Bacteriophages

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, the Escherichia coli PhoE precursor and the bacteriophage PA2 LC precursor were aligned . The alignments were nearly the same as that obtained when BLOSUM45 was used to score substitutions . AVAILABILITY: The C code of the optimization algorithm for searching for the optimal bit-pattern representation of amino acids is available from the authors upon request.

Nucleic Acids Res, 2001 Sep 1, 29(17), 3513 - 9
A relationship between gene expression and protein interactions on the proteome scale: analysis of the bacteriophage T7 and the yeast Saccharomyces cerevisiae; Grigoriev A; The relationship between the similarity of expression patterns for a pair of genes and interaction of the proteins they encode is demonstrated both for the simple genome of the bacteriophage T7 and the considerably more complex genome of the yeast Saccharomyces cerevisiae . Statistical analysis of large-scale gene expression and protein interaction data shows that protein pairs encoded by co-expressed genes interact with each other more frequently than with random proteins . Furthermore, the mean similarity of expression profiles is significantly higher for respective interacting protein pairs than for random ones . Such coupled analysis of gene expression and protein interaction data may allow evaluation of the results of large-scale gene expression and protein interaction screens as demonstrated for several publicly available datasets . The role of this link between expression and interaction in the evolution from monomeric to oligomeric protein structures is also discussed.

Cancer Lett, 2001 Oct 10, 171(2), 153 - 64
Neuroblastoma tumor cell-binding peptides identified through random peptide phage display; Zhang J et al.; Random peptide phage display libraries have been employed widely to identify protein-protein interactions, using as targets either purified proteins, intact cells, or organs . To isolate peptides that bind to human neuroblastoma cells, we have used a phage display approach with the neuroblastoma cell line WAC 2 as the target . In particular, two bacteriophages, t147 and t160, displaying peptides p147 and p160, respectively, were isolated by repeated display cycles . Binding of t147 and t160 to WAC 2 cells was abrogated by pretreatment with the peptides p147 and p160, respectively, which strongly support that cellular binding of both phages is dictated by their displayed peptides . Immunofluorescence analysis by confocal light microscopy revealed that the major proportion of t147 remains on the surface of WAC 2 cells and that only a fraction is taken up into the cells . In contrast, the vast majority of t160 is internalized . K(+) depletion reduced the number of the phages internalized by the cells to approximately 20% for t160 and to 10% for t147, indicating that the phage internalization was through receptor-mediated endocytosis . Phage t147 appears to bind to a range of tumor cell lines, including neuroblastoma, breast cancer, glioblastoma and C-cell carcinoma, but less so to non-tumor lines, such as erythrocytes, lymphocytes, monocytes and epithelial cells . Phage t160 bound to a range of neuroblastoma cell lines and a breast cancer cell line, but not to other tested cell lines . While neither of the displayed peptides conferred a narrow tissue specific binding ability, they do provide a basis for targeted drug delivery in selected experimental or natural tumor systems.

Antonie Van Leeuwenhoek, 2001 Jun, 79(2), 141 - 7
Environmental bacteriophage-host interactions: factors contribution to natural transduction; Miller RV; Over the past two decades the potential for the exchange of bacterial genes in natural environments through transduction (bacteriophage-mediated gene transfer) has been well established . Studies carried out by various laboratories throughout the world have demonstrated that both chromosomal and plasmid DNA can be successfully transduced in natural environments ranging from sewer plants to rivers and lakes . Transduction has been shown to take place in the gills of oysters and the kidneys of mice . Model studies have demonstrated the ability of transduction to maintain genetic material in bacterial gene pools that would otherwise be lost because of negative fitness . Thus, transduction may affect the course of bacterial evolution . Identification of natural transduction has led to the investigation of the dynamics of bacteriophage host interactions in natural aquatic environments and to the exploration of various environmental factors that affect virus-host interactions . Two important environmental factors which affect virus-host interactions are the metabolic state of the host and the exposure of the host to DNA-damaging stresses such as solar UV light . Recent researches on these two areas of virus-host relationships are reviewed.

J Biol Chem, 2001 Nov 2, 276(44), 41128 - 32 Epub 2001 Aug 22.
Conserved regions 4.1 and 4.2 of sigma(70) constitute the recognition sites for the anti-sigma factor AsiA, and AsiA is a dimer free in solution; Urbauer JL et al.; The association of the bacteriophage T4-encoded AsiA protein with the final sigma(70) subunit of the Escherichia coli RNA polymerase is one of the principal events governing transcription of the T4 genome . Analytical ultracentrifugation and NMR studies indicate that free AsiA is a symmetric dimer and the dimers can exchange subunits . Using NMR, the mutual recognition sites on AsiA and final sigma(70) have been elucidated . Residues throughout the N-terminal half of AsiA are involved either directly or indirectly in binding to final sigma(70) whereas the two highly conserved C-terminal regions of final sigma(70), denoted 4.1 and 4.2, constitute the entire AsiA binding domain . Peptides corresponding to these regions bind tightly to AsiA individually and simultaneously . Simultaneous binding promotes structural changes in AsiA that mimic interaction with the complete AsiA binding determinant of final sigma(70) . Moreover, the results suggest that a significant rearrangement of the dimer accompanies peptide binding . Thus, both conserved regions 4.1 and 4.2 are intimately involved in recognition of AsiA by final sigma(70) . The interaction of AsiA with 4.1 provides a potential explanation of the differential abilities of DNA and AsiA to bind to free final sigma(70) and a mechanistic alternative to models of AsiA function that rely on binding to a single site on final sigma(70).

J Mol Biol, 2001 Aug 24, 311(4), 657 - 79
Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages; Clark AJ et al.; HK620 is a temperate lambdoid bacteriophage that adsorbs to the O-antigen of its host, Escherichia coli H . The genome of a temperature-sensitive clear-plaque mutant consists of 38,297 nucleotides in which we recognize 60 open reading frames (orfs) . Eighteen of these lie in a region of the genome that we call the virion structure domain . The other 42 orfs lie in what we call the metabolic domain.Virions of HK620 resemble those of phage P22 . The virion structural orfs encode three kinds of putative proteins relative to the virion proteins of P22: (1) those that are nearly (about 90 %) identical; (2) those that are weakly (about 30 %) identical; and (3) those composed of nearly and weakly identical segments . We hypothesize that these composite proteins form bridges between the virion proteins of the other two kinds.Three of the putative virion proteins that are only weakly identical to P22 proteins are 71, 60 and 79 % identical to proteins encoded by the phage APSE-1, whose virions also resemble those of P22 . Because the hosts of APSE-1 and HK620 have been separated from each other by an estimated 200 My, we propose using the amino acid differences that have accumulated in these proteins to estimate a biological clock for temperate lambdoid phages.The putative transcriptional regulatory gene circuitry of HK620 seems to resemble that of phage lambda . Integration, on the other hand, resembles that of satellite phage P4 in that the attP sequence lies between the leftward promoter and int rather than downstream of int.Comparing the metabolic domains of several lambdoid phage genomes reveals seven short conserved sequences roughly defining boundaries of functional modules . We propose that these boundary sequences are foci of genetic recombination that serve to assort the modules and make the metabolic domain highly mosaic genetically .

Proc Natl Acad Sci U S A, 2001 Aug 28, 98(18), 10220 - 5 Epub 2001 Aug 21.
The tRNA function of SsrA contributes to controlling repression of bacteriophage Mu prophage; Ranquet C et al.; The small regulatory RNA SsrA has both tRNA and mRNA activities . It charges alanine and interacts with stalled ribosomes, allowing for translation to resume on the SsrA mRNA moiety . Hence, unfinished peptides carry a short amino acid tag, which serves as a signal for degradation by energy-dependent proteases . In SsrA-defective Escherichia coli strains, thermoinducible mutants of the transposable bacteriophage Mu (Mucts) are no longer induced at high temperature . Here we show that truncated forms of the key regulator of Mu lysogeny, the repressor Repc, accumulate in the absence of SsrA . These forms resemble C-terminally truncated dominant Mu repressor mutants previously isolated from Mucts, which are no longer thermoinducible and bind operator DNA with a high affinity even at high temperature . Using various ssrA alleles, we demonstrate the importance of SsrA charging on the ribosome for controlling Mu prophage repression . Our results thus substantiate the previous observation that trans-translation is not the only function of the SsrA . The alternative function of SsrA appears to influence the stability of Mu lysogens by controlling the translation of the C-terminal domain of the repressor protein, which modulates the affinity of the protein for DNA and its susceptibility to proteolytic degradation.

Biochemistry, 2001 Aug 28, 40(34), 10342 - 9
Sites of nucleic acid binding in type I-IV intermediate filament subunit proteins; Wang Q et al.; A combination of enzymatic and chemical ladder sequencing of photo-cross-linked protein-single-stranded oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry was employed to identify the amino acid residues responsible for the stable binding of nucleic acids in several intermediate filament (IF) subunit proteins . The IF proteins studied included the type I and type II cytokeratins K8, K18, and K19; the type III proteins desmin, glial fibrillary acidic protein (GFAP), peripherin, and vimentin; and the type IV neurofilament triplet protein L (NF-L) . The site of nucleic acid binding was localized to the non-alpha-helical, amino-terminal head domain of all of the IF proteins tested . GFAP, which has the shortest head domain of the proteins tested, cross-linked via only two amino acid residues . One of these residues was located within a conserved nonapeptide domain that has been shown to be required for filament formation . One or more cross-linked residues were found in a similar location in the other proteins studied . The major binding site for nucleic acids for most of the proteins appears to be localized within the middle of the head domain . The two exceptions to this generalization are GFAP, which lacks these residues, and NF-L, in which a large number of cross-linked residues were found scattered throughout the first half of the head domain . Control experiments were also done with two bacteriophage ssDNA-binding proteins, as well as actin and tubulin . The single sites of cross-linkage observed with the bacteriophage proteins, Phe(183) for the T4 gene 32 protein and Phe(73) for the M13 gene 5 protein, were in good agreement with literature data . Actin and tubulin could not be cross-linked to the oligonucleotide . Aside from the insight into the biological activity of IF proteins that these data provide, they also demonstrate that this analytical method can be employed to study a variety of protein-nucleic acid interactions.

J Biol Chem, 2001 Nov 9, 276(45), 42122 - 30 Epub 2001 Aug 16.
Distinct binding specificity of the multiple PDZ domains of INADL, a human protein with homology to INAD from Drosophila melanogaster; Vaccaro P et al.; PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins . Proteins containing multiple PDZ domains often bind to different trans-membrane and intracellular proteins, playing a central role as organizers of multimeric complexes . To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage lambda . We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster . This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position . Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference . However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide . Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures . For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.

J Biol Chem, 2001 Oct 19, 276(42), 39340 - 9 Epub 2001 Aug 14.
Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme; Alley SC et al.; Assembly of DNA replication systems requires the coordinated actions of many proteins . The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins . By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme . Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase . Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp . Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme . Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.

Biochemistry, 2001 Aug 21, 40(33), 9944 - 9
Shape of Ocr, the gene 0.3 protein of bacteriophage T7: modeling based on light scattering experiments; Blackstock JJ et al.; Ocr, the first protein expressed by bacteriophage T7, inhibits type Iota DNA restriction enzymes by preventing them from binding to DNA . This inhibition allows the phage to successfully infect the host . The shape of ocr is modeled on the basis of static and dynamic light scattering measurements . The static light scattering data confirm previous observations that ocr exists in solution as a dimer . The diffusion constant determined by dynamic light scattering indicates a nonspherical shape of the ocr dimer . Hydrodynamic models of ellipsoids are presented, and it is argued that ocr is best described by a prolate ellipsoid with dimensions of 10.4 nm by 2.6 nm . The size and shape predicted by this model are consistent with ocr acting as a mimic of the DNA structure bound by type Iota restriction enzymes.

