 4 icosahedral arrangement of the gpN capsid protein derived from the P2 helper phage . The precursor procapsids in addition contain an external scaffold made up of the P4-encoded Sid protein . High yields of pure P4 procapsids have been obtained by coexpressing the gpN and Sid proteins from a chimeric plasmid . Biochemical measurements show that the ratio of gpN to Sid in the procapsids is 2:1, corresponding to 120 copies of Sid per procapsid particle . A reconstruction of the P4 procapsid, made from 213 particle images to an effective resolution of about 21 A, greatly improves on the previously determined P4 procapsid structures . The structure shows a T = 4 capsid shell and a unique tandem arrangement of 120 copies of chilli-shaped Sid monomers, which form trimers and dimers on the procapsid surface.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 45 - 9
Phage HK022-based integrative vectors for the insertion of genes in the chromosome of multiply marked Escherichia coli strains; Rossignol M et al.; We constructed a series of plasmids that allow the insertion of cloned DNA in the Escherichia coli chromosome by site-specific integration into the bacteriophage HK022 bacterial attachment site . These plasmids make use of a ColE1 origin of replication, the phage HK022 attachment site attP, antibiotic resistance genes for selection and unique restriction sites . Circularisation of non-replicative fragments containing the HK022 attachment site attP is performed in vitro and site-specific integration of attP containing molecules is ensured by transfer into cells transiently expressing the HK022 integrase gene carried by a thermosensitive replicon . Insertion is very efficient and the inserted fragments are stably maintained without selection pressure . Since integrative fragments carry rarely used antibiotic markers conferring resistance to antibiotics hygromycin or apramycin, they can be used in most E . coli strains in conjunction with many replicative or integrative vectors.

J Chromatogr, 1992 Nov 13, 625(1), 41 - 6
Purification of the C1 repressor of bacteriophage P1 by fast protein liquid chromatography; Velleman M et al.; A fast protein liquid chromatographic method is described for the purification of the C1 repressor of bacteriophage P1 and its truncated form C1* . By using one crude extract, both repressor proteins were purified in parallel to homogeneity and were shown to interact specifically with P1 operator DNA in vitro . The method involves an affinity chromatographic step on heparin-Sepharose, followed by a combination of ion-exchange chromatography on Q Sepharose and S Sepharose . The availability of a homogeneous preparation of the phage repressor is a prerequisite for studies on its structure-function relationship.

FEBS Lett, 2002 Jul 17, 523(1-3), 68 - 72
Murine peripherin gene sequences direct Cre recombinase expression to peripheral neurons in transgenic mice; Zhou L et al.; Spatially and temporally regulated somatic mutations can be achieved by using the Cre/loxP recombination system of bacteriophage P1 . To develop a cell type-specific system of gene targeting in the peripheral nervous system, we generated the transgenic mouse lines expressing Cre recombinase under the control of the mouse peripherin gene promoter . The activity of the Cre recombinase during embryonic development was examined by mating the peripherin-Cre transgenic mice to the knock-in Cre-mediated recombination reporter strain, R26R . Analysis of F1 embryos from this cross showed specific excision of loxP-flanked sequences in the dorsal root ganglia, trigeminal ganglia, and olfactory epithelium, in a pattern very similar to the expression of the endogenous mouse peripherin gene, and the previously reported peripherin-lacZ transgenic mice . Thus, the peripherin-Cre mouse described here will provide a valuable tool for Cre-loxP-mediated conditional expression in the peripheral nervous system.

Chem Commun (Camb), 2002 May 21, (10), 1086 - 7
In vitro inhibition of gene transcription by novel photo-activated polyazaaromatic ruthenium(II) complexes; Pauly M et al.; Under visible irradiation, {Ru(TAP)2(phen)}2+(Cl-)2, {Ru(TAP)2(POQ-Nmet)}2+(Cl-)2 and {Ru(bpy)2(phen)}2+(Cl-)2 were able to dramatically reduce the in vitro transcription of a plasmid DNA template by a bacteriophage RNA polymerase . This photoactivity is related to two different mechanisms of reactivity towards DNA exhibited by these complexes under illumination.

J Biochem Biophys Methods, 2002 Jun 28, 52(1), 31 - 43
An approach to increased polyplex gene delivery by peptides selected from a phage display library; Morpurgo M et al.; Phage display libraries were screened for peptides to be incorporated in nonviral gene delivery vehicles . Cells in culture were incubated with heptamer random peptide libraries displayed on M13 bacteriophages in three to five copies per phage . Surface-adherent phages were removed or inactivated and the cells were fractionated in a nuclear pellet and supernatant . Bacteriophages from each of the two fractions were amplified and reincubated with the cells . Three successive rounds of selection were performed . Eighteen sequenced clones revealed 14 different sequences . Two sequences were homologous to segments of the HIV gp120 protein . For three sequences, the corresponding synthetic peptides were generated and attached via avidin-biotin to polylysine-condensed plasmid DNA containing a reporter gene . The addition of the peptides led to 8-14 times increase in the expression of the reporter.

Biochemistry, 2002 Jul 23, 41(29), 9222 - 8
Histone h1(0) and its carboxyl-terminal domain bind in the major groove of DNA; Mamoon NM et al.; The binding of histone H1(0) to T4 bacteriophage DNA was investigated using thermal denaturation of the DNA titrated with varying concentrations of protein . The H1(0) used was expressed in and purified from a strain of E . coli and is therefore homogeneous with respect to H1 subtype and posttranslational modifications . Two types of T4 DNA were used: wild-type, which contains a modification of the cytosine residues that projects into the major groove: and a mutant type, which lacks the modification of the cytosines . Data were compared to simulated thermal denaturation curves to determine estimates for binding affinity and binding site size in base pairs of the protein . Analysis of the data yielded values of 10(8) M(-1) for K, the binding affinity, and 10 base pairs for n, the number of base pairs covered by one protein, for the mutant T4 DNA . Analysis of the wild-type DNA data suggested that the glucose projecting into the major groove of this DNA decreases the number of sites to which the H1(0) protein can bind, indicating that there are interactions between the protein and the major groove of DNA . The binding site size on this DNA is 10 base pairs, the same as on the unmodified DNA . The affinity for wild-type DNA is slightly higher, 10(9) M(-1) . Data were collected and analyzed for binding of two domains of the protein as well, the carboxyl-terminal domain and the central globular domain . Binding of the carboxyl-terminal domain was quantitatively and qualitatively similar to that of the full-length protein . In contrast, binding of the globular domain was quite different: it binds much more weakly, with a K of 6 x 10(4) M(-1), and covers fewer base pairs, with an n of 3 . Also, there was no evidence that the globular domain interacts with the major groove of DNA.

Infect Immun, 2002 Aug, 70(8), 4282 - 91
One of two copies of the gene for the activatable shiga toxin type 2d in Escherichia coli O91:H21 strain B2F1 is associated with an inducible bacteriophage; Teel LD et al.; Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction . Among Stx2 variants, only stx(2e) from one human STEC isolate has been reported to be carried within a toxin-converting phage . In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages . We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant . Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection . When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant . Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant . Moreover, an stx(2d1)-containing lysogen was isolated from plaques on strain DH5alpha that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C . Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W . Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated . We conclude that despite the sequence similarity of the stx(2d1)- and stx(2d2)-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction.

IUBMB Life, 2002 Mar, 53(3), 161 - 6
LAST motifs and SMART domains in gene 32 protein: an unfolding story of autoregulation?
Karpel RL.
Bacteriophage T4 gene 32 protein is a classical single strand-specific DNA binding protein . It is a single polypeptide chain of 301 amino acid residues that consists of three structural domains, each of which has a binding function . The N-terminal domain is involved in homotypic protein-protein interaction (the basis of binding cooperativity), the core domain binds single strands directly, and the C-terminal domain has a role in heterotypic protein-protein association . The three domains have traditionally been thought to be independent of each other . However, the observation of a striking repetition of a basic, polar sequence (the "LAST" Motif), seen in both the N-terminal and core domains, suggests a linkage between these domains . Moreover, the C-domain and adjoining portion (flap) of the core are highly acidic, and are potential mimics of single-stranded DNA . With these observations, I construct a model in which this flap is associated with the ssDNA binding site in the absence of DNA, and upon cooperative protein binding to DNA, the flap now associates with the N-terminal domain of the adjacent DNA-bound protein . The flap thus acts as a gate, which might slow the binding of the protein to DNA . This could lead to the regulation of the protein's various interactions with other proteins, as well as affect its ability to lower DNA melting temperature.

Mol Microbiol, 2002 Jul, 45(1), 99 - 108
The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage phi X174; Bernhardt TG et al.; Most bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell . The ssDNA phage phi X174 uses a single lysis gene, E, encoding a 91-amino-acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis . Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E-mediated lysis and phi X174 plaque formation . The slyD gene encodes a FKBP-type peptidyl-prolyl cis-trans isomerase (PPIase) . To investigate the molecular basis of this unique FKBP-dependence, spontaneous plaque-forming mutants of phi X174 were isolated on a slyD lawn . All of these Epos ('plates on slyD') suppressors encode proteins with either a R3H or L19F change . The double mutant was also isolated and generated the largest plaques on the slyD lawn . A c-myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function . Western blots and pulse-chase labelling experiments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis-sufficient level of Epos to accumulate . Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect . These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD- dependence phenotype for identifying elements of PPIase selectivity.

Proc Natl Acad Sci U S A, 2002 Jul 9, 99(14), 9139 - 44 Epub 2002 Jul 01.
Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins; Leng F et al.; Transcription by RNA polymerase can stimulate localized DNA supercoiling in Escherichia coli . In vivo, there is extensive experimental support for a "twin-domain" model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase complex and negative supercoils are formed behind it . Negative supercoils accumulate in the template DNA because the positive supercoils are preferentially removed by cellular topoisomerase action . Yet, in vitro, clear and convincing support for the twin-domain mechanism has been lacking . In this article, we reconcile this inconsistency by showing that, in a defined in vitro system with plasmid DNA templates, a variety of sequence-specific DNA-binding proteins, such as the bacteriophage lambda O replication initiator or the E . coli lactose or galactose repressors, strikingly stimulate transcription-coupled DNA supercoiling . We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase . Our data are most consistent with a model in which specific DNA-binding proteins facilitate a twin-domain mechanism to enhance DNA supercoiling during transcription . More precisely, we suggest that some nucleoprotein complexes, perhaps those that contain sharply bent DNA, can form barriers that impede the diffusion and merger of independent chromosomal supercoil domains . Localization of DNA supercoils by nucleoprotein complexes may serve as a general mechanism for modulating DNA transactions that are sensitive to DNA superhelicity.

J Biol Chem, 2002 Sep 6, 277(36), 33041 - 8 Epub 2002 Jun 26.
Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage RB69; Petrov VM et al.; DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor . Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions . We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA . We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb) . Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins . We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.

J Mol Biol, 2002 May 17, 318(5), 1395 - 404
The solution structure of the bacteriophage lambda head-tail joining protein, gpFII; Maxwell KL et al.; The bacteriophage lambda FII protein (gpFII) is a 117 residue structural protein found in the phage particle that is required for the joining of phage heads and tails at the last step of morphogenesis . We have performed biophysical experiments to show that gpFII is stable, monomeric, and reversibly folded . We have also determined the atomic resolution structure of gpFII using NMR spectroscopy . gpFII is shown to possess a novel fold consisting of seven beta-strands and a short alpha-helix . It also displays two large unstructured regions at the N terminus (residues 1-24) and in a large loop near the middle of the protein (residues 46-62) . We speculate that these unstructured regions become structured when gpFII assembles into the phage particle, and that these conformational changes play an important role in regulating the assembly pathway . Alignment of the gpFII sequence with those of homologues from other lambdoid phages has allowed us to putatively identify distinct surfaces on the gpFII structure that mediate binding to the phage head and tail.

J Bacteriol, 2002 Jul, 184(14), 3957 - 64
The bacteriophage T4 transcription activator MotA interacts with the far-C-terminal region of the sigma70 subunit of Escherichia coli RNA polymerase; Pande S et al.; Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA . AsiA binds tightly within the C-terminal portion of the sigma70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at -30, and also interacts with sigma70 . We show here that the N-terminal half of MotA (MotA(NTD)), which is thought to include the activation domain, interacts with the C-terminal region of sigma70 in an E . coli two-hybrid assay . Replacement of the C-terminal 17 residues of sigma70 with comparable sigma38 residues abolishes the interaction with MotA(NTD) in this assay, as does the introduction of the amino acid substitution R608C . Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a sigma70 that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation . We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotA(CTD)) binds DNA with a K(D(app)) that is similar to that of full-length MotA . Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of sigma70 helps to substitute functionally for an interaction between sigma70 and a promoter -35 element.

Biophys J, 2002 Jul, 83(1), 566 - 81
Metal ion-induced lateral aggregation of filamentous viruses fd and M13; Tang JX et al.; We report a detailed comparison between calculations of inter-filament interactions based on Monte-Carlo simulations and experimental features of lateral aggregation of bacteriophages fd and M13 induced by a number of divalent metal ions . The general findings are consistent with the polyelectrolyte nature of the virus filaments and confirm that the solution electrostatics account for most of the experimental features observed . One particularly interesting discovery is resolubilization for bundles of either fd or M13 viruses when the concentration of the bundle-inducing metal ion Mg(2+) or Ca(2+) is increased to large (>100 mM) values . In the range of Mg(2+) or Ca(2+) concentrations where large bundles of the virus filaments are formed, the optimal attractive interaction energy between the virus filaments is estimated to be on the order of 0.01 kT per net charge on the virus surface when a recent analytical prediction to the experimentally defined conditions of resolubilization is applied . We also observed qualitatively distinct behavior between the alkali-earth metal ions and the divalent transition metal ions in their action on the charged viruses . The understanding of metal ions-induced reversible aggregation based on solution electrostatics may lead to potential applications in molecular biology and medicine.

Dis Aquat Organ, 2002 May 10, 49(2), 117 - 22
Segment 8 encodes a structural protein of infectious salmon anaemia virus (ISAV); the co-linear transcript from Segment 7 probably encodes a non-structural or minor structural protein; Bierin E et al.; In this study we present the cloning, expression and partial identification of Genomic Segment 7 of infectious salmon anaemia virus (ISAV) . The nucleotide sequence corresponding to Segment 7 was isolated from a bacteriophage lambda cDNA library and contained 2 overlapping open reading frames (ORFs) of 903 and 522 bases respectively . It also contained an ISAV-specific conserved nucleotide motif in the mRNA 5' region . The co-linear transcript representing the large ORF undergoes a splicing event that removes a 526 nucleotide intron to form a mRNA corresponding to the smaller reading frame . Thus, ISAV Genomic Segment 7 has a similar coding strategy as influenza A virus Segments 7 and 8 . The largest ORF of Segment 7 and the first ORF of Segment 8 was expressed in E . coli as fusion proteins and rabbit antiserum was raised against the recombinant protein from Segment 8 . Immunoblot studies using this antiserum and a serum against purified virus, show that Segment 8 encodes one of the major structural proteins of the virus whereas the co-linear ORF of Segment 7 probably encodes a non- or minor structural protein

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1998, 16(5), 342 - 6
{Cloning and sequence analysis of the gene encoding the partial region CS protein of a Plasmodium falciparum isolate from Yunnan}; Xiao J et al.; AIM: Determining nucleotide sequence of the circumsporozoite protein partial gene of the Plasmodium falciparum PFD-3/YN (Yunnan of China) and finding out the differences of the CS gene sequence between Chinese Plasmodium falciparum isolate and other isolates . METHODS: The circumsporozoite protein gene fragment was amplified by polymerase chain reaction and cloned into M13 bacteriophage . M13-CSP single strand DNAs of the three positive clones were extracted respectively . Then, the nucleotide sequence of the CS gene fragment was determined by the dideoxy chain termination method . PCGENE software was used to compare and analyze the CS gene sequence of the six isolates . RESULTS: Different degrees of diversity of the CS gene sequences were found among P . falciparum PFD-3/YN and other isolates(T4, Wellcome, NF54, 3D7 and 7G8) . A non-silent substitution at the nucleotide level being found in the P . f Th/Tc antigenic epitope region . CONCLUSION: There were differences in the CS gene sequence among P . falciparum PFD-3/YN and those of other isolates.

Nippon Rinsho, 2002 Jun, 60(6), 1077 - 82
{Genetic diversity of enterohemorrhagic Escherichia coli}; Ohnishi M et al.; Enterohemorrhagic Escherichia coli(EHEC) causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome . Genomic comparison of an EHEC O157: H7 strain isolated from the Sakai outbreak and a benign laboratory strain K-12 revealed that acquisition of a large amount of foreign DNA has promoted the genetic diversification of E . coli strains . In the emergence of O157: H7, bacteriophages, in particular, played an important role . EHEC are a group of strains with several serotypes, each belonging to different E . coli lineages . Even in an O157 lineage, significant phenotypic and genetic heterogeneities are observed . Recent knowledge on the significance and the generating mechanism of such heterogeneity in EHEC strains are summarized.

Environ Sci Technol, 2002 Jun 1, 36(11), 2403 - 13
Field and laboratory investigations of inactivation of viruses (PRD1 and MS2) attached to iron oxide-coated quartz sand; Ryan JN et al.; Field and laboratory experiments were conducted to investigate inactivation of viruses attached to mineral surfaces . In a natural gradient transport field experiment, bacteriophage PRD1, radiolabeled with 32P, was injected into a ferric oxyhydroxide-coated sand aquifer with bromide and linear alkylbenzene sulfonates . In a zone of the aquifer contaminated by secondary sewage infiltration, small fractions of infective and 32P-labeled PRD1 broke through with the bromide tracer,followed bythe slow release of 84% of the 32P activity and only 0.011% of the infective PRD1 . In the laboratory experiments, the inactivation of PRD1, labeled with 35S (protein capsid), and MS2, dual radiolabeled with 35S (protein capsid) and 32P (nucleic acid), was monitored in the presence of groundwater and sediment from the contaminated zone of the field site . Release of infective viruses decreased at a much faster rate than release of the radiolabels, indicating that attached viruses were undergoing surface inactivation . Disparities between 32P and 35S release suggest that the inactivated viruses were released in a disintegrated state . Comparison of estimated solution and surface inactivation rates indicates solution inactivation is approximately 3 times as fast as surface inactivation . The actual rate of surface inactivation may be substantially underestimated owing to slow release of inactivated viruses.

Acta Microbiol Immunol Hung, 2002, 49(1), 69 - 80
Fluorescence in situ hybridization (FISH) in the molecular cytogenetics of cancer; Szeles A; In this review, we discuss the developments of fluorescence in situ hybridization (FISH) and place them in the context of their applications in cancer research . These methods are not only very useful for the causal analysis of the development and spread of certain tumors, they are also efficient tools for tumor diagnosis . Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory . Our group contributes to the goal of functional identification of tumor growth antagonizing genes . FISH and molecular analyses have shown that the short arm of human chromosome 3 is frequently deleted in kidney, lung, breast, uterus, testis and ovary carcinomas . Deletion-mapping studies have outlined several separate deletion prone regions in different tumors, namely 3pter-p25, p22-p21.3, p21.1-p14 and p14-p12, which may contain putative tumor suppressor genes (TSGs) . Candidate suppressor genes isolated from frequently deleted regions need to be assayed for possible tumor-antagonizing ability by functional tests . We have developed a functional test system, the microcell hybrid (MCH) based "elimination test" (Et) . The Et is based on the introduction of a single human chromosome into tumor cells of human or murine origin, via microcell fusion . The MCHs were analyzed by FISH painting and PCR for the elimination or retention of specific human chromosome 3 (chr . 3) regions after one or several passages in severe combined immunedeficient (SCID) mice . We have defined a common eliminated region (CER) on chr . 3p21.3 . CER is approximately 1 megabase (Mb) in size . We have covered this region with PACs (bacteriophage PI based artificial chromosome) and used FISH mapping for localization and ordering PACs and cosmids on the chromosome 3 and high-resolution free chromatin/DNA fiber FISH to orient the PAC contig, to measure the lengths of PACs, and to establish their order . Activation of cellular oncogene by chromosomal tanslocation, which brings an oncogene under the influence of a highly active chromosome region, appears to play a pivotal role in the genesis of certain hematopoetic and lymphoid tumors . We have detected specific chromosomal translocations by FISH painting in mouse plamacytoma (MPC), human Burkitt lymphoma (BL) other B-cell derived tumors . We have showed in a murine sarcoma derived line (SEWA) that FISH can be also be used for detection of amplified oncogene (c-myc) and the linked locus (pvt-1) . We have also applied the FISH technique for visualization of integrated and episomal Epstein-Barr virus (EBV) genomes and EBV transcripts in EBV-carrying B-cell derived human cell lines.

Prep Biochem Biotechnol, 2002 May, 32(2), 157 - 72
dUTPase from Escherichia coli; high-level expression and one-step purification; Persson R et al.; The dut gene, which encodes Escherichia coli deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), has been recloned to increase overexpression of the enzyme and to enable simplification of the purification protocol into a one-step procedure . The gene was cloned into the vector pET-3a and expressed in E . coli BL21(DE3) pLysS under the control of a bacteriophage T7 promotor . Induction results in production of dUTPase corresponding to 60% of the extracted protein . Phosphocellulose chromatography at low pH was utilised for one-step purification, resulting in a homogenous preparation of the recombinant protein with a highly specific activity . The yield of purified enzyme is 500 mg per litre of bacterial culture, a significant increase compared to previously employed methods.

Med Sci Monit, 2002 Jun, 8(6), BR212 - 20
Chemoprotection profiles of sodium thiosulfate on methyl methanesulfonate-induced mutagenesis of bacteriophage T4; Malik A et al.; BACKGROUND: The alkylation of nucleic acids is primarily responsible for chemical carcinogenesis . Even during disease treatment, several alkylating drugs interact with nucleic acids and cause severe toxic effects . Thus good chemoprotectants are necessary . For our study we chose a simple model organism, bacteriophage T4 (a nucleoprotenic particle), and alkylating agent methyl methanesulfonate (MMS) to study its lethal effects . Sodium thiosulfate (STS), used as a chemoprotectant, has been tested against alkylating drugs.MATERIAL/METHODS: Bacteriophage T4D(o) were exposed to different molarities of MMS for several pre-termination incubations . Alkylation reactions were stopped with different concentrations of STS at given pre-termination incubation periods and further incubated up to 24 hours . The viability (survival frequency) of phage T4 was studied at various post-termination intervals by plaque count assay.RESULTS: Our results show that the survival frequency is strongly influenced by MMS dosage and exposure time . However, the antidotal effect of STS on MMS-induced lethality directly corresponds to STS dosage . Survival frequencies with 1% quench solution were lower than with 5% quench solution at all molarities of MMS and at different pre- and post-termination periods.CONCLUSIONS: Our studies confirmed the role of STS in the cytoprotection of bacteriophage T4 . In the presence of 1% STS, a moderate inhibition in cytotoxicity was observed, while 5% STS exhibited a significant inhibition against the cytotoxic activity of MMS, presumably due to a rapid covalent binding of the methyl group (carbocation - an electrophile) of MMS with the nucleophilic sulfur atom of STS.

Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8488 - 93 Epub 2002 Jun 17.
Design of protein struts for self-assembling nanoconstructs; Hyman P et al.; Bacteriophage T4 tail fibers have a quaternary structure of bent rigid rods, 3 x 160 nm in size . The four proteins which make up these organelles are able to self-assemble in an essentially irreversible manner . To use the self-assembly domains of these proteins as elements in construction of mesoscale structures, we must be able to rearrange these domains without affecting the self-assembly properties and add internal binding sites for other functional elements . Here we present results on several alterations of the P37 component of the T4 tail fiber that change its length and add novel protein sequences into the protein . One of these sequences is an antibody binding site that is used to inactivate phage carrying the modified gene.

Protein Sci, 2002 Jul, 11(7), 1788 - 99
Characterization of Ejl, the cell-wall amidase coded by the pneumococcal bacteriophage Ej-1; Saiz JL et al.; The Ejl amidase is coded by Ej-1, a temperate phage isolated from the atypical pneumococcus strain 101/87 . Like all the pneumococcal cell-wall lysins, Ejl has a bimodular organization; the catalytic region is located in the N-terminal module, and the C-terminal module attaches the enzyme to the choline residues of the pneumococcal cell wall . The structural features of the Ejl amidase, its interaction with choline, and the structural changes accompanying the ligand binding have been characterized by CD and IR spectroscopies, differential scanning calorimetry, analytical ultracentrifugation, and FPLC . According to prediction and spectroscopic (CD and IR) results, Ejl would be composed of short beta-strands (ca . 36%) connected by long loops (ca . 17%), presenting only two well-predicted alpha-helices (ca . 12%) in the catalytic module . Its polypeptide chain folds into two cooperative domains, corresponding to the N- and C-terminal modules, and exhibits a monomer <--> dimer self-association equilibrium . Choline binding induces small rearrangements in Ejl secondary structure but enhances the amidase self-association by preferential binding to Ejl dimers and tetramers . Comparison of LytA, the major pneumococcal amidase, with Ejl shows that the sequence differences (15% divergence) strongly influence the amidase stability, the organization of the catalytic module in cooperative domains, and the self-association state induced by choline . Moreover, the ligand affinity for the choline-binding locus involved in regulation of the amidase dimerization is reduced by a factor of 10 in Ejl . Present results evidence that sequence differences resulting from the natural variability found in the cell wall amidases coded by pneumococcus and its bacteriophages may significantly alter the protein structure and its attachment to the cell wall.

