Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13671 - 4 Epub 2001 Nov 13. DNA packaging and ejection forces in bacteriophage; Kindt J et al.; We calculate the forces required to package (or, equivalently, acting to eject) DNA into (from) a bacteriophage capsid, as a function of the loaded (ejected) length, under conditions for which the DNA is either self-repelling or self-attracting . Through computer simulation and analytical theory, we find the loading force to increase more than 10-fold (to tens of piconewtons) during the final third of the loading process; correspondingly, the internal pressure drops 10-fold to a few atmospheres (matching the osmotic pressure in the cell) upon ejection of just a small fraction of the phage genome . We also determine an evolution of the arrangement of packaged DNA from toroidal to spool-like structures. Mol Microbiol, 2001 Oct, 42(2), 355 - 68 Determinants of segregational stability of the linear plasmid-prophage N15 of Escherichia coli; Grigoriev PS et al.; N15 is a bacteriophage of Escherichia coli that resembles lambda, but, unlike lambda, it lysogenizes as a linear plasmid . We show that stable maintenance of this unusual plasmid-prophage depends on the parA and parB genes, relatives of the partition genes of F and P1 plasmids . ParB of N15, like its F- and P1-encoded homologues, destabilizes plasmids carrying its target centromere, when present in excess . Within the genome of N15, we identified four unlinked, palindromic sequences that can promote the ParB-mediated destabilization of a moderate-copy vector in cis . They are distant from the parAB operon, unlike the centromeric sites of F and P1 . Each of these palindromes could interact in vivo and in vitro with ParB . Each, when cloned separately, had properties characteristic of centromeric sites: exerted incompatibility against the N15 prophage and mini-N15 plasmids, and stabilized a mini-P1 plasmid depleted of its own partition genes when ParA and ParB of N15 were supplied . A pair of sites was more effective than a single site . Two of the centromeric sites are located in the proximity of promoters of phage genes, suggesting that, in addition to their function in partitioning of N15 prophage, they may control expression of N15 lytic functions. J Biol Chem, 2002 Jan 25, 277(4), 2843 - 51 Epub 2001 Nov 07. DNA transposition of bacteriophage Mu . A quantitative analysis of target site selection in vitro; Haapa-Paananen S et al.; The Mu transpositional DNA recombination machinery selects target sites by assembling a protein-DNA complex that interacts with the target DNA and reacts whenever it locates a favorable sequence composition . Splicing of a transposon into the target generates a 5-bp duplication that reflects the original target site . Preferential usage of different target pentamers was examined with a minimal Mu in vitro system and quantitatively compiled consensus sequences for the most preferred and the least preferred sites were generated . When analyzed as base steps, preferences toward certain steps along the 5-bp target site were detected . We further show that insertion sites can be predicted on the basis of additively calculated base step values . Also surrounding sequences influence the preference of a given pentamer; a symmetrical structural component was revealed, suggesting potential hinges at and around the target site. J Biol Chem, 2002 Jan 18, 277(3), 1719 - 27 Epub 2001 Nov 06. Mechanism underlying replication protein a stimulation of DNA ligase I; Ranalli TA et al.; Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that participates in multiple DNA transactions that include replication and repair . Base excision repair is a central DNA repair pathway, responsible for the removal of damaged bases . We have shown previously that RPA was able to stimulate long patch base excision repair reconstituted in vitro . Herein we show that human RPA stimulates the activity of the base excision repair component human DNA ligase I by approximately 15-fold . Other analyzed single-stranded binding proteins would not substitute, attesting to the specificity of the stimulation . Conversely, RPA was unable to stimulate the functionally homologous ATP-dependent ligase from T4 bacteriophage . Kinetic analyses suggest that catalysis of ligation is enhanced by RPA, as a 4-fold increase in k(cat) is observed, whereas K(m) is not significantly changed . Substrate competition experiments further support the conclusion that RPA does not alter the specificity or rate of substrate binding by DNA ligase I . Additionally, RPA is unable to significantly enhance ligation on substrates containing an unannealed 3'-upstream primer terminus, suggesting that RPA does not stabilize the nick site to enhance ligase recognition . Furthermore when DNA ligase I is pre-bound to the substrate and limited to a single turnover, RPA is still able to stimulate ligation . Overall, the results support a mechanism of stimulation that involves increasing the rate of catalysis of ligation. Mol Phylogenet Evol, 2001 Nov, 21(2), 259 - 69 Detection of homologous recombination among bacteriophage P2 relatives; Nilsson AS et al.; Sequencing of five late genes from 18 isolates of P2-like bacteriophages showed that these are at least 96% identical to the genes of phage P2 . A maximum-parsimony phylogenetic analysis of these genes showed excess homoplasy of a magnitude three to six times higher than that expected . Examination of the distribution of the number of homoplasies at parsimoniously informative sites and incompatibility matrices of such sites revealed a pattern typical for extensive recombination . It has been shown that phage P2 probably incorporated some functionally complete genes or gene modules by recombination with other phages or with different hosts, but homologous recombination within genes has previously not been shown . In this paper we demonstrate that homologous recombination between P2-like bacteriophages occurs randomly at multiple breakpoints in five late genes . The rate of recombination is high but, since some phages were sampled decades apart and in different parts of the world, this has to be viewed on an evolutionary time scale . The applicability of different methods used for detection of recombination breakpoints and estimation of rates of recombination in bacteriophages is discussed . Microbiol Immunol, 2001, 45(9), 657 - 65 DNA substrates influence the recombination efficiency mediated by FLP recombinase expressed in mammalian cells; Nakano M et al.; The FLP recombinase derived from Saccharomyces cerevisiae mediates precise site-specific recombination between a pair of FLP recognition targets (FRTs) . Like the Cre/loxP system derived from bacteriophage P1, the FLP/FRT system has recently been applied to gene regulation systems using an FLP-expressing recombinant adenovirus (rAd) (Nakano et al, Nucleic Acids Res . 29: e40, 2001) . In an attempt to improve the FLP/FRT system by altering its DNA substrates, we compared the recombination efficiency among different substrates by a quantitative in vitro assay using FLP expressed in mammalian cells . Unexpectedly, we found that one linearized DNA substrate showed 4- to >20-fold lower recombination efficiency than other substrates, which phenomenon has not been observed in the Cre/loxP system . The quantitative in vitro assay using truncated DNA substrates suggested that the recombination efficiency seemed to be influenced not only by the linearized position of the substrate, but also by the length between a pair of FRTs . Such substrate preference of FLP expressed in mammalian cells should probably be noted when designing versatile applications of the FLP/FRT system as a gene regulation system in mammalian systems . Fortunately, however, we demonstrated that no substrate preference was observed when using a particular substrate (pCAFNF5) and the preference was reduced when using a certain pair of mutant FRTs (f72), which will also be a promising tool for simultaneous gene regulation in combination with wild-type FRT. Nucleic Acids Res, 2001 Nov 1, 29(21), 4264 - 73 Purification and functional characterization of p16, the ATPase of the bacteriophage Phi29 packaging machinery; Ibarra B et al.; Bacteriophage Phi29 codes for a protein (p16) that is required for viral DNA packaging both in vivo and in vitro . Co-expression of p16 with the chaperonins GroEL and GroES has allowed its purification in a soluble form . Purified p16 shows a weak ATPase activity that is stimulated by either DNA or RNA, irrespective of the presence of any other viral component . The stimulation of ATPase activity of p16, although induced under packaging conditions, is not dependent of the actual DNA packaging and in this respect the Phi29 enzyme is similar to other viral terminases . Protein p16 competes with DNA and RNA in the interaction with the viral prohead, which occurs through the N-terminal region of the connector protein (p10) . In fact, p16 interacts in a nucleotide-dependent fashion with the viral Phi29-encoded RNA (pRNA) involved in DNA packaging, and this binding can be competed with DNA . Our results are consistent with a model for DNA translocation in which p16, bound and organized around the connector, acts as a power stroke to pump the DNA into the prohead, using the hydrolysis of ATP as an energy source. Biochem Biophys Res Commun, 2001 Nov 9, 288(4), 997 - 1000 Purification and crystallization of CII: an unstable transcription activator from phage lambda; Datta AB et al.; The CII protein of the temperate bacteriophage lambda is a transcriptional activator involved in the lysis-lysogeny switch of the phage . It is an unstable protein of 97 amino acids and is known to exist as a tetramer in the native state . The cII gene has been cloned and expressed in Escherichia coli using a T7 promoter based over-expression system . The recombinant CII protein has been purified to homogeneity by ammonium sulfate fractionation followed by two steps of ion-exchange chromatography . The purified protein crystallized at pH 8.2 in hanging-drop vapor diffusion method at 293 K . The crystals diffract to a resolution of 2.8 A and belong to the space group C222 with unit-cell parameters a = 64.10, b = 106.95 and c = 120.16 A . J Biol Chem, 2002 Jan 4, 277(1), 279 - 86 Epub 2001 Oct 30. Bacteriophage T4 Dam DNA-{N6-adenine}methyltransferase . Kinetic evidence for a catalytically essential conformational change in the ternary complex; Evdokimov AA et al.; We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-{N6-adenine}methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex . Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA(Me) and S-adenosyl-l-homocysteine, AdoHcy) release was AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow . A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex . In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation . We propose the following model . Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis . After the chemical step of methyl group transfer from AdoMet to DNA, product DNA(Me) dissociates relatively rapidly (k(off) = 1.7 x s(-1)) from the complex . In contrast, dissociation of product AdoHcy proceeds relatively slowly (k(off) = 0.018 x s(-1)), indicating that its release is the rate-limiting step, consistent with kcat = 0.015 x s(-1) . After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle . We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F . The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues . This route is preferred at high AdoMet concentrations. Biochemistry, 2001 Nov 6, 40(44), 13370 - 7 The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB; Hang JQ et al.; cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging . Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric . That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth . The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN . In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect . In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount . The results indicated that the functional asymmetry of cosNdepends on the presence of cosB . A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB. Biochemistry, 2001 Nov 6, 40(44), 13167 - 76 Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant protein; Duewel HS et al.; When incorporated into proteins, fluorinated amino acids have been utilized as 19F NMR probes of protein structure and protein-ligand interactions, and as subtle structural replacements for their parent amino acids which is not possible using the standard 20-amino acid repertoire . Recent investigations have shown the ability of various fluorinated methionines, such as difluoromethionine (DFM) and trifluoromethionine (TFM), to be bioincorporated into recombinant proteins and to be extremely useful as 19F NMR biophysical probes . Interestingly, in the case of the bacteriophage lambda lysozyme (LaL) which contains only three Met residues (at positions 1, 14, and 107), four 19F NMR resonances are observed when TFM is incorporated into LaL . To elucidate the underlying structural reasons for this anomalous observation and to more fully explore the effect of TFM on protein structure, site-directed mutagenesis was used to assign each 19F NMR resonance . Incorporation of TFM into the M14L mutant resulted in the collapse of the two 19F resonances associated with TFM at position 107 into a single resonance, suggesting that when position 14 in wild-type protein contains TFM, a subtle but different environment exists for the methionine at position 107 . In addition, 19F and {1H-13C}-HMQC NMR experiments have been utilized with paramagnetic line broadening and K2PtCl4 reactivity experiments to obtain information about the probable spatial position of each Met in the protein . These results are compared with the recently determined crystal structure of LaL and allow for a more detailed structural explanation for the effect of fluorination on protein structure. Curr Microbiol, 2001 Oct, 43(4), 299 - 301 Inhibitory effects of gynostemma pentaphyllum on the UV induction of bacteriophage lambda in lysogenic Escherichia coli; Zhu S et al.; Effects of gynostemma pentaphyllum (GP) on the bacteriophage lambda induced by ultraviolet (UV) irradiation have been studied . The results showed that GP could inhibit the UV induction of bacteriophage lambda in lysogenic cells . The inhibitory effects were dependent on the concentration and the reaction time of GP, and were efficient at 40 to approximately 125 microg ml(-1) for 10 min . The inhibitory rate was higher than 70% when the GP concentration was 50 microg ml(-1) . By electron spin resonance (ESR) and spin-trapping techniques, the signals of free radicals were detected in the suspension of the lambda lysogenic bacteria induced by ultraviolet irradiation, but after the addition of GP the signals were decreased . These results indicate that gynostemma pentaphyllum not only is a scavenger of free radicals, but also possesses the biological function of anti-irradiation, and that there is a close relation between the UV irradiation of the bacteriaphage lambda and free radicals. J Mol Evol, 2001 Jul, 53(1), 47 - 54 A general mechanism for viral resistance to suicide gene expression; Bull JJ et al.; Bacteriophage T7 was challenged with either of two toxic genes expressed from plasmids . Each plasmid contained a different gene downstream of a T7 promoter; cells harboring each plasmid caused an infection by wild-type T7 to abort . T7 evolved resistance to both inhibitors by avoidance of the plasmid expression system rather than by blocking or bypassing the effects of the specific toxic gene product . Resistance was due to a combination of mutations in the T7 RNA polymerase and other genes expressed at the same time as the polymerase . Mutations mapped to sites that are unlikely to alter polymerase specificity for its cognate promoter but the basis for discrimination between phage and plasmid promoters in vivo was not resolved . A reporter assay indicated that, relative to wild-type phage, gene expression from the plasmid was diminished several-fold in cells infected by the evolved phages . A recombinant phage, derived from the original mutant but lacking a mutation in the gene for RNA polymerase, exhibited intermediate activity in the reporter assay and intermediate resistance to the toxic gene cassettes . Alterations in both RNA polymerase and a second gene are thus responsible for resistance . These findings have broad evolutionary parallels to other systems in which viral inhibition is activated by viral regulatory signals such as defective-interfering particles, and they may have mechanistic parallels to the general phenomena of position effects and gene silencing. J Clin Microbiol, 2001 Nov, 39(11), 3992 - 8 Isolation of a lysogenic bacteriophage carrying the stx(1(OX3)) gene, which is closely associated with Shiga toxin-producing Escherichia coli strains from sheep and humans; Koch C et al.; A specific PCR for the detection of a variant of the gene encoding Shiga toxin 1 (stx(1)) called stx(1(OX3)) (GenBank accession no . Z36901) was developed . The PCR was used to investigate 148 Stx(1)-producing Escherichia coli strains from human patients (n = 72), cattle (n = 27), sheep (n = 48), and a goat (n = 1) for the presence of the stx(1(OX3)) gene . The stx(1(OX3)) gene was present in 38 Shiga toxin-producing E . coli (STEC) strains from sheep belonging to serogroups O5, O125, O128, O146, and OX3 but was absent from Stx(1)-positive ovine STEC O91 strains . The stx(1(OX3)) gene was also detected in 22 STEC strains from humans with nonbloody diarrhea and from asymptomatic excreters . Serotypes O146:H21 and O128:H2 were most frequently associated with stx(1(OX3))-carrying STEC from sheep and humans . In contrast, Stx(1)-producing STEC strains from cattle and goats and 50 STEC strains from humans were all negative for the stx(1(OX3)) gene . The stx(1(OX3))-negative strains belonged to 13 serotypes which were different from those of the stx(1(OX3))-positive STEC strains . Moreover, the stx(1(OX3)) gene was not associated with STEC belonging to enterohemorrhagic E . coli (EHEC) serogroups O26, O103, O111, O118, O145, and O157 . A bacteriophage carrying the stx(1(OX3)) gene (phage 6220) was isolated from a human STEC O146:H21 strain . The phage was able to lysogenize laboratory E . coli K-12 strain C600 . Phage 6220 shared a similar morphology and a high degree of DNA homology with Stx(2)-encoding phage 933W, which originates from EHEC O157 . In contrast, few similarities were found between phage 6220 and Stx(1)-encoding bacteriophage H-19B from EHEC O26. RNA, 2001 Oct, 7(10), 1486 - 95 Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG; Stepinski J et al.; The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications . However, Pasquinelli et al . {RNA (1995) 1:957-967} found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain . Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs . We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation . The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts . Structures were proven by 1H NMR . Transcripts produced with SP6 polymerase using "anti-reverse" cap analogs (ARCAs) were of the predicted length and indistinguishable in size and homogeneity from those produced with m7GpppG or GpppG . Analysis of the transcripts with RNase T2 and tobacco acid pyrophosphatase indicated that reverse caps were formed with m7GpppG but not with ARCAs . Both of the ARCAs inhibited cell-free translation with a K(I) similar to that of m7GpppG . Finally, the translational efficiency of ARCA-capped transcripts in a rabbit reticulocyte lysate was 2.3- to 2.6-fold higher than that of m7GpppG-capped transcripts . This suggests the presence of reverse caps in conventional in vitro-synthesized mRNAs reduces their translational efficiency. Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1652 - 4 Epub 2001 Oct 25. Crystallization and preliminary X-ray analysis of ocr, the product of gene 0.3 of bacteriophage T7; Sturrock SS et al.; Ocr, the product of gene 0.3 of bacteriophage T7, prevents the action of restriction endonucleases of the host bacteria . The amino-acid sequence of ocr has less than 20% similarity to any protein of known three-dimensional structure . Ocr has been crystallized in a number of different crystal forms and X-ray data for the seleno-L-methionine-substituted form has been collected to a resolution of 1.8 A . The presence of caesium was found to be required for good crystal growth . Anomalous X-ray data was used to identify possible positions for Se and Cs atoms in the unit cell. Biosens Bioelectron, 2001 Dec, 16(9-12), 639 - 46 The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep; Charlton K et al.; The complexity and expense of producing anti-hapten monoclonals via the traditional hybridoma route and the preferential selection of antibodies that recognise the conjugated form of the hapten, over antibodies that specifically recognise free hapten, are two of the more important problems that have limited the development and application of anti-hapten antibodies . The advent of phage display technology allows the rapid isolation of monoclonal antibody fragments from libraries of different antibodies (>10(8)) displayed on the surface of filamentous bacteriophages . Much of the power of this new approach lies in the flexibility with which these libraries can be screened for suitable binders . Using an optimised selection procedure, we have isolated from a sheep antibody phage display library, super-sensitive anti-hapten antibodies specific for the herbicide and environmental pollutant, atrazine . In particular, two phage clones have been isolated that can be expressed cheaply and in quantity in Escherichia coli, demonstrate excellent stability in nonphysiological conditions and are exciting prospects for immunoassay applications including ELISA, dip-stick formats, on-line monitoring and biosensor technologies . In ELISA formats they show low levels of cross reactivity with related molecules and a limit of detection of a 1-2 parts per trillion (p.p.t.), well within the 100 p.p.t . required by EC legislation. J Mol Evol, 2001 Dec, 53(6), 607 - 14 The bacteriophage lambda attachment site in wild strains of Escherichia coli; Kuhn J et al.; The attachment site (attlambda) of bacteriophage lambda was examined in wild strains of Escherichia coli . Although the att region is non-coding, the DNA sequence was invariant in the 13 strains examined . Two other non-coding regions showed nine changes, all associated with a single strain . In four of 33 strains, sequences were inserted in or near the attlambda site and in two of these the insert was related to lambda . Among strains that can be lysogenized by lambda, integration was via the attlambda site in all cases . Some resistant strains can be lysogenized, and these have been termed "lenient." Most of these fail to give normal phage yield after induction . In some cases rare lysogens have been formed in cells that belong to a mutant subpopulation. Protein Expr Purif, 2001 Nov, 23(2), 226 - 32 