J Biol Chem, 2001 Aug 3, 276(31), 28946 - 53
Ligand binding regions in the receptor for urokinase-type plasminogen activator; Liang OD et al.; The interaction between urokinase plasminogen activator (uPA) and its cellular receptor (uPAR) is a key event in cell surface-associated plasminogen activation, relevant for cell migration and invasion . In order to define receptor recognition sites for uPA, we have expressed uPAR fragments as fusion products with the minor coat protein on the surface of M13 bacteriophages . Sequence analysis of cDNA fragments encoding uPA-binding peptides indicated the existence of a composite uPA-binding structure including all three uPAR domains . This finding was confirmed by experiments using an overlapping 15-mer peptide array covering the entire uPAR molecule . Four regions within the uPAR sequence were found to directly bind to uPA: two distinct regions containing amino acids 13--20 and amino acids 74--84 of the uPAR domain I, and regions in the putative loop 3 of the domains II and III . All the uPA-binding fragments from the three domains were shown to have an agonistic effect on uPA binding to immobilized uPAR . Furthermore, uPAR-(154--176) increased uPAR-transfected BAF3-cell adhesion on vitronectin in the presence of uPA, whereas uPAR-(247--276) stimulated the cell adhesion both in the absence or presence of uPA . The latter fragment was also able to augment the binding of vitronectin to uPAR in a purified system, thereby mimicking the effect of uPA on this interaction . These results indicate that uPA binding can take place to particular part(s) on several uPAR molecules and that direct uPAR-uPAR contacts may contribute to receptor activation and ligand binding.

Folia Microbiol (Praha), 2001, 46(1), 37 - 9
The bacteriophages of ruminal prevotellas; Ambrozic J et al.; Rumen bacteriophage-lyzed bacterial strains of the genus Prevotella were isolated and preliminarily characterized . The strain TCl-1 the species P . bryantii was the only prevotella strain successfully infected with filter sterilized rumen fluid from a black-and-white Holstein cow . Two types of plaques were observed, both rather small and turbid . Preliminary electron microscopy observation showed that several morphologically different bacteriophages were present in these plaques . The plaque eluates were further used for the infection of other prevotella strains . The plaques produced by the bacteriophages were observed with two strains, i.e . P . bryantii B(1)4 and P . brevis GA33 . The bacteriophages from both strains were examined by transmission electron microscopy and several morphologically different bacteriophages were observed, among others also a large virion with an icosahedral head with the diameter of approximately 120 nm . The bacteriophage was identified in plaques of bacterial cells of the strain GA33 and has an approximately 800 nm long helical tail, which places it among the largest ruminal bacteriophages described to date . Other bacteriophages from the same indicator strain as well as from P . bryantii B(1)4 strain were smaller and tail structures were not observed in all of them.

Phys Rev Lett . 2001 Aug 20;87(8):088301 . Epub 2001 Aug 01.
Colloidal interactions in suspensions of rods; Lin K et al.; We report direct measurements of entropic interactions of colloidal spheres in suspensions of rodlike fd bacteriophage . We investigate the influence of sphere size, rod concentration, and ionic strength on these interactions . Although the results compare favorably with a recent calculation, small discrepancies reveal entropic effects due to rod flexibility . At high salt concentrations, the potential turns repulsive as a result of viral adsorption on the spheres and viral bridging between the spheres.

RNA, 2001 Aug, 7(8), 1115 - 25
Specific metal-ion binding sites in a model of the P4-P6 triple-helical domain of a group I intron; Lindqvist M et al.; Divalent metal ions play a crucial role in RNA structure and catalysis . Phosphorothioate substitution and manganese rescue experiments can reveal phosphate oxygens interacting specifically with magnesium ions essential for structure and/or activity . In this study, phosphorothioate interference experiments in combination with structural sensitive circular dichroism spectroscopy have been used to probe molecular interactions underlying an important RNA structural motif . We have studied a synthetic model of the P4-P6 triple-helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogous to the Tetrahymena ribozyme . Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3' end of helix P6 (U452) and in the joining region J6/7 (U453) . The effects of sulfur substitution on triple helix formation in the presence of different ratios of magnesium and manganese were studied by the use of difference circular dichroism spectroscopy . The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas no such effect is seen for the pro-Rp oxygen of U452 . The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of manganese could not significantly restore the triple-helical interactions within the isolated substituted model systems . The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hinders the proper formation of the triple helix.

J Mol Biol, 2001 Aug 17, 311(3), 569 - 77
High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding; Morera S et al.; beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA . We report six X-ray structures of the substrate-free and the UDP-bound enzyme . Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+) . The substrate-free BGT structure differs by a domain movement from one previously determined in another space group . Further domain movements are seen in the complex with UDP and the four UDP-metal complexes . Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form . Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis .

J Mol Biol, 2001 Aug 17, 311(3), 445 - 52
Drop-off during ribosome hopping; Herr AJ et al.; Ribosomes bypass a 50 nucleotide non-coding segment of mRNA between the two open reading frames of bacteriophage T4 gene 60 in order to synthesize a topoisomerase subunit . While nearly all ribosomes appear to initiate bypassing, only 50 % resume translation in the second open reading frame . Failure to bypass is shown here to be independent of the stop codon at the end of the first open reading frame and to be amplified by mutant variants of tRNA(Gly)(2) known to diminish bypassing efficiency . Unproductive bypassing may result from premature dissociation of peptidyl-tRNAs from ribosomes (drop-off) or resumption of translation at inappropriate sites . Assessment of the influence of factors known to induce drop-off reveals that ribosome recycling factor accounts for a small fraction of unproductive bypassing products, but none of the other known factors appear to play a significant role . Resumption of translation at inappropriate sites appears to be minimal, which suggests that spontaneous release of the peptidyl-tRNA may account for the remaining unproductive bypassing products and may be inherent to the gene 60 bypassing mechanism .

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9557 - 62 Epub 2001 Jul 31.
Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7; Hollis T et al.; The gene 2.5 protein (gp2.5) of bacteriophage T7 is a single-stranded DNA (ssDNA) binding protein that has essential roles in DNA replication and recombination . In addition to binding DNA, gp2.5 physically interacts with T7 DNA polymerase and T7 primase-helicase during replication to coordinate events at the replication fork . We have determined a 1.9-A crystal structure of gp2.5 and show that it has a conserved OB-fold (oligosaccharide/oligonucleotide binding fold) that is well adapted for interactions with ssDNA . Superposition of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a conserved patch of aromatic residues that stack against the bases of ssDNA in the other crystal structures, suggesting that gp2.5 binds to ssDNA in a similar manner . An acidic C-terminal extension of the gp2.5 protein, which is required for dimer formation and for interactions with the T7 DNA polymerase and the primase-helicase, appears to be flexible and may act as a switch that modulates the DNA binding affinity of gp2.5.

Leukemia, 2001 Aug, 15(8), 1277 - 85
Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin; Lee SC et al.; Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini . The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings . A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor . Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized . Display was used because it allows very 'deep' mutagenesis at selected residues: >10(5) substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized . None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active . Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants . Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.

Mol Carcinog, 2001 Jul, 31(3), 145 - 51
Loss of mismatch repair activity in simian virus 40 large T antigen-immortalized BPH-1 human prostatic epithelial cell line; Yeh CC et al.; Simian virus 40 large T antigen (SVLTAg) has been used to immortalize cells; however, the mechanism leading to immortalization is still unclear . We hypothesize that DNA mismatch repair (MMR) activity is important during SVLTAg-induced immortalization . To test this hypothesis, we used the SVLTAg-immortalized cell line BPH-1 derived from human benign prostate epithelial cells to analyze MMR activity and the expression of MMR genes (hMLH1, hPMS1, hPMS2, hMSH2, hMSH3, and hMSH6) . The results demonstrated that BPH-1 cells were deficient in repairing G:T, A:C, and G:G mispairs in bacteriophage M13mp2 . Reverse-transcription polymerase chain reaction experiments indicated MMR genes (hMSH3, hMSH6, and hPMS1) were expressed at a low level in BPH-1 cells . In contrast, all six MMR genes were expressed in human benign prostate hyperplasia tissues . Downregulation of hMSH3, hMSH6, and hPMS1 genes is not a result of the hypermethylation mechanism because demethylation with 5-aza-2'-deoxycytidine did not restore expression of these genes . Although the hMLH1 gene is expressed in BPH-1 cells, western blotting and exon analyses demonstrated that hMLH1 was mutated and/or deleted in BPH-1 cells .

Biochemistry, 2001 Aug 7, 40(31), 9300 - 10
SELEX selection of high-affinity oligonucleotides for bacteriophage Ff gene 5 protein; Wen JD et al.; The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein . SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 degrees C . Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides . DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs . This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences . The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition . At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion . At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes . The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form . Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers . Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p . Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.

J Mol Biol, 2001 Aug 10, 311(2), 233 - 40
Bacteriophage lambda DNA packaging: DNA site requirements for termination and processivity; Cue D et al.; Bacteriophage lambda chromosomes are processively packaged into preformed shells, using end-to-end multimers of intracellular viral DNA as the packaging substate . A 200 bp long DNA segment, cos, contains all the sequences needed for DNA packaging . The work reported here shows that efficient DNA packaging termination requires cos's I2 segment, in addition to the required termination subsite, cosQ, and the nicking site, cosN . Efficient processivity requires cosB, in addition to cosQ and cosN . An initiation-defective mutant form of cosB sponsored efficient processivity, indicating that the terminase-cosB interactions required for termination are less stringent than those required at initiation . The finding that an initiation-defective form of cosB is functional for processivity allows a re-interpretation of a similar finding, obtained previously, that the initiation-defective cosB of phage 21 is functional for processivity by the lambda packaging machinery . The cosBphi21 result can now be interpreted as indicating that interactions between cosBphi21 and lambda terminase, while insufficient for initiation, function for processivity .

Comb Chem High Throughput Screen, 2001 Aug, 4(5), 417 - 30
Of minibody, camel and bacteriophage; Vaughan CK et al.; This review describes the design process from conception through realisation and optimisation of a minibody'--a minimised antibody . The result was a proteinaceous molecule of novel fold and metal binding activity . We explain how combinatorial approaches, using phage display libraries, were used to randomise loop regions of the minibody . Variants were then selected for desired activities including in vitro inhibition of human interleukin-6 and the protease of the non-structural protein, NS3, of the hepatitis C virus . One such variant was successfully minimised further to produce a cyclic peptide with similar inhibition properties . Thus the work reviewed provides examples of two important processes in protein design and protein minimisation . We conclude by discussing the role of such studies in medical applications and small molecule drug discovery . We also highlight the potential of our work and similar techniques in the post-genomic era.

Microb Comp Genomics, 2000, 5(4), 223 - 31
Comparative analysis of Chlamydia bacteriophages reveals variation localized to a putative receptor binding domain; Read TD et al.; Three recently discovered ssDNA Chlamydia-infecting microviruses, phiCPG1, phiAR39, and Chp2, were compared with the previously characterized phage from avian C . psittaci, Chp1 . Although the four bacteriophages share an identical arrangement of their five main genes, Chpl has diverged significantly in its nucleotide and protein sequences from the other three, which form a closely related group . The VP1 major viral capsid proteins of phiCPG1 and phiAR39 (from guinea pig-infecting C . psittaci and C . pneumoniae, respectively) are almost identical . However, VP1 of ovine C . psittaci phage Chp2 shows a high rate of nucleotide sequence change localized to a region encoding the "IN5" loop of the protein, thought to be a potential receptor-binding site . Phylogenetic analysis suggests that the ORF4 replication initiation protein is evolving faster than the other phage proteins . phiCPG1, phiAR39, and Chp2 are closely related to an ORF4 homolog inserted in the C . pneumoniae chromosome . This sequence analysis opens the way toward understanding the host-range and evolutionary history of these phages.