Biochemistry, 2002 Jun 25, 41(25), 7989 - 97
A unique type II topoisomerase mutant that is hypersensitive to a broad range of cleavage-inducing antitumor agents; O'Reilly EK et al.; Bacteriophage T4 provides a useful model system for dissecting the mechanism of action of antitumor agents that target type II DNA topoisomerases . Many of these inhibitors act by trapping the cleavage complex, a covalent complex of enzyme and broken DNA . Previous analysis showed that a drug-resistant T4 mutant harbored two amino acid substitutions (S79F, G269V) in topoisomerase subunit gp52 . Surprisingly, the single amino acid substitution, G269V, was shown to confer hypersensitivity in vivo to m-AMSA and oxolinic acid {Freudenreich, C . H., et al . (1998) Cancer Res . 58, 1260-1267} . We purified this G269V mutant enzyme and found it to be hypersensitive to a number of cleavage-inducing inhibitors including m-AMSA, VP-16, mitoxantrone, ellipticine, and oxolinic acid . While the mutant enzyme did not exhibit altered DNA cleavage site specificity compared to the wild-type enzyme, it did display an apparent 10-fold increase in drug-independent DNA cleavage . This suggests a novel mechanism of altered drug sensitivity in which the enzyme equilibrium has been shifted to favor the cleavage complex, resulting in an increase in the concentration of cleavage intermediates available to inhibitors . Mutations that alter drug sensitivities tend to cluster within two specific regions of all type II topoisomerases . Residue G269 of gp52 lies outside of these regions, and it is therefore not surprising that G269V leads to a unique mechanism of drug hypersensitivity . We believe that this mutant defines a new category of type II topoisomerase mutants, namely, those that are hypersensitive to all inhibitors that stabilize the cleavage complex.

EMBO J, 2002 Jun 17, 21(12), 3128 - 36
R-loop-dependent rolling-circle replication and a new model for DNA concatemer resolution by mitochondrial plasmid mp1; Backert S; The mitochondrial (mt) plasmid mp1 of Chenopodium album replicates by a rolling-circle (RC) mechanism initiated at two double-stranded replication origins (dso1 and dso2) . Two-dimensional gel electrophoresis and electron microscopy of early mp1 replication intermediates revealed novel spots . Ribonucleotide (R)-loops were identified at dso1, which function as a precursor for the RCs in vivo and in vitro . Bacteriophage T4-like networks of highly branched mp1 concatemers with up to 20 monomer units were mapped and shown to be mainly formed by replicating, invading, recombining and resolving molecules . A new model is proposed in which concatemers were separated into single units by a "snap-back" mechanism and homologous recombination . dso1 is a recombination hotspot, with sequence homology to bacterial Xer recombination cores . mp1 is a unique eukaryotic plasmid that expresses features of phages like T4 and could serve as a model system for replication and maintenance of DNA concatemers.

J Struct Biol, 2002 Jan-Feb, 137(1-2), 236 - 47
Review: conformation and folding of novel beta-structural elements in viral fiber proteins: the triple beta-spiral and triple beta-helix; Mitraki A et al.; Apart from alpha-helical coiled coils and the collagen triple helices, fibrous proteins can contain beta-structure in various conformations . Elongated enzymes such as pectate lyase and the bacteriophage P22 tailspike protein contain single-stranded beta-helices . Virus and bacteriophage fibers, which are often trimeric, have been shown to contain novel triple-stranded beta-structures such as the triple beta-spiral and the triple beta-helix . The conformation and folding of viral fibers containing beta-structure are discussed . (c) 2002 Elsevier Science (USA).

Plant J, 2002 Jun, 30(6), 625 - 37
Six active phage-type RNA polymerase genes in Nicotiana tabacum; Hedtke B et al.; In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme . The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found . Analysis of Nicotiana tabacum, a natural allotetraploid, identified six different RpoT sequences . The study of the progenitor species of tobacco, N . sylvestris and N . tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3) . Interestingly, while the organelles are inherited exclusively from the N . sylvestris maternal parent, all six RpoT genes are expressed in N . tabacum . GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties . Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids . Thus, the dual-targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants . Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP . Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide . Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.

Nucleic Acids Res . 2002 Jun 15;30(12):e59.
Unmodified Cre recombinase crosses the membrane; Will E et al.; Site-specific recombination in genetically modified cells can be achieved by the activity of Cre recombinase from bacteriophage P1 . Commonly an expression vector encoding Cre is introduced into cells; however, this can lead to undesired side-effects . Therefore, we tested whether cell-permeable Cre fusion proteins can be directly used for lox-specific recombination in a cell line tailored to shift from red to green fluorescence after loxP-specific recombination . Comparison of purified recombinant Cre proteins with and without a heterologous 'protein transduction domain' surprisingly showed that the unmodified Cre recombinase already possesses an intrinsic ability to cross the membrane border . Addition of purified recombinant Cre enyzme to primary bone marrow cells isolated from transgenic C/EBPalpha(fl/fl) mice also led to excision of the 'floxed' C/EBPalpha gene, thus demonstrating its potential for in vivo applications . We conclude that Cre enyzme itself or its intrinsic membrane-permeating moiety are attractive tools for direct manipulation of mammalian cells.

J Bacteriol, 2002 Jul, 184(13), 3753 - 5
Phage lambda red-mediated adaptive mutation; Poteete AR et al.; Replacement of the recBCD genes of Escherichia coli with the red recombination genes of bacteriophage lambda results in a strain in which adaptive mutation occurs at an elevated frequency . Like RecBCD-dependent adaptive mutation, Red-mediated adaptive mutation is dependent upon recA and ruvABC functions.

J Mol Biol, 2002 Jun 7, 319(3), 603 - 14
Delineating the site of interaction on the pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli; Deng LW et al.; The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus . pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end . Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E . coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane . Surface residues on the D2 domain of pIII were replaced systematically with alanine . Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E . coli F(+) cells infected with the mutants . A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili . This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili . These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein . (c) 2002 Elsevier Science Ltd.

Transgenic Res, 2002 Apr, 11(2), 151 - 9
Expression of a 434:VP16 chimeric activator leads to high-level activation of gene expression in stable transformants of Arabidopsis; Storgaard M et al.; The performance of an expression system based on a fusion of the bacteriophage 434-repressor to the VP16 activation domain of Herpes simplex virus type 1 (434:VP16) was tested after stable integration into Arabidopsis . A special feature of this system was the use of the monocot maize ubiquitin1 and rice actin1 promoters to drive the expression of the 434:VP16 activator and 434-repressor, respectively . Our results demonstrate that the maize ubiquitin1 and the rice actin1 promoters, each of which contain introns, are active in Arabidopsis and can be used to express genes in this dicot species . Activation of gene expression after co-integration of the activator and reporter cassettes into the same genomic locus resulted in a higher activation level (84-fold activation) compared to crossing individual lines expressing only the activator or the operator reporter cassette alone (9-fold activation) . Increasing the number of operator elements in the reporter cassette from 1 to 4 increased the activation level in cross-activated lines to an average of 281-fold with one combination of parental lines giving a 900-fold activation . Simultaneous expression of the 434-repressor protein driven by the rice actin promoter resulted in a significant decrease in the 434:VP16 mediated reporter gene expression . Nevertheless, an overall induction via 434:VP16 was possible even in the presence of the 434-repressor protein . This feature is important for genes which need to be absolutely repressed except under activating conditions . To our knowledge this investigation is the first report on the use of the 434:VP16 chimeric activator in an expression system in stably transformed plant lines.

FEMS Microbiol Lett, 2002 May 21, 211(1), 77 - 83
Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes; Morita M et al.; A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample . The phage concentration in a swine stool, estimated by plaque assay on E . coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample . PP01 infects strains of E . coli O157:H7 but does not infect E . coli strains of other O-serogroups and K-12 strains . Infection of an E . coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis . The further incubation of the culture for 7 h produced phage-resistant E . coli O157:H7 mutant . One PP01-resistant E . coli O157:H7 mutant had lost the major outer membrane protein OmpC . Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor . DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.

J Mol Biol, 2002 May 24, 319(1), 37 - 51
Effects of substitutions in a conserved DX(2)GR sequence motif, found in many DNA-dependent nucleotide polymerases, on transcription by T7 RNA polymerase; Imburgio D et al.; The region in bacteriophage T7 RNA polymerase (RNAP) comprising residues 421-425 contains a sequence motif (DX(2)GR) that is conserved among many DNA-dependent nucleotide polymerases . We have found that alterations in this motif result in enzymes that display weaker retention of the RNA product during transcript initiation, a decreased ability to make the transition to a stable elongation complex, and changes in substrate binding and catalytic activity . Many of these defects are coupled with an altered response to the presence or absence of the non-template strand . The observed constellation of defects supports a role for the motif in interacting with and stabilizing the RNA:DNA hybrid during the early stages of transcript initiation . This is consistent with the position of the motif in a T7 RNAP initiation complex . Although a conserved DX(2)GR sequence motif is also observed in multisubunit RNAPs, the structural organization of the motif and the manner in which it interacts with the RNA:DNA hybrid in the latter enzymes is different from that in T7 RNAP . However, another element in the multisubunit RNAPs that contains a highly conserved arginine residue may play the same role as R425 in T7 RNAP . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 31, 319(2), 289 - 304
A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4 terminase (gp17) and late sigma factor (gp55) interaction; Malys N et al.; HOC and SOC are dispensable T4 capsid proteins that can be used for phage display of multiple copies of peptides and proteins . A bipartite phage T4 peptide library was created by displaying on tetra-alanine linker peptides five randomized amino acids from the carboxyl-terminus of SOC and five randomized amino acids from the amino terminus of HOC . The bipartite library was biopanned against the phage T4 terminase large subunit gp17 to identify T4 gene products that may interact with the terminase . The sequences of selected phages displayed matches to those T4 gene products previously known by genetic and biochemical criteria to interact with gp17: gp20 (portal protein), gp32 (single-stranded DNA binding protein), gp16 (terminase small subunit), and gp17 (self) . In addition, matches were found to gp55 (T4 late sigma factor), gp45 (sliding clamp), gp44 (clamp loader), gp2 (DNA end protein), and gp23 (major capsid protein) . Abundant amino acid sequence matches were found to aa region 118-134 of gp55 . Immunoprecipitation and affinity column chromatography demonstrated direct binding of gp17 and gp55; moreover, gp17 bound specifically to a column-coupled peptide corresponding to gp55 residues 111-136 . Measurements of gene 17 and other mRNA levels in mutant-infected bacteria did not support a role of gp17-gp55 interaction in regulation of terminase or other late gene transcription . However, whereas DNA concatemers that accumulate in prohead and terminase defective phage T4 infections could be packaged in vitro to approximately 10% wild-type efficiency, 55am33am defective concatemeric DNA was packaged at least 100-fold less efficiently . Moreover, gp55 residues 111-136 peptide specifically blocked DNA packaging in vitro . These results suggest that the T4 terminase interaction with T4 late sigma factor gp55 plays a role in DNA packaging in vivo . The gp55 interaction may function to load the terminase onto DNA for packaging .

J Mol Biol, 2002 Apr 26, 318(2), 557 - 69
Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5; Plancon L et al.; Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA . A histidine-tagged form of pb5 was overproduced and purified . Isolated pb5 is monomeric and organized mostly as beta-sheets (51%) . pb5 functionality was attested in vivo by its ability to impair infection of E . coli cells by phage T5 and Phi80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source . pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5 . However, pb5 alone was not sufficient for the conversion of FhuA into an open channel . Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography . The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent . SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 degrees C; (ii) thermal denaturation of the complex occurred at 85 degrees C, while pb5 and FhuA were denatured at 45 degrees C and 74 degrees C, respectively . The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 Apr 26, 318(2), 321 - 31
RNase E and polyadenyl polymerase I are involved in maturation of CI RNA, the P4 phage immunity factor; Briani F et al.; Bacteriophage P4 immunity is controlled by a small stable RNA (CI RNA) that derives from the processing of primary transcripts . In previous works, we observed that the endonuclease RNase P is required for the maturation of CI RNA 5'-end; moreover, we found that polynucleotide phosphorylase (PNPase), a 3' to 5' RNA-degrading enzyme, is required for efficient 5'-end processing of CI RNA, suggesting that 3'-end degradation of the primary transcript might be involved in the production of proper RNase P substrates . Here, we demonstrate that another Escherichia coli nuclease, RNase E, would appear to be involved in this process . We found that transcripts of the P4 immunity region are modified by the post-transcriptional addition of short poly(A) tails and heteropolymeric tails with prevalence of A residues . Most oligoadenylated transcripts encompass the whole cI locus and are thus compatible as intermediates in the CI RNA maturation pathway . On the contrary, in a polynucleotide phosphorylase (PNPase)-defective host, adenylation occurred most frequently within cI, implying that such transcripts are targeted for degradation . We did not find polyadenylation in a pcnB mutant, suggesting that the pcnB-encoded polyadenyl polymerase I (PAP I) is the only enzyme responsible for modification of P4 immunity transcripts . Maturation of CI RNA 5'-end in such a mutant was impaired, further supporting the idea that processing of the 3'-end of primary transcripts is an important step for efficient maturation of CI RNA by RNase P . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 Apr 26, 318(2), 305 - 20
UTP allosterically regulates transcription by Escherichia coli RNA polymerase from the bacteriophage T7 A1 promoter; Johnson RS et al.; In the case of Escherichia coli RNA polymerase, UTP at elevated concentrations suppresses terminated transcript accumulation during multiple-round transcription from a DNA construct containing the T7 A1 promoter and T(e) terminator . The step that is affected by UTP at elevated concentrations is promoter clearance . In an attempt to understand better the mechanism by which UTP regulates this step, we analyzed the effect of UTP on the formation of pppApU in the presence of only UTP and ATP . At elevated concentrations, UTP is a non-competitive inhibitor with respect to ATP in the formation of pppApU . This indicates that the effect of UTP on the formation of pppApU is mediated through an allosteric site . Moreover, the magnitude of the inhibition of pppApU formation is sufficient to account for the decrease in terminated transcript accumulation at elevated UTP concentrations . Thus, it appears that UTP modulates terminated transcript accumulation during multiple-round transcription from this DNA construct by allosteric regulation of promoter clearance at the point of transcription initiation . (c) 2002 Elsevier Science Ltd.

Mech Dev, 2002 Jul, 115(1-2), 53 - 62
E-cadherin is a survival factor for the lactating mouse mammary gland; Boussadia O et al.; The Ca(++)-dependent cell adhesion molecule E-cadherin is expressed throughout mouse development and in adult tissues . Classical gene targeting has demonstrated that E-cadherin-deficient embryos die at the blastocyst stage . To study the involvement of E-cadherin in organogenesis, a conditional gene inactivation scheme was undertaken using the bacteriophage P1 recombinase Cre/loxP system . Mice with homozygous loxP sites in both alleles of the E-cadherin (Cdh1) gene were generated and these mice were crossed with transgenic mice with the Cre recombinase under the control of the hormone-inducible MMTV promoter . This resulted in deletion of the E-cadherin gene in the differentiating alveolar epithelial cells of the mammary gland . The mutant mammary gland developed normally up to 16-18 days of pregnancy but exhibited a dramatic phenotype around parturition . The production of milk proteins was so drastically reduced that adult mutant mothers could not suckle their offspring . Thus, the lack of E-cadherin affected the terminal differentiation program of the lactating mammary gland . In concordance with this finding, the prolactin-dependent activation of the transcription factor Stat5a was initiated but not maintained in the mutant gland . Instead, without E-cadherin massive cell death was observed at parturition and the mutant mammary gland at this stage resembled that of the involuted gland normally seen after weaning . These results demonstrate an essential role for E-cadherin in the function of differentiated alveolar epithelial cells . No tumors were detected in mutant glands lacking E-cadherin.

Biochem J, 2002 Jun 15, 364(Pt 3), 857 - 62
Composition of the lambda plasmid heritable replication complex; Potrykus K et al.; Previous studies indicated during replication of plasmids derived from bacteriophage lambda (the so-called lambda plasmids), that, once assembled, replication complex can be inherited by one of the two daughter plasmid copies after each replication round, and may function in subsequent replication rounds . It seems that similar processes occur during replication of other DNA molecules, including chromosomes of the yeast Saccharomyces cerevisiae . However, apart from some suggestions based on genetic experiments, composition of the lambda heritable replication complex remains unknown . In amino acid-starved Escherichia coli relA mutants, replication of lambda plasmid DNA is carried out exclusively by the heritable replication complex as assembly of new complexes is impaired due to inhibition of protein synthesis . Here, using a procedure based on in vivo cross-linking, cell lysis, immunoprecipitation with specific sera, de-cross-linking and PCR analysis, we demonstrate that the lambda heritable replication complex consists of O, P, DnaB and, perhaps surprisingly, DnaK proteins.

Vopr Virusol, 2002 Mar-Apr, 47(2), 31 - 4
{Localization of tick-borne encephalitis virus protein E antigenic determinant recognized by antihemagglutinating monoclonal antibodies using a phage-display peptide library}; Loktev AV et al.; Bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (MAb) 10H10 to tick-borne encephalitis (TBE) virus protein E were selected from a phage-display peptide library by affinity selection and enzyme immunoassay . The library contained randomized peptides that are 6 amino acids long, fused with protein pIII and exposed on the surface of the bacteriophage . No significant homology between the sequences of selected peptides and TBE virus protein E was detected . Computer software was created to locate the conformation epitopes on the surface of protein E . Amino acids R73, C74, T76, M77, N103, C105, L107, and S112 were found to form a discontinuous epitope recognized by MAb 10H10 . These amino acids are remote in the protein sequence but close in the tertiary structure and form a whole epitope located in the structural domain II of protein E . Presumably the localized amino acids bind to the cellular receptor for TBE virus.

Microbiol Mol Biol Rev, 2002 Jun, 66(2), 179 - 202
T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis; Sullivan CS et al.; Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated . The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40 . Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle . Deciphering how a single protein can perform such numerous and diverse functions has remained elusive . Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain . The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes . This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis . The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen-a most amazing molecule.

Adv Space Res, 2000, 26(12), 1983 - 94
Quantification of biologically effective environmental UV irradiance; Horneck G; To determine the impact of environmental UV radiation on human health and ecosystems demands monitoring systems that weight the spectral irradiance according to the biological responses under consideration . In general, there are three different approaches to quantify a biologically effective solar irradiance . (i) weighted spectroradiometry where the biologically weighted radiometric quantities are derived from spectral data by multiplication with an action spectrum of a relevant photobiological reaction, e.g . erythema, DNA damage, skin cancer, reduced productivity of terrestrial plants and aquatic foodweb, (ii) wavelength integrating chemical-based or physical dosimetric systems with spectral sensitivities similar to a biological response curve, and (iii) biological dosimeters that directly weight the incident UV components of sunlight in relation to the effectiveness of the different wavelengths and to interactions between them . Most biological dosimeters, such as bacteria, bacteriophages, or biomolecules, are based on the UV sensitivity of DNA . If precisely characterized, biological dosimeters are applicable as field and personal dosimeters . c2001 COSPAR Published by Elsevier Science Ltd . All rights reserved.

Virology, 2002 Apr 10, 295(2), 266 - 71
Characterization of phi12, a bacteriophage related to phi6: nucleotide sequence of the large double-stranded RNA; Gottlieb P et al.; The isolation of additional bacteriophages besides phi6 containing segmented double-stranded RNA genomes (dsRNA) has expanded the Cystoviridae family to nine members . Comparing the genomic sequences of these viruses has allowed evaluation of important genetic as well as structural motifs . These comparative studies are resulting in greater understanding of viral evolution and the role played by genetic and structural variation in the assembly mechanisms of the cystoviruses . In this regard, the large double-stranded RNA genomic segment of bacteriophage phi12 was copied as cDNA and its nucleotide sequence determined . This genome's organization is similar to that of the large segment of bacteriophages phi6, phi8, and phi13 . In the amino acid sequence of the viral RNA-dependent RNA polymerase (P2), similarity was found to the comparable proteins of phi6, phi8, and phi13 . Amino acid sequence similarity was also noted in the nucleotide triphosphate phosphorylase (P4) to the comparable proteins of phi8 and phi13 . (c) 2002 Elsevier Science (USA).

EMBO J, 2002 Jun 3, 21(11), 2827 - 32
Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining; Chappell C et al.; The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability . In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ) . DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ . Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus . Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes . Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK . This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.

J Food Prot, 2002 May, 65(5), 861 - 3
Control of Brochothrix thermosphacta spoilage of pork adipose tissue using bacteriophages; Greer GG et al.; Adipose tissue discs were coinoculated with Brochothrix thermosphacta and homologous bacteriophages (phages) to determine the effects these had on phage multiplication, bacterial growth, and off-odor development during storage at 2 degrees C or under simulated retail display at 6 degrees C . In the presence of about 10(5) bacteria/cm2 and an equivalent number of phages, there was a 3-log increase in phage numbers and a 2-log decrease in bacterial numbers, and objectionable off-odors were suppressed during refrigerated storage . Up to 68% of the surviving bacterial population were resistant to phages . The storage life of adipose tissue could be increased from 4 days in controls to 8 days in phage-treated samples by preventing the development of off-odors associated with the growth of B . thermosphacta . Phages may provide a novel approach to extending the storage quality of chilled meats.

J Bacteriol, 2002 Jun, 184(12), 3416 - 8
Requirement for NusG for transcription antitermination in vivo by the lambda N protein; Zhou Y et al.; Transcription antitermination by the bacteriophage lambda N protein is stimulated in vitro by the Escherichia coli NusG protein . Earlier work suggested that NusG was not required for N activity in vivo . Here we present evidence that NusG also stimulates N-mediated transcription antitermination in intact cells.

Microb Ecol, 2001 Oct, 42(3), 407 - 415
Distribution of virus-like Particles in an Oligotrophic Marine Environment (Alboran Sea, Western Mediterranean); Alonso MC et al.; Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude . In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment . Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM) . The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses . In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20 . VLP (virus-like particle) counts were not significantly correlated (p > 0.001) with chlorophyll a concentration or the abundance of cyanobacteria . However, there was a positive and significant correlation with bacterial abundance (p <0.001) . THE ANALYSIS OF SIZE AND MORPHOLOGY OF VIRAL PARTICLES BY TEM AND THE CORRELATION OBTAINED BETWEEN THE NUMBERS OF VLP AND BACTERIA SUGGEST THAT THE MAJORITY OF THE VIRAL PARTICLES IN THE ALBORAN SEA ARE BACTERIOPHAGES . NONE OF THE INDIRECT EVIDENCE SUGGESTED THAT EUKARYOTIC ALGAE OR CYANOBACTERIA WERE IMPORTANT HOST ORGANISMS IN THESE WATERS.

Microb Ecol, 2001 Oct, 42(3), 395 - 406
Effects of Bacteriophages on the Population Dynamics of Four Strains of Pelagic Marine Bacteria; Middelboe M et al.; Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities . In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model . By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages . In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species . Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation . Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments . The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones . The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks) . However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis . Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems.

RNA, 2002 May, 8(5), 686 - 97
T7 RNA polymerase-directed transcripts are processed in yeast and link 3' end formation to mRNA nuclear export; Dower K et al.; We have characterized transcripts synthesized in vivo by bacteriophage T7 RNA polymerase to investigate yeast mRNA processing . T7 transcripts are not capped, consistent with capping being tightly coupled to RNA polymerase II (pol II) transcription . In contrast to higher eukaryotic non-pol II transcripts, yeast T7 transcripts are spliced as well as cleaved and polyadenylated . However, T7 and pol II transcripts are affected differently in cleavage and polyadenylation mutant strains, indicating that pol II may have a role in yeast 3' end formation . T7 transcripts with 3' ends directed by a polyadenylation signal are exported from the nucleus, and this export is dependent on the canonical cleavage and polyadenylation machinery . Importantly, transcripts with T7 terminator-directed 3' ends are unadenylated and predominantly nuclear in wild-type cells . Our results suggest that transcription by pol II is required for neither the nuclear export of an in vivo-transcribed mRNA nor for the retention of transcripts with aberrant 3' ends . Moreover, proper 3' end formation may be necessary and sufficient to promote mRNA export in yeast.

Genetics, 2002 May, 161(1), 21 - 31
Alterations of the portal protein, gpB, of bacteriophage lambda suppress mutations in cosQ, the site required for termination of DNA packaging; Wieczorek DJ et al.; The cosQ site of bacteriophage lambda is required for DNA packaging termination . Previous studies have shown that cosQ mutations can be suppressed in three ways: by a local suppressor within cosQ, an increase in the length of the lambda chromosome, and missense mutations affecting the prohead's portal protein, gpB . In the present work, revertants of a set of lethal cosQ mutants were screened for suppressors . Seven new cosQ suppressors affected gene B, which encodes the portal protein of the prohead . All seven were allele-nonspecific suppressors of cosQ mutations . Experiments with several phages having two cosQ suppressors showed that the suppression effects were additive . Furthermore, these double suppressors had minimal effects on the growth of cosQ(+) phages . These trans-acting suppressors affecting the portal protein are proposed to allow the mutant cosQ site to be more efficiently recognized, due to the slowing of the rate of translocation.