Expression in Escherichia coli of the death domain of the human p55 tumor necrosis factor receptor; De Wilde G et al.; The p55 tumor necrosis factor receptor (TNF-RI) is the main receptor by which TNF exerts its effects . The signaling capacity largely depends on the presence of an intact C-terminal protein-protein interaction domain, a so-called death domain (DD) . Here we report the expression and purification of the human TNF-RI DD as a fusion with the Escherichia coli thioredoxin A (TRX) protein . When expressed under control of the bacteriophage T7 promoter, TRX-DD accumulates as a soluble protein in the cytoplasm of E . coli . The TRX-DD protein was released from the cells into the periplasmic fraction after osmotic shock . Due to self-association of the DD, a large part of the material appeared as multimers; it could be removed by selective precipitation and a combination of ion-exchange and size-exclusion chromatography . This purification protocol yielded 30 mg of purified, monomeric protein from 1 liter of shake-flask culture . The purified TRX-DD was found to be functional as it still bound to the TNF-RI-associated DD protein and the intracellular part of TNF-RI . We conclude that TRX-DD is correctly folded and can be used for further structure/function analysis . Arch Virol, 2001 Aug, 146(8), 1553 - 70 Intracellular hepatitis C virus RNA-dependent RNA polymerase activity; Goobar-Larsson L et al.; Studies of intracellular hepatitis C virus (HCV) RNA-dependent RNA polymerase activity (RdRp activity) have been limited by the poor replicative capacity of HCV in cell culture . We have developed a method that allows for the measurement of HCV specific RdRp activity in eukaryotic cells . This method is based on the transient expression of the HCV polymerase and its templates under the control of the T7 promoter in the presence of an infection with recombinant vaccinia virus (vTF7-3) expressing the bacteriophage T7 DNA-dependent RNA polymerase . Both negative-strand and positive-strand RNA synthesis were characterised, and the role of the other HCV non-structural proteins for polymerase activity was assessed . With this assay we were able to show that: a) Intracellular HCV RdRp activity is not restricted to, but is higher for templates containing HCV specific sequences, b) The HCV polymerase is active within the polyprotein precursor, c) Cleavage of NS5b from the polyprotein precursor does not determine template specificity, and d) HCV RdRp activity is higher in the presence of the other HCV non-structural proteins and lower within a protease-deficient polyprotein precursor . This method allows the measurement of intracellular HCV polymerase activity and may be used to test substances against the HCV polymerase in search of potential drugs for anti-HCV therapy. Arch Virol, 2001 Aug, 146(8), 1487 - 98 Rapid degradation of bacteriophage lambda O protein by ClpP/ClpX protease influences the lysis-versus-lysogenization decision of the phage under certain growth conditions of the host cells; Czyz A et al.; The initiator of bacteriophage lambda DNA replication, the O protein, is rapidly degraded in Escherichia coli by the ClpP/ClpX protease encoded by the host . Although the biochemical mechanism of this degradation has been investigated intensively, a physiological role for this process remained unknown since little effect of dysfunction of clpP and clpX genes on the lytic development of the phage was observed . Here we demonstrate that activities of clpP and clpX genes influence the lysis-versus-lysogenization decision of bacteriophage lambda under certain growth conditions of the host cells . This decision is influenced specifically by ClpP/ClpX-mediated O degradation and resultant inhibition of early lambda DNA replication because mutations in clpP and clpX genes have little effect on stability of other lambda proteins involved in the regulation of the phage developmental switch. Gene, 2001 Aug 22, 274(1-2), 209 - 16 Detection of rare DNA targets by isothermal ramification amplification; Zhang DY et al.; We described previously a novel DNA amplification technique, termed ramification amplification (RAM) (Zhang et al., Gene 211 (1998) 277) . This method was designed to utilize a circular probe (C-probe) that is covalently linked by a DNA ligase when it hybridizes to a target . Then, a DNA polymerase extends the bound forward primer along the C-probe and continuously displaces a downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to in vivo 'rolling circle' replication of bacteriophage . This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex, and resulting in an exponential amplification . Previously, we were able to achieve a significant amplification using phi29 DNA polymerase that has a high processivity and strong displacement activity . However, due to the intrinsic limitations of the polymerase, we only achieved a sensitivity of 10,000 target molecules, which is insufficient for most practical uses . Therefore, we tested several DNA polymerases and found that exo(-) Bst DNA polymerase meets the requirement for high sensitivity . By further improving the assay condition and format, we are able to detect fewer than ten targets in 1 h and to apply successfully this method for detection of Epstein-Barr virus in human lymphoma specimens. J Biol Chem, 2001 Dec 28, 276(52), 49419 - 26 Epub 2001 Oct 22. Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7; Lee SJ et al.; At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase . In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage . The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively . T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN . T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases . To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases . The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites . Two lysine residues, Lys-122 and Lys-128, are essential for phage growth . The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein . The altered primases are unable to either synthesize or extend an oligoribonucleotide . However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase . Other lysines in the vicinity are not essential for the synthesis of primers. J Biol Chem, 2002 Jan 4, 277(1), 155 - 60 Epub 2001 Oct 22. Proteolytic sensitivity and helper T-cell epitope immunodominance associated with the mobile loop in Hsp10s; Carmicle S et al.; Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes . The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences . Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S . Each protease preferentially cleaved the Hsp10s in the mobile loop . Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites . Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis . The two largest deletions effectively inhibited proteolysis by several proteases . Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10 . Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity. Prog Nucleic Acid Res Mol Biol, 2001, 70, 77 - 118 A tale of two HSV-1 helicases: roles of phage and animal virus helicases in DNA replication and recombination; Marintcheva B et al.; Helicases play essential roles in many important biological processes such as DNA replication, repair, recombination, transcription, splicing, and translation . Many bacteriophages and plant and animal viruses encode one or more helicases, and these enzymes have been shown to play many roles in their respective viral life cycles . In this review we concentrate primarily on the roles of helicases in DNA replication and recombination with special emphasis on the bacteriophages T4, T7, and A as model systems . We explore comparisons between these model systems and the herpesviruses--primarily herpes simplex virus . Bacteriophage utilize various pathways of recombination-dependent DNA replication during the replication of their genomes . In fact the study of recombination in the phage systems has greatly enhanced our understanding of the importance of recombination in the replication strategies of bacteria, yeast, and higher eukaryotes . The ability to "restart" the replication process after a replication fork has stalled or has become disrupted for other reasons is a critical feature in the replication of all organisms studied . Phage helicases and other recombination proteins play critical roles in the "restart" process . Parallels between DNA replication and recombination in phage and in the herpesviruses is explored . We and others have proposed that recombination plays an important role in the life cycle of the herpesviruses, and in this review, we discuss models for herpes simplex virus type 1 (HSV-1) DNA replication . HSV-1 encodes two helicases . UL9 binds specifically to the origins of replication and is believed to initiate HSV DNA replication by unwinding at the origin; the heterotrimeric helicase-primase complex, encoded by UL5, UL8, and UL52 genes, is believed to unwind duplex viral DNA at replication forks . Structure-function analyses of UL9 and the helicase-primase are discussed with attention to the roles these proteins might play during HSV replication. Nature, 2001 Oct 18, 413(6857), 748 - 52 The bacteriophage straight phi29 portal motor can package DNA against a large internal force; Smith DE et al.; As part of the viral infection cycle, viruses must package their newly replicated genomes for delivery to other host cells . Bacteriophage straight phi29 packages its 6.6-microm long, double-stranded DNA into a 42 x 54 nm capsid by means of a portal complex that hydrolyses ATP . This process is remarkable because entropic, electrostatic and bending energies of the DNA must be overcome to package the DNA to near-crystalline density . Here we use optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force-generating motor . This motor can work against loads of up to 57 pN on average, making it one of the strongest molecular motors reported to date . Movements of over 5 microm are observed, indicating high processivity . Pauses and slips also occur, particularly at higher forces . We establish the force-velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads . Notably, the packaging rate decreases as the prohead is filled, indicating that an internal force builds up to approximately 50 pN owing to DNA confinement . Our data suggest that this force may be available for initiating the ejection of the DNA from the capsid during infection. Mol Biol Evol, 2001 Nov, 18(11), 2067 - 82 The DIRS1 group of retrotransposons; Goodwin TJ et al.; Only three retrotransposons of the DIRS1 group have previously been described: DIRS1 from the slime mold Dictyostelium discoideum, PAT from the nematode Panagrellus redivivus, and Prt1 from the zygomycetous fungus Phycomyces blakesleeanus . Analyses of the reverse transcriptase sequences encoded by these elements suggest that they are related to the long terminal repeat (LTR) retroelements, such as the Ty3/gypsy retrotransposons and the vertebrate retroviruses . The DIRS1-group elements, however, have several unusual structural features which distinguish them from typical LTR elements: (1) they lack the capacity to encode DDE-type integrases or aspartic proteases; (2) they have open reading frames (ORFs) of unknown function; (3) they integrate without creating duplications of their target sites; and (4) although they are bordered by terminal repeats, these sequences differ from typical LTRs in that they are either inverted repeats or "split" direct repeats . Because of the small number of DIRS1-like elements described, and the