Gene, 2001 Jul 11, 272(1-2), 227 - 35
Cell toxicity caused by products of the p(L) operon of bacteriophage lambda; Sergueev K et al.; Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon . We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing . Viability assays and flow cytometry were used to monitor cell death and filamentation . The kil gene was shown to cause cell death and filamentation as described previously . Another killing activity was mapped within the p(L) operon to the gam gene . Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam . In both cases, the shorter of the two possible products could cause cell killing . The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions . The expression level of the p(L) operon is down-regulated by Cro repressor . In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal . The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.

Mutat Res, 2001 Aug 8, 479(1-2), 121 - 30
Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11; Johnson JM 3rd et al.; Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant . This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host . Genetic mapping located the probable mutation to a region of the E . coli chromosome containing a newly described gene called tas . To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage . Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed . These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA . Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site . Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E . coli, when compared with the published E . coli K-12 sequence . A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions . Yet, none of the changes altered the predicted amino acid sequences . These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.

Mol Diagn, 2001 Jun, 6(2), 141 - 50
Ramification amplification: a novel isothermal DNA amplification method; Zhang DY et al.; We have developed a novel isothermal DNA amplification method with an amplification mechanism quite different from conventional PCR . This method uses a specially designed circular probe (C-probe) in which the 3' and 5' ends are brought together in juxtaposition by hybridization to a target . The two ends are then covalently linked by a T4 DNA ligase in a target-dependent manner, producing a closed DNA circle . In the presence of an excess of primers (forward and reverse primers), a DNA polymerase extends the bound forward primer along the C-probe and displaces the downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to the "rolling circle" replication of bacteriophages in vivo . 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 . This ramification process continues until all ssDNAs become double-stranded, resulting in an exponential amplification that distinguishes itself from the previously described nonexponential rolling circle amplification . In this report, we prove the principle of ramification amplification . By using a unique bacteriophage DNA polymerase, O29 DNA Polymerase, that has an intrinsic high processivity, we are able to achieve significant amplification within 1 hour at 35 degrees C . In addition, we applied this technique for in situ detection of Epstein-Barr viral sequences in Raji cells.

Biochemistry, 2001 Jul 31, 40(30), 8962 - 70
Kinetic and calorimetric evidence for two distinct scaffolding protein binding populations within the bacteriophage P22 procapsid; Parker MH et al.; A wide variety of viruses require the transient presence of scaffolding proteins to direct capsid assembly . In the case of bacteriophage P22, a model in which the scaffolding protein selectively stabilizes on-pathway growing intermediates has been proposed . The stoichiometry and thermodynamics of binding of the bacteriophage P22 scaffolding protein within the procapsid were analyzed by light scattering and isothermal titration calorimetry . Calorimetric experiments carried out between 10 and 37 degrees C were consistent with the presence of at least two distinct populations of binding sites, in agreement with kinetic evidence obtained by a light scattering assay . Binding to the high-affinity sites occurred at 20 degrees C with a stoichiometry of approximately 60 scaffolding molecules per procapsid and an apparent K(d) of approximately 100-300 nM and was almost completely enthalpy-driven . For the second binding population, precise fitting of the data was impossible due to small heats of binding, but the thermodynamics of binding were clearly distinct from the high-affinity phase . The heat capacity change (DeltaC(p)()) of binding was large for the high-affinity sites and negative for both sets of sites . Addition of sodium chloride (1 M) greatly reduced the magnitude of the apparent DeltaH, in agreement with previous evidence that electrostatic interactions play a major role in binding . A mutant scaffolding protein that forms covalent dimers (R74C/L177I) bound only to the high-affinity sites . These data comprise the first quantitative measurements of the energetics of the coat protein/scaffolding protein interaction.

J Bacteriol, 2001 Aug, 183(16), 4771 - 8
Transduction by phiBB-1, a bacteriophage of Borrelia burgdorferi; Eggers CH et al.; We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated phiBB-1 . This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia . To demonstrate the ability of phiBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B . burgdorferi CA-11.2A cells . The kan cassette recombined into a resident cp32 and was stably maintained . The cp32 containing the kan cassette was packaged by phiBB-1 released from this B . burgdorferi strain . phiBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B . burgdorferi . This is the first direct evidence of a mechanism for lateral gene transfer in B . burgdorferi.

Clin Immunol, 2001 Aug, 100(2), 181 - 90
Long-term low-dose IL-2 enhances immune function in common variable immunodeficiency; Cunningham-Rundles C et al.; Common variable immunodeficiency (CVID) is a primary immunodeficiency disease characterized by hypogammaglobulinemia and lack of antibody production . Numerous T cell defects have been described, including reduced gene expression and production of IL-2 . Since some of the T cell defects could be explained by lack of IL-2, we have been investigating the effects of in vivo IL-2 treatment . Here, a long-acting form of IL-2, PEG-IL-2, was given for 12-18 months to 15 randomly chosen CVID subjects, in comparison to 39 CVID subjects who served as controls . After 6 to 12 months of treatment, T cell proliferative responses to mitogens and to IL-2 were significantly enhanced; proliferative responses to tetanus and candida antigens increased up to 50-fold . Four of eight subjects immunized with the neoantigen bacteriophage φX 174 displayed increased antibody responses after treatment . Treated subjects recorded reduced, but not overall statistically significant, days of bronchitis, diarrhea, and joint pain . These data indicate that IL-2 might serve as an adjuvant to therapy in some subjects with CVID, enhancing T cell functions and reversing T cell anergy in most .

Phys Rev E Stat Nonlin Soft Matter Phys . 2001 Jul;64(1 Pt 1):011906 . Epub 2001 Jun 18.
Modeling translocation of particles on one-dimensional polymer lattices; Wang XJ et al.; We introduce a general random walk model that is an extension of the random walk model proposed by Berg . The model can be used to describe a particle's translocation along a polymeric lattice with a nonuniform distribution of obstacles . These obstacles are representative of DNA-bound proteins, of drugs, and of a DNA packing environment . Using this model in the bacteriophage replication process, we show the effects of random obstacles on an ATP-driven particle's translocation along single-stranded DNA . The principal finding is that the average statistical time of the translocation process decreases with the increase of an obstacle's strength . We also find an interesting relation between the average statistical time and the DNA chain length . Our results can be used to explain some physiological phenomena . They show the usefulness of our model in an analysis of the effect of random obstacles on particles' translocation along one-dimensional polymer lattices.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8461 - 8
DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination; Kuzminov A; Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized . Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions . There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro . The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared . In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8411 - 8
Assembly of RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis; Gasior SL et al.; Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA) . Biochemical studies have shown that assembly of recombinase involves accessory factors . These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb) . In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites . Here we briefly review mediated recombinase assembly and present results of new in vivo experiments . Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity . Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases . In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells . Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30 degrees C but not 20 degrees C, accounting for the cold sensitivity of rad55 null mutants . Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G(1), consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8368 - 75
Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer; Trakselis MA et al.; The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process . A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner . Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication . Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly . By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly . Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces . On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP . Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation . The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly . This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43 . This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8312 - 8
Bacteriophage T4 gene 41 helicase and gene 59 helicase-loading protein: a versatile couple with roles in replication and recombination; Jones CE et al.; Bacteriophage T4 uses two modes of replication initiation: origin-dependent replication early in infection and recombination-dependent replication at later times . The same relatively simple complex of T4 replication proteins is responsible for both modes of DNA synthesis . Thus the mechanism for loading the T4 41 helicase must be versatile enough to allow it to be loaded on R loops created by transcription at several origins, on D loops created by recombination, and on stalled replication forks . T4 59 helicase-loading protein is a small, basic, almost completely alpha-helical protein whose N-terminal domain has structural similarity to high mobility group family proteins . In this paper we review recent evidence that 59 protein recognizes specific structures rather than specific sequences . It binds and loads the helicase on replication forks and on three- and four-stranded (Holliday junction) recombination structures, without sequence specificity . We summarize our experiments showing that purified T4 enzymes catalyze complete unidirectional replication of a plasmid containing the T4 ori(uvsY) origin, with a preformed R loop at the position of the R loop identified at this origin in vivo . This replication depends on the 41 helicase and is strongly stimulated by 59 protein . Moreover, the helicase-loading protein helps to coordinate leading and lagging strand synthesis by blocking replication on the ori(uvsY) R loop plasmid until the helicase is loaded . The T4 enzymes also can replicate plasmids with R loops that do not have a T4 origin sequence, but only if the R loops are within an easily unwound DNA sequence.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8306 - 11
Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer; Mosig G et al.; Two major pathways of recombination-dependent DNA replication, "join-copy" and "join-cut-copy," can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway . In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA . In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis . The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay . Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways . These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions . Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate . The resulting sequence divergence generates barriers to formation of viable recombinants . The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8290 - 7
The tight linkage between DNA replication and double-strand break repair in bacteriophage T4; George JW et al.; Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4 . Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs . DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology . A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR) . This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery . We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair . We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks . First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes . The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR . Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin . Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin . Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8247 - 54
Handoff from recombinase to replisome: insights from transposition; Nakai H et al.; Bacteriophage Mu replicates as a transposable element, exploiting host enzymes to promote initiation of DNA synthesis . The phage-encoded transposase MuA, assembled into an oligomeric transpososome, promotes transfer of Mu ends to target DNA, creating a fork at each end, and then remains tightly bound to both forks . In the transition to DNA synthesis, the molecular chaperone ClpX acts first to weaken the transpososome's interaction with DNA, apparently activating its function as a molecular matchmaker . This activated transpososome promotes formation of a new nucleoprotein complex (prereplisome) by yet unidentified host factors {Mu replication factors (MRF alpha 2)}, which displace the transpososome in an ATP-dependent reaction . Primosome assembly proteins PriA, PriB, DnaT, and the DnaB--DnaC complex then promote the binding of the replicative helicase DnaB on the lagging strand template of the Mu fork . PriA helicase plays an important role in opening the DNA duplex for DnaB binding, which leads to assembly of DNA polymerase III holoenzyme to form the replisome . The MRF alpha 2 transition factors, assembled into a prereplisome, not only protect the fork from action by nonspecific host enzymes but also appear to aid in replisome assembly by helping to activate PriA's helicase activity . They consist of at least two separable components, one heat stable and the other heat labile . Although the MRF alpha 2 components are apparently not encoded by currently known homologous recombination genes such as recA, recF, recO, and recR, they may fulfill an important function in assembling replisomes on arrested replication forks and products of homologous strand exchange.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8241 - 6
Single-strand interruptions in replicating chromosomes cause double-strand breaks; Kuzminov A; Replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template DNA and collapse, generating double-strand ends . To model replication fork collapse in vivo, I constructed phage lambda chromosomes carrying the nicking site of M13 bacteriophage and infected with these substrates Escherichia coli cells, producing M13 nicking enzyme . I detected double-strand breaks at the nicking sites in lambda DNA purified from these cells . The double-strand breakage depends on (i) the presence of the nicking site; (ii) the production of the nicking enzyme; and (iii) replication of the nick-containing chromosome . Replication fork collapse at nicks in template DNA explains diverse phenomena, including eukaryotic cell killing by DNA topoisomerase inhibitors and inviability of recombination-deficient vertebrate cell lines.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8173 - 80
Historical overview: searching for replication help in all of the rec places; Cox MM; For several decades, research into the mechanisms of genetic recombination proceeded without a complete understanding of its cellular function or its place in DNA metabolism . Many lines of research recently have coalesced to reveal a thorough integration of most aspects of DNA metabolism, including recombination . In bacteria, the primary function of homologous genetic recombination is the repair of stalled or collapsed replication forks . Recombinational DNA repair of replication forks is a surprisingly common process, even under normal growth conditions . The new results feature multiple pathways for repair and the involvement of many enzymatic systems . The long-recognized integration of replication and recombination in the DNA metabolism of bacteriophage T4 has moved into the spotlight with its clear mechanistic precedents . In eukaryotes, a similar integration of replication and recombination is seen in meiotic recombination as well as in the repair of replication forks and double-strand breaks generated by environmental abuse . Basic mechanisms for replication fork repair can now inform continued research into other aspects of recombination . This overview attempts to trace the history of the search for recombination function in bacteria and their bacteriophages, as well as some of the parallel paths taken in eukaryotic recombination research.