Genetics, 2002 May, 161(1), 11 - 20
Distinguishing between selection and population expansion in an experimental lineage of bacteriophage T7; Hahn MW et al.; Experimental evolution of short-lived organisms offers the opportunity to study the dynamics of polymorphism over time in a controlled environment . Here, we characterize DNA polymorphism data over time for four genes in bacteriophage T7 . Our experiment ran for 2500 generations and populations were sampled after 500, 2000, and 2500 generations . We detect positive selection, purifying ("negative") selection, and population expansion in our experiment . We also present a statistical test that is able to distinguish demographic from selective events, processes that are hard to identify individually because both often produce an excess of rare mutations . Our "heterogeneity test" modifies common statistics measuring the frequency spectrum of polymorphism (e.g., Fu and Li's D) by looking for processes producing different patterns on nonsynonymous and synonymous mutations . Test results agree with the known conditions of the experiment, and we are therefore confident that this test offers a tool to evaluate natural populations . Our results suggest that instances of segregating deleterious mutations may be common, but as yet undetected, in nature.

Arch Virol, 2002 May, 147(5), 981 - 93
Novel peptides that inhibit the propagation of Newcastle disease virus; Ramanujam P et al.; A disulfide constrained random heptapeptide library displayed on filamentous bacteriophage M13 was applied to select specific ligands that interact with Newcastle disease virus (NDV) . A fusion phage carrying the amino acid sequence TLTTKLY was selected from the panning procedure . An antibody competition assay showed that the selected phage was capable of competing with the polyclonal antibodies raised against NDV for binding sites on the virus . Determination of the binding affinity of this phage with NDV by an equilibrium binding assay in solution revealed two different dissociation constants, suggesting that there could be two distinct binding sites for the phage on NDV . Synthetic peptides with the sequence CTLTTKLYC, either in linear or cyclic conformations inhibited the binding of phage bearing the same sequence to NDV . These peptides also inhibited the hemolytic activity of the virus as well as its propagation in embryonated chicken eggs.

J Biol Chem, 2002 Aug 2, 277(31), 28143 - 9 Epub 2002 May 20.
Mutations in the yeast mitochondrial RNA polymerase specificity factor, Mtf1, verify an essential role in promoter utilization; Karlok MA et al.; The yeast mitochondrial RNA polymerase (RNAP) is a two-subunit enzyme composed of a catalytic core (Rpo41) and a specificity factor (Mtf1) encoded by nuclear genes . Neither subunit on its own interacts with promoter DNA, but the combined holo-RNAP recognizes and selectively initiates from promoters related to the consensus sequence ATATAAGTA . To pursue the question of why Rpo41, which resembles the single polypeptide RNAPs from bacteriophage T7 and T3, requires a separate specificity factor, we analyzed a collection of Mtf1 point mutations that confer an in vivo petite phenotype . These mutant proteins are able to interact with Rpo41 and are capable of nearly wild type levels of initiation in vitro with a consensus promoter-containing template (14 S rRNA) . However, the petite phenotype of two mutants can be explained by the fact that they exhibit dramatic transcriptional defects on non-consensus promoters . Y54F is incapable of transcribing the weak tRNA(Cys) promoter, and C192F cannot transcribe either tRNA(Cys) or the variant COX2 promoter from linear DNA templates . Transcription of the tRNA(Cys) promoter by both mutants was significantly corrected by addition of an initiating dinucleotide primer or by supercoiling the DNA template . These results establish the critical role of Mtf1 in promoter recognition and initiation of transcription.

Biopolymers, 2002, 67(4-5), 214 - 25
New structural insights from Raman spectroscopy of proteins and their assemblies; Thomas GJ Jr; Protein structure and stability are sensitive to and dependent on the local interactions of amino acid side chains . A diverse and important type of side-chain interaction is the hydrogen bond . Although numerous hydrogen bonds are resolved in protein 3-dimensional structures, those of the cysteine sulfhydryl group (S-H) are elusive to high-resolution X-ray and NMR methods . However, the nature and strength of sulfhydryl hydrogen bonds (S-H* * *X) are amenable to investigation by Raman spectroscopy . The power of the Raman method for characterizing S-H* * *X interactions is illustrated by resolving the Raman S-H stretching band for each of the eight cysteines per 666-residue subunit in the trimeric tailspike of icosahedral bacteriophage P22 . The Raman sulfhydryl signatures of the wild-type tailspike and eight single-site cysteine to serine mutants reveal a heretofore unrecognized diversity of S-H hydrogen bonds in a native protein . The use of Raman spectroscopy to identify the non-hydrogen-bonded state of the tyrosine phenoxyl group is also described . This unusual and unexpected state occurs for all tyrosines in the assembled capsids of filamentous viruses Ff and Pf1 . The Raman spectral signature of the non-hydrogen-bonded tyrosine phenoxyl, which is characterized by an extraordinary Raman Fermi doublet intensity ratio (I850/I830 = 6.7), extends and refines the existing correlation for hydrogen-bonded tyrosines . Finally, a novel Raman signature for tryptophan in the Pf3 filamentous virus is identified, which is proposed as diagnostic of "cation-pi interaction" involving the guanidinium group of Arg 37 as a cation donor and the indolyl ring of Trp 38 as a pi-electron acceptor . These studies demonstrate the power of Raman spectroscopy for investigating the interactions of key side chains in native protein assemblies .

Mol Microbiol, 2002 May, 44(4), 957 - 70
Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli; Wagner PL et al.; The stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the lambda bacteriophage family . In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1) . Growth of H-19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production . Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized . The studies reported here identify the factors that contribute to Stx1 production after induction of the H-19B prophage . We found that replication of the phage genome, with the associated increase in stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production . Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions . Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage phi 361, was found to be unnecessary for high-level Stx1 production after phage induction . Finally, we present evidence that phage-mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release . By amplifying stx(1)AB copy number, regulating stx(1)AB transcription and allowing for Stx1 release, the biology of the Stx-encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.

Int J Med Microbiol, 2002 Mar, 291(8), 625 - 31
The bacteriophage-associated ehly1 and ehly2 determinants from Escherichia coli O26:H- strains do not encode enterohemolysins per se but cause release of the ClyA cytolysin; Oscarsson J et al.; This report presents evidence that the bacteriophage-associated proteins Ehlyl and Ehly2 (enterohemolysin) of non-verotoxigenic E . coli O26:H- are not hemolysins per se . Ehly1 and Ehly2 conferred a hemolytic phenotype on wild-type but not on clyA knockout strains of E . coli K-12 when introduced in trans on plasmids . According to immunoblot analyses, and studies of the expression from a chromosomal clyA::luxAB fusion, the production of cytolysin A (ClyA) was not enhanced by the expression of Ehly1 or Ehly2 . However, the analysis provided evidence that there was a low basal level expression of ClyA by E . coli K-12 strains that could be detected phenotypically when the bacteria were subject to lytic effects by bacteriophages or their gene products . In experiments when lysis of the bacteria was caused deliberately either by using lytic bacteriophages (P1 or phiW), or by triggering the SOS response with mitomycin C treatment, sufficient amounts of ClyA were released to cause detectable lysis on blood agar plates . Taken together, our findings support a model where Ehlyl and Ehly2 cause a hemolyric phenotype on blood agar as a result of their lytic and lethal effects on the bacterial hosts.

Life Sci Space Res, 1979, 17, 129 - 32
Genetic effects of cosmic radiation on bacteriophage T4Br+ (on materials of biological experiment "Soyuz-Apollo"); Yurov SS et al.; During the experiment "Spore-ring Forming Fungi Biorhythm" of the Apollo-Soyuz test project the Rhythm-1 apparatus contained a dried film culture of bacteriophage T4Br+, growing cultures of Actinomyces and plastic nuclear particle detectors . The following were studied: the frequency of induction of r mutations in the bacteriophage film per 2 X 10(4) surviving particles, the spectrum of mutant types obtained (rI, rII, rIII), and the possible molecular mechanisms for the occurrence of rII mutants with due regard to the registered tracks of heavy nuclear particles . The studies showed that the local radiation due to heavy nuclear particle tracks plays a major role in space radiation damage.

Biotechnol Bioeng, 2002 Jun 30, 78(7), 722 - 30
Connection between gene dosage and protein stability revealed by a high-yield production of recombinant proteins in an E . coli LexA1(Ind-) background; Medina MG et al.; Bacterial production of a plasmid-encoded bacteriophage P22 tailspike protein shows different yield and impact on cell viability in RecA+ LexA+, RecA- LexA+ and RecA+ LexA1(Ind-) backgrounds . In a LexA1(Ind-) context, we have observed lesser toxicity and higher productivity than in the wild-type strain, in which the bacterial growth was inhibited after induction of recombinant gene expression . Also, a negative effect of the incubation temperature on the growth of producing cells was also detected . By exploring the molecular basis of these inhibitory events, we found a connection between the dosage of the recombinant gene and the proteolytic stability of the encoded protein . Under both genetic and environmental conditions favoring higher plasmid copy number and consequently increasing the synthesis rate of the recombinant protein, enhanced protein degradation was observed in parallel with an important growth inhibition . Altogether, the obtained data suggest the existence of a critical concentration of recombinant protein over which cell proteolysis is stimulated at rates not compatible with optimal physiological conditions for bacterial growth .

J Contam Hydrol, 2002 Mar, 55(1-2), 113 - 35
Kinetic modeling of virus transport at the field scale; Schijven JF et al.; Bacteriophage removal by soil passage in two field studies was re-analyzed with the goal to investigate differences between one- and two-dimensional modeling approaches, differences between one- and two-site kinetic sorption models, and the role of heterogeneities in the soil properties . The first study involved removal of bacteriophages MS2 and PRDI by dune recharge, while the second study represented removal of MS2 by deep well injection . In both studies, removal was higher during the first meters of soil passage than thereafter . The software packages HYDRUS-ID and HYDRUS-2D, which simulate water flow and solute transport in one- and two-dimensional variably saturated porous media, respectively, were used . The two codes were modified by incorporating reversible adsorption to two types of kinetic sites . Tracer concentrations were used first to calibrate flow and transport parameters of both models before analyzing transport of bacteriophages . The one-dimensional one-site model did not fully describe the tails of the measured breakthrough curves of MS2 and PRD1 from the dune recharge study . While the one-dimensional one-site model predicted a sudden decrease in virus concentrations immediately after the peaks, measured data displayed much smoother decline and tailing . The one-dimensional two-site model simulated the overall behavior of the breakthrough curves very well . The two-dimensional one-site model predicted a more gradual decrease in virus concentrations after the peaks than the one-dimensional one-site model, but not as good as the one-dimensional two-site model . The dimensionality of the problem hence can partly explain the smooth decrease in concentration after peak breakthrough . The two-dimensional two-site model provided the best results . Values for k(att2) and k(det2) could not be determined at the last two of four monitoring wells, thus suggesting that either a second type of kinetic sites is present in the first few meters of dune passage and not beyond the second monitoring well, or that effects of soil heterogeneity and dimensionality of the problem overshadowed this process . Variations between single collector efficiencies were relatively small, whereas collision efficiencies varied greatly . This implies that the nonlinear removal of MS2 and PRD1 is mainly caused by variations in interactions between grain and virus surfaces rather than by physical heterogeneity of the porous medium . Similarly, a two-site model performed better than the one-site model in describing MS2 concentrations for the deep well injection study . However, the concentration data were too sparse in this study to have much confidence in the fitted parameters.

Poult Sci, 2002 Apr, 81(4), 437 - 41
Prevention of Escherichia coli respiratory infection in broiler chickens with bacteriophage (SPR02); Huff WE et al.; Bacteriophages are viruses that can infect and kill bacteria . Three studies were conducted to determine the efficacy of bacteriophage to prevent an Escherichia coli respiratory infection in broiler chickens . In the first study 3-d-old-birds were challenged with an air sac inoculation of 10(3) cfu of E . coli per mL mixed with either 10(3) or 10(6) pfu of bacteriophage, or 10(4) cfu E . coli mixed with 10(4) or 10(8) pfu of bacteriophage . In the second study, drinking water of birds to 1 wk of age was treated with 10(3) or 10(4) pfu of bacteriophage per mL and birds were air sac challenged with 10(3) cfu of E . coli, or water was treated with 10(4) or 10(6) pfu of bacteriophage per milliliter and birds were challenged with 10(4) cfu of E . coli . In the third study, birds were air sac challenged at 1 wk of age with 10(4) cfu of E . coli and given 10(5) or 10(6) pfu of bacteriophage per mL of water from 1 d of age to 2 wk of age . In Studies 1 and 2, there were two replicate pens per treatment with 10 birds per pen, and in Study 3, there were four replicate pens per treatment with 10 birds per pen . The studies were all concluded when the birds were 3 wk of age . In Study 1, BW was decreased at 1 and 2 wk of age in the birds that were challenged with 10(3) or 10(4) cfu of E . coli and was decreased at 2 wk of age in the birds challenged with 10(4) cfu of E . coli mixed with 10(4) pfu of the bacteriophage . Mortality was decreased from 80% in the birds challenged with 10(3) cfu of E . coli to 25 and 5% when mixed with 10(3) or 10(6) pfu of the bacteriophage, respectively . Mortality was decreased from 85% in birds challenged with 10(4) cfu of E . coli to 35% when mixed with 10(4) pfu of the bacteriophage, and no mortality occurred when mixed with 10(8) pfu of bacteriophage . There was essentially no protection observed in Studies 2 and 3 when the birds were challenged with 10(3) or 10(4) cfu of E . coli with bacteriophage present in their drinking water at any level . These data suggest that bacteriophage can protect birds from a respiratory challenge with E . coli, but that adding the bacteriophage to the drinking water offered no protection to the birds . The complete protection of the birds observed in Study 1 suggests that bacteriophage may possibly be developed as an alternative to antibiotic use in poultry.

Biochemistry (Mosc), 2002 Apr, 67(4), 498 - 502
Some properties of site-specific nickase BspD6I and the possibility of its use in hybridization analysis of DNA; Zheleznaya LA et al.; A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand . The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55 degrees C) . The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target . Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule . The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon) . The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19 . Unfortunately, nickase BspD6I does not cleave DNA in the RNA-DNA duplexes and therefore cannot be used for detection of RNA targets.

Acta Vet Scand Suppl, 2001, 95, 79 - 84
Zoonotic Escherichia coli; Wasteson Y; Escherichia coli is a normal inhabitant of the gastrointestinal tract of all warm-blooded animals, but variants of this species is also among the important etiological agents of enteritis and several extraintestinal diseases . The E . coli strains that cause diarrhoeal illness are categorised into pathogenicity groups based on virulence properties, mechanisms of pathogenicity, clinical symptoms and serology . The five main categories include enterotoxinogenic E . coli (ETEC), enteropathogenic E . coli (EPEC), enteroaggregative E . coli (EAggEC), enteroinvasive E . coli (EIEC) and Shiga (Vero) toxin-producing E . coli (STEC/VTEC) . From a zoonotic point of view, STEC is the only E . coli pathogenicity group of major interest, as the shiga toxin-producing strains are able to cause severe disease in humans when being transmitted through the food chain from their animal reservoirs . The focus of this manuscript is therefore on STEC; pathogenicity factors, disease, the reservoirs and on-farm ecology, transmission into the food chain, growth and survival in food and in the environment, and the shiga toxin-encoding bacteriophages.

Mol Microbiol, 2002 May, 44(3), 749 - 60
Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis; Leggate DR et al.; Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli . Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active . A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product . Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS-PAGE but, under native PAGE conditions, was not observed to form the approximately 250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested . Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata-lytic mechanism of bacteriophage E endosialidase and those of exosialidases.

Mol Microbiol, 2002 May, 44(3), 695 - 708
The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB; Walburger A et al.; The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins . It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages . To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two-hybrid screen using the different components of the Tol/Pal system and colicin A . Using this system, we confirmed the already known interactions and identified several new interactions . TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB . Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C-terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII . The TolA/TolB interaction was confirmed by cross-linking experiments on purified proteins . Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity . These results demonstrate that the TolA/TolB interaction allows the formation of a trans-envelope complex that brings the inner and outer membranes in close proximity.

Toxicol Lett, 2002 May 28, 131(3), 181 - 9
Bilirubin/biliverdin-Cu(II) induced DNA breakage; reaction mechanism and biological significance; Asad SF et al.; Bilirubin and its metabolic precursor biliverdin are heme degradation products but have been proposed as physiological antioxidants . Reports from another laboratory as well as from ours have shown bilirubin to form a complex with the transition metal ion-Cu(II) . Such a complex was shown by us to cause oxidative DNA damage . Further, biliverdin was also shown to be capable of causing similar DNA damage . In the present studies we have aimed to elucidate the mechanism of DNA breakage reaction by these bile pigments . Absorption and fluorescence studies indicate binding of bile pigments to DNA and copper ions . Cu(II) is reduced by these compounds to Cu(I) which is an essential intermediate in the DNA breakage reaction . Redox recycling of Cu(II) leads to generation of reactive oxygen species . Strand scission by the bile pigments-Cu(II) system is found to be biologically significant as assayed by bacteriophage inactivation . Our results, therefore are suggestive of one of the mechanisms through which endogenous DNA damage may occur.

J Virol, 2002 Jun, 76(11), 5784 - 92
Evolution of bacteriophage in continuous culture: a model system to test antiviral gene therapies for the emergence of phage escape mutants; Lindemann BF et al.; The emergence of viral escape mutants is usually a highly undesirable phenomenon . This phenomenon is frequently observed in antiviral drug applications for the treatment of viral infections and can undermine long-term therapeutic success . Here, we propose a strategy for evaluating a given antiviral approach in terms of its potential to provoke the appearance of resistant virus mutants . By use of Q beta RNA phage as a model system, the effect of an antiviral gene therapy, i.e., a virus-specific repressor protein expressed by a recombinant Escherichia coli host, was studied over the course of more than 100 generations . In 13 experiments carried out in parallel, 12 phage populations became resistant and 1 became extinct . Sequence analysis revealed that only two distinct phage mutants emerged in the 12 surviving phage populations . For both escape mutants, sequence variations located in the repressor binding site of the viral genomic RNA, which decrease affinity for the repressor protein, conferred resistance to translational repression . The results clearly suggest the feasibility of the proposed strategy for the evaluation of antiviral approaches in terms of their potential to allow resistant mutants to appear . In addition, the strategy proved to be a valuable tool for observing virus-specific molecular targets under the impact of antiviral drugs.

RNA, 2002 Apr, 8(4), 542 - 7
MALDI-TOF mass spectrometry methods for evaluation of in vitro aminoacyl tRNA production; Petersson EJ et al.; Unnatural amino acid mutagenesis requires the in vitro production of aminoacyl tRNAs . Bacteriophage T4 RNA ligase is used to ligate a-amino-protected dCA amino acids to 74mer tRNA . Previously, there has been no facile method for evaluating the efficiency of this reaction prior to using the tRNA in translation . We report a novel use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in monitoring the formation of aminoacyl 76mer tRNA . This method is more efficient and precise than the traditional technique of gel electrophoresis . These MALDI conditions should also prove useful for analyzing aminoacyl tRNAs produced through aminoacyl tRNA synthetases and other methods.

Anal Sci, 2001 Mar, 17(3), 387 - 90
Novel biosensor for the rapid measurement of estrogen based on a ligand-receptor interaction; Murata M et al.; A bioaffinity sensor was developed aiming at the detection of estrogen . This biosensor system is based on the specific binding of estrogen to its receptor immobilized on a gold disk electrode . The recombinant DNA encoding human estrogen receptor ligand-binding domain was expressed in bacteria using the histidine-tag fusion system . The expression of the fusion protein was under control of a bacteriophage T7 promoter, and the protein was purified under native conditions by affinity chromatography, which is based on a specific interaction between a histidine-tag, located in the N-terminus of the protein, and the Ni(II) chelate adsorbent . The protein was immobilized on an Au-electrode with Ni(II)-mediated chemisorption using a histidine tag and thiol-modified iminodiacetic acid . Cyclic voltammetric measurements showed that the reversible electrochemical reaction of a ferrocyanide/ferricyanide redox couple was suppressed by the presence of estrogen in a concentration-dependent manner . It seems reasonable to suppose that the electrostatic property of the protein layer on the electrode surface was altered by complexation with estrogen . These data suggest that this biosensor is applicable to the evaluation binding activities of the chemicals toward the human estrogen receptor.

Science, 2002 May 3, 296(5569), 892 - 5
Ordering of quantum dots using genetically engineered viruses; Lee SW et al.; A liquid crystal system was used for the fabrication of a highly ordered composite material from genetically engineered M13 bacteriophage and zinc sulfide (ZnS) nanocrystals . The bacteriophage, which formed the basis of the self-ordering system, were selected to have a specific recognition moiety for ZnS crystal surfaces . The bacteriophage were coupled with ZnS solution precursors and spontaneously evolved a self-supporting hybrid film material that was ordered at the nanoscale and at the micrometer scale into approximately 72-micrometer domains, which were continuous over a centimeter length scale . In addition, suspensions were prepared in which the lyotropic liquid crystalline phase behavior of the hybrid material was controlled by solvent concentration and by the use of a magnetic field.

Microbiology, 2002 May, 148(Pt 5), 1533 - 42
The cell surface protein Ag43 facilitates phage infection of Escherichia coli in the presence of bile salts and carbohydrates; Gabig M et al.; It was found that infection of Escherichia coli by bacteriophage lambda is inhibited in the presence of certain bile salts and carbohydrates when cells are in the "OFF" state for production of the phase-variable cell surface protein antigen 43 (Ag43) . The inhibition of phage growth was found to be due to a significant impairment in the process of phage adsorption . Expression of the gene encoding Ag43 (agn43) from a plasmid or inactivation of the oxyR gene (encoding an activator of genes important for defence against oxidative stress) suppressed this inhibition . A mutation, rpoA341, in the gene encoding the alpha subunit of RNA polymerase also facilitated phage adsorption in the presence of bile salts and carbohydrates . The rpoA341 mutation promoted efficient production of Ag43 in a genetic background that would otherwise be in the "OFF" phase for expression of the agn43 gene . Analysis of a reporter gene fusion demonstrated that the promoter for the agn43 gene was more active in the rpoA341 mutant than in the otherwise isogenic rpoA(+) strain . The combined inhibitory action of bile salts and carbohydrates on phage adsorption and the abolition of this inhibition by production of Ag43 was not restricted to lambda, as a similar phenomenon was observed for the coliphages P1 and T4.

J Biol Chem, 2002 Jul 5, 277(27), 24340 - 5 Epub 2002 May 01.
Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta; Lu X et al.; The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now . In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA . We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125 . A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50 . A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments . The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA . These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.

Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 6005 - 10
Observation by fluorescence microscopy of transcription on single combed DNA; Gueroui Z et al.; Molecular combing is a powerful procedure for aligning a large array of DNA molecules onto a surface . This technique usually leads to an overstretching of about 150% of the molecules' contour length . By changing the magnitude of capillary forces during the combing process, we were able to reduce the relative extension of the DNA molecules . Thus we achieved combing of T7 DNA with an extension close to its molecule contour length . We checked the ability of combed DNA to interact with DNA binding proteins . Using the T7 bacteriophage transcription system, we investigated the transcription activity of RNA polymerase on combed DNA by direct visualization of newly synthesized fluorescent RNAs . Our experiments show that no transcription activity occurs on overstretched DNA molecules, whereas we observe a transcription activity for nonoverstretched molecules . This activity is observed both in multiple initiation experiments and for one immobilized T7 RNA polymerase per promoter . These results open possibilities for the study of single enzyme actions on combed DNA by optical methods.

J Immunol Methods, 2002 Apr 1, 262(1-2), 191 - 204
A model system for optimising the selection of membrane antigen-specific human antibodies on intact cells using phage antibody display technology; Hegmans JP et al.; The functional expression of human antibody fragments on the surface of filamentous bacteriophage, and selection of phage antibodies (PhAbs) with antigens, has provided a powerful tool for generating novel antibodies . Applications of phage antibody display technology have increased over the past decade . Successful isolation of phage antibodies has been reported mostly using purified antigens . Isolation has proven to be more complicated with complex mixtures of antigens, such as intact cells . A given cell type contains thousands of different epitopes, each capable in theory of binding phage antibodies . Often antigens are not known or cannot be purified without disrupting their conformational integrity . To overcome problems involving phage antibody selections on intact cells, we have developed an experimental model system that allows for optimisation and comparison of various selection strategies . The model system comprises labelling of intact cells with the fluorescently labelled phospholipid fluorescein-DHPE . Upon incubation, this phospholipid is readily incorporated in the membrane of any cell type . Labelling intensity is regulated by varying the phospholipid concentration . After optimisation of key steps in the selection procedure, we were able to isolate fluorescein-DHPE specific phage from a synthetic library using intact cells . This model system can be applied to any cell type and we demonstrate that it can be used to efficiently compare and optimise selection strategies.

