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