Proc Natl Acad Sci U S A, 2001 Jul 31, 98(16), 9348 - 52 Epub 2001 Jul 17.
Holins kill without warning; Grundling A et al.; Holins comprise the most diverse functional group of proteins known . They are small bacteriophage-encoded proteins that accumulate during the period of late-protein synthesis after infection and cause lysis of the host cell at a precise genetically programmed time . It is unknown how holins achieve temporal precision, but a conserved feature of their function is that energy poisons subvert the normal scheduling mechanism and instantly trigger membrane disruption . On this basis, timing has been proposed to involve a progressive decrease in the energized state of the membrane until a critical triggering level is reached . Here, we report that membrane integrity is not compromised after the induction of holin synthesis until seconds before lysis . The proton motive force was monitored by the rotation of individual cells tethered by a single flagellum . The results suggest an alternative explanation for the lysis "clock," in which holin concentrations build to a critical level that leads to formation of an oligomeric complex that disrupts the membrane.

J Am Chem Soc, 2001 Jan 31, 123(4), 633 - 40
Field- and phage-induced dipolar couplings in a homodimeric DNA quadruplex: relative orientation of G.(C-A) triad and G-tetrad motifs and direct determination of C2 symmetry axis orientation; Al-Hashimi HM et al.; We present a new NMR procedure for determining the three-dimensional fold of C2-symmetric nucleic acid homodimers that relies on long-range orientational constraints derived from the measurement of two independent sets of residual dipolar couplings under two alignment conditions . The application is demonstrated on an (15)N/(13)C-enriched deoxyoligonucleotide sequence, d(G-G-G-T-T-C-A-G-G), shown previously to dimerize into a quadruplex in solution and form a pair of G.(C-A) triads and G-G-G-G tetrads (G-tetrad) motifs . One-bond (1)H-(15)N ((1)D(NH)) and (1)H-(13)C ((1)D(CH)) residual dipolar couplings have been measured between nuclei in the bases of these motifs using bacteriophage as an ordering medium, and under direct magnetic field alignment (800 MHz) . By combining the two dipolar data sets in an order matrix analysis, the orientation of the G.(C-A) triad relative to the G-tetrad within a contiguous monomeric unit can directly be determined, even in the presence of interstrand/intrastrand NOE ambiguity . We further demonstrate that the orientation of the C2-axis of molecular symmetry in the homodimer relative to the G.(C-A) triad and G-tetrad motifs can unambiguously be determined using the two sets of independent dipolar coupling measurements . The three-dimensional fold of the homodimer determined using this procedure is very regular and in excellent agreement with a previously determined high-resolution NOE-based NMR structure, where interstrand/intrastrand NOEs were treated as ambiguous and where noncrystallographic symmetry constraints were implicitly imposed during the structure calculation.

J Biol Chem, 2001 Sep 14, 276(37), 34905 - 12 Epub 2001 Jul 13.
Role of the C-terminal residue of the DNA polymerase of bacteriophage T7; Kumar JK et al.; The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand . Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo . Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced . Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase . However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis . Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.

Cell Res, 2001 Jun, 11(2), 95 - 100
The expression and antigenicity identification of recombinant rat TGF-beta1 in bacteria; Gao CF et al.; In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria . Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase . This system allowed an active and selective synthesis of recombinant TGF-beta1 . The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD . Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity . Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data . In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody . The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

Nucleic Acids Res, 2001 Jul 15, 29(14), 3059 - 68
Characterisation of the structure of ocr, the gene 0.3 protein of bacteriophage T7; Atanasiu C et al.; The product of gene 0.3 of bacteriophage T7, ocr, is a potent inhibitor of type I DNA restriction and modification enzymes . We have used biophysical methods to examine the mass, stability, shape and surface charge distribution of ocr . Ocr is a dimeric protein with hydrodynamic behaviour equivalent to a prolate ellipsoid of axial ratio 4.3 +/- 0.7:1 and mass of 27 kDa . The protein is resistant to denaturation but removal of the C-terminal region reduces stability substantially . Six amino acids, N4, D25, N43, D62, S68 and W94, are all located on the surface of the protein and N4 and S68 are also located at the interface between the two 116 amino acid monomers . Negatively charged amino acid side chains surround W94 but these side chains are not part of the highly acidic C-terminus after W94 . Ocr is able to displace a short DNA duplex from the binding site of a type I enzyme with a dissociation constant of the order of 100 pM or better . These results suggest that ocr is of a suitable size and shape to effectively block the DNA binding site of a type I enzyme and has a large negatively charged patch on its surface . This charge distribution may be complementary to the charge distribution within the DNA binding site of type I DNA restriction and modification enzymes.

Chem Biol, 2001 Jul, 8(7), 661 - 71
Filamentous bacteriophage stability in non-aqueous media; Olofsson L et al.; BACKGROUND: Filamentous bacteriophage are used as general cloning vectors as well as phage display vectors in order to study ligand-receptor interactions . Exposure to biphasic chloroform-water interface leads to specific contraction of phage, to non-infective I- or S-forms . RESULTS: Upon exposure, phage were inactivated (non-infective) at methanol, ethanol and 1-propanol concentrations inversely dependent upon alcohol hydrophobicity . Infectivity loss of phage at certain concentrations of 1-propanol or ethanol coincided with changes in the spectral properties of the f1 virion in ultraviolet fluorescence and circular dichroism studies . CONCLUSIONS: The alcohols inactivate filamentous phage by a general mechanism--solvation of coat protein--thereby disrupting the capsid in a manner quite different from the previously reported I- and S-forms . The infectivity retention of phagemid pG8H6 in 99% acetonitrile and the relatively high general solvent resistance of the phage strains studied here open up the possibility of employing phage display in non-aqueous media.

J Hum Genet, 2001, 46(7), 372 - 7
Definition of a 1-Mb homozygous deletion at 9q32-q33 in a human bladder-cancer cell line; Fujiwara H et al.; We performed detailed molecular analyses of a suspected homozygous deletion on chromosome 9q32-q33 in a bladder-cancer cell line (KYBTDS) derived from a superficial papillary transitional cell carcinoma (TCC) . We examined 13 sequence-tagged site (STS) markers mapped along 9q32-q33 by polymerase chain reaction (PCR), and used 13 bacterial artificial chromosome (BAC)/bacteriophage P1-derived artificial chromosome (PAC) genomic clone probes representing these STS markers as probes for dual-color fluorescence in situ hybridization (FISH) analyses to define the deleted region cytogenetically and at the molecular level . Southern blotting confirmed the findings . This combination of techniques revealed that the homozygous deletion in the KYBTDS cell line involved less than 1 megabase of DNA, flanked by markers A003P42 and SGC33380 . This interval overlaps part of a common region of deletion observed in a number of primary bladder cancers; moreover, the DNA sequence within the 1-Mb segment corresponds to part of a YAC genomic clone that encompasses a putative tumor suppressor gene, DBCCR1.

Mol Gen Mikrobiol Virusol, 2001, (2), 19 - 24
{Use of phage display for detecting single-nucleotide differences in genes}; Denisov SG et al.; Experimental strategy has been developed for selection of mismatched DNA binding phages from library of E . coli f1 filamentous phages carrying random peptide inserts on the surface of bacteriophage particles . The strategy is based on the use of phage display technique, DNA heteroduplexes (with single nucleotide variations), and paramagnetic beads . DNA heteroduplexes have been obtained from biotin-labeled PCR product . During the first stage the phage particles were incubated with DNA heteroduplexes possessing mismatched nucleotides . The next step after elimination of free phages and separation of bound phages from DNA heteroduplexes was subtraction of phages binding with DNA heteroduplexes (without mismatched nucleotides) . Phages selected by this method were capable of discriminating DNA heteroduplexes with single nucleotide variations from DNA homoduplexes . Phages immobilized on solid base retain their activity and specificity, and therefore can be used for developing a new screening automated method for detecting point mutations and gene polymorphism.

Biofizika, 2001 May-Jun, 46(3), 486 - 93
{Photoinactivation of bacteriophage PM2 by cyanine dyes}; Gonikberg EM et al.; Photoinactivation of the lipid-containing bacteriophage PM2 by visible light and cyanine dyes (carbo- and dicarbocyanines), aluminum phtalocyanine tetrasulfonate and methylene blue was studied . It was concluded that cyanine dye aggregates adsorbed on phage particles and oxygen are essential for phage photoinactivation.

Virology, 2001 Jul 20, 286(1), 113 - 8
Reverse genetics and recombination in Phi8, a dsRNA bacteriophage; Onodera S et al.; Bacteriophage Phi8 has a genome of three dsRNA segments . It is able to acquire plasmid transcripts of cDNA copies of the genomic segments as replacements of its resident chromosomes . It is also able to effect recombination between the plasmid transcripts and the resident chromosomes . Depending upon the extent of sequence identity between the plasmid transcript and the resident chromosome, the recombination can be homologous or heterologous . Homologous recombination has not previously been reported for viruses with double-stranded RNA genomes .

Anal Biochem, 2001 Jul 15, 294(2), 108 - 17
External surface display of proteins linked to DNA-binding domains; McGregor DP et al.; A novel system (DBDX) was developed which allows the external surface display on filamentous bacteriophage of proteins fused to either the N- or the C-terminus of a DNA-binding protein . In conjunction with helper phage infection, expression of proteins fused to the estrogen receptor DNA-binding domain (DBD) in a phagemid vector containing the DNA sequence recognized by the DBD resulted in the production of phage particles which display the fusion protein through the phage pVIII coat on the external surface of the particle . The viability of the technique was established with several model systems: particles displaying the C-terminal domain of N-cadherin or the biotinylation domain of propionyl coenzyme A carboxylase fused to the C-terminus of the DBD were found to be bound specifically by antibody or streptavidin, respectively . Human kappa constant region cDNA was selected from a N-terminal DBD fusion lymphocyte cDNA library after two rounds of selection with anti-kappa antibody . This display system may complement currently available bacterial selection techniques .

Mol Microbiol, 2001 Jun, 40(6), 1371 - 9
SeqA, the Escherichia coli origin sequestration protein, is also a specific transcription factor; Slominska M et al.; The SeqA protein is a negative regulator of initiation of DNA replication in the Escherichia coli chromosome . Here, we demonstrate that SeqA stimulates transcription from the bacteriophage lambda pR promoter both in vivo and in vitro . The activity of the lambda pL promoter was found not to be affected by this protein . SeqA-mediated stimulation of pR was dependent on the state of template methylation: transcription was activated on fully methylated and hemimethylated templates but not on an unmethylated template . Using electrophoretic mobility shift assay and electron microscopy, we demonstrated that SeqA interacts specifically with a pR promoter region located on both fully methylated and hemimethylated DNA molecules, but not on unmethylated DNA . The activity of SeqA was found to affect the initiation of lambda plasmid replication positively in vivo, probably via pR-dependent expression of lambda replication genes and transcriptional activation of ori lambda . We conclude that, apart from its function in the control of DNA replication, SeqA is also a specific transcription factor.

J Mol Biol, 2001 Jul 13, 310(3), 603 - 15
A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold; Desiderio A et al.; We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability . Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5x10(7) independent scFvs, cloned in a phagemid vector . The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens . Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target . High yields of both soluble and phage antibodies were obtained in Escherichia coli . Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm . The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications .

J Virol, 2001 Aug, 75(15), 7107 - 13
Phage display of adenovirus type 5 fiber knob as a tool for specific ligand selection and validation; Pereboev A et al.; Adenovirus (Ad) vectors are most potent for use as gene delivery vehicles to infect human cells in vitro and in vivo with high efficiency . The main limitation in utilization of Ad as a gene transfer vector is the lack of specificity . Genetic modifications of Ad capsid proteins resulting in incorporation of foreign polypeptide ligand sequences can redirect the vector towards target cells . However, in many cases the incorporated ligands lose specificity or lead to conformational changes influencing virion integrity . In order to select target-specific ligands a priori structurally compatible with Ad, we propose a system for displaying polypeptide sequences in the context of the Ad fiber knob on the surfaces of filamentous bacteriophages . To establish this concept, we displayed the wild-type Ad serotype 5 knob and knobs containing c-Myc epitopes and six-histidine sequences in the pJuFo phage system . The knobs remained trimeric and bound the coxsackievirus-Ad receptor, and the phage knob-displayed ligands recognized and bound their cognates in the phage-displayed knob context . Further development of this system may be useful for candidate ligand fidelity and Ad structural compatibility validation prior to Ad modification.

Mol Biotechnol, 2001 Mar, 17(3), 219 - 24
Bacteriophage lambda-based expression vectors; Christensen AC; Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector . The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector . A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size . Some lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes . Screening lambda libraries with antibodies or ligands is a powerful way of identifying novel genes.

Nucleic Acids Res, 2001 Jul 1, 29(13), 2829 - 35
Measurement of steady-state kinetic parameters for DNA unwinding by the bacteriophage T4 Dda helicase: use of peptide nucleic acids to trap single-stranded DNA products of helicase reactions; Nanduri B et al.; Measurement of steady-state rates of unwinding of double-stranded oligonucleotides by helicases is hampered due to rapid reannealing of the single-stranded DNA products . Including an oligonucleotide in the reaction mixture which can hybridize with one of the single strands can prevent reannealing . However, helicases bind to single-stranded DNA, therefore the additional oligonucleotide can sequester the enzyme, leading to slower observed rates for unwinding . To circumvent this problem, the oligonucleotide that serves as a trap was replaced with a strand of peptide nucleic acid (PNA) . Fluorescence polarization was used to determine that a 15mer PNA strand does not bind to the bacteriophage T4 Dda helicase . Steady-state kinetic parameters of unwinding catalyzed by Dda were determined by using PNA as a trapping strand . The substrate consisted of a partial duplex with 15 nt of single-stranded DNA and 15 bp . In the presence of 250 nM substrate and 1 nM Dda, the rate of unwinding in the presence of the DNA trapping strand was 0.30 nM s(-1) whereas the rate was 1.34 nM s(-1) in the presence of the PNA trapping strand . PNA prevents reannealing of single-stranded DNA products, but does not sequester the helicase . This assay will prove useful in defining the complete kinetic mechanism for unwinding of oligonucleotide substrates by this helicase.

Nat Genet, 2001 Jul, 28(3), 200 - 1
A helicase is born; Moraes CT; One of three loci previously associated with autosomal dominant progressive external ophthalmoplegia (adPEO) encodes ANT1, a mitochondrial nucleotide transporter . Now, mutations in two other genes are found in people with adPEO . One of these encodes a new helicase, Twinkle, which is related to the product of bacteriophage T7 gene 4, and co-localizes with mitochondrial DNA . The identification of Twinkle adds a new star to the expanding constellation of 'helicase diseases'.

Microbiology, 2001 Jul, 147(Pt 7), 1929 - 36
Mosaic structure of Shiga-toxin-2-encoding phages isolated from Escherichia coli O157:H7 indicates frequent gene exchange between lambdoid phage genomes; Johansen BK et al.; Shiga-toxin-2 (stx(2))-encoding bacteriophages were isolated from Norwegian Escherichia coli O157:H7 isolates of cattle and human origin . The phages were characterized by restriction enzyme analysis, hybridization with probes for toxin genes and selected phage DNA such as the P gene, integrase gene and IS1203, and by PCR studies and partial sequencing of selected DNA regions in the integrase to stx(2) region of the phages . The stx(2)-phage-containing bacteria from which inducible phages were detected produced similar amounts of toxin, as shown by a Vero cell assay . The results indicate that the population of stx(2)-carrying phages is heterogeneous, although the phages from epidemiologically linked E . coli O157:H7 isolates were similar . There appears to have been frequent recombination of stx(2) phages with other lambdoid phages.

Microbiology, 2001 Jul, 147(Pt 7), 1923 - 8
The double mechanism of incompatibility between lambda plasmids and Escherichia coli dnaA(ts) host cells; Glinkowska M et al.; For plasmids derived from bacteriophage lambda, the initiation of bidirectional DNA replication from orilambda depends on the stimulation of transcription from the p(R) promoter by the host replication initiator protein DnaA . Certain Escherichia coli dnaA(ts) mutants cannot be transformed by wild-type lambda plasmids even at the temperature permissive to cell growth . This plasmid-host incompatibility appeared to be due to inefficient stimulation of transcription from the p(R) promoter by the mutant DnaA protein . This paper shows that there is a second mechanism for the incompatibility between lambda plasmids and dnaA(ts) hosts, exemplified in this study by the dnaA46 mutant . This is based on the competition between the lambda P protein and the host DnaA and DnaC proteins for DnaB helicase . Both mechanisms must be operative for the incompatibility.

Environ Mol Mutagen, 2001, 37(4), 290 - 6
Just how does the cII selection system work in Muta Mouse?
Swiger RR.
The lambda CII protein is an essential component in the lytic vs . lysogeny decision a bacteriophage makes upon infection of a host at low temperatures . The protein interacts with numerous phage promoters modulating the expression of the CI repressor, thus providing the mechanism for lysogenization soon after infection . The Big Blue and Muta Mouse are two widely used in vivo mutational model systems . The assays rely on retrievable lambda-based transgenes housing mutational targets (lacI or lacZ, respectively) . The transgenes provide an elegant vehicle for the quantification of mutations sustained in virtually any tissue of the rodent . The use of the bacteriophage cII locus as an alternative, or additional mutational target for use with the Big Blue rodent system was first reported by Jakubczak et al . ({1996}: Proc Natl Acad Sci USA 93:9073-9078) . More recently, this selection assay has been applied successfully to the Muta Mouse (Swiger et al . {1999}: Environ Mol Mutagen 33:201-207) . The use of an Hfl bacterial strain and low temperature allows the determination of mutations sustained at the cII locus in either system, with high fidelity . The cII selection assay in the Big Blue relies on the presence of the lambda repressor protein CI . In contrast, the recombinant construct used to make the Muta Mouse transgene lacks functional CI protein . Nevertheless, we report an excellent system for quantifying mutations at the cII locus in Muta Mouse . Just how does cII selection work in the Muta Mouse? Written in the context of lambda recombinant genetics, this paper explores the question further .

Biochemistry (Mosc), 2001 Jun, 66(6), 693 - 7
Properties of bacteriophage T4 baseplate protein encoded by gene 8; Shneider MM et al.; Gene product 8 (gp8, 344 amino acids per monomer) of bacteriophage T4 is one of the baseplate structural proteins . We constructed an expression vector of gp8 and developed a method for purification of recombinant protein . CD spectroscopy showed that gp8 is an alpha/beta type structural protein . Its polypeptide chain consists of nearly 40% beta-structure and 15% alpha-helix . These data agree with results of prediction of secondary structure based on the amino acid sequence of the protein . The sedimentation coefficient under standard conditions (S20,w) is 4.6S . Analytical ultracentrifugation results demonstrated that gp8 in solution has two types of oligomers--dimer and tetramer . The tetramer of gp8 may be included in the wedge (1/6 of the baseplate), and the dimer may be an intermediate product of association.

J Mol Biol, 2001 Jun 29, 310(1), 51 - 67
A small protein-protein interaction domain common to KlcB and global regulators KorA and TrbA of promiscuous IncP plasmids; Bhattacharyya A et al.; The kor regulon of broad host-range, incompatibility group P (IncP) plasmids uses the KorA, KorB, and KorC repressors to regulate expression of genes for replication, conjugation, segregation, and host range . One operon, kilC, encodes the KorC repressor and two genes of unknown function (klcA and klcB) . The predicted sequences of the 51.1 kDa KlcB protein, the 11.3 kDa KorA repressor, and another small (13.5 kDa) regulatory protein, TrbA, show a highly related 35 amino acid residue segment (V-L-P domain) . We found that induction of the klcB gene is toxic to Escherichia coli host cells harboring an IncP plasmid . We confirmed a model in which the V-L-P domain of KlcB interacts directly with the V-L-P domain of KorA to derepress KorA-regulated operons, thereby allowing expression of toxic genes . First, a lacZ reporter fused to the kleA promoter, which is regulated by KorA and KorC, revealed that klcB induction specifically releases KorA-repression but has no effect on KorC repression . Second, induced expression of the V-L-P domains from KorA or KlcB is sufficient to release KorA-repression at the kleA promoter . Third, purified GST-KlcB fusion protein interacts specifically with His-tagged KorA . Fourth, fusion of the V-L-P domains of KorA and TrbA and full-length KlcB polypeptide to the DNA-binding domain of bacteriophage lambda repressor leads to the formation of functional, dimeric repressors, and mutations that alter conserved residues of the V-L-P domain adversely affect dimerization . Fifth, crosslinking experiments demonstrated that the V-L-P domain of KorA is able to dimerize in solution and form heterodimers in mixtures with full-length KorA polypeptide . These findings show that the V-L-P domain is a protein-protein interaction module that is likely to be responsible for dimerization of KorA and TrbA, and important for KlcB dimerization . We speculate on the possible significance of KlcB-KorA heterodimers in IncP plasmid maintenance .

J Bacteriol, 2001 Jul, 183(14), 4105 - 9
Dual regulatory control of a particle maturation function of bacteriophage P1; Lehnherr H et al.; A unique arrangement of promoter elements was found upstream of the bacteriophage P1 particle maturation gene (mat) . A P1-specific late-promoter sequence with conserved elements located at positions -22 and -10 was expected from the function of the gene in phage morphogenesis . In addition to a late-promoter sequence, a -35 element and an operator sequence for the major repressor protein, C1, were found . The -35 and -10 elements constituted an active Escherichia coli sigma(70) consensus promoter, which was converted into a P1-regulated early promoter by the superimposition of a C1 operator . This combination of early- and late-promoter elements regulates and fine-tunes the expression of the particle maturation gene . During lysogenic growth the gene is turned off by P1 immunity functions . Upon induction of lytic growth, the expression of mat starts simultaneously with the expression of other C1-regulated P1 early functions . However, while most of the latter functions are downregulated during late stages of lytic growth the expression of mat continues throughout the entire lytic growth cycle of bacteriophage P1 . Thus, the maturation function has a head start on the structural components of the phage particle.

FEBS Lett, 2001 Jun 15, 499(1-2), 147 - 53
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.

Genesis, 2001 Jun, 30(2), 65 - 9
Hypomorphic phenotype in mice with pituitary-specific knockout of steroidogenic factor 1; Zhao L et al.; The bacteriophage Cre recombinase provides a powerful approach for tissue-specific gene inactivation . Using a Cre transgene driven by the common alpha subunit of glycoprotein hormones (alphaGSU-Cre), we have previously inactivated steroidogenic factor 1 (SF-1) in the anterior pituitary, causing hypogonadotropic hypogonadism with sexual infantilism, sterility, and severe gonadal hypoplasia . We now explore the molecular mechanisms underlying a hypomorphic gonadal phenotype in mice carrying two floxed SF-1 alleles (F/F) relative to mice carrying one recombined and one floxed allele (F/R) . Because their Cre-mediated disruption of the locus encoding SF-1 was less efficient, alphaGSU-Cre, F/F mice retained some gonadotropin-expressing cells in the anterior pituitary, thereby stimulating some gonadal function . This novel in vivo model for exploring the effects of differing levels of gonadotropins on gonadal development highlights the need for careful genotype-phenotype comparisons in studies using Cre recombinase to produce tissue-specific knockouts.