J Biol Chem, 2002 Jun 28, 277(26), 23181 - 5 Epub 2002 Apr 25.
The gene e.1 (nudE.1) of T4 bacteriophage designates a new member of the Nudix hydrolase superfamily active on flavin adenine dinucleotide, adenosine 5'-triphospho-5'-adenosine, and ADP-ribose; Xu W et al.; The T4 bacteriophage gene e.1 was cloned into an expression vector and expressed in Escherichia coli, and the purified protein was identified as a Nudix hydrolase active on FAD, adenosine 5'-triphospho-5'-adenosine (Ap(3)A), and ADP-ribose . Typical of members of the Nudix hydrolases, the enzyme has an alkaline pH optimum (pH 8) and requires a divalent cation for activity that can be satisfied by Mg(2+) or Mn(2+) . For all substrates, AMP is one of the products, and unlike most of the other enzymes active on Ap(3)A, the T4 enzyme hydrolyzes higher homologues including Ap(4-6)A . This is the first member of the Nudix hydrolase gene superfamily identified in bacterial viruses and the only one present in T4 . Although the protein was predicted to be orthologous to E . coli MutT on the basis of a sequence homology search, the properties of the gene and of the purified protein do not support this notion because of the following . (a) The purified enzyme hydrolyzes substrates not acted upon by MutT, and it does not hydrolyze canonical MutT substrates . (b) The e.1 gene does not complement mutT1 in vivo . (c) The deletion of e.1 does not increase the spontaneous mutation frequency of T4 phage . The properties of the enzyme most closely resemble those of Orf186 of E . coli, the product of the nudE gene, and we therefore propose the mnemonic nudE.1 for the T4 phage orthologue.

J Bacteriol, 2002 May, 184(10), 2789 - 804
Snapshot of the genome of the pseudo-T-even bacteriophage RB49; Desplats C et al.; RB49 is a virulent bacteriophage that infects Escherichia coli . Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage . To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the approximately 170-kb genome . Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis . However, when translated, about 70% of them encoded proteins with homology to T4 proteins . Among these sequences were the numerous components of the virion and the phage DNA replication apparatus . Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome . The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication . This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues . Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences . The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies . For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters . These results indicate that RB49 and T4 have diverged substantially from their last common ancestor . The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes.

Genetics, 2002 Apr, 160(4), 1273 - 81
Dependence of epistasis on environment and mutation severity as revealed by in silico mutagenesis of phage t7; You L et al.; Understanding how interactions among deleterious mutations affect fitness may shed light on a variety of fundamental biological phenomena, including the evolution of sex, the buffering of genetic variations, and the topography of fitness landscapes . It remains an open question under what conditions and to what extent such interactions may be synergistic or antagonistic . To address this question, we employed a computer model for the intracellular growth of bacteriophage T7 . We created in silico 90,000 mutants of phage T7, each carrying from 1 to 30 mutations, and evaluated the fitness of each by simulating its growth cycle . The simulations sought to account for the severity of single deleterious mutations on T7 growth, as well as the effect of the resource environment on our fitness measures . We found that mildly deleterious mutations interacted synergistically in poor-resource environments but antagonistically in rich-resource environments . However, severely deleterious mutations always interacted antagonistically, irrespective of environment . These results suggest that synergistic epistasis may be difficult to experimentally distinguish from nonepistasis because its effects appear to be most pronounced when the effects of mutations on fitness are most challenging to measure . Our approach demonstrates how computer simulations of developmental processes can be used to quantitatively study genetic interactions at the population level.

Mol Microbiol, 2002 Apr, 44(2), 489 - 500
Genetics of the phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2); Sumby P et al.; The phage growth limitation (Pgl) system, encoded by Streptomyces coelicolor A3(2), confers protection against the temperate bacteriophage phiC31 and its homoimmune relatives . The Pgl phenotype is characterized by the ability of Pgl+ hosts to support a phage burst on initial infection but subsequent cycles are severely attenuated . Previously, two adjacent genes pglY and pglZ were shown to be required for Pgl . It had been shown by Southern blotting that Streptomyces lividans, a close relative of S . coelicolor and naturally Pgl-, does not contain homologues of pglYZ and that introduction of pglYZ into S . lividans is not sufficient to confer a Pgl+ phenotype . Moreover, the mechanism of the Pgl+<--> Pgl- phase variation associated with this phenotype is also not understood . Here we describe two novel genes, pglW and pglX, that were shown to be part of this system by complementation of Pgl- mutants and by insertional mutagenesis . pglW encodes a 169 kDa protein that includes putative motifs for both serine/threonine protein kinase activity and DNA binding . pglX encodes a 136 kDa protein with putative adenine-specific DNA methyltransferase activity . pglW and pglX have overlapping stop-start codons suggesting transcriptional and translational coupling . S1 mapping of transcripts initiating up-stream of pglW indicated that, like pglYZ, pglWX is expressed in uninfected cultures . A homologue of pglX with 76% amino acid identity was identified in S . coelicolor, and insertional mutagenesis indicated that this gene was not required for the Pgl+ phenotype . Southern blots indicated that S . lividans does not contain homologues of pglW or pglX . A plasmid encoding pglWXYZ was able to confer the Pgl+ phenotype to S . lividans implying that these four genes constitute the whole system.

Nucleic Acids Res . 2002 May 1;30(9):e40.
A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases; Brunet E et al.; In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous phages . The proteins are generally expressed as C-terminal fusions with the N-terminus of the minor coat protein p3 for display on phages . As full-length cDNAs generally contain several stop codons near their 3' end, this approach cannot be used for their expression on the surface of phages . Here we show that in vitro selection for catalytic activity is compatible with a system for expression of proteins as N-terminal fusions on the surface of bacteriophages . It is highlighted for the Stoffel fragment of Taq DNA polymerase I and makes use of (p3-Jun/Fos-Stoffel fragment) fusions . The efficiency of the selection is measured by an enrichment factor found to be about 55 for a phage polymerase versus a phage not expressing a polymerase . This approach could provide a method for the functional cloning of nucleotidyl transferases from cDNA libraries using filamentous phage display.

Life Sci Space Res, 1980, 18, 159 - 65
Genetic effects of space hadrons on bacteriophage under Alpine conditions; Yurov SS et al.; A dried film culture of bacteriophage T4Br + was kept in a lead bioblock for 366 days under Alpine conditions at an altitude of 6100 m above sea level to study the genetic effect of space hadrons . In the gelatin-like film under study we discovered some film plots with markedly reduced bacteriophage survival . In such plots, the mutation frequency exceeded the spontaneous background mutation rate 60-100 times . The spectrum of r mutations as classified into standard groups rI, rII and rIII differed from that found for other model radiation systems such as gamma-ray radiation in buffer or nutrient broth, and hadron and HZE particle radiation under space flight conditions . Reversion analysis of 159 rII mutants showed that 54.4% had small and elongated deletions, 23.16% had point mutations, and 22.5% of all the mutants had both small deletion and point mutations.

Curr Biol, 2002 Apr 16, 12(8), R276 - 8
Selfish DNA: new abode for homing endonucleases; Edgell DR; A 30-year old conundrum concerning the genetics of T-even bacteriophages has at last been solved, the answer turning out to involve free-standing homologs of intron-encoded homing endonucleases.

Mol Microbiol, 2002 Apr, 44(1), 283 - 96
HF2: a double-stranded DNA tailed haloarchaeal virus with a mosaic genome; Tang SL et al.; HF2 is a haloarchaeal virus infecting two Halorubrum species (Family Halobacteriaceae) . It is lytic, has a head-and-tail morphology and belongs to the Myoviridae (contractile tails) . The linear double-stranded DNA genome was sequenced and found to be 77 670 bp in length, with a mol% G+C of 55.8 . A total of 121 likely open reading frames (ORFs) were identified, of which 37 overlapped at start and stop codons . The predicted proteins were usually acidic (average pI of 4.8), and less than about 12% of them had homologues in the sequence databases . Four complete tRNA-like sequences (tRNA-Arg, -Asx, -Pro and -Tyr) and an incomplete tRNA-Thr were detected . A transcription map showed that most of the genome was transcribed and that the synthesis of transcripts occurred in a highly organized and reproducible pattern over a 5 h infection cycle . Transcripts often spanned multiple ORFs, suggesting that viral genes were organized into operons . The predicted ORF and observed transcript directions matched well and showed that transcription is mainly directed inwards from the genome termini, meeting at about 45-48 kb, and this was also a turning point in a cumulative GC-skew plot . The low point in cumulative GC-skew, near the left end, was a region rich in short repeats and lacking ORFs, which is likely to be an origin of replication . The HF2 genome is a mosaic of components from widely different sources, demonstrating clearly that viruses of haloarchaea, like their bacteriophage counterparts, are vectors for the exchange and transmission of genetic material between wide taxonomic distances, even across domains.

Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5675 - 9
Filamentous phage as vector-mediated antibody delivery to the brain; Frenkel D et al.; Early diagnosis of Alzheimer's disease is prevented by lack of means to visualize and target beta amyloid plaques in the brains of affected people . There are many methods of detecting amyloid plaques by staining postmortem brain tissue, but none are available for monitoring in living patients . We propose anti-beta amyloid antibodies as a highly specific probe to monitor amyloid plaque formation in living patients . Intranasal administration of filamentous phage as delivery vector of anti-beta amyloid antibody fragment into Alzheimer's APP transgenic mice enables in vivo targeting of beta amyloid plaques . The plaques were co-visualized both by thioflavin-S and fluorescent-labeled anti-phage antibodies in the olfactory bulb and the hippocampus region . The genetically engineered filamentous bacteriophage proved to be an efficient and nontoxic viral delivery vector to the brain, offering an obvious advantage over other mammalian vectors . The ability to image A beta deposits in vivo would arguably provide the most useful diagnostic and monitoring test for early diagnosis of Alzheimer's disease.

Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5373 - 7
Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in phage capsids; Arsuaga J et al.; When linear double-stranded DNA is packed inside bacteriophage capsids, it becomes highly compacted . However, the phage is believed to be fully effective only if the DNA is not entangled . Nevertheless, when DNA is extracted from a tailless mutant of the P4 phage, DNA is found to be cyclic and knotted (probability of 0.95) . The knot spectrum is very complex, and most of the knots have a large number of crossings . We quantified the frequency and crossing numbers of these knots and concluded that, for the P4 tailless mutant, at least half the knotted molecules are formed while the DNA is still inside the viral capsid rather than during extraction . To analyze the origin of the knots formed inside the capsid, we compared our experimental results to Monte Carlo simulations of random knotting of equilateral polygons in confined volumes . These simulations showed that confinement of closed chains to tightly restricted volumes results in high knotting probabilities and the formation of knots with large crossing numbers . We conclude that the formation of the knots inside the viral capsid is driven mainly by the effects of confinement.

Curr Opin Struct Biol, 2002 Apr, 12(2), 217 - 24
Clamp loaders and sliding clamps; Jeruzalmi D et al.; A coherent view of the structure and function of DNA polymerase processivity factors (sliding clamps and clamp loaders) is emerging from recent structural studies . Crystal structures of sliding clamps from the T4 and RB69 bacteriophages, and from an archaebacterium expand the gallery of ring-shaped processivity factors and clarify how the clamp interacts with the DNA polymerase . Crystallographic and electron microscopic views of clamp loaders from bacteria, archaebacteria and eukaryotes emphasize their common architecture and have produced models of how ATPbinding might be coupled to clamp opening/loading.

FEBS Lett, 1970 Mar 16, 7(1), 83 - 85

Braunitzer G, Braig S, Krug F, Hobom G.
Spontaneous mutants of the bacteriophage fd and mutants resulting from fd-infected cultures of E . coli grown in the presence of 2.7-diaminofluoren and proflavin were isolated by means of free flow electrophoresis.The amino acid analyses of the mutant coat proteins (B-proteins) show significant differences in comparison with the amino acid analyses of wild type coat protein.

Dev Biol, 2002 Apr 15, 244(2), 305 - 18
Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse; Hayashi S et al.; In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse . In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero . In the current work, we have generated a transgenic mouse line in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages . We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection . In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled . Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line . This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration .

Biochemistry, 2002 Apr 16, 41(15), 4771 - 8
Manganese substantially alters the dynamics of translesion DNA synthesis; Hays H et al.; The effect of metal ion substitution on the dynamics of translesion DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase was quantitatively evaluated through steady-state and transient kinetic techniques . Substitution of Mn(2+) for Mg(2+) enhances the steady-state rate of dNMP misinsertion opposite an abasic site by 11-34-fold . At the molecular level, the enhancement in translesion DNA synthesis reflects a substantial increase in the rate of the conformational change preceding phosphoryl transfer for all dNTPs that were tested . This is best illustrated by the biphasic pre-steady-state time course of dAMP insertion opposite an abasic site which indicates that a step after chemistry is rate-limiting for steady-state enzyme turnover . Furthermore, the k(pol) value of 40 s(-1) measured under single-turnover reaction conditions is 20-fold greater than the k(cat) value of 2 s(-1) measured for steady-state enzyme turnover . Finally, the low elemental effect ( approximately 2.4-fold reduction in k(pol)) measured by substituting the alpha-thiotriphosphate analogue for dATP further argues that chemistry is not rate-limiting . In contrast to the biphasic insertion of dAMP, pre-steady-state time courses for the insertion of dCMP, dGMP, or dTMP opposite an abasic site were linear . Nearly identical k(pol) values ( approximately 1 s(-1)) were measured for the insertion of dCMP, dGMP, and dTMP opposite the abasic site using single-turnover conditions . However, the large elemental effects of 27 and 70 measured by substituting the alpha-thiotriphosphate analogues for dCTP and dGTP, respectively, suggest that phosphoryl transfer may be the rate-limiting step for their insertion opposite the abasic site . Various models are discussed in an attempt to explain the effect of metal ion substitution on the dynamics of translesion DNA replication.

FEMS Immunol Med Microbiol, 2002 Feb 18, 32(3), 205 - 9
The antibody response to bacteriophage is linked to the lymphopenia gene in congenic BioBreeding rats; Clark L et al.; Congenic BioBreeding (BB) rats, homozygous for the autosomal lymphopenia (Lyp) gene (Lyp/Lyp), heterozygous (Lyp/+), or wild-type (+/+), were immunized with the T cell-dependent bacteriophage PhiX174 to determine effects of Lyp on primary and secondary antibody responses . The primary PhiX174 antibody response did not differ between the three different genotypes . In contrast, the secondary immune response, expressed as the peak neutralizing titer, was markedly reduced in Lyp/Lyp (9.9+/-3.2; mean value+/-S.E.M . for seven rats) compared to both Lyp/+ (51+/-12; n=13; P=0.006) and +/+ (100+/-20; n=7; P=0.004) BB rats . We suggest that the secondary antibody response to the T cell-dependent neoantigen PhiX174 is linked in a recessive manner to genetic factor(s) in the Lyp gene region.

J Virol, 2002 May, 76(9), 4634 - 42
Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin; Yang X et al.; The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane proteins . Soluble gp140 glycoproteins composed of the uncleaved ectodomains of gp120 and gp41 form unstable, heterogeneous oligomers, but soluble gp140 trimers can be stabilized by fusion with a C-terminal, trimeric GCN4 motif (X . Yang et al., J . Virol . 74:5716-5725, 2000) . To understand the influence of the C-terminal trimerization domain on the properties of soluble HIV-1 envelope glycoprotein trimers, uncleaved, soluble gp140 glycoproteins were stabilized by fusion with another trimeric motif derived from T4 bacteriophage fibritin . The fibritin construct was more stable to heat and reducing conditions than the GCN4 construct . Both GCN4- and fibritin-stabilized soluble gp140 glycoproteins exhibited patterns of neutralizing and nonneutralizing antibody binding expected for the functional envelope glycoprotein spike . Of note, two potently neutralizing antibodies, immunoglobulin G1b12 and 2G12, exhibited the greatest recognition of the stabilized, soluble trimers, relative to recognition of the gp120 monomer . The observed similarities between the GCN4 and fibritin constructs indicate that the HIV-1 envelope glycoprotein ectodomains dictate many of the antigenic and structural features of these fusion proteins . The melting temperatures and ligand recognition properties of the GCN4- and fibritin-stabilized soluble gp140 glycoproteins suggest that these molecules assume conformations distinct from that of the fusion-active, six-helix bundle.

Mol Cell, 2002 Mar, 9(3), 541 - 52
Nucleosome remodeling induced by RNA polymerase II: loss of the H2A/H2B dimer during transcription; Kireeva ML et al.; RNA polymerase II (Pol II) must transcribe genes in a chromatin environment in vivo . We examined transcription by Pol II through nucleosome cores in vitro . At physiological and lower ionic strengths, a mononucleosome imposes a strong block to elongation, which is relieved at increased ionic strength . Passage of Pol II causes a quantitative loss of one H2A/H2B dimer but does not alter the location of the nucleosome . In contrast, bacteriophage SP6 RNA polymerase (RNAP) efficiently transcribes through the same nucleosome under physiological conditions, and the histone octamer is transferred behind SP6 RNAP . Thus, the mechanisms for transcription through the nucleosome by Pol II and SP6 RNAP are clearly different . Moreover, Pol II leaves behind an imprint of disrupted chromatin structure.

Biol Chem, 2002 Jan, 383(1), 149 - 58
Random peptide bacteriophage display as a probe for urokinase receptor ligands; Fong S et al.; The urokinase receptor is a multi-functional protein that plays a central role in cell surface plasminogen activation, cell migration, and cell adhesion . We previously demonstrated that high affinity peptide ligands for the urokinase receptor, which are urokinase competitors, can be obtained from a 15mer peptide library (Goodson et al., 1994) . In order to probe for additional urokinase receptor binding sites we affinity selected the same bacteriophage library on complexes of soluble urokinase receptor (suPAR) and the receptor binding domain of urokinase, residues 1-48 (uPA1-48) . Bacteriophage were isolated which bound to suPAR and suPAR:uPA1-48 complexes with high yield . The peptide sequences encoded by these bacteriophage were distinct from those obtained previously on urokinase receptor expressing cells, and comprise two groups based upon effects on su-PAR:1-anilino-8-napthalene sulfonate (ANS) fluorescence, and vitronectin binding competition . Alanine scanning mutagensis of the soluble peptides was used to define minimal regions and key residues for suPAR binding by competition with the parent bacteriophage . A comparison of these results with sequences of domains of both vitronectin and integrin alpha-chains, which have been reported to be important for urokinase receptor binding, suggests that the homology with the peptide sequences selected is functionally significant.

J Biol Chem, 2002 Jun 7, 277(23), 20555 - 62 Epub 2002 Apr 01.
Assembly of the bacteriophage T4 helicase: architecture and stoichiometry of the gp41-gp59 complex; Ishmael FT et al.; The bacteriophage T4 59 protein (gp59) plays an essential role in recombination and replication by mediating the assembly of the gene 41 helicase (gp41) onto DNA . gp59 is required to displace the gp32 single-stranded binding protein on the lagging strand to expose a site for helicase binding . To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex were investigated . Both the N and C termini of gp41 were found to lie close to or in the gp41-gp41 subunit interface and interact with gp59 . The site of interaction of gp41 on gp59 is proximal to Cys-215 of gp59 . Binding of gp41 to gp59 stimulates a conformational change in the protein resulting in hexamer formation of gp59, and gp59 likewise stimulates oligomer formation of gp41 . The gp59 subunits in this complex are arranged in a head to head orientation, such that Cys-42 of one subunit is in close proximity to Cys-42 on an adjacent subunit, and Cys-215 on one subunit is close to Cys-215 on a neighboring subunit . As the helicase is loaded onto DNA, a conformational change in the gp41-gp59 complex occurs, which may serve to displace gp32 from the lagging strand and load the hexameric helicase in its place.

J Clin Microbiol, 2002 Apr, 40(4), 1441 - 6
Identification, characterization, and distribution of a Shiga toxin 1 gene variant (stx(1c)) in Escherichia coli strains isolated from humans; Zhang W et al.; By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J . We designated this variant "Stx1c" and developed stxB(1) restriction fragment length polymorphism and stx(1c)-specific PCR strategies to determine the frequency and distribution of stx(1c) among 212 STEC strains isolated from humans . stx(1c) was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea . stx(1c) was most frequently (in 23 STEC strains {63.9%}) associated with stx(2d), but 12 (33.3%) of the 36 STEC strains possessed stx(1c) only . A single STEC strain possessed stx(1c) together with stx(2) and was isolated from a patient with hemolytic-uremic syndrome . All 36 stx(1c)-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145 . Stx1c was produced by all stx(1c)-containing STEC strains, but reacted weakly with a commercial immunoassay . We conclude that STEC strains harboring the stx(1c) variant account for a significant proportion of human STEC isolates . The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx(1c) among clinical STEC isolates and their association with human disease in prospective studies.

Mol Microbiol, 2002 Mar, 43(5), 1079 - 88
The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain: the 'double wing' motif; Li N et al.; MotA is a transcription factor from bacteriophage T4 that helps adapt the host Escherichia coli transcription apparatus to T4 middle promoters . We have determined the crystal structure of the C-terminal DNA-binding domain of MotA (MotCF) to 1.6 A resolution using multiwavelength, anomalous diffraction methods . The structure reveals a novel DNA-binding alpha/beta motif that contains an exposed beta-sheet surface that mediates interactions with the DNA . Independent biochemical experiments have shown that MotCF binds to one surface of a single turn of DNA through interactions in adjacent major and minor grooves . We present a model of the interaction in which beta-ribbons at opposite corners of the six-stranded beta-sheet penetrate the DNA grooves, and call the motif a 'double wing' to emphasize similarities to the 'winged-helix' motif . The model is consistent with data on how MotA functions at middle promoters, and provides an explanation for why MotA can form non-specific multimers on DNA.

J Bacteriol, 2002 Apr, 184(8), 2155 - 66
Use of the Caulobacter crescentus genome sequence to develop a method for systematic genetic mapping; West L et al.; The functional analysis of sequenced genomes will be facilitated by the development of tools for the rapid mapping of mutations . We have developed a systematic approach to genetic mapping in Caulobacter crescentus that is based on bacteriophage-mediated transduction of strategically placed antibiotic resistance markers . The genomic DNA sequence was used to identify sites distributed evenly around the chromosome at which plasmids could be nondisruptively integrated . DNA fragments from these sites were amplified by PCR and cloned into a kanamycin-resistant (Kan(r)) suicide vector . Delivery of these plasmids into C . crescentus resulted in integration via homologous recombination . A set of 41 strains containing Kan(r) markers at 100-kb intervals was thereby generated . These strains serve as donors for generalized transduction using bacteriophage phiCr30, which can transduce at least 120 kb of DNA . Transductants are selected with kanamycin and screened for loss of the mutant phenotype to assess linkage between the marker and the site of the mutation . The dependence of cotransduction frequency on sequence distance was evaluated using several markers and mutant strains . With these data as a standard, previously unmapped mutations were readily localized to DNA sequence intervals equivalent to less than 1% of the genome . Candidate genes within the interval were then examined further by subcloning and complementation analysis . Mutations resulting in sensitivity to ampicillin, in nutritional auxotrophies, or temperature-sensitive growth were mapped . This approach to genetic mapping should be applicable to other bacteria with sequenced genomes for which generalized transducing phage are available.

J Bacteriol, 2002 Apr, 184(8), 2088 - 99
Xis protein binding to the left arm stimulates excision of conjugative transposon Tn916; Connolly KM et al.; Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((Tn916)Int) and excisionase ((Tn916)Xis) proteins . In this study, we have investigated the role of the (Tn916)Xis protein in stimulating excisive recombination . The functional relevance of (Tn916)Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay . Our results indicate that in Escherichia coli the stimulatory effect of the (Tn916)Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon . These sites lie in between the core and arm sites recognized by (Tn916)Int, suggesting that the (Tn916)Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage lambda, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis . However, our finding that excision in E . coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage lambda recombination.

BMC Genomics . 2002 Mar 21;3(1):8 . Epub 2002 Mar 21.
Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52; Iyer LM et al.; BACKGROUND: The DNA single-strand annealing proteins (SSAPs), such as RecT, Redbeta, ERF and Rad52, function in RecA-dependent and RecA-independent DNA recombination pathways . Recently, they have been shown to form similar helical quaternary superstructures . However, despite the functional similarities between these diverse SSAPs, their actual evolutionary affinities are poorly understood . RESULTS: Using sensitive computational sequence analysis, we show that the RecT and Redbeta proteins, along with several other bacterial proteins, form a distinct superfamily . The ERF and Rad52 families show no direct evolutionary relationship to these proteins and define novel superfamilies of their own . We identify several previously unknown members of each of these superfamilies and also report, for the first time, bacterial and viral homologs of Rad52 . Additionally, we predict the presence of aberrant HhH modules in RAD52 that are likely to be involved in DNA-binding . Using the contextual information obtained from the analysis of gene neighborhoods, we provide evidence of the interaction of the bacterial members of each of these SSAP superfamilies with a similar set of DNA repair/recombination protein . These include different nucleases or Holliday junction resolvases, the ABC ATPase SbcC and the single-strand-binding protein . We also present evidence of independent assembly of some of the predicted operons encoding SSAPs and in situ displacement of functionally similar genes . CONCLUSIONS: There are three evolutionarily distinct superfamilies of SSAPs, namely the RecT/Redbeta, ERF, and RAD52, that have different sequence conservation patterns and predicted folds . All these SSAPs appear to be primarily of bacteriophage origin and have been acquired by numerous phylogenetically distant cellular genomes . They generally occur in predicted operons encoding one or more of a set of conserved DNA recombination proteins that appear to be the principal functional partners of the SSAPs.