unusual structures of these elements, little is known about their evolution, distribution, and replication mechanisms . Here, we report the identification of several new DIRS1-like retrotransposons, including elements from nematodes, sea urchins, fish, and amphibia . We also present evidence for the existence of DIRS1-like sequences in the human genome . In addition, we show that the lack of DDE-type integrase genes from elements of the DIRS1 group is explained by the finding that the previously uncharacterized ORFs of these elements encode proteins related to the site-specific recombinase of bacteriophage lambda . The presence of lambda-recombinase-like genes in DIRS1 elements also accounts for the lack of target-site duplications for these elements and may be related to the unusual structures of their terminal repeats. Mol Biol (Mosk), 2001 Sep-Oct, 35(5), 844 - 56 {Interaction of HIV-1 reverse transcriptase and bacteriophage T7 RNA polymerase with NTP phosphonate analogs and inorganic pyrophosphate}; Andreeva OI et al.; We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhydrolyzable bisphosphonate groups . We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups . The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated . One of PPi analogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT . The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases . It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups . The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV-1 RT is more sensitive to this type of compounds . On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme . The peculiarities observed in the model correlate well with the experimental data on inhibition. J Virol, 2001 Nov, 75(22), 11088 - 95 Comparison of polymerase subunits from double-stranded RNA bacteriophages; Yang H et al.; The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes . We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage phi6, and shown that the protein can catalyze RNA synthesis in vitro . In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3' termini from the phi6 minus strands are favored . This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from phi6 dsRNA segments in vivo . To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the phi6-related bacteriophages phi8 and phi13 and assayed their polymerase activities in vitro . The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates . However, they differ from each other as well as from phi6 Pol in certain biochemical properties . Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3'-terminal sequences from the virus-specific minus strands . This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit. J Virol, 2001 Nov, 75(22), 10923 - 32 The UL6 gene product forms the portal for entry of DNA into the herpes simplex virus capsid; Newcomb WW et al.; During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled . Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids . Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid . Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6 . After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices . Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6 . In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 +/- 2.6 . Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene . After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 +/- 1.1 and 5.0 +/- 0.7 nm, respectively, and a height of 19.5 +/- 1.9 nm . The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12 . The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass . The HSV-1 portal is the first identified in a virus infecting a eukaryote . In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as phi29, T4, and P22 . This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved. J Biol Chem, 2002 Jan 4, 277(1), 161 - 8 Epub 2001 Oct 15. Structural basis for helper T-cell and antibody epitope immunodominance in bacteriophage T4 Hsp10 . Role of disordered loops; Dai G et al.; Antigen three-dimensional structure potentially limits the access of endoproteolytic processing enzymes to cleavage sites and of class II major histocompatibility antigen-presenting proteins to helper T-cell epitopes . Helper T-cell epitopes in bacteriophage T4 Hsp10 have been mapped by restimulation of splenocytes from CBA/J and C57BL/6J mice immunized in conjunction with mutant (R192G) heat-labile enterotoxin from Escherichia coli . Promiscuously immunogenic sequences were associated with unstable loops in the three-dimensional structure of T4 Hsp10 . The immunodominant sequence lies on the N-terminal flank of the 22-residue mobile loop, which is sensitive to proteolysis in divergent Hsp10s . Several mobile loop deletions that inhibited proteolysis in vitro caused global changes in the helper T-cell epitope map . A mobile loop deletion that strongly stabilized the protein dramatically reduced the immunogenicity of the flanking immunodominant helper T-cell epitope, although the protein retained good overall immunogenicity . Antisera against the mobile loop deletion variants exhibited increased cross-reactivity, most especially the antisera against the strongly stabilized variant . The results support the hypothesis that unstable loops promote the presentation of flanking epitopes and suggest that loop deletion could be a general strategy to increase the breadth and strength of an immune response. Exp Hematol, 2001 Oct, 29(10), 1136 - 46 Protein-protein interactions in hematology and phage display; Mullaney BP et al.; Phage display, which exploits fundamental tools and principles of immune repertoire diversity, antigen-antibody interactions, and clonal and immunologic selection, is used increasingly to advance experimental and clinical hematology . Phage display is based on the ability of bacteriophage to present engineered proteins on their surface coat . Diverse libraries of proteins such as peptides, antibody fragments, and protein domains corresponding to gene fragments or cDNAs may be displayed . Interactions between phage-displayed proteins and target antigens can be identified rapidly and characterized using high throughput methodologies . Peptide and gene fragment libraries are particularly useful to characterize binding interactions between proteins, such as ligand-receptor interactions . This approach allows rapid generation of human antibodies, often against nonimmunogenic, conserved proteins . Phage antibodies against surface and intracellular antigens are used as reagents for flow cytometry, in vivo imaging, and therapeutic targeting . Phage-derived antibodies also facilitate analyses of the humoral antibody response . Finally, cellular delivery of phage-displayed peptides and gene fragments can be used to modulate functional pathways and molecules in vitro and in vivo . The combinatorial power of phage display enables identification of candidate epitopes without knowledge of the protein interaction, a priori . Overall, these capabilities provide a versatile, high-throughput approach to develop tools and reagents useful for a plethora of experimental hematology applications . This paper focuses on current and future applications of antibody and epitope phage display technology in hematology. J Mol Biol, 2001 Oct 12, 313(1), 181 - 95 Genetic selection for and molecular dynamic modeling of a protein transmembrane domain multimerization motif from a random Escherichia coli genomic library; Leeds JA et al.; In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacteriophage lambda cI repressor DNA-binding domain . Sequences were isolated using a modified lambda cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E . coli inner membrane . Screening resulted in the identification of several novel sequences that appear to mediate helix-helix interactions . One sequence, representing the predicted sixth transmembrane domain (TM6) of the E . coli protein YjiO, was chosen for further analysis . Using site-directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated . This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly . J Biol Chem, 2001 Dec 14, 276(50), 46941 - 5 Epub 2001 Oct 11. DNA unwinding mechanism for the transcriptional activation of momP1 promoter by the transactivator protein C of bacteriophage Mu; Basak S et al.; Transcription factor-induced conformational changes in DNA are one of the mechanisms of transcription activation . C protein of bacteriophage Mu appears to transactivate the mom gene of the phage by this mode . DNA binding by C to its site leads to torsional changes that seem to compensate for a weak momP1 promoter having a suboptimal spacing of 19 bp between the poor -35 and -10 elements . The C-mediated unwinding could realign the promoter elements for RNA polymerase recruitment to the reoriented promoter . In this study, the model has been tested by mutational analysis of the spacer region of momP1 and by assessing the strength of the mutant promoters . The response to C-mediated transactivation was dependent on the spacer length of the promoters . Mutants with 17-bp spacing between the two promoter elements showed reduced activity in the presence of the transactivator as compared with their basal level . A synthetic promoter with near consensus promoter elements and optimal 17-bp spacing was also tested to evaluate the model . The results imply a role for C-mediated unwinding in mom transcription activation. Trends Microbiol, 2001 Oct, 9(10), 481 - 5 Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Ohnishi M, Kurokawa K, Hayashi T. Determination of the genome sequence of enterohemorrhagic Escherichia coli O157 Sakai and genomic comparison with the laboratory strain K-12 has revealed that the two strains share a highly conserved 4.1-Mb sequence and that each also contains a large amount of strain-specific sequence . The analysis also revealed the presence of a surprisingly large number of prophages in O157, most of which are lambda-like phages that resemble each other . Based on these results, we discuss how the E . coli strains have diverged from a common ancestral strain, and how bacteriophages contributed to this process . We also describe possible mechanisms by which O157 acquired many closely related phages, and raise the possibility that such bacteria might function as 'phage factories', releasing a variety of chimeric or mosaic phages into the environment. Horm Res, 2000, 54(5-6), 296 - 300 Targeted somatic mutagenesis in mouse epidermis; Indra AK et al.; Gene targeting in the mouse is a powerful tool to study mammalian gene function . The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases . To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the human keratin 14 (K14) promoter . We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice . Furthermore, Tamoxifen administration to adult K14-Cre-ER(T2) mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment . These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes . Structure (Camb), 2001 Oct, 9(10), 917 - 30 Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions; Martin CS et al.; BACKGROUND: The dsDNA bacteriophage PRD1 has a membrane inside its icosahedral capsid . While its large size (66 MDa) hinders the study of the complete virion at atomic resolution, a 1.65-A crystallographic structure of its major coat protein, P3, is available . Cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction have shown the capsid at 20-28 A resolution . Striking architectural similarities between PRD1 and the mammalian adenovirus indicate a common ancestor . RESULTS: The P3 atomic structure has been fitted into improved cryo-EM reconstructions for three types of PRD1 particles: the wild-type virion, a packaging mutant without DNA, and a P3-shell lacking the membrane and the vertices . Establishing the absolute EM scale was crucial for an accurate match . The resulting "quasi-atomic" models of the capsid define the residues involved in the major P3 interactions, within the quasi-equivalent interfaces and with the membrane, and show how these are altered upon DNA packaging . CONCLUSIONS: The new cryo-EM reconstructions reveal the structure of the PRD1 vertex and the concentric packing of DNA . The capsid is essentially unchanged upon DNA packaging, with alterations limited to those P3 residues involved in membrane contacts . These are restricted to a few of the N termini along the icosahedral edges in the empty particle; DNA packaging leads to a 4-fold increase in the number of contacts, including almost all copies of the N terminus and the loop between the two beta barrels . Analysis of the P3 residues in each quasi-equivalent interface suggests two sites for minor proteins in the capsid edges, analogous to those in adenovirus. Biochemistry, 2001 Oct 16, 40(41), 12321 - 8 Fluorescence resonance energy transfer shows a close helix-helix distance in the transmembrane M13 procoat protein; Eisenhawer M et al.; During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other . Previous studies on the molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process . We investigated the intramolecular distance between the two helices of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix . Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers . The results obtained from the FRET measurements, combined with molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm . The present approach might be of general interest for determining the topology and the folding of membrane proteins. Mem Inst Oswaldo Cruz, 2001, 96 Suppl, 131 - 5 r-Sm14 - pRSETA efficacy in experimental animals; Ramos CR et al.; Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega) . The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein . Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S . mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials . Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E . coli expression systems which would be more suitable for scale up and downstream processing . We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids . The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin . The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S . mansoni soluble secreted/excreted proteins in Western-Blot . Both proteins were also protective against S . mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system. J Mol Biol, 2001 Oct 5, 312(5), 999 - 1009 Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently; Yang S et al.; The UvsX protein from bacteriophage T4 is a member of the RecA/Rad51/RadA family of recombinases active in homologous genetic recombination . Like RecA, Rad51 and RadA, UvsX forms helical filaments on DNA . We have used electron microscopy and a novel method for image analysis of helical filaments to show that UvsX-DNA filaments exist in two different conformations: an ADP state and an ATP state . As with RecA protein, these two states have a large difference in pitch . Remarkably, even though UvsX is only weakly homologous to RecA, both UvsX filament states are more similar to the RecA crystal structure than are RecA-DNA filaments . We use this similarity to fit the RecA crystal structure into the UvsX filament, and show that two of the three previously described blocks of similarity between UvsX and RecA are involved in the subunit-subunit interface in both the UvsX filament and the RecA crystal filament . Conversely, we show that human Rad51-DNA filaments have a different subunit-subunit interface than is present in the RecA crystal, and this interface involves two blocks of sequence similarity between Rad51 and RecA that do not overlap with those found between UvsX and RecA . This suggests that helical filaments in the RecA/Rad51/RadA family may have arisen from convergent evolution, with a conserved core structure that has assembled into multimeric filaments in a number of different ways . Genes Chromosomes Cancer, 2001 Nov, 32(3), 265 - 74 Detection of illegitimate rearrangement within the immunoglobulin locus on 14q32.3 in B-cell malignancies using end-sequenced probes; Poulsen TS et al.; Translocation involving the immunoglobulin heavy chain (IGH) locus is a recurring event in B-cell oncogenesis . The aim of this study was to characterize clones from bacterial artificial chromosome (BAC) libraries and/or bacteriophage P1 artificial chromosome libraries spanning the IGH locus for detection of illegitimate rearrangement within the region by fluorescence in situ hybridization (FISH) . In silico analysis of the IGH variable (IGHV) DNA sequence (NT_001716.v1) was performed to identify BAC probes located within the IGHV cluster . Clones of the constant (IGHC) cluster were found in the literature or at Validation, orientation, and overlap of these probes were confirmed using interphase-, metaphase-, and fiber-FISH . We have identified seven BAC end-sequenced probes (3087C18, 47P23, 76N15, 12F16, 101G24, 112H5, and 151B17) covering 612 kb of the distal IGHV cluster, which, together with probes covering the IGHC cluster (11771 and 998D24), could be used in interphase nuclei and metaphase chromosome analysis . A visual split of the IGHV and IGHC clusters indicating a translocation was analyzed by dual-color FISH in a series of 21 cell lines of different origins . Translocations were found, as expected, in eight of eight myelomas, four of four lymphomas, none of five leukemias, and none of four Epstein-Barr virus-transformed B-lymphoblastoid cell lines . To summarize, we have established a set of IGHV and IGHC probes that can be used for universal screening of illegitimate rearrangement within the IGH locus in B-cell malignancies . These probes allow for routine FISH analysis to detect this early central oncogenic event . J Biol Chem, 2001 Dec 7, 276(49), 46187 - 95 Epub 2001 Sep 27. Solution structure of bacteriophage PRD1 vertex complex; Sokolova A et al.; Bacteriophage PRD1 is a prototype of viruses with an internal membrane . The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus . The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies . The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31 . The vertex complex mediates host cell binding and controls double-stranded DNA delivery . Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering . Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data . Protein P5 is a long trimer that resembles the adenovirus spike protein pIV . The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart . P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII . P5 further polymerize into a nonameric form ((P5(3))(3)) . In the presence of P31, P5 associates into a P5(6):P31 complex . The constructed models of these assemblies provided support for a model of vertex assembly onto the virion . Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed. FEBS Lett, 2001 Sep 21, 505(3), 467 - 73 Using an in vivo phagemid system to identify non-compatible loxP sequences; Siegel RW et al.; The site-specific recombination system of bacteriophage P1 is composed of the Cre recombinase that recognizes a 34-bp loxP site . The Cre/loxP system has been extensively used to manipulate eukaryotic genomes for functional genomic investigations . The creation of additional heterologous loxP sequences potentially expands the utility of this system, but only if these loxP sequences do not recombine with one another . We have developed a stringent in vivo assay to examine the degree of recombination between all combinations of each previously published heterologous loxP sequence . As expected, homologous loxP sequences efficiently underwent Cre-mediated recombination . However, many of the heterologous loxP pairs were able to support recombination with rates varying from 5 to 100% . Some of these loxP sequences have previously been reported to be non-compatible with one another . Our study also confirmed other heterologous loxP pairs that had previously been shown to be non-compatible, as well as defined additional combinations that could be used in designing new recombination vectors. Can J Microbiol, 2001 Aug, 47(8), 722 - 6 Precise excision of bacteriophage Mu DNA; Abbes C et al.; The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations . Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely . In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction . In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated . The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability. Nucleic Acids Res, 2001 Oct 1, 29(19), 3955 - 64 Protein and DNA requirements of the bacteriophage HP1 recombination system: a model for intasome formation; Esposito D et al.; A fundamental step in site-specific recombination reactions involves the formation of properly arranged protein-DNA structures termed intasomes . The contributions of various proteins and DNA binding sites in the intasome determine not only whether recombination can occur, but also in which direction the reaction is likely to proceed and how fast the reaction will go . By mutating individual DNA binding sites and observing the effects of various mixtures of recombination proteins on the mutated substrates, we have begun to categorize the requirements for intasome formation in the site-specific recombination system of bacteriophage HP1 . These experiments define the binding site occupancies in both integrative and excessive recombination for the three recombination proteins: HP1 integrase, HP1 Cox and IHF . This data has allowed us to create a model which explains many of the biochemical features of HP1 recombination, demonstrates the importance of intasome components on the directionality of the reaction and predicts further ways in which the role of the intasome can be explored. EMBO J, 2001 Oct 1, 20(19), 5453 - 60 Design and development of a catalytic ribonucleoprotein; Atsumi S et al.; Ribonucleoproteins (RNPs) consisting of derivatives of a ribozyme and an RNA-binding protein were designed and constructed based upon high-resolution structures of the corresponding prototype molecules, the Tetrahymena group I self-splicing intron RNA and two proteins (bacteriophage lambdaN and HIV Rev proteins) containing RNA-binding motifs . The splicing reaction proceeds efficiently only when the designed RNA associates with the designed protein either in vivo or in vitro . In vivo mutagenic protein selection was effective for improving the capability of the protein . Kinetic analyses indicate that the protein promotes RNA folding to establish an active conformation . The fact that the conversion of a ribozyme to an RNP can be accomplished by simple molecular design supports the RNA world hypothesis and suggests that a natural active RNP might have evolved readily from a ribozyme. Gene, 2001 Sep 5, 275(1), 73 - 81 Human cell lines expressing hormone regulated T7 RNA polymerase localized at distinct intranuclear sites; Yarovoi SV et al.; Although several systems are now available for the controlled expression of eukaryotic genes transcribed by RNA polymerase II, regulated expression has been more difficult to achieve in the case of genes transcribed by RNA polymerase III . In the present study the gene for bacteriophage T7 RNA polymerase, implanted