Bioorg Med Chem Lett, 2001 Jun 18, 11(12), 1501 - 5
Methodology for optimizing functional miniature proteins based on avian pancreatic polypeptide using phage display; Chin JW et al.; Synthetic genes for avian pancreatic polypeptide (aPP) and for the miniature DNA binding protein PPBR4 were cloned and expressed on the surface of M13 bacteriophage . We anticipate that these constructs will have utility optimizing the properties of miniature proteins based on aPP that result from our previously described protein grafting procedure.

Biochemistry, 2001 Jun 26, 40(25), 7651 - 61
Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein; Xu H et al.; The gene 59 protein (gp59) of bacteriophage T4 performs a vital function in phage DNA replication by directing the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA at nascent replication forks . The helicase assembly activity of gp59 is required for optimum efficiency of helicase acquisition by the replication fork during strand displacement DNA synthesis and is essential for helicase and primosome assembly during T4 recombination-dependent DNA replication transactions . Of central importance is the ability of gp59 to load the gp41 helicase onto ssDNA previously coated with cooperatively bound molecules of gp32, the T4 ssDNA binding protein . Gp59 heteroassociations with ssDNA, gp32, and gp41 all appear to be essential for this loading reaction . Previous studies demonstrated that a tripartite complex containing gp59 and gp32 simultaneously cooccupying ssDNA is an essential intermediate in gp59-dependent helicase loading; however, the biochemical and structural parameters of gp59-gp32 complexes with or without ssDNA are currently unknown . To better understand gp59-gp32 interactions, we performed fluorescence anisotropy and analytical ultracentrifugation experiments employing native or rhodamine-labeled gp59 species in combination with altered forms of gp32, allowing us to determine their binding parameters, shape parameters, and other hydrodynamic properties . Two truncated forms of gp32 were used: gp32-B, which lacks the N-terminal B-domain required for cooperative binding to ssDNA and for stable self-association, and A-domain fragment, which is the C-terminal peptide of gp32 lacking ssDNA binding ability . Results indicate that gp59 binds with high affinity to either gp32 derivative to form a 1:1 heterodimer . In both cases, heterodimer formation is accompanied by a conformational change in gp59 which correlates with decreased gp59-DNA binding affinity . Hydrodynamic modeling suggests an asymmetric prolate ellipsoid shape for gp59, consistent with its X-ray crystallographic structure, and this asymmetry appears to increase upon binding of gp32 derivatives . Implications of our findings for the structure and function of gp59 and gp59-gp32 complexes in T4 replication are discussed.

Genetics, 2001 Jun, 158(2), 507 - 17
Identification of important amino acid residues that modulate binding of Escherichia coli GroEL to its various cochaperones; Klein G et al.; Genetic experiments have shown that the GroEL/GroES chaperone machine of Escherichia coli is absolutely essential, not only for bacterial growth but also for the propagation of many bacteriophages including lambda . The virulent bacteriophages T4 and RB49 are independent of the host GroES function, because they encode their own cochaperone proteins, Gp31 and CocO, respectively . E . coli groEL44 mutant bacteria do not form colonies above 42 degrees nor do they propagate bacteriophages lambda, T4, or RB49 . We found that the vast majority (40/46) of spontaneous groEL44 temperature-resistant colonies at 43 degrees were due to the presence of an intragenic suppressor mutation . These suppressors define 21 different amino acid substitutions in GroEL, each affecting one of 13 different amino acid residues . All of these amino acid residues are located at or near the hinge, which regulates the large en bloc movements of the GroEL apical domain . All of these intragenic suppressors support bacteriophages lambda, T4, and RB49 growth to various extents in the presence of the groEL44 allele . Since it is known that the GroEL44 mutant protein does not interact effectively with Gp31, the suppressor mutations should enhance cochaperone binding . Analogous intragenic suppressor studies were conducted with the groEL673 temperature-sensitive allele.

Genetics, 2001 Jun, 158(2), 495 - 506
Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging; Wieczorek DJ et al.; Bacteriophage lambda is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos . A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging . During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking . In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ . A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal . Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging . Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required . The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN . The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.

Gene, 2001 May 30, 270(1-2), 201 - 9
Isolation and characterization of the murine transforming growth factor-beta2 promoter; Wilder PJ et al.; This report describes the isolation and characterization of the 5' flanking region of the murine transforming growth factor beta-2 (TGF-beta2) gene . A genomic clone containing the promoter region of the gene was isolated after screening a bacteriophage P1 genomic library . The resulting clone was sequenced and compared to promoters for the human and chicken TGF-beta2 genes . The sequence located near the transcription start site is highly conserved . It includes a TATA box, an E-box, and a largely conserved CRE/ATF site . A series of murine TGF-beta2 promoter/reporter constructs was generated to identify regulatory regions of the gene . As in the case of the human TGF-beta2 gene, sequences just upstream of the TATA box, including the CRE/ATF site, actively stimulate the murine TGF-beta2 promoter . However, unlike the human TGF-beta2 gene, the 5' flanking region of the murine TGF-beta2 gene contains a long alternating purine/pyrimidine repeat that unexpectedly exerts a strong positive effect on its promoter . This is of particular interest since alternating purine/pyrimidine repeats in other promoters have been observed to be inhibitory.

J Cell Biochem, 2001 Apr 2-27, 82(1), 145 - 54
Non-natural CBP2 binding peptides and peptomers modulate carcinoma cell adhesion and invasion; Hebert C et al.; A combinatorial approach that utilized a repertoire of bacteriophage-peptides has identified a number of non-natural CBP2 binding peptides . Moreover, co-localization of some of these peptides with CBP2 in a number of tumor cell lines demonstrated that the peptides were directed to an intracellular location spatially coincident with the normal distribution of CBP2 {Sauk et al., 2000} . From among these sequences WHYPWFQNWAMA and LDSRYSLQAAMY were the most effective CBP2 binding peptides and best fulfilled the combinatorial motif containing deep hydrophobic pockets . When the hydropathic profiles of collagen alpha1(IV) and alpha2 (IV) were compared with these dodecapeptides, the hydropathic profiles of WHYPWFQNWAMA and LDSRYSLQAAMY closely matched those of alpha1(IV) 414-452 and alpha1(IV)531-543 . These peptides were shown to be functional peptidomimics and possessed the ability to alter cell adhesion and invasion of human squamous cell carcinoma cell lines . Peptomers were formed of these non-natural peptides to explore the role that a repetitive peptide may have on cell adhesion . The enhanced cell adhesion observed with the peptomers required both CBP2 antibodies and integrin antibodies for inhibition . The enhanced adhesion observed even in the face of combined antibody inhibition was consistent with such complexes possessing correspondingly slower dissociation rates . Thus, suggesting that peptomers may function in a like manner to multimeric peptide MHC complexes (tetramers) binding more than one cell receptor on a specific cell . These findings evoke both peptidomimics of native ligands and their peptomers as potential reagents by which to target tumor cells for chemotherapy, imaging, or retargeting viral vectors for gene therapy .

J Biol Chem, 2001 Aug 17, 276(33), 31092 - 8 Epub 2001 Jun 13.
Directional resolution of synthetic holliday structures by the Cre recombinase; Lee L et al.; The Cre recombinase of bacteriophage P1 cleaves its target site, loxP, in a defined order . Recombination is initiated on one pair of strands to form a Holliday intermediate, which is then resolved by cleavage and exchange of the other pair of strands to yield recombinant products . To investigate the influence of the loxP sequence on the directionality of resolution, we constructed synthetic Holliday (chi) structures containing either wild-type or mutant lox sites . We found that Cre preferentially resolved the synthetic wild-type chi structures on a particular pair of strands . The bias in the direction of resolution was dictated by the asymmetric loxP sequence since the resolution bias was abolished with symmetric lox sites . Systematic substitutions of the loxP site revealed that the bases immediately 5' to the scissile phosphodiester bonds were primarily responsible for the directionality of resolution . Interchanging these base pairs was sufficient to reverse the resolution bias . The Cre-lox co-crystal structures show that Lys(86) makes a base-specific contact with guanine immediately 5' to one of the scissile phosphates . Substituting Lys(86) with alanine resulted in a reduction of the resolution bias, indicating that this amino acid is important for establishing the bias in resolution.

Biochemistry, 2001 Jun 19, 40(24), 7180 - 91
Dynamics of translesion DNA synthesis catalyzed by the bacteriophage T4 exonuclease-deficient DNA polymerase; Berdis AJ; The mechanism and dynamics of translesion DNA synthesis were evaluated using primer/templates containing a tetrahydrofuran moiety designed to mimic an abasic site . Steady-state kinetic analysis reveals that the T4 DNA polymerase preferentially incorporates dATP across from the abasic site with 100-fold higher efficiency than the other nucleoside triphosphates . Under steady-state conditions, the catalytic efficiency of dATP incorporation across from an abasic site is only 220-fold lower than that across from T . Surprisingly, misincorporation across from T is favored 4-6-fold versus replication across an abasic site, suggesting that the dynamics of the polymerization cycle are differentially affected by formation of aberrant base pairs as opposed to the lack of base-pairing capabilities afforded by the abasic site . Linear pre-steady-state time courses were obtained for the incorporation of any dNTP across from an abasic site, indicating that chemistry or a step prior to chemistry is rate-limiting for the polymerization cycle . Low elemental effects (<3) measured by substituting the alpha-thiotriphosphate analogues for dATP, dCTP, and dGTP indicate that chemistry is not solely rate-limiting . Single-turnover experiments yield kpol/Kd values that are essentially identical to kcat/Km values and provide further evidence that the conformational change preceding chemistry is rate-limiting . Extension beyond an A:abasic mispair is approximately 20-fold and 100-fold faster than extension beyond a G:abasic mispair or C:abasic mispair, respectively . Extension from the G:abasic or A:abasic site mispair generates significant elemental effects (between 5 and 20) and suggests that chemistry is at least partially rate-limiting for extension beyond either mispair.

J Mol Biol, 2001 Jun 22, 309(5), 1029 - 48
Analysis of the roles of tRNA structure, ribosomal protein L9, and the bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA; Herr AJ et al.; A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames . In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame . Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing . To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping) . Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing . In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing . These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing.Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context . Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing .

J Mol Biol, 2001 Jun 15, 309(4), 869 - 91
Dissection of the ATP-driven reaction cycle of the bacteriophage T4 DNA replication processivity clamp loading system; Pietroni P et al.; Processive DNA replication requires the loading of a multisubunit ring-shaped protein complex, known as a sliding or processivity clamp, onto the primer-template (p/t) DNA . This clamp then binds to the replication polymerase to form a processive polymerase holoenzyme . The processivity of the holoenzyme derives from the topological properties of the clamp, which encircles the DNA without actually binding to it . Multisubunit complexes known as clamp-loaders utilize ATP to drive the placement of this ring around the DNA . To further understand the role of ATP binding and hydrolysis in driving clamp-loading in the DNA replication system of bacteriophage T4, we report the results of a series of presteady-state and steady-state kinetic ATPase experiments involving the various components of the reconstituted system . The results obtained are consistent with a mechanism in which a slow step, which involves the binary ATP-bound clamp-clamp loader complex, activates this complex and permits p/t DNA to bind and stimulate ATP hydrolysis . ATP hydrolysis itself, as well as the subsequent (after clamp-loading) dissociation of the clamp-loader and the slippage of the loaded clamp from the p/t DNA construct, are shown to be fast steps . A second slow step occurs after ATP hydrolysis . This step involves the dissociated clamp loader complex and may reflect ADP release . Only one molecule of ATP is hydrolyzed per clamp-loading event . Rate constants for each step, and an overall reaction mechanism for the T4 clamp-loading system, are derived from these data and from other results in the literature . The principles that emerge fit into a general framework that can apply to many biological processes involving ATP-driven reaction cycles .