Biochemistry, 2002 Apr 2, 41(13), 4399 - 406
Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase; Mandal SS et al.; The fluorescence of the base analogue 2-aminopurine (2AP) was used to detect physical changes in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase . Fluorescent enzyme-DNA complexes were formed with 2AP placed in the template strand opposite the primer terminus (the n position) and placed one template position 5' to the primer terminus (the n + 1 position) . The fluorescence enhancement for 2AP at the n position was shown to be due to formation of the editing complex, which indicates that the 2AP-T terminal base pair is recognized primarily as a mismatch . 2AP fluorescence at the n + 1 position, however, was a reporter for DNA interactions in the polymerase active center that induce intrastrand base unstacking . T4 DNA polymerase produced base unstacking at the n + 1 position following formation of the phosphodiester bond . Thus, the increase in fluorescence intensity for 2AP at the n + 1 position could be used to measure the nucleotide incorporation rate in primer extension reactions in which 2AP was placed initially at the n + 2 position . Primer extension occurred at the rate of about 314 s(-1) . The amount of base unstacking at the template n + 1 position was sensitive to the local DNA sequence . More base unstacking was detected for DNA substrates with an A-T base pair at the primer terminus compared to C-G or G-C base pairs . Since proofreading is also increased by A-T base pairs compared to G-C base pairs at the primer terminus, we propose that base unstacking may provide an opportunity for the DNA polymerase to reexamine the primer terminus.

Proc Natl Acad Sci U S A, 2002 Apr 2, 99(7), 4185 - 90 Epub 2002 Mar 19.
Directed evolution of the site specificity of Cre recombinase; Santoro SW et al.; Cre recombinase from bacteriophage P1 recognizes a 34-bp recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA . To better understand the molecular basis of protein-DNA recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxP sites . To be selected, members of a library of Cre variants produced by targeted random mutagenesis must rapidly catalyze recombination, in vivo, between two variant loxP sites that are located on a reporter plasmid . Recombination results in an altered pattern of fluorescent protein expression that can be identified by flow cytometry . Fluorescence-activated cell sorting can be used either to screen positively for recombinase variants that recognize a novel loxP site, or negatively for variants that cannot recognize the wild-type loxP site . The use of positive screening alone resulted in a relaxation of recombination site specificity, whereas a combination of positive and negative screening resulted in a switching of specificity . One of the identified recombinases selectively recombines a novel recombination site and operates at a rate identical to that of wild-type Cre . Analysis of the sequences of the resulting Cre variants provides insight into the evolution of these altered specificities . This and other systems should contribute to our understanding of protein-DNA recognition and may eventually be used to evolve custom-tailored recombinases that can be used for gene study and inactivation.

J Mol Biol, 2002 Mar 22, 317(2), 179 - 90
Identification of two middle promoters upstream DNA ligase gene 30 of bacteriophage T4; Truncaite L et al.; Bacteriophage T4 DNA ligase gene 30 lies in the cluster of prereplicative genes located counterclockwise from map units 149 to 121 . Based on the early transcription studies this gene has been considered as a typical early gene of bacteriophage T4 . In agreement with this assignment, two strong T4 early promoters, P(E )30.8 (128.6) and P(E )30.7 (128.2), located about 3.1 and 2.7 kb upstream from gene 30 have been revealed by promoter mapping and sequence analysis . In addition, the existence of a putative early promoter just upstream of gene 30 was proposed from the sequence data . However, here we show that the putative early promoter just upstream of gene 30 is, in fact, a T4 middle promoter . Furthermore, we detected one more middle promoter located in the genomic region between early promoter P(E )30.7 (128.2) and DNA ligase gene 30 in the coding region of gene 30.3 . Both new middle promoters have differences from the consensus MotA box, while their -10 regions match the sigma(70) consensus sequence very well . The 5' ends of MotA-dependent transcripts directed from these promoters, as well as the kinetics of 5' end accumulation in the cells, have been determined by primer extension analysis . The results of these analyses indicate that both MotA-dependent and MotA-independent promoters control the transcription of T4 DNA ligase gene 30 in vivo . Moreover, we show that the first transcripts for gene 30 are directed from its own middle promoter, P(M)30 .

J Biol Chem, 2002 Jun 14, 277(24), 21630 - 8 Epub 2002 Mar 18.
Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library; Choi JY et al.; Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain . A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner . The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding . SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif . These motifs are classified based on the positions of conserved hydrophobic residues . To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments . The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants . Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.

Science, 2002 Mar 15, 295(5562), 2091 - 4
Reverse transcriptase-mediated tropism switching in Bordetella bacteriophage; Liu M et al.; Host-pathogen interactions are often driven by mechanisms that promote genetic variability . We have identified a group of temperate bacteriophages that generate diversity in a gene, designated mtd (major tropism determinant), which specifies tropism for receptor molecules on host Bordetella species . Tropism switching is the result of a template-dependent, reverse transcriptase-mediated process that introduces nucleotide substitutions at defined locations within mtd . This cassette-based mechanism is capable of providing a vast repertoire of potential ligand-receptor interactions.

Infect Immun, 2002 Apr, 70(4), 1896 - 908
The nucleotide sequence of Shiga toxin (Stx) 2e-encoding phage phiP27 is not related to other Stx phage genomes, but the modular genetic structure is conserved; Recktenwald J et al.; In this study we determined the complete nucleotide sequence of Shiga toxin 2e-encoding bacteriophage phi P27, isolated from the Shiga toxin-producing Escherichia coli patient isolate 2771/97 . phi P27 is integrated as a prophage in the chromosomal yecE gene . This integration generates identity segments of attL and attR sites with lengths of 11 nucleotides . The integrated prophage genome has a size of 42,575 bp . We identified 58 open reading frames (ORFs), each with a length of >150 nucleotides . The deduced proteins of 44 ORFs showed significant homologies to other proteins present in sequence databases, whereas 14 putative proteins did not . For 29 proteins, we could deduce a putative function . Most of these are related to the basic phage propagation cycle . The phi P27 genome represents a mosaic composed of genetic elements which are obviously derived from related and unrelated phages . We identified five short linker sequences of 22 to 151 bp in the phi P27 sequence which have also been detected in a couple of other lambdoid phages . These linkers are located between functional modules in the phage genome and are thought to play a role in genetic recombination . Although the overall DNA sequence of phi P27 is not highly related to other known phages, the data obtained demonstrate a typical lambdoid genome structure.

EMBO J, 1983, 2(3), 345 - 52
Bacteriophage Mu DNA circularizes following infection of Escherichia coli; Puspurs AH et al.; Mu DNA, isolated from infected cells or minicells, has been shown to be held by proteins in twisted and open circular forms . Circularization does not require protein synthesis in the infected cells . A 64,000-dalton polypeptide is injected into the infected cell with Mu DNA and co-sediments with Mu DNA through sucrose gradients . Circularization of the infecting Mu DNA does not require removal of the Escherichia coli DNA sequences which are attached to both ends of the Mu genome in the viral particle.

EMBO J, 1983, 2(1), 67 - 71
IS2 insertion is a major cause of spontaneous mutagenesis of the bacteriophage P1: non-random distribution of target sites; Sengstag C et al.; Insertion mutations arising spontaneously in the P1 prophage and affecting vegetative phage reproduction were screened for the presence of insertion sequence 2 (IS2) . Filter hybridization identified 28 out of 44 independent insertions as IS2 . Their target specificity is not random . A region that amounts to < 2% of the phage genome had trapped 15 of the 28 IS2 elements . However, precise mapping of nine mutants in this hot spot segment revealed no preferred insertion site . Rather, the nine IS2 are distributed over the whole target segment and IS2 are found in both orientations . Sequence data indicate that at least two sequence variants of IS2 participated in mutagenesis of the phage genome . The detectable transposition of IS2 from the host chromosome to the prophage occurs with a frequency of 3 x 10(-5) per cell per generation under the particular experimental conditions . It is concluded that IS2, a natural resident of Escherichia coli K12 strains, is an important agent for spontaneous mutagenesis and exerts this action non-randomly along the genome.

EMBO J, 1983, 2(9), 1521 - 6
The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein; Karnik S et al.; Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor . Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis . Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis . Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function . Expression of other cistrons in addition to the A2 cistron does not enhance host lysis . Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis . This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.

EMBO J, 1983, 2(9), 1515 - 9
Accuracy of DNA polymerase-alpha in copying natural DNA; Grosse F et al.; The fidelity of DNA polymerase-alpha from calf thymus (9S enzyme) in copying bacteriophage phi174am16 DNA in vitro has been determined from the frequency of production of different revertants . In the self-priming reaction we were able to measure the frequencies of base pairing mismatches during the course of replication on biasing the ratios of deoxynucleoside triphosphates . The frequency of dGTP:T, dGTP:G and dATP:G mismatches were 7.6 x 10(-5), 4.4 x 10(-5) and 2.8 x 10(-5), respectively, at equal concentrations of the deoxynucleoside triphosphates . dCTP:A, dGTP:A, dCTP:T and dTTP:T mismatches were below the limit of detection (<5 x 10(-6)) . A synthetic dodecamer primer with a 3' end covering the first two bases of the amber codon was used to determine the misinsertion frequency of the first nucleotide incorporated . This gave a misinsertion frequency of 1.5 x 10(-4) for the dGTP:T mismatch, which is slightly higher than that observed from the pool bias studies . Further, it showed no sensitivity to biasing the nucleotide pool, suggesting a different mechanism for the incorporation of the first nucleotide . These data do not support 'energy-relay'-like models for achieving high accuracy in eukaryotes . The observed misinsertion frequencies were corrected for mismatch repair of the heteroduplexes during the transfection experiments by parallel experiments using a mismatched primer . This was synthesized to have the same G:T mismatch as produced in the preceding experiment.

EMBO J, 2002 Mar 15, 21(6), 1477 - 86
Dynamics of a protein polymer: the assembly and disassembly pathways of the MuB transposition target complex; Greene EC et al.; MuB assembles into a polymer on DNA in the presence of ATP and is directly involved in the selection of an appropriate site on the Escherichia coli chromosome for the insertion of the bacteriophage Mu genome . We have developed an assay using fluorescently tagged proteins to monitor the polymeric state of MuB via fluorescence resonance energy transfer . We show that polymer assembly is initiated by the formation of an ATP-MuB complex . MuB then self-associates into a protomer before binding to DNA . Upon binding to DNA, a dramatic increase in energy transfer is observed, suggesting a conformational change within MuB . Polymer disassembly is much slower than assembly and is greatly stimulated by the MuA transposase . Additionally, MuB is readily exchanged between polymers, and ATP hydrolysis is directly coupled to polymer disassembly . Our data support a model in which a combination of rapid polymer assembly, MuA-mediated disassembly, followed by rapid reassembly of the polymer allows MuB to sample multiple DNA targets until an appropriate site is located for the insertion of the bacteriophage genome.

J Virol, 2002 Apr, 76(7), 3482 - 92
Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides; Bressanelli S et al.; We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions . An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 A away from the catalytic site . This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation . The electron density map at 1.7-A resolution clearly shows the mode of binding of the guanosine moiety to the enzyme . In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides . Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage phi6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms . No equivalent of the protein stacking platform observed for the priming nucleotide in the phi6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.

J Virol, 2002 Apr, 76(7), 3276 - 81
Distribution of spontaneous mutants and inferences about the replication mode of the RNA bacteriophage phi6; Chao L et al.; When a parent virus replicates inside its host, it must first use its own genome as the template for replication . However, once progeny genomes are produced, the progeny can in turn act as templates . Depending on whether the progeny genomes become templates, the distribution of mutants produced by an infection varies greatly . While information on the distribution is important for many population genetic models, it is also useful for inferring the replication mode of a virus . We have analyzed the distribution of mutants emerging from single bursts in the RNA bacteriophage phi6 and find that the distribution closely matches a Poisson distribution . The match suggests that replication in this bacteriophage is effectively by a stamping machine model in which the parental genome is the main template used for replication . However, because the distribution deviates slightly from a Poisson distribution, the stamping machine is not perfect and some progeny genomes must replicate . By fitting our data to a replication model in which the progeny genomes become replicative at a given rate or probability per round of replication, we estimated the rate to be very low and on the on the order of 10(-4) . We discuss whether different replication modes may confer an adaptive advantage to viruses.

Photochem Photobiol, 2002 Feb, 75(2), 85 - 91
Chlorella virus pyrimidine dimer glycosylase excises ultraviolet radiation- and hydroxyl radical-induced products 4,6-diamino-5-formamidopyrimidine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from DNA; Jaruga P et al.; A DNA glycosylase specific for UV radiation-induced pyrimidine dimers has been identified from the Chlorella virus Paramecium Bursaria Chlorella virus-1 . This enzyme (Chlorella virus pyrimidine dimer glycosylase {cv-pdg}) exhibits a 41% amino acid identity with endonuclease V from bacteriophage T4 (T4 pyrimidine dimer glycosylase {T4-pdg}), which is also specific for pyrimidine dimers . However, cv-pdg possesses a higher catalytic efficiency and broader substrate specificity than T4-pdg . The latter excises 4,6-diamino-5-formamidopyrimidine (FapyAde), a UV radiation- and hydroxyl radical-induced monomeric product of adenine in DNA . Using gas chromatography-isotope-dilution mass spectrometry and y-irradiated DNA, we show in this work that cv-pdg also displays a catalytic activity for excision of FapyAde and, in addition, it excises 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) . Kinetic data show that FapyAde is a better substrate for cv-pdg than FapyGua . On the other hand, cv-pdg possesses a greater efficiency for the extension of FapyAde than T4-pdg . These two enzymes exhibit different substrate specificities despite substantial structural similarities.

Virology, 2001 Nov 25, 290(2), 199 - 210
Characterization of the developmental switch region of bacteriophage P2 Hy dis; Renberg-Eriksson SK et al.; In this work, the DNA sequence of the transcriptional switch that affects the development of the P2 Hy dis bacteriophage was determined . The switch contains two face-to-face-located promoters and two repressors, Cox and C . The locations of the Pc and Pe promoters were determined by primer extension analysis . The P2 Hy dis homolog of the P2 multifunctional Cox protein was shown to be able to substitute for P2 Cox in repression of the P2 Pc promoter, excision of the P2 prophage, and activation of the satellite phage P4 PLL promoter . A directly repeated sequence, flanking the--35 region of the Pe promoter, was found to be important for C repressor binding as well as for repression . The P4 E protein was shown to derepress the developmental switch of P2 Hy dis in a plasmid-based derepression assay.

Res Microbiol, 2002 Jan-Feb, 153(1), 13 - 8
Bacteriophage therapy of infectious diseases in aquaculture; Nakai T et al.; Bacteriophages may be candidates as therapeutic agents in bacterial infections . Here we describe the protective effects of phages against experimentally induced bacterial infections of cultured fish and discuss the potential for phage therapy in aquaculture.

J Biol Chem, 2002 May 10, 277(19), 17117 - 24 Epub 2002 Feb 27.
Bacteriophage phi 6 RNA-dependent RNA polymerase: molecular details of initiating nucleic acid synthesis without primer; Laurila MR et al.; Like most RNA polymerases, the polymerase of double-strand RNA bacteriophage phi6 (phi6pol) is capable of primer-independent initiation . Based on the recently solved phi6pol initiation complex structure, a four-amino acid-long loop (amino acids 630-633) has been suggested to stabilize the first two incoming NTPs through stacking interactions with tyrosine, Tyr(630) . A similar loop is also present in the hepatitis C virus polymerase, another enzyme capable of de novo initiation . Here, we use a series of phi6pol mutants to address the role of this element . As predicted, mutants at the Tyr(630) position are inefficient in initiation de novo . Unexpectedly, when the loop is disordered by changing Tyr(630)-Lys(631)-Trp(632) to GSG, phi6pol becomes a primer-dependent enzyme, either extending complementary oligonucleotide or, when the template 3' terminus can adopt a hairpin-like conformation, utilizing a "copy-back" initiation mechanism . In contrast to the wild-type phi6pol, the GSG mutant does not require high GTP concentration for its optimal activity . These findings suggest a general model for the initiation of de novo RNA synthesis.

Tidsskr Nor Laegeforen, 2001 Nov 10, 121(27), 3197 - 200
{Bacteria-killing viruses, Stalinists and "superbugs"}; Olsen I et al.; In June 2000, the WHO warned that the level of resistance to drugs used to treat common infectious diseases is now reaching a crisis point . If world governments do not control infections better in order to slow down the development of drug resistance, entire populations could be wiped out by superbugs against which there is no efficient treatment . Development of resistance is due to both underuse and overuse of drugs, and strategies have been worked out, to slow down the development of resistance for instance by the Norwegian Ministry of Health and Social Affairs . The present article deals with an old principle, mainly developed behind the Iron Curtain, which is now attracting renewed attention in the west: the application of bacterial viruses (bacteriophages) in the fight against bacteria . According to clinical trials in Eastern Europe, mostly uncontrolled, phages have been used successfully in treatments against antibiotic-resistant bacteria, for instance in suppurative wound infections, gastroenteritis, sepsis, osteomyelitis and pneumonia . These encouraging data are supported by recent findings in well-controlled animal models demonstrating that phages can rescue animals from a variety of fatal infections . The present review discusses possible advantages and limitations of phage treatment in humans.

Biotechnol Bioeng, 2002 Apr 20, 78(2), 203 - 16
Understanding viral partitioning in two-phase aqueous nonionic micellar systems: 2 . Effect of entrained micelle-poor domains; Kamei DT et al.; Unlike the partitioning behavior of hydrophilic, water-soluble proteins, the partitioning behavior of viruses in the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C10E4) micellar system cannot be fully explained using the excluded-volume theory developed recently by our group . A central assumption underlying the excluded-volume theory--that macroscopic phase separation equilibrium is attained--was therefore challenged experimentally and theoretically . Photographs of the two-phase aqueous C10E4 micellar system were taken for different volume ratios to demonstrate that the entrainment of micelle-poor (virus-rich) domains in the macroscopic, top, micelle-rich phase decreases with a decrease in the volume ratio . Partitioning experiments were then conducted with the model virus bacteriophage P22 and the model protein cytochrome c at different operating temperatures for different volume ratios . For bacteriophage P22, the measured viral partition coefficient at each temperature decreased by about an order of magnitude when the volume ratio was decreased from 10 to 0.1, which clearly indicated that entrainment is an important factor influencing viral partitioning . For cytochrome c, the measured protein partition coefficient did not change, which demonstrated that this entrainment effect negligibly influences protein partitioning . A new theoretical description of partitioning was also developed that combines the excluded-volume theory with this entrainment effect . In this theory, one fitted parameter--the volume fraction of entrained micelle-poor domains in the macroscopic, top, micelle-rich phase--is used to account for the entrainment . To fit this parameter, only a single partitioning experiment is required for a given volume ratio, irrespectively of the partitioning solute . The new theoretical description of partitioning yielded very good quantitative predictions of the viral partition coefficients . Accordingly, it can be concluded that the primary mechanisms governing viral partitioning in the two-phase aqueous C10E4 micellar system are the entrainment of micelle-poor (virus-rich) domains in the macroscopic, top, micelle-rich phase and the excluded-volume interactions that operate between the viruses and the micelles .

Biotechnol Bioeng, 2002 Apr 20, 78(2), 190 - 202
Understanding viral partitioning in two-phase aqueous nonionic micellar systems: 1 . Role of attractive interactions between viruses and micelles; Kamei DT et al.; The partitioning behavior of viruses in the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C10E4) micellar system cannot be fully explained by considering solely the repulsive, steric, excluded-volume interactions that operate between the viruses and the nonionic C10E4 micelles . Specifically, an excluded-volume theory developed recently by our group is not able to quantitatively predict the observed viral partition coefficients, even though this theory is capable of providing reasonable quantitative predictions of protein partition coefficients . To shed light on the discrepancy between the theoretically predicted and the experimentally measured viral partition coefficients, a central assumption underlying the excluded-volume theory that the viruses and the C10E4 micelles interact solely through repulsive, excluded-volume interactions was challenged in this study . In particular, utilizing bacteriophage P22 as a model virus, a competitive inhibition test and a partitioning study of the capsids of bacteriophage P22 were conducted . Based on the results of these two experimental studies, it was concluded that any attractive interactions between the tailspikes of bacteriophage P22 and the C10E4 micelles are negligible . Another experimental study was carried out wherein the partition coefficients of the model viruses, bacteriophages P22 and T4, were measured at various temperatures, and compared with those previously obtained for bacteriophage phiX174 . This comparison also indicated that possible attractive, electromagnetic-induced interactions between the bacteriophage particles and the C10E4 micelles cannot be invoked to rationalize the observed discrepancy between the theoretically predicted and the experimentally measured viral partition coefficients .

Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3770 - 5 Epub 2002 Feb 26.
Genetic instability favoring transversions associated with ErbB2-induced mammary tumorigenesis; Liu S et al.; It has been argued that genetic instability is required to generate the myriad mutations that fuel tumor initiation and progression and, in fact, patients with heritable cancer susceptibility syndromes harbor defects in specific genes that normally maintain DNA integrity . However, the vast majority of human cancers arise sporadically, in the absence of deficiencies in known "mutator" genes . We used a cII-based mutation detection assay to show that the mean frequency of forward mutations in primary mammary adenocarcinomas arising in mouse mammary tumor virus-c-erbB2 transgenic mice harboring multiple copies of the lambda bacteriophage genome was significantly higher than in aged-matched, wild-type mammary tissue . Analysis of the cII mutational spectrum within the mammary tumor genomic DNA demonstrated a >6-fold elevation in transversion mutation frequency, resulting in a highly unusual inversion of the transition/transversion ratio characteristic of normal epithelium; frameshift mutation frequencies were unaltered . Arising oncogenic point mutations within the c-erbB2 transgene of such tumors were predominantly transversions as well . Data from this model system support the notion that elaboration of a mutator phenotype is a consequential event in breast cancer and suggest that a novel DNA replication/repair gene is a relatively early mutational target in c-erbB2-induced mammary tumorigenesis.

J Mol Biol, 2002 Feb 22, 316(3), 547 - 61
The large subunit of bacteriophage lambda's terminase plays a role in DNA translocation and packaging termination; Duffy C et al.; The DNA packaging enzyme of bacteriophage lambda, terminase, is a heteromultimer composed of a small subunit, gpNu1, and a large subunit, gpA, products of the Nu1 and A genes, respectively . The role of terminase in the initial stages of packaging involving the site-specific binding and cutting of the DNA has been well characterized . While it is believed that terminase plays an active role in later post-cleavage stages of packaging, such as the translocation of DNA into the head shell, this has not been demonstrated . Accordingly, we undertook a generalized mutagenesis of lambda's A gene and found ten lethal mutations, nine of which cause post-cleavage packaging defects . All were located in the amino-terminal two-thirds of gpA, separate from the carboxy-terminal region where mutations affecting the protein's endonuclease activity have been found . The mutants fall into five groups according to their packaging phenotypes: (1) two mutants package part of the lambda chromosome, (2) one mutant packages the entire chromosome, but very slowly compared to wild-type, (3) two mutants do not package any DNA, (4) four mutants, though inviable, package the entire lambda chromosome, and (5) one mutant may be defective in both early and late stages of DNA packaging . These results indicate that gpA is actively involved in late stages of packaging, including DNA translocation, and that this enzyme contains separate functional domains for its early and late packaging activities .

Mol Biol Evol, 2002 Mar, 19(3), 230 - 8
Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus; Rokyta D et al.; Deletion of the viral ligase gene drastically reduced the fitness of bacteriophage T7 on a ligase-deficient host . Viral evolution recovered much of this fitness during long-term passage, but the final fitness remained below that of the intact virus . Compensatory changes occurred chiefly in genes involved in DNA metabolism: the viral endonuclease, helicase, and DNA polymerase . Two other compensatory changes of unknown function also occurred . Using a method to distinguish compensatory mutations from other beneficial mutations, five additional substitutions from the recovery were shown to enhance adaptation to culture conditions and were not compensatory for the deletion . In contrast to the few previous studies of viral recovery from deletions, the compensatory changes in T7 did not restore the deletion or duplicate major regions of the genome . The ability of this deleted genome to recover much of the lost fitness via mutations in its remaining genes reveals a considerable evolutionary potential to modify the interactions of its elements in maintaining an essential set of functions.

J Struct Biol, 2001 Oct, 136(1), 20 - 9
Correlation of topographic surface and volume data from three-dimensional electron microscopy; Dimmeler E et al.; Three-dimensional(3D) reconstructions from tilt series in an electron microscope show in general an anisotropic resolution due to an instrumentally limited tilt angle . As a consequence, the information in the z direction is blurred, thus making it difficult to detect the boundary of the reconstructed structures . In contrast, high-resolution topography data from microscopic surface techniques provide exactly complementary information . The combination of topographic surface and volume data leads to a better understanding of the 3D structure . The new correlation procedure presented determines both the height scaling of the topographic surface and the relative position of surface and volume data, thus allowing information to be combined . Experimental data for crystalline T4 bacteriophage polyheads were used to test the new method . Three-dimensional volume data were reconstructed from a negatively stained tilt series . Topographic data for both surfaces were obtained by surface relief reconstruction of electron micrographs of freeze-dried and unidirectionally metal-shadowed polyheads . The combined visualization of volume data with the scaled and aligned surface data shows that the correlation technique yields meaningful results . The reported correlation method may be applied to surface data obtained by any microscopic technique yielding topographic data . (C) 2001 Elsevier Science (USA).