with a eukaryotic nuclear localization signal, was linked to a 5'-flanking ecdysone-responsive promoter and stably transformed human cell lines were constructed in which the ecdysone promoter-T7 RNA polymerase gene had been integrated intact, as demonstrated by a polymerase chain reaction assay . Exposure of these cells to the ecdysone analog ponasterone A resulted in the appearance of a single protein having the expected size of T7 RNA polymerase in immunoblots of cell extracts probed with an affinity purified antibody raised against the C-terminus of T7 RNA polymerase . The induced T7 RNA polymerase was exclusively localized in the nucleus of induced cells and was undetectable in uninduced cells either by immunoblotting or immunofluorescence . The induced T7 RNA polymerase was present at numerous punctate foci dispersed throughout the nucleoplasmic regions of the nucleus and was also present in the nucleoli . Both of these observed intranuclear localizations have relevance to the potential applications of this system. Anal Chem, 2001 Sep 1, 73(17), 4196 - 201 Microfabricated polycarbonate CE devices for DNA analysis; Liu Y et al.; The microchip capillary electrophoresis (CE) devices were fabricated in polycarbonate (PC) plastic material by compression molding . The molded devices were enclosed utilizing thermal bonding to another PC wafer . These thermal bonds do not yield up to an applied force equivalent to 150 psi . Aqueous fluid transport inside the plastic CE devices was enhanced by UV irradiation treatment of the hydrophobic polycarbonate plastic surfaces prior to thermal bonding . In comparison to glass microchannels, electroosmotic flow (EOF) in native PC channels is low and is independent of buffer pH at pH 7 and 9 . UV irradiation of PC surfaces increases surface hydrophilicity and increases EOF . CE DNA separation was demonstrated in these PC CE devices with good resolution and run-to-run reproducibility . The on-chip PCR/CE analysis of a 500-bp region of bacteriophage lambda DNA was also demonstrated. Mol Immunol, 2001 Aug, 38(4), 313 - 26 Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries; Nuttall SD et al.; The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule . To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein . To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages . On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis . One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E . coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein . Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues . Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries. Biochemistry (Mosc), 2001 Aug, 66(8), 875 - 84 Principles of selective inactivation of a viral genome . Comparative kinetic study of modification of the viral RNA and model protein with oligoaziridines; Tsvetkova EA et al.; Comparative kinetic analysis of inactivation of bacteriophage MS2 infectivity and aminoalkylation of a model protein (trypsin inhibitor) with oligoaziridines was performed in order to evaluate the selectivity of viral RNA modification with oligocationic reagents . The transition from ethyleneimine monomer to di-, tri-, and tetramer leads to a sharp increase in the rate constant of infectivity inactivation, whereas the rate constant of protein modification changes insignificantly . The selectivity coefficient of the phage RNA aminoalkylation relative to trypsin inhibitor modification increases in this series by more than an order of magnitude . This effect is probably associated with the strengthening of the reagent binding to the nucleic acid, which implies a reaction mechanism that involves the formation of a reactive intermediate . The latter might be an electrostatic complex of the oligocationic reagent and RNA, the only polyanion in the virion . A pronounced decrease in the rate constant of infectivity inactivation in the presence of multiply charged anions (in phosphate buffer) and a biogenic polyamine (spermine) favors this hypothesis . Increasing the reaction temperature increases the rate constant of infectivity inactivation and decreases selectivity of the viral RNA modification. J Biomol NMR, 2001 Aug, 20(4), 365 - 77 Characterization of molecular alignment in aqueous suspensions of Pf1 bacteriophage; Zweckstetter M et al.; The phase diagram of Pf1 solutions has been studied indirectly by observation of 2H quadrupole splittings of the solvent signal and measurement of dipolar couplings in solute macromolecules . At low volume fractions of Pf1 and at high ionic strength, alignment of both the phage and the solute depends strongly on the strength of the magnetic field . Both the theoretical and experimentally determined phase diagram of Pf1 show that at low concentrations and high ionic strengths the solution becomes isotropic . However, just below the nematic phase boundary the behavior of the system is paranematic, with cooperative alignment which depends on the strength of the applied magnetic field . Above 16 mg/ml Pf1 is fully nematic up to 600 mM NaCl . Alignment of proteins with a significant electric dipole moment, which tends to be strong in Pf1, can be reduced by either high ionic strength or low phage concentration . Because ionic strength modulates both the orientation and magnitude of the alignment tensor in Pf1 medium, measurement at two ionic strengths can yield linearly independent alignment tensors. J Struct Biol, 2001 Jul, 135(1), 38 - 46 Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage phi29; Morais MC et al.; A method has been developed for three-dimensional image reconstruction of symmetry-mismatched components in tailed phages . Although the method described here addresses the specific case where differing symmetry axes are coincident, the method is more generally applicable, for instance, to the reconstruction of images of viral particles that deviate from icosahedral symmetry . Particles are initially oriented according to their dominant symmetry, thus reducing the search space for determining the orientation of the less dominant, symmetry-mismatched component . This procedure produced an improved reconstruction of the sixfold-symmetric tail assembly that is attached to the fivefold-symmetric prolate head of phi29, demonstrating that this method is capable of detecting and reconstructing an object that included a symmetry mismatch . A reconstruction of phi29 prohead particles using the methods described here establishes that the pRNA molecule has fivefold symmetry when attached to the prohead, consistent with its proposed role as a component of the stator in the phi29 DNA packaging motor . AIDS Res Hum Retroviruses, 2001 Sep 1, 17(13), 1249 - 55 Rapid genetic selection of inhibitor-resistant protease mutants: clinically relevant and novel mutants of the HIV protease; Sices HJ et al.; Site-specific proteolysis is an important regulatory mechanism in many basic cellular processes as well as playing critical roles in the life cycle of viruses and other pathogenic organisms . For the human immunodeficiency virus (HIV) the encoded protease is required for the replication of the virus and has been the target of novel antiviral therapeutics . However, the emergence of inhibitor-resistant viral strains has become an increasingly significant clinical problem . Using a bacteriophage-based genetic selection, a bank of inhibitor-resistant mutants in this protease has been isolated that includes mutations that correlate with resistant clinical isolates . The rapid selection of such mutations has implications for the prediction of relevant mutations and may be applicable to other viral systems. Nucleic Acids Res, 2001 Sep 15, 29(18), 3742 - 56 Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution; Kobayashi I; Restriction-modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase . RM systems sometimes behave as discrete units of life, like viruses and transposons . RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells . However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome . This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage . There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference . They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons . The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements . Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements . The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks . Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems . RM systems compete with each other in several ways . One is competition for recognition sequences in post-segregational killing . Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity . The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes. J Mol Biol, 2001 Sep 14, 312(2), 311 - 22 Conformational dynamics of a transposition repressor in modulating DNA binding; Rai SS et al.; The repressor of bacteriophage Mu functions in the establishment and maintenance of lysogeny by binding to Mu operator DNA to shut down transposition . A domain at its N terminus functions in DNA binding, and temperature-sensitive mutations in this domain can be suppressed by truncations at the C terminus . To understand the role of the C-terminal tail in DNA binding, a fluorescent probe was attached to the C terminus to examine its environment and its movement with respect to the DNA binding domain . The emission spectrum of this probe indicated that the C terminus was in a relatively hydrophobic environment, comparable to the environment of the probe attached within the DNA-binding domain . Fluorescence of two tryptophan residues located within the DNA-binding domain was quenched by the probe attached to the C terminus, indicating that the C terminus is in close proximity to this domain . Addition of DNA, even when it did not contain operator DNA, reduced quenching of tryptophan fluorescence, indicating that the tail moves away from the DNA-binding domain as it interacts with DNA . The presence of the tail also produced a trypsin hypersensitive site within the DNA-binding domain; mutant repressors with an altered or truncated C terminus were relatively resistant to cleavage at this site . Interaction of the wild-type repressor with DNA greatly reduced cleavage at the site . A repressor with a temperature-sensitive mutation in the DNA-binding domain was especially sensitive to cleavage by trypsin even in the presence of DNA, and the C-terminal tail failed to move in the presence of DNA at elevated temperatures . These results indicate that the tail sterically inhibits DNA binding and that it moves during establishment of repression . Such conformational changes are likely to be involved in communication between repressor protomers for cooperative DNA binding . Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11450 - 5 Epub 2001 Sep 11. Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo; Pfeifer A et al.; The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells . We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo . Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells . To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Cre-dependent manner . Thus, the activity of Cre terminates its own expression (self-deleting) . We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors . In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity . Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo. J Biol Chem, 2001 Dec 7, 276(49), 46151 - 9 Epub 2001 Sep 10. A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive; Kumar JK et al.; Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage replication . A single point mutation in gene 5 confers temperature sensitivity for phage growth . The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain . Upon infection of Escherichia coli by the temperature-sensitive phage at 42 degrees C, there is no detectable T7 DNA synthesis in vivo . DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degrees C or 42 degrees C . Upon infection at 30 degrees C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degrees C or 42 degrees C approach levels observed using wild-type T7 phage . The amount of soluble gene 5 protein produced at 42 degrees C is comparable to that produced at 30 degrees C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility . Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive . Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 degrees C closely resemble that of wild-type T7 DNA polymerase . It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway. Trends Biochem Sci, 2001 Sep, 26(9), 566 - 72 Dynamic protein interactions in the bacteriophage T4 replisome; Trakselis MA et al.; The bacteriophage T4 DNA replisome is a complex dynamic system employing a variety of proteins to orchestrate the synthesis of DNA on both the leading and lagging strands . Assembly of the protein complexes responsible for DNA synthesis and priming requires the coordination of transient biomolecular interactions . This interplay of proteins has been dissected through the use of small molecules including fluorescent probes and crosslinkers, enabling the development of a complex dynamic structural and kinetic model for DNA polymerase holoenzyme assembly and primosome formation. J Biomed Sci, 2001 Sep, 8(5), 430 - 6 Binding of phage-displayed HIV-1 Tat to TAR RNA in the presence of cyclin T1; Jonas G et al.; The transactivator protein (Tat) of the human immunodeficiency virus (HIV) is a key regulatory protein in the viral replication cycle . Together with cellular cyclin T1 and an RNA element (transactivation response; TAR) located at the 5' end of all viral transcripts, it forms a ternary complex that ultimately enhances the expression of all viral genes . In this ternary complex, cyclin T1 interacts directly with Tat and TAR . The presence of cyclin T1 is essential for high TAR RNA affinity and specificity of Tat . To study protein-protein and protein-RNA interaction, we developed a phage display system that displays functional Tat on the surface of bacteriophage M13 . The addition of recombinant cyclin T1 to the selections yielded a phage display system that mirrors all binding properties of the cyclin T1-Tat-TAR complex known from cell assays and biochemical studies . Phage-displayed Tat protein as well as the cyclin T1 are fully functional . The relative binding capabilities of wild-type- and mutant Tat-displaying phages show that the presence of cyclin T1 significantly reduces the importance of basic residues in the basic sequence region of Tat for its binding to TAR . J Mol Microbiol Biotechnol, 2001 Oct, 3(4), 513 - 7 Vaccinia virus-free recovery of vesicular stomatitis virus; Harty RN et al.; The advent of reverse-genetics represents a powerful new approach to elucidate aspects of negative-sense RNA virus replication . The reverse-genetics system established previously for vesicular stomatitis virus (VSV) required four plasmids encoding the nucleoprotein (N), phosphoprotein (P), polymerase (L), and the full-length, anti-genomic RNA . Transcription to yield the antigenomic RNA as well as the N, P, and L, mRNAs was initiated by bacteriophage T7 polymerase expressed from a recombinant Vaccinia virus . In this report, we describe the successful recovery of infectious VSV in the absence of Vaccinia virus . The N, P, and L genes of VSV were inserted downstream of both the T7 promoter and an internal ribosomal entry site (IRES element) . T7 polymerase was expressed constitutively from BSR-T7/5 cells . RTPCR was used to confirm that the recovered VSV was derived from transfected DNA . Virion protein profile, CPE in tissue culture, and virus titer of the recombinant VSV were indistinguishable from those of parental VSV . Thus, the need for Vaccinia virus is eliminated with this system, making it an attractive, alternative approach for the recovery of infectious VSV from DNA. J Microbiol Methods, 1991, 13, 87 - 97 Acridine orange staining reaction as an index of physiological activity in Escherichia coli; McFeters GA et al.; The assumption that the acridine orange (AO) color reaction may be used as an index of physiological activity was investigated in laboratory grown Escherichia coli . Spectrofluorometric observations of purified nucleic acids, ribosomes and the microscopic color of bacteriophage-infected cells stained with AO confirmed the theory that single-stranded nucleic acids emit orange to red fluorescence while those that are double-stranded fluoresce green in vivo . Bacteria growing actively in a rich medium could be distinguished from cells in stationary phase by the AO reaction . Cells from log phase appeared red, whereas those in stationary phase were green . However, this differentiation was not seen when the bacteria were grown in a minimal medium or when a variation of the staining method was used . Also, shifting bacteria in stationary phase to starvation conditions rapidly changed their AO staining reaction . Boiling and exposure to lethal concentrations of azide and formalin resulted in stationary-phase cells that appeared red after staining but bacteria killed with chlorine remained green . These findings indicate that the AO staining reaction may be suggestive of physiological activity under defined conditions . However, variables in staining and fixation procedures as well as uncertainties associated with mixed bacterial populations in environmental samples may produce results that are not consistent with the classical interpretation of this reaction . The importance of validating the putative physiological implications of this staining reaction is stressed. Adv Space Res, 1983, 3(8), 61 - 4 The effect of alpha particles on bacteriophage T4Br+; Leont'eva GA et al.; It is generally accepted that heavy charged particles play an important part in generating the secondary flux of nuclear particles formed by the interaction of space hadrons with nuclei . It is assumed that these particles are responsible for the high biological efficiency of space hadrons in causing cellular damage by their strong interactions . To examine this assumption we investigated the effects of 5.3 MeV alpha particles on bacteriophage T4 . This energy provides a LET value of 88.6 KeV/micrometer lying in the range of the highest biological efficiency. Adv Space Res, 1983, 3(8), 51 - 60 Effect of HZE particles and space hadrons on bacteriophages; Yurov SS et al.; The effect of high energy (HZE) particles and high energy hadrons on T4Br+ bacteriophage was analyzed . The experiments were done in orbital flight, on high mountains, on an accelerator, and with an alpha particle source . We studied the survival rate of the bacteriophage, the mutation frequency, the mutation spectrum and the revertability under the action of chemical mutagens with a known mechanism of action on DNA . It was found that the biological efficiency of HZE particles and high energy hadrons is greater than that of gamma radiation . The spectra of mutations produced by these mutations and the mechanisms of their action are also different . These effects were local, because of the mode of interaction of the radiant energy with biological objects, and depended on the linear energy transfer (LET) . The modes have now been experimentally defined. Adv Space Res, 1981, 1(14), 75 - 81 A review and comparative analysis of the biological damage induced during space flight by HZE particles and space hadrons; Akoev IG et al.; We have studied the somatic and genetic effects of heavy ions (HZE particles) and the very high energy hadrons of space radiation on various organisms ranging in complexity from bacteriophage to man . Experimental data were obtained in space, on high mountains and in a proton accelerator at energies of 76 GeV . In all these experiments local micro- and macroradiational damage was observed . This damage was characterized by severity over large local regions and for the most part was due to cascades of secondary particle bundles resulting from the collision of very high energy space hadrons with atomic nuclei rather than from cellular hits from relatively low energy single HZE particles . At present there does not appear to be any effective way to provide shielding against these cosmic hadrons. ASGSB Bull, 1991 Jul, 4(2), 151 - 260 Summary of biological spaceflight experiments with cells; Dickson KJ; Numerous biological experiments with cells have been conducted in space, and the importance of these experiments and this area of study is continually becoming evident . This contribution is a compilation of available information about spaceflight experiments with cells for the purpose of providing a single source of information for those interested in space gravitational cell biology . Experiments focused on a study of the effects of gravity and its absence on cells, cell function, and basic cellular processes have been included . Experiments include those involving viruses, bacteriophage, unicellular organisms, lower fungi, and animal and plant cell and tissue cultures, but exclude experiments with cells that were carried on a flight as part of a whole organism and later removed for study, and experiments with fertilized eggs . In addition, experiments in biotechnology, in which the microgravity environment is employed to study cell purification, cell fusion, protein crystallization, and similar processes, have not been included . Spaceflight experiments conducted by scientists from the U.S., U.S.S.R., and other countries and flown onboard sounding rockets (TEXUS, MAXUS, Consort), biosatellites (Biosatellite II, Cosmos), and various crewed spacecraft including the space shuttle (STS) and Soyuz, and space stations (Salyut, Mir) have been included, as well as high altitude balloon flights . Balloon flights are not spaceflights but can and are used as controls for the effects of space radiation, since organisms carried on balloons may be exposed to some of the same radiation as those taken into space, yet continue to be exposed to Earth's gravitational force . Parabolic flights on aircraft during which periods of microgravity of less than a minute are achieved have arbitrarily been excluded, because even though numerous experiments have been conducted, few results have been published. Biomol Eng, 2001 Sep, 18(2), 57 - 63 Engineering M13 for phage display; Sidhu SS; Phage display is achieved by fusing polypeptide libraries to phage coat proteins . The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA . Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA . The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms . Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions . Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms . These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display . These improvements expand the utility of phage display as a powerful tool in modern biotechnology. Anal Chem, 2001 Aug 15, 73(16), 3935 - 9 Direct and quantitative detection of bacteriophage by "hearing" surface detachment using a quartz crystal microbalance; Dultsev FN et al.; We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM) . This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface . There is no interference from nonspecifically adsorbed phage . The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage . The method has potential as a sensitive and low-cost method for virus detection. Mol Cell Biol, 2001 Oct, 21(19), 6585 - 97 Polycomb group repression reduces DNA accessibility; Fitzgerald DP et al.; The Polycomb group proteins are responsible for long-term repression of a number of genes in Drosophila melanogaster, including the homeotic genes of the bithorax complex . The Polycomb protein is thought to alter the chromatin structure of its target genes, but there has been little direct evidence for this model . In this study, the chromatin structure of the bithorax complex was probed with three separate assays for DNA accessibility: (i) activation of polymerase II (Pol II) transcription by Gal4, (ii) transcription by the bacteriophage T7 RNA polymerase (T7RNAP), and (iii) FLP-mediated site-specific recombination . All three processes are restricted or blocked in Polycomb-repressed segments . In contrast, control test sites outside of the bithorax complex permitted Gal4, T7RNAP, and FLP activities throughout the embryo . Several P insertions in the bithorax complex were tested, providing evidence that the Polycomb-induced effect is widespread over target genes . This accessibility effect is similar to that seen for SIR silencing in Saccharomyces cerevisiae . In contrast to SIR silencing, however, episomes excised from Polycomb-repressed chromosomal sites do not show an altered superhelix density. Mol Microbiol, 2001 Aug, 41(4), 885 - 96 A chimeric activator of transcription that uses two DNA-binding domains to make simultaneous contact with pairs of recognition sites; Langdon RC et al.; Many well-known transcriptional regulatory proteins are composed of at least two independently folding domains and, typically, only one of these is a DNA-binding domain . However, some transcriptional regulators have been described that have more than one DNA-binding domain . Regulators with a single DNA-binding domain often bind co-operatively to the DNA in homotypic or heterotypic combinations, and two or more DNA-binding domains of a single regulatory protein can also bind co-operatively to suitably positioned recognition sequences . Here, we examine the behaviour of a chimeric activator of transcription with two different DNA-binding domains, that of the bacteriophage lambda cI protein and that of the Escherichia coli cyclic AMP receptor protein . We show that these two DNA-binding moieties, when present in the same molecule, can bind co-operatively to a pair of cognate recognition sites located upstream of a test promoter, thereby permitting the chimera to function as a particularly strong activator of transcription from this promoter . Our results show how such a bivalent DNA-binding protein can be used to regulate transcription differentially from promoters that bear either one or both recognition sites. Mol Microbiol, 2001 Aug, 41(3), 601 - 10 Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage phi C31; Cowlishaw DA et al.; Mutants of Streptomyces coelicolor A3(2) J1929 (Delta pglY) were isolated that were resistant to the Streptomyces temperate phage phi C31 . These strains could be transfected with phi C31 DNA, but could not act as infective centres after exposure to phage . Thus, it was concluded that infection was blocked at the adsorption/DNA injection step . The mutants fell into three classes . Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S . coelicolor A3(2) . The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate-D-mannose:protein O-D-mannosyltransferases . Concanavalin A (ConA) inhibited phi C31 infection of S . coelicolor J1929, and this could be partially reversed by the addition of the sugar, alpha-D-methyl-pyranoside . Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots . Class I and II mutants were sensitive to phi C31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h . A phage with the same phenotype, phi DT4002, was isolated independently, and a missense mutation was found in a putative tail gene . It is proposed that the phi C31 receptor is a cell wall glycoprotein, and that the phi C31h mutation compensates for the lack of glycosylation of the receptor. J Mol Biol, 2001 Aug 31, 311(5), 951 - 6 The primase active site is on the outside of the hexameric bacteriophage T7 gene 4 helicase-primase ring; VanLoock MS et al.; Gene 4 of bacteriophage T7 encodes a protein (gp4) that can translocate along single-stranded DNA, couple the unwinding of duplex DNA with the hydrolysis of dTTP, and catalyze the synthesis of short RNA oligoribonucleotides for use as primers by T7 DNA polymerase . Electron microscopic studies have shown that gp4 forms hexameric rings, and X-ray crystal structures of the gp4 helicase domain and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined . Earlier biochemical studies have shown that when single-stranded DNA is bound to the hexameric ring, the primase domain remains accessible to free DNA . Given these results, a model was suggested in which the primase active site in the gp4 hexamer is located on the outside of the hexameric ring . We have used electron microscopy and single-particle image analysis to examine T7 gp4, and have determined that the primase active site is located on the outside of the hexameric ring, and therefore provide direct structural support for this model . Biol Chem, 2001 Jul, 382(7), 1049 - 55 Identification and crystallisation of a heat- and protease-stable fragment of the bacteriophage T4 short tail fibre; van Raaij MJ et al.; Irreversible binding of T-even bacteriophages to Escherichia coli is mediated by the short tail fibres, which serve as inextensible stays during DNA injection . Short tail fibres are exceptionally stable elongated trimers of gene product 12 (gp12), a 56 kDa protein . The N-terminal region of gp12 is important for phage attachment, the central region forms a long shaft, while a C-terminal globular region is implicated in binding to the bacterial lipopolysaccharide core . When gp12 was treated with stoichiometric amounts of trypsin or chymotrypsin at 37 degrees C, an N-terminally shortened fragment of 52 kDa resulted . If the protein was incubated at 56 degrees C before trypsin treatment at 37 degrees C, we obtained a stable trimeric fragment of 3 x 33 kDa lacking residues from both the N- and C-termini . Apparently, the protein unfolds partially at 56 degrees C, thereby exposing protease-sensitive sites in the C-terminal region and extra sites in the N-terminal region . Well-diffracting crystals of this fragment could be grown . Our results indicate that gp12 carries a stable central region, consisting of the C-terminal part of the shaft and the attached N-terminal half of the globular region . Implications for structure determination of the gp12 protein and its folding are discussed. Curr Pharm Biotechnol, 2001 Sep, 2(3), 217 - 23 Phage displayed biomolecules as preventive and therapeutic agents; Manoutcharian K et al.; Phage display is a powerful technology for selecting and engineering peptides and proteins expressed on the surface of filamentous bacteriophage . The advantages of phage display technology over other research tools and its' great potential have been demonstrated by successful application of phage display in diverse fields of biomedical/clinical research . In this review we will describe some recent developments in phage display, including new expression vectors, display formats, bioselection strategies and applications in pharmaceutical biotechnology . We highlight some important applications of phage display to identify disease- and pathogen-specific biomolecules, making particular emphasis on development of phage display-derived preventive and therapeutic vaccines. Acta Crystallogr D Biol Crystallogr, 2001 Sep, 57(Pt 9), 1260 - 9 Epub 2001 Aug 23. Structure determination of the head-tail connector of bacteriophage phi29; Simpson AA et al.; The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry . It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads . This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions . DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle . The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA . The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy . The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening . Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative . The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map . The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule . One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2 . The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector . This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging. J Biol Chem, 2001 Nov 2, 276(44), 40457 - 63 Epub 2001 Aug 28. Two active site asparagines are essential for the reaction mechanism of the class III anaerobic ribonucleotide reductase from bacteriophage T4; Andersson J et al.; Class III ribonucleotide reductase is an anaerobic enzyme that uses a glycyl radical to catalyze the reduction of ribonucleotides to deoxyribonucleotides and formate as ultimate reductant . The reaction mechanism of class III ribonucleotide reductases requires two cysteines within the active site, Cys-79 and Cys-290 in bacteriophage T4 NrdD numbering . Cys-290 is believed to form a transient thiyl radical that initiates the reaction with substrate and Cys-79 to take part as a transient thiyl radical in later steps of the reductive reaction . The recently solved three-dimensional structure of class III ribonucleotide reductase (RNR) from bacteriophage T4 shows that two highly conserved asparagines, Asn-78 and Asn-311, are positioned close to the essential Cys-79 . We have investigated the function of Asn-78 and Asn-311 by site-directed mutagenesis and measured enzyme activity and glycyl radical formation in five single (N78(A/C/D) and N311(A/C)) and one double (N78A/N311A) mutant proteins . Our results suggest that both asparagines are important for the catalytic mechanism of class III RNR and that one asparagine can partially compensate for the lack of the other functional group in the single Asn --> Ala mutant proteins . A plausible role for these two asparagines could be in positioning formate in the active site to orient it toward the proposed thiyl radical of Cys-79 . This would also control the highly reactive carbon dioxide radical anion form of formate within the active site before it is released as carbon dioxide . A detailed reaction scheme including the function of the two asparagines and two formate molecules is proposed for class III RNRs. Bioinformatics, 2001 Aug, 17(8), 676 - 85 Representation of amino acids as five-bit or three-bit patterns for filtering protein databases; Coghlan A et al.; MOTIVATION: We propose representing amino acids by bit-patterns so they may be used in a filter algorithm for similarity searches over protein databases, to rapidly eliminate non-homologous regions of database sequences . The filter algorithm would be based on dynamic programming optimization . It would have the advantage over previous filter algorithms that its substitution scoring function distinguishes between conservative and non-conservative amino acid substitutions . RESULTS: Simulated annealing was used to search for the best five-bit or three-bit patterns to represent amino acids, where similar amino acids were given similar bit-patterns . The similarity between amino acids was estimated from the BLOSUM45 matrix . Representing amino acids by these five-bit and three-bit patterns,