J Mol Biol, 2001 Jun 8, 309(3), 573 - 87
Repression of transcription initiation at 434 P(R) by 434 repressor: effects on transition of a closed to an open promoter complex; Xu J et al.; The lambdoid bacteriophage repressors function both as transcription activators and repressors . Regulation of transcription at the adjacent, but divergent promoters, P(RM) and P(R), determines the phage's choice between the lytic and lysogenic development pathways . Here, we demonstrate that 434 repressor bound at 434 O(R)1 alone is not sufficient to repress transcription from 434 P(R,) but that 434 repressor bound at 434 O(R)2 alone is necessary and sufficient to repress P(R )transcription . This is different from what occurs in the related bacteriophage lambda, in which binding of lambda repressor to either lambdaO(R)1 or lambdaO(R)2 represses transcription from lambdaP(R) . The combined results of gel mobility shift and KMnO(4) footprinting assays show that while 434 repressor binding to 434 O(R)2 does not preclude RNA polymerase binding at the P(R) promoter, it does prevent it from forming open complexes at this promoter . The RNA polymerase-P(R) complexes that form in the presence of repressor are heparin-resistant and the DNA is not melted . This observation indicates that 434 repressor bound at 434 O(R)2 inhibits transcription initiation at the P(R) promoter by "locking" the RNA polymerase-P(R) complex into an inactive state instead of "blocking" the access of RNA polymerase to promoter DNA .

Biochem Biophys Res Commun, 2001 Jun 15, 284(3), 798 - 807
Expression of unphosphorylated form of human double-stranded RNA-activated protein kinase in Escherichia coli; Matsui T et al.; Interferon (IFN)-inducible, double-stranded (dsRNA)-activated protein kinase (PKR) is a key mediator of the antiviral and antiproliferative effects of IFN . PKR is present within cells in a latent state . In response to binding dsRNA, the enzyme becomes activated, causing autophosphorylation and an increase in specific kinase activity . In order to study PKR and its inhibitors, a large amount of the enzyme in its latent, unphosphorylated state is required . When PKR is fused to glutathione S-transferase (GST-PKR) and the fusion protein is expressed in Escherichia coli, the PKR obtained is fully activated by autophosphorylation . Therefore, we have developed an expression plasmid in which both GST-PKR and bacteriophage lambda protein phosphatase (lambda-PPase) genes were placed downstream of a T7 promoter . After induction of expression, unphosphorylated GST-PKR was obtained in good yield, and purified to near homogeneity . The purified enzyme has dsRNA-dependent activation and phosphorylates the translation initiation factor eIF2 alpha . Using the recombinant protein, we analyzed the inhibition mechanisms of two viral inhibitors, vaccinia virus K3L protein and adenovirus virus-associated RNA I (VAI RNA) . K3L inhibited both autophosphorylation of PKR and phosphorylation of eIF2 alpha, whereas VAI RNA inhibited only autophosphorylation . The separation of autophosphorylation and catalytic activity shows that the recombinant PKR is useful in analyzing the functions of PKR, its inhibitors, and its regulatory molecules . The coexpression system of protein kinase with lambda-PPase described here will be applicable to obtaining unphosphorylated and unactivated forms of other protein kinases .

J Biol Chem, 2001 Sep 28, 276(39), 36168 - 73 Epub 2001 Jun 06.
Neural model of the genetic network; Vohradsky J; Many cell control processes consist of networks of interacting elements that affect the state of each other over time . Such an arrangement resembles the principles of artificial neural networks, in which the state of a particular node depends on the combination of the states of other neurons . The lambda bacteriophage lysis/lysogeny decision circuit can be represented by such a network . It is used here as a model for testing the validity of a neural approach to the analysis of genetic networks . The model considers multigenic regulation including positive and negative feedback . It is used to simulate the dynamics of the lambda phage regulatory system; the results are compared with experimental observation . The comparison proves that the neural network model describes behavior of the system in full agreement with experiments; moreover, it predicts its function in experimentally inaccessible situations and explains the experimental observations . The application of the principles of neural networks to the cell control system leads to conclusions about the stability and redundancy of genetic networks and the cell functionality . Reverse engineering of the biochemical pathways from proteomics and DNA micro array data using the suggested neural network model is discussed.

J Biol Chem, 2001 Aug 17, 276(33), 31257 - 64 Epub 2001 Jun 06.
The disordered mobile loop of GroES folds into a defined beta-hairpin upon binding GroEL; Shewmaker F et al.; The GroES mobile loop is a stretch of approximately 16 amino acids that exhibits a high degree of flexible disorder in the free protein . This loop is responsible for the interaction between GroES and GroEL, and it undergoes a folding transition upon binding to GroEL . Results derived from a combination of transferred nuclear Overhauser effect NMR experiments and molecular dynamics simulations indicate that the mobile loop adopts a beta-hairpin structure with a Type I, G1 Bulge turn . This structure is distinct from the conformation of the loop in the co-crystal of GroES with GroEL-ADP but identical to the conformation of the bacteriophage-panned "strongly binding peptide" in the co-crystal with GroEL . Analysis of sequence conservation suggests that sequences of the mobile loop and strongly binding peptide were selected for the ability to adopt this hairpin conformation.

Genes Chromosomes Cancer, 2001 Jul, 31(3), 228 - 39
Smallest region of overlapping deletion in 1p36 in human neuroblastoma: a 1 Mbp cosmid and PAC contig; Bauer A et al.; In human neuroblastomas, the distal portion of 1p is frequently deleted, as if one or more tumor suppressor genes from this region were involved in neuroblastoma tumorigenesis . Earlier studies had identified a smallest region of overlapping deletion (SRO) spanning approximately 23 cM between the most distally retained D1S80 and by the proximally retained D1S244 . In pursuit of generating a refined delineation of the minimally deleted region, we have analyzed 49 neuroblastomas of different stages for loss of heterozygosity (LOH) from 1pter to 1p35 by employing 26 simple sequence length polymorphisms . Fifteen of the 49 tumors (31%) had LOH; homozygous deletion was not detected . Seven tumors had LOH at all informative loci analyzed, and eight tumors showed a terminal or an interstitial allelic loss of 1p . One small terminal and one interstitial deletion defined a new 1.7 cM SRO, approximately 1 Mbp in physical length, deleted in all tumors between the retained D1S2731 (distal) and D1S2666 (proximal) . To determine the genomic complexity of the deleted region shared among tumors, we assembled a physical map of the I Mbp SRO consisting predominantly of bacteriophage P1-derived artificial chromosome (PAC) clones . A total of 55 sequence-tagged site (STS) markers (23 published STSs and short tandem repeats and 32 newly identified STSs from the insert ends of PACs and cosmids) were assembled in a contig, resulting in a sequence-ready physical map with approximately one STS per 20 Kbp . Twelve genes (41BB, CD30, DFFA, DJ1, DR3, FRAP, HKR3, MASP2, MTHFR, RIZ, TNR2, TP73) previously mapped to 1p36 are localized outside this SRO . On the basis of this study, they would be excluded as candidate genes for neuroblastoma tumorigenesis . Ten expressed sequence tags were integrated in the contig, of which five are located outside the SRO . The other five from within the SRO may provide an entrance point for the cloning of candidate genes for neuroblastoma .

Protein Eng, 2001 Apr, 14(4), 287 - 96
A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs; Short MK et al.; We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin . Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin . The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H:100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10:digoxin co-crystal structure . Several mutant Fabs obtained following panning on digoxin-BSA showed increased affinity for digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100 . Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs . Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs than to digoxin . This specificity change was unexpected, as C16 lies on the opposite side of digoxin from H:CDR3 . Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.

J Immunol, 2001 Jun 15, 166(12), 7250 - 9
Novel G protein-coupled responses in leukocytes elicited by a chemotactic bacteriophage displaying a cell type-selective binding peptide; Jaye DL et al.; Recently, we identified a neutrophil-binding phage displaying a novel peptide motif, GPNLTGRW . It was determined that this peptide, when displayed on bacteriophage (FGP phage), elicits a transient increase in cytosolic calcium . Here, we show that FGP phage stimulate neutrophil chemotaxis and induce a pertussis toxin-sensitive rise in cytosolic calcium in monocytes as well as in neutrophils . In contrast to the calcium response elicited by classical chemoattractants fMLP and IL-8, the FGP phage-elicited response in neutrophils is dependent on extracellular calcium and is mediated by receptor-activated, divalent cation channels . Consistent with G protein-coupled receptor signaling, FGP phage effect homologous and reciprocal heterologous desensitization with fMLP- and IL-8-stimulated calcium responses . Like non-G protein-coupled responses, the FGP-elicited calcium transient is abolished with phosphoinositide-3-kinase inactivation . Nonetheless, specific binding of GTP to neutrophil membranes follows stimulation with FGP phage, further supporting involvement of G proteins . However, FGP phage neither bind to nor elicit a calcium response from transfectant cells harboring known candidate G protein-coupled receptors . These data together suggest that the elicited responses are mediated by a novel G protein-coupled receptor or represent novel responses of a known receptor.

J Biol Chem, 2001 Aug 3, 276(31), 29559 - 66 Epub 2001 Jun 04.
Conditional coupling of leading-strand and lagging-strand DNA synthesis at bacteriophage T4 replication forks; Kadyrov FA et al.; Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA . We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution . If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed . Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis . A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis . Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA . It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase . Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.

Mol Cell, 2001 May, 7(5), 993 - 1001
Modification of the properties of elongating RNA polymerase by persistent association with nascent antiterminator RNA; Sen R et al.; Nascent RNA encoded by putL, a cis-acting antitermination site of bacteriophage HK022, increases readthrough of terminators by directly modifying the transcript elongation complex . To characterize the interaction between the antiterminator RNA and RNA polymerase, we stalled the elongation complex downstream of putL and determined the sensitivity of the transcript to ribonuclease cleavage . Part of PutL RNA was protected from cleavage by wild-type polymerase, but not by a mutant with a defect in put-dependent antitermination . We also exposed the stalled complex to oligonucleotides complementary to putL RNA, restarted transcription, and measured antitermination . Some, but not all, complementary oligonucleotides inhibited antitermination . Finally, cleavage of the RNA between putL and the 3'-end released putL RNA from the stalled complex and prevented antitermination.

Nat Biotechnol, 2001 Jun, 19(6), 537 - 42
Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS); Chen G et al.; Periplasmic expression with cytometric screening (PECS) is a powerful and rapid "display-less" technology for isolating ligand-binding proteins from diverse libraries . Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand . Under the proper conditions, ligands as large as about 10 kDa can equilibrate within the periplasmic space without compromising the cell's integrity or viability . The bacterial cell envelope effectively serves as a dialysis bag to selectively retain receptor-fluorescent probe complexes but not free ligand . Cells displaying increased fluorescence are then isolated by flow cytometry . We demonstrate that scFv antibodies with both very high and low affinity to digoxigenin can be isolated from libraries screened by PECS using a benchtop flow cytometer . We also show that preexisting libraries constructed for display on filamentous bacteriophage can be screened by PECS without the need for subcloning . In fact, PECS was found to select for proteins that could be missed by conventional phage panning and screening methods.