J Biol Chem, 2002 Apr 26, 277(17), 14501 - 8 Epub 2002 Feb 20.
Transcription termination: primary intermediates and secondary adducts; Kashlev M et al.; In living organisms, stable elongation complexes of RNA polymerase dissociate at specific template positions in a process of transcription termination . It has been suggested that the dissociation is not the immediate cause of termination but is preceded by catalytic inactivation of the elongation complex . In vitro reducing ionic strength can be used to stabilize very unstable and catalytically inactive complex at the point of termination; the previous biochemical characterization of this complex has led to important conclusions regarding termination mechanism . Here we analyze in detail the complexes formed between DNA template, nascent RNA, and Escherichia coli RNA polymerase during transcription through the tR2 terminator of bacteriophage lambda . At low ionic strength, the majority of elongation complexes fall apart upon reaching the terminator . Released RNA and DNA efficiently rebind RNA polymerase (RNAP) and form binary RNAP.RNA and RNAP.DNA complexes, which are indistinguishable from binary complexes obtained by direct mixing of the purified nucleic acids and the enzyme . A small fraction of elongation complexes that reach termination point escapes dissociation because RNA polymerase has backtracked from the terminator to an upstream DNA position . Thus, transcription elongation to a terminator site produces no termination intermediates that withstand dissociation in the time scale appropriate for biochemical studies.

J Biol Chem, 2002 May 10, 277(19), 16365 - 70 Epub 2002 Feb 19.
Complementary combining site contact residue mutations of the anti-digoxin Fab 26-10 permit high affinity wild-type binding; Short MK et al.; Antibody 26-10, obtained in a secondary immune response, binds digoxin with high affinity (K(a) = 1.3 x 10(10) M(-1)) because of extensive shape complementarity . We demonstrated previously that mutations of the hapten contact residue HTrp-100 to Arg (where H refers to the heavy chain) resulted in increased specificity for digoxin analogs substituted at the cardenolide 16 position . However, mutagenesis of H:CDR1 did not result in such a specificity change despite the proximity of the H:CDR1 hapten contact residue Asn-35 to the cardenolide 16 position . Here we constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 in a 26-10 mutant containing Arg-100 (26-10-RRALD) . Phage were selected by panning against digoxin, gitoxin (16-OH), and 16-acetylgitoxin coupled to bovine serum albumin . Clones that retained wild-type Asn at position 35 showed preferred binding to gitoxin, like the 26-10-RRALD parent . In contrast, clones containing Val-35 selected mainly on digoxin-bovine serum albumin demonstrated a shift back to wild-type specificity . Several clones containing Val-35 bound digoxin with increased affinity, approaching that of the wild type in a few instances, in contrast to the mutation Val-35 in the wild-type 26-10 background, which reduces affinity for digoxin 90-fold . It has therefore proven possible to reorder the 26-10 binding site by mutations including two major contact residues on opposite sides of the site and yet to retain high affinity for binding for digoxin . Thus, even among antibodies that have undergone affinity maturation in vivo, different structural solutions to high affinity binding may be revealed.

Infect Immun, 2002 Mar, 70(3), 1219 - 24
Use of bacteriophage Ba1 to identify properties associated with Bordetella avium virulence; Shelton CB et al.; Bordetella avium causes bordetellosis, an upper respiratory disease of birds . Commercially raised turkeys are particularly susceptible . We report here on the use of a recently described B . avium bacteriophage, Ba1, as a tool for investigating the effects of lysogeny and phage resistance on virulence . We found that lysogeny had no effect on any of the in vivo or in vitro measurements of virulence we employed . However, two-thirds (six of nine) spontaneous phage-resistant mutants of our virulent laboratory strain, 197N, were attenuated . Phage resistance was associated, in all cases, with an inability of the mutants to bind phage . Further tests of the mutants revealed that all had increased sensitivities to surfactants, and increased amounts of incomplete (O-antigen-deficient) lipopolysaccharide (LPS) compared to 197N . Hot phenol-water-extracted 197N LPS inactivated phage in a specific and dose-dependent manner . Acid hydrolysis and removal of lipid A had little effect upon the ability of isolated LPS to inactivate Ba1, suggesting that the core region and possibly the O antigen were required for phage binding . All of the mutants, with one exception, were significantly more sensitive to naive turkey serum and, without exception, significantly less able to bind to tracheal rings in vitro than 197N . Interestingly, the three phage-resistant mutants that remained virulent appeared to be O antigen deficient and were among the mutants that were the most serum sensitive and least able to bind turkey tracheal rings in vitro . This observation allowed us to conclude that even severe defects in tracheal ring binding and serum resistance manifested in vitro were not necessarily indicative of attenuation and that complete LPS may not be required for virulence.

Virology, 2002 Feb 1, 293(1), 182 - 91
Uptake and processing of modified bacteriophage M13 in mice: implications for phage display; Molenaar TJ et al.; Internalization and degradation of filamentous bacteriophage M13 by a specific target cell may have major consequences for the recovery of phage in in vivo biopanning of phage libraries . Therefore, we investigated the pharmacokinetics and processing of native and receptor-targeted phage in mice . (35)S-radiolabeled M13 was chemically modified by conjugation of either galactose (lacM13) or succinic acid groups (sucM13) to the coat protein of the phage to stimulate uptake by galactose recognizing hepatic receptors and scavenger receptors, respectively . Receptor-mediated endocytosis of modified phage reduced the plasma half-life of native M13 (t(1/2) = 4.5 h) to 18 min for lactosylated and 1.5 min for succinylated bacterophage . Internalization of sucM13 was complete within 30 min after injection and resulted in up to 5000-fold reduction of bioactive phage within 90 min . In conclusion, these data provide information on the in vivo behavior of wild-type and receptor-targeted M13, which has important implications for future in vivo phage display experiments and for the potential use of M13 as a viral gene delivery vehicle.

Virology, 2002 Feb 1, 293(1), 118 - 24
Characterization of phi 12, a bacteriophage related to phi 6: nucleotide sequence of the small and middle double-stranded RNA; Gottlieb P et al.; The isolation of additional bacteriophages containing segmented double-stranded RNA genomes has expanded the Cystoviridae family to nine members . Comparing the genomic sequences of these viruses has allowed evaluation of important genetic as well as structural motifs . These comparative studies are resulting in greater understanding of viral evolution and the role played by genetic and structural variation in the assembly mechanisms of the cystoviruses . In this regard, the small and middle double-stranded RNA genomic segments of bacteriophage phi 12 were copied as cDNA and their nucleotide sequences determined . This genome's organization is similar to that of the small and middle segments of bacteriophages phi 6, phi 8, and phi 13 . Although there is little similarity in the nucleotide sequences, similarity exists in the amino acid sequence of the lysis cassette proteins to those of phi 6 . The host cell attachment proteins are found to have marked similarity to the phi 13 attachment proteins.

Biochemistry, 2002 Feb 26, 41(8), 2589 - 98
Macromolecular import into Escherichia coli: the TolA C-terminal domain changes conformation when interacting with the colicin A toxin; Deprez C et al.; Various macromolecules such as bacteriotoxins and phage DNA parasitize some envelope proteins of Escherichia coli to infect the bacteria . A two-step import mechanism involves the primary interaction with an outer membrane receptor or with a pilus followed by the translocation across the outer membrane . However, this second step is poorly understood . It was shown that the TolA, TolQ, and TolR proteins play a critical role in the translocation of group A colicins and filamentous bacteriophage minor coat proteins (g3p) . Translocation of these proteins requires the interaction of their N-terminal domain with the C-terminal domain of TolA (TolAIII) . In this work, short soluble TolAIII domains were overproduced in the cytoplasm and in the periplasm of E . coli . In TolAIII, the two cysteine residues were found to be reduced in the cytoplasmic form and oxidized in the periplasmic form . The interaction of TolAIII with the N-terminal domain of colicin A (ATh) is observed in the presence and in the absence of the disulfide bridge . The complex formation of TolAIII and ATh was found to be independent of the ionic strength . An NMR study of TolAIII, both free and bound, shows a significant structural change when interacting with ATh, in the presence or absence of the disulfide bridge . In contrast, such a structural modification was not observed when TolAIII interacts with g3p N1 . These results suggest that bacteriotoxins and Ff bacteriophages parasitize E . coli using different interactions between TolA and the translocation domain of the colicin and g3p protein, respectively.

Hum Antibodies, 2001, 10(3-4), 95 - 9
Phage antibodies from combinatorial library neutralize vaccinia virus; Tikunova NV et al.; The library of human scFv antibodies displayed on the surface of bacteriophages was panned against Vaccinia virus (VACV), strain Elstree . 75% binding with Vaccinia virus . 5 clones were characterized for their binding with VACV and their ability to neutralize VACV in plaque reduction neutralization test (PRNT) . Antibodies from the clones were obtained as soluble individual molecules and their binding activities were confirmed in ELISA.

Eur J Biochem, 2002 Feb, 269(3), 833 - 41
Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein; Boudko SP et al.; Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac . This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly . The short C-terminal region has a beta-annulus-like structure . We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements . The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)) . These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively . The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1) . The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively . This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size . The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region . According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation . In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell . The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation . These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.

J Mol Biol, 2001 Nov 30, 314(3), 401 - 11
The N-terminal ATPase site in the large terminase protein gp17 is critically required for DNA packaging in bacteriophage T4; Rao VB et al.; Double-stranded DNA packaging in bacteriophages is apparently driven by the most powerful molecular motor ever measured . Although it is widely accepted that a translocating ATPase powers the DNA packaging machine, the identity of the ATPase that generates this driving force is unknown . Evidence suggests that the large terminase protein gp17, which possesses two consensus ATP binding motifs and an ATPase activity, is a strong candidate for the translocating ATPase in bacteriophage T4 . This hypothesis was tested by a PCR-directed combinatorial mutagenesis approach in which mutant libraries consisting of all possible codon combinations were constructed at the signature residues of the ATP binding motifs . The impact on gp17 function of each randomly selected mutant was evaluated by phenotypic analysis following recombinational transfer into the viral genome . The precise mutation giving rise to a particular phenotype was determined by DNA sequencing . The data showed that the N-terminal ATP binding site I (SRQLGKT(161-167)), but not the ATP binding site II (TAAVEGKS(299-306)), is critical for gp17 function . Even conservative substitutions such as G165A, K166R, and T167A were not tolerated at the GKT signature residues, which are predicted to interact with the ATP substrate . Biochemical analyses of the mutants showed a complete loss of in vitro DNA packaging activity but not the terminase (DNA-cutting) activity . The purified K166G mutant showed a loss of gp17-ATPase activity . The data, for the first time, implicated a specific ATPase center in the viral dsDNA packaging .

Biol Chem, 2001 Dec, 382(12), 1669 - 77
Ligand-mediated protection against phage lysis as a positive selection strategy for the enrichment of epitopes displayed on the surface of E . coli cells; Camaj P et al.; We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E . coli cells by a combination of ligand-mediated protection and phage-mediated selection . The effectiveness of this new approach was demonstrated by displaying the T7.tag on the surface of E . coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3 . A monoclonal T7.tag antibody was used as protective ligand for T7.tag-displaying cells and phage K3 for the elimination of unprotected cells . When populations of bacteria, containing between 6 to 10,000 cells displaying the T7.tag and approximately 10(8) cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibody-dependent survival of T7.tag displaying cells was observed, yielding an enrichment factor of up to 10(7)-fold . The CISTEM technology was used to select sequences from a T7.tag-based, randomised library and the results were compared to those obtained from selection by MACS with the same library . Together, these results reveal a novel in vivo screening strategy in which an E . coli phage receptor is used as display plafform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage . Extentions and modifications of the basic strategy should lead to novel applications for the identification of protein-ligand interactions.

Nucleic Acids Res, 2002 Feb 15, 30(4), 950 - 7
Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase; Tackett AJ et al.; Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone . Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion . We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda . Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda . However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA . Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson-Crick binding of PNA to ssDNA . Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence . To study the basis for this phenomenon, we examined self-aggregation by PNAs . The 15mer PNA readily self-aggregates to the point of precipitation . Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA . Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications.

Curr Biol, 2002 Feb 5, 12(3), R96 - 8
DNA packaging: a new class of molecular motors; Moore SD et al.; DNA is packaged into preformed bacteriophage capsids to liquid crystalline density by the action of a portal protein complex . Single molecule packaging studies indicate that this is a new and extremely powerful class of molecular motors.

Curr Biol, 2002 Feb 5, 12(3), R87 - 9
The lambda switch: cI closes the gap in autoregulation; Hochschild A; The bacteriophage lambda genetic switch is still yielding surprises . A recent study reveals that a long-range interaction involving proteins bound 2.4 kilobases away from one another on the phage genome mediates negative autoregulation, solving a long-standing puzzle concerning the regulation of lysogeny.

J Mol Biol, 2002 Feb 8, 316(1), 19 - 34
Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins; Lee J et al.; The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template . A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides . Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments . The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range . Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated . The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites . In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs . The size of the Okazaki fragments is not affected by the number of recognition sites on the template .

Chembiochem, 2001 Mar 2, 2(3), 212 - 9
An error-prone T7 RNA polymerase mutant generated by directed evolution; Brakmann S et al.; Viruses replicate their genomes at exceptionally high mutation rates . Their offspring evolve rapidly and therefore, are able to evade common immunological and chemical antiviral agents . In parallel, virus genomes cannot tolerate a further increase in mutation rate: Experimental evidence exists that even few additional mutations are sufficient for the extinction of a viral population . A future antiviral strategy might therefore aim at increasing the error-producing capacity of viral replication enzymes . We employed the principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity . A mutant T7 RNA polymerase with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type was selected . This enzyme synthesized highly heterogeneous RNA products in vitro or in vivo and also decreased the replication efficiency of wild-type bacteriophage T7 during infection.

J Biol Chem, 2002 Apr 26, 277(17), 14530 - 8 Epub 2002 Feb 04.
Site-specific photo-cross-linking between lambda integrase and its DNA recombination target; Kovach MJ et al.; The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed . We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position . When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically . The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20% . The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly . Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.

Genes Dev, 2002 Feb 1, 16(3), 351 - 62
Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns; Belle A et al.; All genetic markers from phage T2 are partially excluded from the progeny of mixed infections with the related phage T4 (general, or phage exclusion) . Several loci, including gene 56 of T2, are more dramatically excluded, being present in only approximately 1% of the progeny . This phenomenon is referred to as localized marker exclusion . Gene 69 is adjacent to gene 56 of T4 but is absent in T2, being replaced by completely nonhomologous DNA . We describe SegF, a novel site-specific DNA endonuclease encoded by gene 69, which is similar to GIY-YIG homing endonucleases of group I introns . Interestingly, SegF preferentially cleaves gene 56 of T2, both in vitro and in vivo, compared with that of phage T4 . Repair of the double-strand break (DSB) results in the predominance of T4 genes 56 and segF in the progeny, with exclusion of the corresponding T2 sequences . Localized exclusion of T2 gene 56 is dependent on full-length SegF and is likely analogous to group I intron homing, in which repair of a DSB results in coconversion of markers in the flanking DNA . Phage T4 has many optional homing endonuclease genes similar to segF, whereas similar endonuclease genes are relatively rare in other members of the T-even family of bacteriophages . We propose that the general advantage enjoyed by T4 phage, over almost all of its relatives, is a cumulative effect of many of these localized events.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 54 - 63
Structural characterization of bacteriophage M13 solubilization by amphiphiles; Stopar D et al.; The structural properties of bacteriophage M13 during disassembly were studied in different membrane model systems, composed of a homologue series of the detergents sodium octyl sulfate, sodium decyl sulfate, and sodium dodecyl sulfate . The structural changes during phage disruption were monitored by spin-labeled electron spin resonance (ESR) and circular dichroism spectroscopy . For the purpose of ESR spectroscopy the major coat protein mutants V31C and G38C were site-directed spin labeled in the intact phage particle . These mutants were selected because the mutated sites are located in the hydrophobic part of the protein, and provide good reporting locations for phage integrity . All amphiphiles studied were capable of phage disruption . However, no significant phage disruption was detected below the critical micelle concentration of the amphiphile used . Based on this finding and the linear dependence of phage disruption by amphiphiles on the phage concentration, it is suggested that the solubilization of the proteins of the phage coat by amphiphiles starts with an attachment to and penetration of amphiphile molecules into the phage particle . The amphiphile concentration in the phage increases in proportion to the amphiphile concentration in the aqueous phase . Incorporation of the amphiphile in the phage particle is accompanied with a change in local mobility of the spin-labeled part of the coat protein and its secondary structure . With increasing the amphiphile concentration in the phage particle, a concentration is reached where the concentration of the amphiphile in the aqueous phase is around its critical micelle concentration . A further increase in amphiphile concentration results in massive phage disruption . Phage disruption by amphiphiles appears to be dependent on the phage coat mutations . It is concluded that phage disruption is dependent on a hydrophobic effect, since phage solubilization could significantly be increased by keeping the hydrophilic part of the amphiphile constant, while increasing its hydrophobic part.

Nature, 2002 Jan 31, 415(6871), 553 - 7
Structure of the cell-puncturing device of bacteriophage T4; Kanamaru S et al.; Bacteriophage T4 has a very efficient mechanism for infecting cells . The key component of this process is the baseplate, located at the end of the phage tail, which regulates the interaction of the tail fibres and the DNA ejection machine . A complex of gene product (gp) 5 (63K) and gp27 (44K), the central part of the baseplate, is required to penetrate the outer cell membrane of Escherichia coli and to disrupt the intermembrane peptidoglycan layer, promoting subsequent entry of phage DNA into the host . We present here a crystal structure of the (gp5-gp27)3 321K complex, determined to 2.9 A resolution and fitted into a cryo-electron microscopy map at 17 A resolution of the baseplate-tail tube assembly . The carboxy-terminal domain of gp5 is a triple-stranded beta-helix that forms an equilateral triangular prism, which acts as a membrane-puncturing needle . The middle lysozyme domain of gp5, situated on the periphery of the prism, serves to digest the peptidoglycan layer . The amino-terminal, antiparallel beta-barrel domain of gp5 is inserted into a cylinder formed by three gp27 monomers, which may serve as a channel for DNA ejection.

World J Gastroenterol, 2000 Apr, 6(2), 220 - 222
Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3; Jiang RL et al.; AIM:To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector cosmid pTM3 and to express HCV core antigen in HepG2 cells.METHODS:Core gene cDNA of HCV was introduced into eukaryotic expression vector cosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2 cells were transfected with the recombinant plasmid pTM3-Q534 by lipofectin.RESULTS:From the transfected bacteria Top10F', 2 pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10 ampicillin resistant colonies . By reverse transcription PCR and indirect immunofluorescence technique, HCV RNA and core protein was identified in HepG2 cells transfected with the recombinant plasmid.CONCLUSION:The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 was successful.

Bioorg Med Chem, 2002 Mar, 10(3), 743 - 51
Novel angular furo and thieno-quinolinones: synthesis and preliminary photobiological studies; Fossa P et al.; A number of new furo and thienoquinolinones carrying an electron-withdrawing function or unsubstituted at the position 3 were synthesized in order to obtain new potential photochemotherapeutic agents with increased antiproliferative activity and decreased toxic side effects . Our interest in studying the SAR of these derivatives also prompted us to investigate the influence of N-methylation on biological activity, by preparing N-methyl derivatives . The antiproliferative activity of all the newly synthesized compounds was evaluated and compared to 8-methoxypsoralen (8-MOP), the drug widely used in PUVA-therapy . The 3-unsubstituted thienoquinolinones were generally the most potent derivatives, followed by the furo-analogues . In particular, the unsubstituted thieno{2,3-h}quinoline-2(1H)one showed the highest activity in T2 bacteriophage, HeLa cells and Ehrlich cells tests . All the compounds, assayed on Escherichia coli WP2 TM9, showed a similar mutagenic activity, very close to that of 8-MOP . Except for 2-oxo-1,2-dihydrothieno{2,3-h}quinoline-3-carboxylic acid, which appeared to be very effective, all compounds generated singlet oxygen to slightly larger amounts when compared to 8-MOP . The N-methyl analogues only induced moderate skin erythemas on albino guinea pigs, while all other derivatives appeared to be entirely inactive . On the basis of these results, the unsubstituted thieno{2,3h}quinoline 2(1H)one seems to be the most interesting potential drug for PUVA photochemotherapy and photopheresis.

J Mol Biol, 2002 Jan 25, 315(4), 663 - 76
Detailed architecture of a DNA translocating machine: the high-resolution structure of the bacteriophage phi29 connector particle; Guasch A et al.; The three-dimensional crystal structure of the bacteriophage phi29 connector has been solved and refined to 2.1A resolution . This 422 kDa oligomeric protein connects the head of the phage to its tail and translocates the DNA into the prohead during packaging . Each monomer has an elongated shape and is composed of a central, mainly alpha-helical domain that includes a three-helix bundle, a distal alpha/beta domain and a proximal six-stranded SH3-like domain . The protomers assemble into a 12-mer, propeller-like, super-structure with a 35 A wide central channel . The surface of the channel is mainly electronegative, but it includes two lysine rings 20 A apart . On the external surface of the particle a hydrophobic belt extends to the concave area below the SH3-like domain, which forms a crown that retains the particle in the head . The lipophilic belt contacts the non-matching symmetry vertex of the capsid and forms a bearing for the connector rotation . The structure suggests a translocation mechanism in which the longitudinal displacement of the DNA along its axis is coupled to connector spinning .

J Mol Biol, 2002 Jan 25, 315(4), 541 - 9
Characterization of the small antisense CI RNA that regulates bacteriophage P4 immunity; Forti F et al.; In the immune state bacteriophage P4 prevents expression of the replication functions by premature termination of transcription . A small RNA, the CI RNA, is the trans acting factor that regulates P4 immunity, by pairing to complementary target sequences and causing premature transcription termination . The CI RNA is matured by RNAse P and PNPase from the leader region of the same operon it regulates . In this work we better characterize this molecule . CI RNA copy number was determined to be around 500 molecules per lysogenic cell . By S(1) mapping we defined the 3'-end at 8423(+/-1); thus CI RNA is 79(+/-1) nt long . The minimum region for correct processing requires two bases upstream of the CI RNA 5'-end and the CCA sequence at the 3'-end . Computer analysis by FOLD RNA of CI RNA sequence predicts a cloverleaf-like structure formed by a double-stranded stalk, a minor and a major stem loop, and a single-stranded bulge . We analysed several cI mutations, which fall either in the single or double-stranded CI RNA regions . Base substitutions in the main loop and in the single-stranded bulge apparently did not change CI RNA structure, but affected its activity by altering the complementarity with the target sequences, whereas a mutation in the secondary stem had a disruptive effect on CI RNA secondary structure . The effects of this latter mutation were suppressed by a base substitution that restored the complementarity with the corresponding base in the stem . Base substitutions in the main stem caused only local alterations in the secondary structure of CI . However, when the substitutions concerned either G8501 or its complementary base at the bottom of the stem, CI RNA was not correctly processed .

Biotechniques, 2002 Jan, 32(1), 88 - 90, 92
Detection and evaluation of non-recombinants in cDNA libraries by multiple cloning region PCR; Bandyopadhyay D et al.; Bacteriophages that are routinely used in cDNA libraries do not require any biological selection for forming plaques . Thus parental non-recombinant phages are always found in variable proportions together with recombinant ones in all cDNA libraries . The presence of non-recombinants in significant proportions dilutes the abundance of rare cDNA species and makes library screening difficult . If the exact proportion of non-recombinants in a library were known, then one would screen proportionately more plaques to get a positive clone . In the absence of such information, screening is conventionally conducted on a number that is based on the titer of the library . We have devised a method using the flanking sequences from either side of the multiple cloning region (MCR) of all lambda phage vector derivatives as primers for PCR amplification . A non-recombinant phage produces a fragment equal to the size of the MCR, whereas a recombinant phage produces a fragment larger than the MCR, which is an MCR+ fragment . All cDNA libraries that we have studied show the presence of the MCR fragment (indicating non-recombinants) at variable proportions ranging between 6% and 36% of the total phages present . We also show that their presence negatively influences the retrieval of target cDNA sequences.

BMC Biotechnol . 2001;1(1):13 . Epub 2001 Dec 28.
A combined in vitro/in vivo selection for polymerases with novel promoter specificities; Chelliserrykattil J et al.; BACKGROUND: The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter . A combined in vitro/in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities . Large (10(3)-10(6)) polymerase libraries were made and cloned downstream of variant promoters . Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendant mRNAs in vivo . Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection . RESULTS AND CONCLUSIONS: A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified . A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity . This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides.

Genetics, 2002 Jan, 160(1), 5 - 12
Multiple mechanisms for degradation of bacteriophage T4 soc mRNA; Kai T et al.; The dmd gene of bacteriophage T4 is required for regulation of mRNA stability in a stage-dependent manner during infection . When this gene is mutated, late genes are globally silenced because of rapid degradation of mRNAs . To investigate the mechanism of such mRNA degradation, we analyzed the late gene soc transcripts . The degradation of soc mRNA was remarkably stabilized when its ability to be translated was impaired; either disruption of translation initiation signals or elimination of termination codons was effective in stabilization of soc mRNA and removal of elongation modestly stabilized it . Even in the absence of translation, however, the residual activity was still significant . These results suggested that the degradation of soc transcripts was promoted by two different mechanisms; one is dependent on translation and the other independent of translation . We found several cleavages introduced into soc RNA specifically when the dmd gene was mutated; some of them could be linked to polypeptide chain elongation and termination, suggesting the correlation with ribosomal action, and the others were independent of translation.

Mol Cell, 2002 Jan, 9(1), 187 - 94
Structure of Ocr from bacteriophage T7, a protein that mimics B-form DNA; Walkinshaw MD et al.; We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA . This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA . We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes . Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule . This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

Water Sci Technol, 2001, 44(11-12), 599 - 605
River water quality improvement by natural and constructed wetland systems in the tropical semi-arid region of northeastern Brazil; de Ceballos BS et al.; The efficiencies of a natural Typha spp wetland (Wn) formed on a river bed and its effluent treatment in a constructed wetland (Wc, subsurface horizontal flow) were investigated in northeastern Brazil (Paraiba State) . The Wc system (12 tanks with stone gravel, 4.13 m2, 0.22 m3, 20 Typha spp rhizomes, m(-2) each, with 38, 29, and 19 mm x d(-1) hydraulic loadings, and 5, 7, and 10 days HRT) was fed daily with effluent from a Wn . Wn removal presented the highest values after Typha spp were cut during the 5th week . Removal values were (1st and 2nd periods or before and after cutting): 75% and 81% BOD5; 10-53% total phosphorus; 13%-55% ammonia; 89%-91% FC; 90-96% coliphages and bacteriophages . Wc removals increased with time with best results on 10 d HRT . Removals were also higher in the 2nd period: 74%-78% BOD5; 58%-82% ammonia; 90% FC; 94-98% FS; and 92%-96% coliphages and bacteriophages . Despite the high remaining values of FC (1.4 x 10(4) CFU/100 ml) and FX (4 x 10(3) CFU/100 ml), the removals were satisfactory and HRT dependent, suggesting a gradual optimization of the system with time . The Wc exhibited good efficiency for improving water quality from polluted river.

Eur J Cancer, 2002 Jan, 38(2), 231 - 9
Molecular approaches to chemo-radiotherapy; Marples B et al.; Although radiotherapy is used to treat many solid tumours, normal tissue tolerance and inherent tumour radioresistance can hinder successful outcome . Cancer gene therapy is one approach being developed to address this problem . However, the potential of many strategies are not realised owing to poor gene delivery and a lack of tumour specificity . The use of treatment-, condition- or tumour-specific promoters to control gene-directed enzyme prodrug therapy (GDEPT) is one such method for targeting gene expression to the tumour . Here, we describe two systems that make use of GDEPT, regulated by radiation or hypoxic-responsive promoters . To ensure that the radiation-responsive promoter is be activated by clinically relevant doses of radiation, we have designed synthetic promoters based on radiation responsive CArG elements derived from the Early Growth Response 1 (Egr1) gene . Use of these promoters in several tumour cell lines resulted in a 2-3-fold activation after a single dose of 3 Gy . Furthermore, use of these CArG promoters to control the expression of the herpes simplex virus (HSV) thymidine kinase (tk) gene in combination with the prodrug ganciclovir (GCV) resulted in substantially more cytotoxicity than seen with radiation or GCV treatment alone . Effectiveness was further improved by incorporating the GDEPT strategy into a novel molecular switch system using the Cre/loxP recombinase system of bacteriophage P1 . The level of GDEPT bystander cell killing was notably increased by the use of a fusion protein of the HSVtk enzyme and the HSV intercellular transport protein vp22 . Since hypoxia is also a common feature of many tumours, promoters containing hypoxic-responsive elements (HREs) for use with GDEPT are described . The development of such strategies that achieve tumour targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy.

J Electron Microsc (Tokyo), 2001, 50(5), 417 - 22
Atomic force microscopy analysis of bacteriophages phiKZ and T4; Matsko N et al.; Bacteriophage phiKZ was investigated by atomic force microscopy (AFM) and transmission electron microscopy (TEM) . The well-known phage T4 was used as a reference sample . Reproducible contact mode AFM images of native and partially disintegrated particles of bacteriophage phiKZ in air were obtained . It was demonstrated that the heads of phiKZ and T4 phages were compressed differently and depended on adsorption onto highly ordered pyrolytic graphite and mica . We have established a procedure to partially disintegrate the viral particles after which the internal protein body, which is a helical structure with a cylinder-like form, was easily observable inside the bacteriophage phiKZ head.

Vet Microbiol, 2002 Feb 26, 85(1), 37 - 46
Virulence factors of Escherichia coli isolated from bovine clinical mastitis; Kaipainen T et al.; Escherichia coli isolates from bovine mastitis were examined for a selection of virulence factors . The strains originated from Finland and Israel, which have differences in the proportion of mastitis caused by E . coli, clinical pictures of coliform mastitis, environmental conditions and herd management . The genes of nine virulence factors were detected by polymerase chain reaction . Presence of K1 and K5 capsules was assessed by use of specific bacteriophages . Serum resistance was tested by a turbidimetric assay . Out of 160 Finnish isolates, 37% had traT, 14% cnf2, 8% cnf1, 11% aer, 9% f17, 8% sfa, 7% pap, 1% afa8D and 1% afa8E . Out of 113 Israeli isolates, 41% had traT, 4% aer, 3% cnf2, 1% cnf1, 1% sfa and 1% f17 . Some of the genes were distributed among two major pathotype groups, with either f17 family or sfa, pap and cnf1 as major determinants . Genes for F17a, CS31A, Afa7D and Afa7E were not detected . Altogether 49% of Finnish and 42% of Israeli isolates had at least one virulence gene, but genes other than traT were present in only 24% of Finnish and 5% of Israeli isolates . Serum resistance was more common among Finnish (94/160) than Israeli isolates (19/113) . K1 and K5 capsules were not detected.

Cell, 2002 Jan 11, 108(1), 13 - 6
Phage genomics: small is beautiful; Brussow H et al.; The Age of Genomics dawned only gradually for bacteriophages . It was 1977 when the genome of phage phi X174 was published and 1983 when the "large" genome of phage lambda hit the streets . More recently, the pace has quickened, so that we now have over 100 complete phage genomes and can expect thousands in a very few years . These sequences have been marvelously informative for the biology of the individual phages, but with the advent of high volume sequencing technology, the real excitement for phage biology is that it is now possible to analyze the sequences together and thereby address--for the first time at whole genome resolution--a set of fundamental biological questions related to populations: What is the structure of the global phage population? What are its dynamics? How do phages evolve? This is Comparative Genomics with a capital "C".

Biochemistry, 2002 Jan 22, 41(3), 1051 - 9
Inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors; Reiter NJ et al.; Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy . Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdaPP, albeit with inhibition constants (K(i)) that range over 5 orders of magnitude . In addition, small organic anions were tested as inhibitors . Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP . Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme . This suggests a change in the bridging interaction between Mn(2+) ions of the dimer due to protonation or replacement of a bridging ligand . Inhibitor binding also induces several spectral shifts . Hyperfine splitting characteristic of a spin-coupled (Mn(2+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP . These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.

Biochemistry, 2002 Jan 22, 41(3), 914 - 21
Oxidation of 7,8-dihydro-8-oxoguanine affords lesions that are potent sources of replication errors in vivo; Henderson PT et al.; Three single-stranded DNA genomes have been constructed that contain the 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) oxidation products oxaluric acid, oxazalone, and cyanuric acid . Oligonucleotides containing each lesion were synthesized by treating an oligonucleotide containing a single 8-oxodG with peroxynitrite, and the desired products were isolated by HPLC . The modified oligonucleotides were ligated into M13mp7L2 bacteriophage DNA in such a way that the lesion was situated at a known site in the lacZ gene fragment of the viral genome . The circular genomes were transfected into wild-type AB1157 Escherichia coli . The relative efficiency of lesion bypass by DNA polymerase was determined by counting the number of initial independent infections produced by each genome relative to that of an unmodified DNA control . Viral progeny were analyzed for mutation frequency and type by PCR amplification of the insert region followed by a recently developed post-labeling assay . All three secondary lesions were readily bypassed, causing G --> T transversions at frequencies at least an order of magnitude higher than 8-oxodG . These data establish a model whereby the modestly mutagenic primary lesion 8-oxodG is oxidized in vivo to much more highly mutagenic secondary lesions.

Genetics, 2001 Dec, 159(4), 1393 - 404
Profiles of adaptation in two similar viruses; Holder KK et al.; The related bacteriophages phiX174 and G4 were adapted to the inhibitory temperature of 44 degrees and monitored for nucleotide changes throughout the genome . Phage were evolved by serial transfer at low multiplicity of infection on rapidly dividing bacteria to select genotypes with the fastest rates of reproduction . Both phage showed overall greater fitness effects per substitution during the early stages of adaptation . The fitness of phiX174 improved from -0.7 to 5.6 doublings of phage concentration per generation . Five missense mutations were observed . The earliest two mutations accounted for 85% of the ultimate fitness gain . In contrast, G4 required adaptation to the intermediate temperature of 41.5 degrees before it could be maintained at 44 degrees . Its fitness at 44 degrees increased from -2.7 to 3.2, nearly the same net gain as in phiX174, but with three times the opportunity for adaptation . Seventeen mutations were observed in G4: 14 missense, 2 silent, and 1 intergenic . The first 3 missense substitutions accounted for over half the ultimate fitness increase . Although the expected pattern of periodic selective sweeps was the most common one for both phage, some mutations were lost after becoming frequent, and long-term polymorphism was observed . This study provides the greatest detail yet in combining fitness profiles with the underlying pattern of genetic changes, and the results support recent theories on the range of fitness effects of substitutions fixed during adaptation.

Zhonghua Jie He He Hu Xi Za Zhi, 2000 Aug, 23(8), 480 - 4
{Rapid rifampicin susceptibility test by using recombinant mycobacteriophages}; Lu B et al.; OBJECTIVE: To set up a fast, sensitive and specific way to detect mycobacteria and rifampicin susceptibility to mycobacteria by using recombinant mycobacteriophages and bioluminescent method . METHODS: Firstly detected the luciferase activity in different bacteria by using mycobacteriophages, then assessed the best drug concentration of rifampicin for drug susceptibility test in luciferase reporter assay, and lastly used the above conditions to decide rifampicin susceptibilities to different mycobacteria and compare the result with L-J medium culture . RESULTS: Bacteriophages had high light production specifically in mycobacteria and only very low light production in E . coli, the difference being obvious . The light production of rifampicin-resisitant mycobacterium strains was much higher than that of sensitive strains in culture with rifampicin(P < 0.05) . Different drug concentrations of rifampicin were used to optimize drug concentration for rifampicin drug susceptibility test and the optimum concentration was found to be 2 micrograms/ml . The correlation of drug susceptibility test between luciferase reporter phages to L-J medium in standard strains and clinical isolates was the sarce . Temperature sensitive Phage 88 was more sensitive than Phage 40(P < 0.05) . The time for drug resistance test was 72 hours . CONCLUSIONS: Luciferase reporter phage can detect mycobacteria specifically and can be used in rifampicin susceptibility test . This assay is a fast, sensitive and specific method to detect mycobacterium strains and their resistance to rifampicin.

J Mol Biol, 2002 Jan 4, 315(1), 63 - 72
Transmembrane domain mediated self-assembly of major coat protein subunits from Ff bacteriophage; Melnyk RA et al.; The 50-residue major coat protein (MCP) of Ff bacteriophage exists as a single-spanning membrane protein in the Escherichia coli host inner membrane prior to assembly into lipid-free virions . Here, the molecular bases for the specificity and stoichiometry that govern the protein-protein interactions of MCP in the host membrane are investigated in detergent micelles . To address these structural issues, as well as to circumvent viability requirements in mutants of the intact protein, peptides corresponding to the effective alpha-helical TM segment of wild-type and mutant bacteriophage MCPs were synthesized . Fluorescence resonance energy transfer (FRET) experiments on the dansyl and dabcyl-labeled MCP TM domain peptides in detergent micelles demonstrated that the peptides specifically associate into non-covalent homodimers, as postulated for the biologically relevant membrane-embedded MCP oligomer . MCP peptides labeled with short-range pyrene fluorophores at the N terminus displayed excimer fluorescence consistent with homodimerization occurring in a parallel fashion . Variant peptides synthesized with single substitutions at helix-interactive positions displayed a wide range of dimer/monomer ratios on SDS-PAGE gels, which are interpreted in terms of steric volume, presence or absence of beta-branching, and the effect of polar substituents . The overall results indicate discrete roles for helix-helix interfacial residues as packing recognition elements in the membrane-inserted state, and suggest a possible correlation between phage viability and efficacy of MCP TM-TM interactions .

Novartis Found Symp, 2002, 241, 159 - 68; discussion 168-72, 226-32
Sodium channels in primary sensory neurons: relationship to pain states; Wood JN et al.; Electrophysiological studies of dorsal root ganglion (DRG) neurons, and the results of PCR, Northern blot and in situ hybridization analyses have demonstrated the molecular diversity of Na+ channels that operate in sensory neurons . Several subtypes of alpha-subunit have been detected in DRG neurons and transcripts encoding all three beta-subunits are also present . Interestingly, one alpha subunit, Na(v)1.8, is selectively expressed in C-fibre and Adelta fibre associated sensory neurons that are predominantly involved in damage sensing . Another channel, Na(v).3, is selectively up regulated in a variety of models of neuropathic pain . In this review we focus on Na+ channels that are selectively expressed in DRG neurons as potential analgesic drug targets . In the absence of subtype specific inhibitors, the production of null mutant mice provides useful information on the specialized functions of particular Na+ channels . A refinement of this approach is to delete Na+ channel genes flanked by lox-P sites in the sensory ganglia of adult animals, using viruses to deliver the bacteriophage Cre recombinase enzyme.

Mol Biol (Mosk), 2001 Nov-Dec, 35(6), 996 - 1000
{Sequence-determined conformational changes in the coding region of the promoter DNA on transcription complex formation}; Masulis IS et al.; Chemical footprinting was used to study the spatial structure of bacteriophage T7 promoter D upon formation of the transcriptionally active complex with Escherichia coli RNA polymerase . Enzyme binding was shown to induce conformational changes in sites located at positions 43 and 57, several helix turns away from the transcription start . This was the first finding of a structural deformation induced by assembly of the transcription complex . The deformation was associated with specific features of the promoter nucleotide sequence, and suggested high cooperativity in the organization of the transcription complex and substantial energy perturbations caused by the enzyme.

Can J Microbiol, 2001 Nov, 47(11), 1033 - 41
Efficient and predictable recovery of viruses from water by small scale ultrafiltration systems; Winona LJ et al.; Current methods to concentrate viruses from large volumes of water are prone to inconsistent results and are costly and complex procedurally . Ultrafiltration can utilize size exclusion rather than adsorption and (or) elution to concentrate viruses and, therefore, may offer greater flexibility in developing methods that can provide more consistent recoveries among different viruses and widely varying water conditions . Two small scale ultrafiltration systems (hollow fiber and tangential flow) were tested with a virus suspended in 2 L of reagent grade, tap, ground, or surface water . Three model viruses were used (bacteriophages PP7 and T1 and poliovirus) to compare and characterize the recovery of viruses with the two ultrafiltration systems . Pretreatment of the ultrafilters with blocking agents and the use of elution agents can serve to prevent viral adsorption to the filter surface or to elute bound virus and keep viral agents suspended in the retentate . The use of a blocking and elution step concentrated viruses (>60% recovery) from widely varying water qualities, including surface water, such that a single method can be used to efficiently concentrate viruses from all of the water types tested . Both ultrafiltration systems appear to be able to efficiently recover viruses; however, the hollow fiber systems provided slightly better results in the 2-L volumes tested.

J Biol Chem, 2002 Mar 8, 277(10), 7703 - 12 Epub 2001 Dec 27.
Sequence and positional requirements for DNA sites in a mu transpososome; Goldhaber-Gordon I et al.; Transposition of bacteriophage Mu uses two DNA cleavage sites and six transposase recognition sites, with each recognition site divided into two half-sites . The recognition sites can activate transposition of non-Mu DNA sequences if a complete set of Mu sequences is not available . We have analyzed 18 sequences from a non-Mu DNA molecule, selected in a functional assay for the ability to be transposed by MuA transposase . These sequences are remarkably diverse . Nonetheless, when viewed as a group they resemble a Mu DNA end, with a cleavage site and a single recognition site . Analysis of these "pseudo-Mu ends" indicates that most positions in the cleavage and recognition sites contribute sequence-specific information that helps drive transposition, though only the strongest contributors are apparent from mutagenesis data . The sequence analysis also suggests variability in the alignment of recognition half-sites . Transposition assays of specifically designed DNA substrates support the conclusion that the transposition machinery is flexible enough to permit variability in half-site spacing and also perhaps variability in the placement of the recognition site with respect to the cleavage site . This variability causes only local perturbations in the protein-DNA complex, as indicated by experiments in which altered and unaltered DNA substrates are paired.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 39 - 59 Epub 2001 Dec 21.
The X-ray crystal structure of P3, the major coat protein of the lipid-containing bacteriophage PRD1, at 1.65 A resolution; Benson SD et al.; P3 has been imaged with X-ray crystallography to reveal a trimeric molecule with strikingly similar characteristics to hexon, the major coat protein of adenovirus . The structure of native P3 has now been extended to 1.65 A resolution (R(work) = 19.0% and R(free) = 20.8%) . The new high-resolution model shows that P3 forms crystals through hydrophobic patches solvated by 2-methyl-2,4-pentanediol molecules . It reveals details of how the molecule's high stability may be achieved through ordered solvent in addition to intra- and intersubunit interactions . Of particular importance is a 'puddle' at the top of the molecule containing a four-layer deep hydration shell that cross-links a complex structural feature formed by 'trimerization loops' . These loops also link subunits by extending over a neighbor to reach the third subunit in the trimer . As each subunit has two eight-stranded viral jelly rolls, the trimer has a pseudo-hexagonal shape to allow close packing in its 240 hexavalent capsid positions . Flexible regions in P3 facilitate these interactions within the capsid and with the underlying membrane . A selenometh-ionine P3 derivative, with which the structure was solved, has been refined to 2.2 A resolution (R(work) = 20.1% and R(free) = 22.8%) . The derivatized molecule is essentially unchanged, although synchrotron radiation has the curious effect of causing it to rotate about its threefold axis . P3 is a second example of a trimeric 'double-barrel' protein that forms a stable building block with optimal shape for constructing a large icosahedral viral capsid . A major difference is that hexon has long variable loops that distinguish different adenovirus species . The short loops in P3 and the severe constraints of its various interactions explain why the PRD1 family has highly conserved coat proteins.

J Antimicrob Chemother, 2001 Dec, 48(6), 757 - 67
Novel non-nucleoside inhibitors of cytomegaloviruses (BAY 38-4766): in vitro and in vivo antiviral activity and mechanism of action; Reefschlaeger J et al.; For two decades it has been impossible to develop drugs with novel mechanisms of action against herpesviruses, and treatment has been confined largely to the use of inhibitors of viral DNA polymerase . As a representative of a novel inhibitory approach, the non-nucleosidic BAY 38-4766 was identified as a highly selective inhibitor of human cytomegalovirus (HCMV) . The compound selectively inhibits not only HCMV strains, including ganciclovir-resistant, ganciclovir/foscarnet and ganciclovir/cidofovir double-resistant clinical isolates, but also a number of monkey and rodent cytomegaloviruses . In a murine cytomegalovirus (MCMV) pathogenicity model in mice, antiviral efficacy and excellent tolerability were demonstrated . BAY 38-4766-resistant HCMV and MCMV strains are not cross-resistant to the nucleoside analogues ganciclovir and cidofovir or the pyrophosphate analogue foscarnet, indicating a different mode of action . Mechanistic studies demonstrated that the high selectivity of this drug class is most likely due to the inhibition of a late stage of the viral replication cycle . Sequence analyses of resistant HCMV and MCMV strains revealed mutations in UL89 and UL104, proteins known to be involved in viral DNA cleavage and packaging . Consequently, the drug is highly specific for the viral as opposed to cellular functions, since UL89 is related to a bacteriophage terminase and no human equivalent exists . In addition, because some of the genes of the viral DNA cleavage and packaging complex are highly conserved among herpesviruses, development of broad-spectrum agents covering additional human herpesviruses might be possible using this approach.

Environ Mol Mutagen, 2001, 38(2-3), 159 - 65
Frequencies and relative levels of clustered damages in DNA exposed to gamma rays in radioquenching vs . nonradioquenching conditions; Sutherland BM et al.; Clustered damage induced by ionizing radiation--two or more oxidized bases, abasic sites, or strand breaks within a few DNA helical turns--have been postulated to be major lethal and/or mutagenic sites . Although they have recently been shown to be induced in genomic DNAs by ionizing photons and particles, little is known of the factors that affect their yields or the relative levels of the classes of clusters . Toward this aim we have investigated the effect of DNA milieu, specifically, a nonradioquenching (phosphate) or radioquenching (Tris) solution, upon the generation of clustered lesions in a well-defined molecule, T7 bacteriophage DNA . Irradiation of DNA in Tris reduces the yields of all clustered damages to 1-3% of the levels formed in phosphate . Further, although the percentage of the total clusters in oxidized purine clusters is largely unchanged, and the level of abasic clusters decreases, the frequencies of double-strand breaks and oxidized pyrimidine clusters increase in the radioquenching solution . The ratio of the level of oxidized pyrimidine clusters to double-strand breaks in a DNA in radioquenching solution is similar to that obtained in DNA in human cells, also a radioquenching environment.

J Biol Chem, 2002 Mar 1, 277(9), 6943 - 8 Epub 2001 Dec 13.
ATPase activity of the terminase subunit pUL56 of human cytomegalovirus; Hwang JS et al.; Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids . This process is believed to be mediated by two packaging proteins, the terminase subunits . In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit . In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP . In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography . ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56 . The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics . Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis . Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected . This interaction was confirmed by co-immunoprecipitations of infected cells . Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity.

J Mol Biol, 2001 Dec 14, 314(5), 1137 - 46
Crystal structure of a heat and protease-stable part of the bacteriophage T4 short tail fibre; van Raaij MJ et al.; Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres . After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipopolysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube . The short tail fibres consist of a parallel, in-register, trimer of gene product 12 (gp12) . The 1.9 A crystal structure of a heat and protease-stable fragment of gp12 reveals three new folds: a central right-handed triple beta-helix, a globular C-terminal domain containing a beta-sandwich and an N-terminal beta-structure reminiscent of but different from the adenovirus triple beta-spiral . The centre of the C-terminal domain shows weak homology to gp11, a trimeric protein connecting the short fibre to the base-plate, suggesting that the trimerisation motifs of gp11 and gp12 are similar . Repeating sequence motifs suggest that the N-terminal beta-structure extends further towards the N terminus and is conserved in the long tail fibre proteins gp34 and gp37 .

J Mol Biol, 2001 Dec 14, 314(5), 1007 - 15
Mutational changes of conserved residues in the Q-loop region of transcription factor Rho greatly reduce secondary site RNA-binding; Wei RR et al.; Transcription factor Rho of Eschericia coli is a ring-shaped homohexameric protein that terminates transcripts by its action on nascent RNAs . To test the functional importance of the phylogenetically highly conserved residues of the Q-loop region, four mutant Rho proteins, S281A, K283A, T286A and D290A, were isolated and analyzed for their biochemical properties . All four proteins were very defective in terminating transcripts in vitro at the bacteriophage lambda tR1 terminator and had corresponding defects in ATP hydrolysis activated by lambda cro RNA . Although the four proteins were normal or near normal in their sensitivity to cleavage with H(2)O(2) in the presence of Fe-EDTA and in their ability to bind to lambda cro RNA and ATP, they were defective in RNA-specific, secondary site interactions . This was indicated by the lack of protection from cleavage at their Q-loops by oligo(C) in the presence of poly(dC), and their defects in ATP hydrolysis activated by oligo(C) in the presence of poly(dC) . This evidence, together with the observations that cleavage of the Q-loop residues is protected specifically by RNA, suggests that the Q-loop makes interactions with RNA that are essential for activation of ATP hydrolysis and the termination of transcription .

EMBO J, 2001 Dec 17, 20(24), 7149 - 59
Flipping a genetic switch by subunit exchange; Lambert LJ et al.; The bacteriophage T4 AsiA protein is a multifunctional protein that simultaneously acts as both a repressor and activator of gene expression during the phage life cycle . These dual roles with opposing transcriptional consequences are achieved by modification of the host RNA polymerase in which AsiA binds to conserved region 4 (SR4) of sigma(70), altering the pathway of promoter selection by the holoenzyme . The mechanism by which AsiA flips this genetic switch has now been revealed, in part, from the three-dimensional structure of AsiA and the elucidation of its interaction with SR4 . The structure of AsiA is that of a novel homodimer in which each monomer is constructed as a seven-helix bundle arranged in four overlapping helix-loop-helix elements . Identification of the protein interfaces for both the AsiA homodimer and the AsiA-sigma(70) complex reveals that these interfaces are coincident . Thus, the AsiA interaction with sigma(70) necessitates that the AsiA homodimer dissociate to form an AsiA-SR4 heterodimer, exchanging one protein subunit for another to alter promoter choice by RNA polymerase.

J Bacteriol, 2002 Jan, 184(1), 104 - 10
The lytic enzyme of bacteriophage PRD1 is associated with the viral membrane; Rydman PS et al.; Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity . Protein P15 is a soluble beta-1,4-N-acetylmuramidase that causes phage-induced host cell lysis . We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane . Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane . The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used . The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 1106 - 13
Dual targeting of phage-type RNA polymerase to both mitochondria and plastids is due to alternative translation initiation in single transcripts; Kobayashi Y et al.; We isolated and sequenced a nuclear gene and cDNA encoding a bacteriophage T7-type RNA polymerase, NsRpoT-B, from Nicotiana sylvestris . The gene, NsRpoT-B, consists of 19 exons and 18 introns and encodes a polypeptide of 1020 amino acid residues . The predicted NsRpoT-B protein shows 71% amino acid identity with NsRpoT-A which is a mitochondrial protein . Quantitative RT-PCR revealed that steady-state NsRpoT-B mRNA accumulation is highest in the mature leaves and lowest in the cotyledons . Transient expression assays in protoplasts from N . sylvestris leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-B encodes dual targeting signals directing the protein into mitochondria and plastids . This strongly suggests that NsRpoT-B functions as an RNA polymerase transcribing genes from two different plant organelle genomes . NsRpoT-B transcripts have two potential translation initiation codons . An in vitro translation assay indicated that a chimeric mRNA encoding the N-terminal NsRpoT-B fused to an sGFP produced two polypeptides translated from the first and second initiation codons . This implies that the dual targeting of NsRpoT-B protein is regulated, in part, at the level of translation . We have designated this protein NsRpoTpm.

Biochemistry, 2001 Dec 18, 40(50), 15074 - 85
A zinc ribbon protein in DNA replication: primer synthesis and macromolecular interactions by the bacteriophage T4 primase; Valentine AM et al.; The gene product 61 primase protein from bacteriophage T4 was expressed as an intein fusion and purified to homogeneity . The primase binds one zinc ion, which is coordinated by four cysteine residues to form a zinc ribbon motif . Factors that influence the rate of priming were investigated, and a physiologically relevant priming rate of approximately 1 primer per second per primosome was achieved . Primase binding to the single-stranded binding protein (1 primase:4 gp32 monomers; K(d) approximately 860 nM) and to the helicase protein in the presence of DNA and ATP-gamma-S (1 primase:1 helicase monomer; K(d) approximately 100 nM) was investigated by isothermal titration calorimetry (ITC) . Because the helicase is hexameric, the inferred stoichiometry of primase binding as part of the primosome is helicase hexamer:primase in a ratio of 1:6, suggesting that the active primase, like the helicase, might have a ring-like structure . The primase is a monomer in solution but binds to single-stranded DNA (ssDNA) primarily as a trimer (K(d) approximately 50-100 nM) as demonstrated by ITC and chemical cross-linking . Magnesium is required for primase-ssDNA binding . The minimum length of ssDNA required for stable binding is 22-24 bases, although cross-linking reveals transient interactions on oligonucleotides as short as 8 bases . The association is endothermic at physiologically relevant temperatures, which suggests an overall gain in entropy upon binding . Some possible sources of this gain in entropy are discussed.

Plasmid, 2001 Nov, 46(3), 210 - 22
Genetic organization of the Francisella plasmid pFNL10; Pomerantsev AP et al.; We report here the molecular characterization of pFNL10, a 3990-bp cryptic plasmid of Francisella novicida-like F6168 . The plasmid was maintained in F . novicida Utah 112 and F . tularensis LVS strains . We sequenced the entire plasmid and found six open reading frames (ORFs)-ORF1, ORF2, ORF3, ORF4, ORF5, and ORFm . ORF3, ORF4, ORF5, and ORFm are located on the same strand, and we designated it the plus strand . ORF1 and ORF2 are on the complementary strand . The ORFs appear to be arranged in two operons, one comprising ORF5 and ORF4 and the other ORF1 and ORF2 . There exist two distinct promoters similar to the Escherichia coli sigma(70) promoter, one 5' to ORF1-ORF2 operon and the other 5' to ORF5-ORF4 operon . We found that in both promoters the transcriptional start is an adenosine . ORF3 is positioned in tandem with ORF5-ORF4, but has its own transcriptional start, a thymidine . However, sequence analysis revealed no recognizable promoter in physical proximity to ORF3 . Sequence analysis revealed transcriptional terminators immediately downstream of the two operons . Experimental results showed that the ORF1-ORF2 terminator is authentic . But we could not definitively confirm the ORF5-ORF4 terminator . Two sets of direct repeats, one 31 and the other 13 bp, characteristic of ori are positioned between the two promoters . ORF1 encodes a protein that bears homology to the replication initiation protein RepA of various bacteria, and disruption of this ORF indeed blocked pFNL10 replication . In contrast, ORF2 disruption caused formation of plasmid multimers, suggesting aberrant replication . Our analysis also suggests that pFNL10 replicates by the theta mode . The ORF5-ORF4 operon resembles the phd-doc operon of Escherichia coli bacteriophage P1, but the significance of this similarity is unclear .

Biochem Biophys Res Commun, 2001 Dec 14, 289(4), 908 - 15
The chaperonins of Synechocystis PCC 6803 differ in heat inducibility and chaperone activity; Kovacs E et al.; The chaperonins GroEL and Cpn60 were isolated from the cyanobacterium Synechocystis PCC 6803 and characterized . In cells grown under optimal conditions their ratio was about one to one . However, the amount of GroEL increased considerably more than that of Cpn60 in response to heat stress . The labile chaperonin oligomer required stabilization by MgATP or glycerol during isolation . Use of the E . coli mutant strain, groEL44 revealed that the functional properties of the two cyanobacterial chaperonins are strikingly different . Overexpression of cyanobacterial GroEL in the E . coli mutant strain allowed growth at elevated temperature, the formation of mature bacteriophage T4, and active Rubisco enzyme assembly . In contrast, Cpn60 partially complemented the temperature-sensitive phenotype, the Rubisco assembly defect and did not promote the growth of the bacteriophage T4 . The difference in chaperone activity of the two cyanobacterial chaperonins very probably reflects the unique chaperonin properties required during the life of Synechocystis PCC 6803.

J Mol Biol, 2001 Dec 7, 314(4), 807 - 22
Crystal structure of an antigen-binding fragment bound to single-stranded DNA; Tanner JJ et al.; Antibodies to DNA are characteristic of the autoimmune disease systemic lupus erythematosus (SLE) and they also serve as models for the study of protein-DNA recognition . Anti-DNA antibodies often play an important role in disease pathogenesis by mediating kidney damage via antibody-DNA immune complex formation . The structural underpinnings of anti-DNA antibody pathogenicity and antibody-DNA recognition, however, are not well understood, due in part to the lack of direct, experimental three-dimensional structural information on antibody-DNA complexes . To address these issues for anti-single-stranded DNA antibodies, we have determined the 2.1 A crystal structure of a recombinant Fab (DNA-1) in complex with dT5 . DNA-1 was previously isolated from a bacteriophage Fab display library from the immunoglobulin repertoire of an SLE-prone mouse . The structure shows that DNA-1 binds oligo(dT) primarily by sandwiching thymine bases between Tyr side-chains, which allows the bases to make sequence-specific hydrogen bonds . The critical stacking Tyr residues are L32, L49, H100, and H100A, while His L91 and Asn L50 contribute hydrogen bonds . Comparison of the DNA-1 structure to other anti-nucleic acid Fab structures reveals a common ssDNA recognition module consisting of Tyr L32, a hydrogen bonding residue at position L91, and an aromatic side-chain from the tip of complementarity determining region H3 . The structure also provides a framework for interpreting previously determined thermodynamics data, and this analysis suggests that hydrophobic desolvation might underlie the observed negative enthalpy of binding . Finally, Arg side-chains from complementarity determining region H3 appear to play a novel role in DNA-1 . Rather than forming ion pairs with dT5, Arg contributes to oligo(dT) recognition by helping to maintain the structural integrity of the combining site . This result is significant because antibody pathogenicity is thought to be correlated to the Arg content of anti-DNA antibody hypervariable loops .

J Mol Biol, 2001 Dec 7, 314(4), 649 - 54
Modifying the specificity of an RNA backbone contact; Dertinger D et al.; The interaction between the MS2 bacteriophage coat protein homodimer and its cognate RNA hairpin is facilitated by 21 different RNA-protein contacts . In one of these contacts, the 2'-hydroxyl group at ribose -5 of the RNA acts as a hydrogen bond donor to Glu63 in one subunit of the protein . Previous experiments showed that substitution of ribose -5 with deoxyribose resulted in a 24-fold decrease in binding affinity between RNA and protein . Using a protein where the two MS2 monomers were fused to increase stability, the contribution of this contact to the overall binding affinity was investigated by site-directed mutagenesis . When Glu63 was substituted with glutamine, aspartate, or alanine, the binding affinity of the hairpin for the protein was weakened by 12 to 100-fold, similar to that observed with deoxyribose at position -5 . However, the specificity of the three mutant proteins for RNAs with various modifications at the 2'-position of ribose -5 differed dramatically . While the Glu63Asp protein resembled the wild-type protein in preferring the 2'-hydroxyl group over a proton or a bulky 2'-substituent, both the Glu63Ala and Glu63Gln proteins preferred bulky 2'-substituents over the 2'-hydroxyl group by more than 100-fold . These experiments emphasize the ease with which the specificity of a protein-nucleic acid interaction can be changed at thermodynamically important sites .

Int J Parasitol, 2001 Dec, 31(14), 1659 - 68
Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries; Beghetto E et al.; Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection . With the aim of identifying the immunodominant epitopes of T . gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach . A library of cDNA fragments from T . gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D . The lambda D-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite . Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions . In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).

Nucleic Acids Res, 2001 Dec 1, 29(23), 4892 - 900
The P1 phage replication protein RepA contacts an otherwise inaccessible thymine N3 proton by DNA distortion or base flipping; Lyakhov IG et al.; The RepA protein from bacteriophage P1 binds DNA to initiate replication . RepA covers one face of the DNA and the binding site has a completely conserved T that directly faces RepA from the minor groove at position +7 . Although all four bases can be distinguished through contacts in the major groove of B-form DNA, contacts in the minor groove cannot easily distinguish between A and T bases . Therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching into the minor groove of B-form DNA . RepA binding sites with modified base pairs at position +7 were used to investigate contacts with RepA . The data show that RepA contacts the N3 proton of T at position +7 and that the T=A hydrogen bonds are already broken in the DNA before RepA binds . To accommodate the N3 proton contact the T(+7 )/A(+7)((')) base pair must be distorted . One possibility is that T(+7) is flipped out of the helix . The energetics of the contact allows RepA to distinguish between all four bases, accounting for the observed high sequence conservation . After protein binding, base pair distortion or base flipping could initiate DNA melting as the second step in DNA replication.

Nucleic Acids Res, 2001 Dec 1, 29(23), 4881 - 91
Strong minor groove base conservation in sequence logos implies DNA distortion or base flipping during replication and transcription initiation; Schneider TD; The sequence logo for DNA binding sites of the bacteriophage P1 replication protein RepA shows unusually high sequence conservation ( approximately 2 bits) at a minor groove that faces RepA . However, B-form DNA can support only 1 bit of sequence conservation via contacts into the minor groove . The high conservation in RepA sites therefore implies a distorted DNA helix with direct or indirect contacts to the protein . Here I show that a high minor groove conservation signature also appears in sequence logos of sites for other replication origin binding proteins (Rts1, DnaA, P4 alpha, EBNA1, ORC) and promoter binding proteins (sigma(70), sigma(D) factors) . This finding implies that DNA binding proteins generally use non-B-form DNA distortion such as base flipping to initiate replication and transcription.

Life Support Biosph Sci, 2001, 8(1), 9 - 14
Selecting a sensitive bacteriophage assay for evaluation of a prototype water recycling system; Brion GM et al.; A rapid, simple, and direct (RSD) assay of eluate from filter concentration was developed to enumerate low numbers of MS2 bacteriophage, used as a surrogate for enteric viruses, from samples collected from a prototype-sized water recycling system . The RSD assay utilized a 50-ml eluate volume in a modified single-layer assay, neutralizing eluate pH by buffered, double-strength agar . The RSD assay developed was simpler and minimized sample-handling steps compared with another published method . The RSD assay method showed greater sensitivity than the other published method for recovering phage from filter eluate while avoiding pH shifts, which can inactivate phage . Grant numbers: NAGW 2356.

Gene, 2001 Nov 14, 279(1), 33 - 40
Genomic organization and organ-specific expression of a nuclear gene encoding phage-type RNA polymerase in Nicotiana sylvestris; Kobayashi Y et al.; We have isolated and sequenced a nuclear gene and cDNA encoding bacteriophage T7-type single subunit RNA polymerase, NsRpoT-A, from Nicotiana sylvestris . NsRpoT-A consists of 19 exons and 18 introns; the first intron is 17 kb, the longest yet identified in a plant gene . Genomic Southern analysis indicated that N . sylvestris contains a small family of NsRpoT genes . Quantitative RT-PCR revealed that steady-state mRNA levels are highest in the leaves and lowest in the cotyledons . Phylogenetic analysis of NsRpoT-A and the RpoT proteins of other plant species suggested that NsRpoT-A is a mitochondrial protein . The TargetP program predicted localization of the NsRpoT-A gene product to the mitochondria . Using a transient expression assay and protoplasts from N . sylvestris mesophyll cells, we clearly demonstrated that the N-terminal sequence of NsRpoT-A targets the protein to the mitochondria . We therefore named this protein NsRpoTm.

Mol Microbiol, 2001 Nov, 42(3), 767 - 76
Bacteriophage T7 protein kinase phosphorylates RNase E and stabilizes mRNAs synthesized by T7 RNA polymerase; Marchand I et al.; The T7 protein encoded by the early gene 0.7 exhibits bifunctional activity . Whereas its C-terminal one-third participates in host transcription shut-off, the N-terminal two-thirds bears a protein kinase ('PK') activity that can phosphorylate a number of host proteins in addition to itself . Here, we show that, when PK is expressed in uninfected Escherichia coli cells, the C-terminal half of RNase E and the associated RNA helicase RhlB are heavily phosphorylated . Meanwhile, a subset of RNase E substrates, including the lac and cat mRNAs synthesized by bacteriophage T7 RNA polymerase (RNAP), are stabilized . These mRNAs are genuinely less stable than their counterparts synthesized by E . coli RNAP, because T7 RNAP outpaces translating ribosomes, creating naked, RNase E-sensitive mRNA stretches behind itself . Thus, PK alleviates this effect of desynchronizing transcription and translation . The relationship between the modification of RNase E and RhlB and these mRNA stabilization effects, which may be relevant to the stability of late T7 mRNAs during infection, is discussed.

J Struct Biol, 2001 Sep, 135(3), 270 - 80
Fluorescence microscopy of single viral capsids; Huang S et al.; To build a foundation for the single-molecule fluorescence microscopy of protein complexes, the present study achieved fluorescence microscopy of single, nucleic acid-free protein capsids of bacteriophage T7 . The capsids were stained with Alexa 488 (green emission) . Manipulation of the capsids' thermal motion was achieved in three dimensions . The procedure for manipulation included embedding the capsids in an agarose gel . The data indicate that the thermal motion of capsids is reduced by the sieving of the gel . The thermal motion can be reduced to any desired level . A semilogarithmic plot of an effective diffusion constant as a function of gel concentration is linear . Single, diffusing T7 capsids were also visualized in the presence of single DNA molecules that had been both stretched and immobilized by gel-embedding . The DNA molecules were stained with ethidium (orange emission) . This study shows that single-molecule (protein and DNA) analysis is possible for both packaging of DNA in a bacteriophage capsid and other events of DNA metabolism . The major problem is the maintenance of biochemical activity . (c)2001 Elsevier Science.

Biophys J, 2001 Dec, 81(6), 3398 - 408
Partially condensed DNA conformations observed by single molecule fluorescence microscopy; Serwer P et al.; To detect partially condensed conformations of a double-stranded DNA molecule, single molecule fluorescence microscopy is performed here . The single DNA molecules are ethidium stained, 670 kilobase pair bacteriophage G genomes that are observed both during and after expulsion from capsids . Expulsion occurs in an agarose gel . Just after expulsion, the entire G DNA molecule typically has a partially condensed conformation not previously described (called a balloon) . A balloon subsequently extrudes a filamentous segment of DNA . The filamentous segment becomes gently elongated via diffusion into the network that forms the agarose gel . The elongated DNA molecule usually has bright spots that undergo both appearance/disappearance and apparent motion . These spots are called dynamic spots . A dynamic spot is assumed to be the image of a zone of partially condensed DNA segments (globule) . The positions of globules along an elongated DNA molecule 1) are restricted primarily to time-stable regions with comparatively high thermal motion-induced, micrometer-scale bending of the DNA molecule and 2) move within a given region on a time scale smaller than the time scale of recording . Less mobile globules are observed when either magnesium cation or ethanol is added before gel-embedding DNA molecules . These observations are explained by globules induced at equilibrium by a bending-dependent, inter-DNA segment force . Theory has previously predicted that globules are induced by electrostatic forces along an electrically charged polymer at equilibrium . The hypothesis is proposed that intracellular DNA globules assist action-at-a-distance during DNA metabolism.

Cancer Res, 2001 Nov 15, 61(22), 8110 - 2
Modulation of the immune response by systemic targeting of antigens to lymph nodes; Trepel M et al.; Factors that determine the immunogenicity of an antigen in vivo are still largely unknown . Direct administration of antigens into lymphatic organs appears to enhance immune response . We hypothesized that systemically targeting antigens to lymphatic tissue in vivo might modulate immunity . To test this hypothesis, we measured the humoral immune response elicited by bacteriophage vaccination . We show that the responses against a lymph node-targeted phage are significantly higher than those against control untargeted phage; the effect is specific because it is inhibited by coadministration of the cognate synthetic peptides displayed . Our data suggest that systemic targeting of antigens to lymph nodes through the circulation modulates humoral immune response . This strategy may have broad applications in the development of vaccines, production of antibodies, and immunotherapy.

Biol Res, 2001, 34(3-4), 207 - 16
The bacteriophage lambda DNA packaging enzyme: identification of four structural domains of the gpNu1 subunit using limited proteolysis; Araya P et al.; Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA . Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its insolubility in aqueous solutions . Therefore, to identify smaller and potentially water-soluble domains of gpNu1, we analyzed the nature of the products obtained by limited digestion of the protein with several proteases . The gpNu1 subunit was obtained from E . coli cells transfected with the plasmid pH6-Nu1 that overproduces the protein . Incubation of gpNu1 solubized in 2.5 M guanidinium chloride with chymotrypsin resulted in the formation of at least eight discrete protein bands, while treatment with endoproteinase glu-C and bromelain yielded three and one major bands, respectively . The peptides generated by digestion with the various proteases were separated by two-dimensional gel electrophoresis and transferred to Immobilon membranes . Amino acid sequencing of the peptides allowed for the precise assignment of their N-terminal amino acid, while their estimated molecular weights permitted the identification of their C-terminal ends . The results reveal that in the presence of 2.5 M guanidinium chloride, gpNu1 is partially folded in at least four distinct structural domains that correspond to functional domains as determined by previously reported genetic experiments . This information is key to design new plasmids to overproduce these domains for further structural analysis.

Nucleic Acids Res . 2001 Nov 15;29(22):E113.
Isolation of viral coat protein mutants with altered assembly and aggregation properties; Peabody DS et al.; A method was developed to screen bacteria for synthesis of mutant proteins with altered assembly and solubility properties using bacteriophage MS2 coat protein as a model self-associating protein . Colonies expressing coat protein from a plasmid were covered with an agarose overlay under conditions that caused the lysis of some of the cells in each colony . The proteins thus liberated diffused through the overlay at rates depending on their molecular sizes . After transfer of the proteins to a nitrocellulose membrane, probing with coat protein-specific antiserum revealed spots whose sizes and intensities were related to the aggregation state of coat protein . The method was employed in the isolation of assembly defective mutants and to find soluble variants of an aggregation-prone coat protein mutant.

Nucleic Acids Res, 2001 Nov 15, 29(22), 4736 - 43
Sea urchin mtDBP is a two-faced transcription termination factor with a biased polarity depending on the RNA polymerase; Fernandez-Silva P et al.; The sea urchin mitochondrial displacement (D)-loop binding protein mtDBP has been previously identified and cloned . The polypeptide (348 amino acids) displays a significant homology with the human mitochondrial transcription termination factor mTERF . This similarity, and the observation that the 3' ends of mitochondrial RNAs coded by opposite strands mapped in correspondence of mtDBP-binding sites, suggested that mtDBP could function as transcription termination factor in sea urchin mitochondria . To investigate such a role we tested the capability of mtDBP bound to its target sequence in the main non-coding region to affect RNA elongation by mitochondrial and bacteriophage T3 and T7 RNA polymerases . We show that mtDBP was able to terminate transcription bidirectionally when initiated by human mitochondrial RNA polymerase but only unidirectionally when initiated by T3 or T7 RNA polymerases . Time-course experiments indicated that mtDBP promotes true transcription termination rather than transcription pausing . These results indicate that mtDBP is able to function as a bipolar transcription termination factor in sea urchin mitochondria . The functional significance of such an activity could be linked to the previously proposed dual role of the protein in modulating mitochondrial DNA transcription and replication.

Genes Dev, 2001 Nov 15, 15(22), 3013 - 22
Octamerization of lambda CI repressor is needed for effective repression of P(RM) and efficient switching from lysogeny; Dodd IB et al.; The CI repressor of bacteriophage lambda is a model for the role of cooperativity in the efficient functioning of genetic switches . Pairs of CI dimers interact to cooperatively occupy adjacent operator sites at O(R) and at O(L) . These CI tetramers repress the lytic promoters and activate transcription of the cI gene from P(RM) . CI is also able to octamerize, forming a large DNA loop between O(R) and O(L), but the physiological role of this is unclear . Another puzzle is that, although a dimer of CI is able to repress P(RM) by binding to the third operator at O(R), O(R)3, this binding seems too weak to affect CI production in the lysogenic state . Here we show that repression of P(RM) at lysogenic CI concentrations is absolutely dependent on O(L), in this case 3.8 kb away . A mutant defective in this CI negative autoregulation forms a lysogen with elevated CI levels that cannot efficiently switch from lysogeny to lytic development . Our results invalidate previous evidence that Cro binding to O(R)3 is important in prophage induction . We propose the octameric CI:O(R)-O(L) complex increases the affinity of CI for O(R)3 by allowing a CI tetramer to link O(R)3 and the third operator at O(L), O(L)3.

Ann N Y Acad Sci, 2001 Sep, 945, 276 - 88
Foreign DNA integration--perturbations of the genome--oncogenesis; Doerfler W et al.; We have been interested in the consequences of foreign DNA insertion into established mammalian genomes and have initially studied this problem in adenovirus type 12 (Ad12)-transformed cells or in Ad12-induced hamster tumors . Since integrates are frequently methylated de novo, it appears that they might be modified by an ancient defense mechanism against foreign DNA . In cells transgenic for the DNA of Ad12 or for the DNA of bacteriophage lambda, changes in cellular methylation and transcription patterns have been observed . Thus, the insertion of foreign DNA can have important functional consequences that are not limited to the site of foreign DNA insertion . These findings appear to be relevant also for tumor biology and for the interpretation of data derived from experiments with transgenic organisms . For most animals, the main portal of entry for foreign DNA is the gastrointestinal tract . Large amounts of foreign DNA are regularly ingested with the supply of nutrients . Starting in 1987/1988, we have been investigating the fate of orally administered foreign DNA in mice . Naked DNA of bacteriophage M13 and the cloned gene for the green fluorescent protein (GFP) of Aequorea victoria have been used as test molecules . Moreover, the plant-specific gene for the ribulose-1,5-bisphosphate carboxylase (rubisco) has been followed in mice after feeding soybean leaves . At least transiently, food-ingested DNA can be traced to different organs and, after transplacental transfer, to fetuses and newborns . There is no evidence for germ line transmission or for the expression of orally administered GFP DNA.

Biogerontology, 2000, 1(1), 67 - 78
Applying phage display technology in aging research; Kristensen P et al.; Filamentous bacteriophage offers the possibility of linking genotype with phenotype in one genetic package . By creating a library of different proteins as fusion to one of the coat proteins of filamentous bacteriophage it becomes possible to isolate proteins based on their binding characteristics . Phage displayed libraries of varying formats, ranging from small peptides to antibody fragments, have been selected successfully for ligands toward a large panel of targets . These peptides and antibody fragments are applicable in analysis of the behaviour of certain proteins with respect to changes in biological processes such as aging, thus providing valuable tools for a general understanding of the aging process . Alternatively, display of antibody fragments or peptides on filamentous bacteriophage can be instrumental in the discovery of novel antigens as exemplified by selections on cell surfaces or on complex protein mixtures such as sera from aging individuals . Although phage display has been applied successfully in a large number of studies relating to cancer, viral infections and other biological processes, its application in the field of aging research is yet to be realisedPublication Types:

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