Genome Res, 2001 Jun, 11(6), 946 - 58
Evolutionary role of restriction/modification systems as revealed by comparative genome analysis; Rocha EP et al.; Type II restriction modification systems (RMSs) have been regarded either as defense tools or as molecular parasites of bacteria . We extensively analyzed their evolutionary role from the study of their impact in the complete genomes of 26 bacteria and 35 phages in terms of palindrome avoidance . This analysis reveals that palindrome avoidance is not universally spread among bacterial species and that it does not correlate with taxonomic proximity . Palindrome avoidance is also not universal among bacteriophage, even when their hosts code for RMSs, and depends strongly on the genetic material of the phage . Interestingly, palindrome avoidance is intimately correlated with the infective behavior of the phage . We observe that the degree of palindrome and restriction site avoidance is significantly and consistently less important in phages than in their bacterial hosts . This result brings to the fore a larger selective load for palindrome and restriction site avoidance on the bacterial hosts than on their infecting phages . It is then consistent with a view where type II RMSs are considered as parasites possibly at the verge of mutualism . As a consequence, RMSs constitute a nontrivial third player in the host-parasite relationship between bacteria and phages.

Nucleic Acids Res, 2001 Jun 1, 29(11), 2361 - 9
A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-{N6-adenine}-methyltransferase; Malygin EG et al.; The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase . With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix . Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes . In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex . Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se . We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base . Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor . The results of pre-steady-state methylation kinetics are consistent with this model.

Microbiol Res, 2001, 156(1), 35 - 40
Phage spread dynamics in clonal bacterial populations is depending on features of the founder cell; Ramirez E et al.; Plate-cultured bacterial colonies are intriguing models to study host-parasite interactions in senescent populations . During the growth of bacteriophage-infected colonies there is a synchronous prophage induction episode among lysogenic cells that allows a dramatic but time-restricted amplification of viral particles . We report here that the dynamics of phage spread depends on the history of the lysogenic cell that establishes the clonal population, the duration of the pre-burst period being shorter when the founder, infected cell derives from older colonies . These results offer a physiologic explanation for the self-contained progression of the viral spread in closed environments, that ensures both viral dissemination but also survival of most of the host cells.

Mol Genet Genomics, 2001 Mar, 265(1), 95 - 101
Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host; Ozawa H et al.; To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum . The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R . solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R . solancacearum M4S cells . The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined . We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA . This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa . The bacteriolytic gene was placed under the control of an inducible promoter . and the plasmid was transformed into Escherichia coli NM522 . The soluble proteins extracted from E.coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R . solanacearum isolated in various districts in Japan . DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282 . These observations indicate that the bacteriolytic protein shows nonspecific activity against R . solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages . These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.

Protein Sci, 2001 Jun, 10(6), 1150 - 9
Structure of a malaria parasite antigenic determinant displayed on filamentous bacteriophage determined by NMR spectroscopy: implications for the structure of continuous peptide epitopes of proteins; Monette M et al.; The NANP repeating sequence of the circumsporozoite protein of Plasmodium falciparum was displayed on the surface of fd filamentous bacteriophage as a 12-residue insert (NANP)(3) in the N-terminal region of the major coat protein (pVIII) . The structure of the epitope determined by multidimensional solution NMR spectroscopy of the modified pVIII protein in lipid micelles was shown to be a twofold repeat of an extended and non-hydrogen-bonded loop based on the sequence NPNA, demonstrating that the repeating sequence is NPNA, not NANP . Further, high resolution solid-state NMR spectra of intact hybrid virions containing the modified pVIII proteins demonstrate that the peptides displayed on the surface of the virion adopt a single, stable conformation; this is consistent with their pronounced immunogenicity as well as their ability to mimic the antigenicity of their native parent proteins.

Nucleic Acids Res, 2001 May 15, 29(10), E50 - 0
The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd; Enshell-Seijffers D et al.; Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides . Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII . The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages . 'Type 88' vectors contain two pVIII genes in one phage genome . In this study a novel 'type 88' expression vector has been rationally designed and constructed . Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene . It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome . The deletions were independent of recA yet required a functional F-episome . Transcription was also found to be a positive factor for deletion . Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages . The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

Nucleic Acids Res, 2001 May 15, 29(10), 2088 - 96
Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72; Rossler OG et al.; RNA helicases, like their DNA-specific counterparts, can function as processive enzymes, unwinding RNA with a defined step size in a unidirectional fashion . Recombinant nuclear DEAD-box protein p68 and its close relative p72 are reported here to function in a similar fashion, though the processivity of both RNA helicases appears to be limited to only a few consecutive catalytic steps . The two proteins resemble each other also with regard to other biochemical properties . We have found that both proteins exhibit an RNA annealing in addition to their helicase activity . By using both these activities the enzymes are able in vitro to catalyse rearrangements of RNA secondary structures that otherwise are too stable to be resolved by their low processive helicase activities . RNA rearrangement proceeds via protein induced formation and subsequent resolution of RNA branch migration structures, whereby the latter step is dependent on ATP hydrolysis . The analysed DEAD-box proteins are reminiscent of certain DNA helicases, for example those found in bacteriophages T4 and T7, that catalyse homologous DNA strand exchange in cooperation with the annealing activity of specific single strand binding proteins.

Mol Genet Genomics, 2001 Apr, 265(2), 225 - 33
On the fate of plant or other foreign genes upon the uptake in food or after intramuscular injection in mice; Hohlweg U et al.; Uptake and persistence of the DNA of bacteriophage M13 and the cloned gene for the green fluorescent protein (GFP) as test genes for food-ingested DNA have previously been traced from the intestinal contents, via the gut wall, Peyer's patches and peripheral white blood cells to spleen and liver, and via the placenta to fetuses and newborn animals . We have now chosen a natural scenario and fed soybean leaves to mice . The distribution of the plant-specific, nucleus-encoded ribulose-1,5-bisphosphate carboxylase (Rubisco) gene has been studied in the mouse . The Rubisco gene or fragments of it can be recovered in the intestine from 2 h up to 49 h after feeding, and in the cecum up to 121 h after ingestion . Thus, plant-associated, naturally fed DNA is more stable in the intestinal tract than naked DNA . Rubisco gene-specific PCR products have also been amplified from spleen and liver DNA . There is no evidence for the expression of orally administered genes, as assessed by the RT-PCR method . Moreover, mice have been continuously fed daily with GFP DNA for 8 generations and have been examined for the transgenic state by assaying DNA isolated from tail tips, occasionally from internal organs of the animals, by PCR . The results have been uniformly negative and argue against the germline transfer of orally administered DNA . Upon the intramuscular injection of GFP DNA, authentic GFP DNA fragments have been amplified by PCR from DNA from muscle for up to 17 months post-injection, and from DNA from organs remote from the site of injection up to 24 h post injection . GFP fragments can also be retrieved from the intestinal contents up to 6 h post injection . The organism apparently eliminates injected foreign DNA via the liver-bile-intestinal route.

J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 361 - 70
Facilitation of bacteriophage lambda DNA injection by inner membrane proteins of the bacterial phosphoenol-pyruvate: carbohydrate phosphotransferase system (PTS); Esquinas-Rychen M et al.; Infection of Escherichia coli by bacteriophage lambda depends on two membrane protein complexes: (i) maltoporin (LamB) in the outer membrane for adsorption and (ii) the IIC(Man)-IID(Man) complex of the mannose transporter in the inner membrane for DNA penetration . IIC(Man) and IID(Man) are components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) which together with the IIAB(Man) subunit mediate transport and phosphorylation of sugars . To identify structural determinants important for penetration of lambda DNA, the homologous IIC-IID complexes of E . coli, K . pneumoniae and B . subtilis, and chimeric complexes between the IIC and IID were characterized . All three complexes support sugar transport in E . coli . Only IIC-IID of E . coli and B . subtilis also support bacteriophage lambda infection . The six chimeric complexes had lost transport activity, but three containing IIC of E . coli or B . subtilis continue to support bacteriophage lambda infection . Complexes containing IIC(Man) and fusion proteins between truncated IID(Man) and alkaline phosphatase or beta-galactosidase support penetration of lambda DNA if less than 100 residues are missing from the C-terminus of IID(Man) . Truncation of IIC(Man) renders the complex unstable . Taken together, these results suggest, that IIC is the major specificity determinant for lambda infection but that the IIC subunit is stably expressed only in a complex with the IID subunit . Lambda DNA in transit across the periplasmic space, but not transforming plasmid DNA, is inaccessible to the non-specific nuclease NucA of Anabaena sp . targeted to the periplasmic space either in soluble form or as a fusion protein to the C-terminus of IID(Man).

Dis Markers, 2000, 16(1-2), 83 - 90
Use of phage antibodies to distinguish closely related species of protozoan parasites; Paget T et al.; Acanthamoeba are typically identified in the laboratory using culture and microscopic observation . In this paper we describe the isolation and specificity of antibody fragments that can be used for the identification of Acanthamoeba . A phage library expressing a large repertoire (approx . 5 x 10(9)) of antibody fragments was used to generate two libraries one enriched for bacteriophage that exhibit genus specific binding and the other containing bacteriophage that bind specifically to pathogenic Acanthamoeba . Individual clones were isolated on the basis of binding by ELISA, and then flow cytometry and immunofluorescence were used for further characterisation . Four monoclonal antibodies were isolated, specific for Acanthamoeba at the generic level with clone HPPG6 exhibiting the highest level of binding . Furthermore clone HPPG55 was specific for pathogenic species of Acanthamoeba.

Dis Markers, 2000, 16(1-2), 53 - 62
Clinical applications of phage-derived sFvs and sFv fusion proteins; Chester KA et al.; Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice . MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas . MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery . Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer . We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNF alpha aims to reduce sequestration and increase tumor concentrations of systemically administered TNF alpha.

Eur J Biochem, 2001 May, 268(10), 3099 - 107
De novo identification of cell-type specific antibody-antigen pairs by phage display subtraction . Isolation of a human single chain antibody fragment against human keratin 14; Stausbol-Gron B et al.; The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific antigens from a phage display antibody repertoire by using phage display subtraction . Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles . It allows the interaction between two cognate molecules to be analysed through affinity selections . Recently, large repertoires of phage displayed human antibody fragments have been constructed . From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology . A novel subtractive strategy for selecting antibodies from phage libraries was applied . Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand . A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly non-differentially expressed antigens . A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis . The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database . In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis . Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.

Electrophoresis, 2001 Apr, 22(6), 981 - 9
Application of the concept of an electrophoretic ratchet; Griess GA et al.; Fractionation via a gel electrophoretic ratchet has previously succeeded for comparatively large (radius R > or = 95 nm) spheres (Serwer, P, Griess, G.A., Anal . Chim . Acta 1998, 372, 299-306) . The electrical oscillations are the following electrical field pulses: high field --> low field --> high field, etc . The field is inverted after each pulse; the time-integral of the field can be zero . Response to the ratchet is caused by steric trapping in the high field-direction, but not in the low field-direction . Trapping and, therefore, response to the ratchet decrease as R decreases . The smaller spheres do not respond to the ratchet . In the present study, spheres with R values smaller than 95 nm are made, for the first time, to respond to a similar gel electrophoretic ratchet . To achieve this objective, the heterogeneity of pore size is increased for the gel used . The heterogeneity of pore size is increased by (i) forming the gel with degraded hydroxyethyl agarose, and (ii) gelling at comparatively high temperature . If a particle still does not respond to the ratchet (because the particle is too small), this particle has a net migration in the high field-direction, when the above-described pulsed field is biased in the high field-direction . If a particle does respond to the improved ratchet, the particle has a net migration in the low field-direction . Here, the R of ratchet-responding spheres is reduced to 30-50 nm . These ratchet-responding spheres include both intact bacteriophage particles (R = 30 nm) and latex spheres . The smaller ratchet-responding spheres have an electrophoretic mobility that decreases in magnitude as the electrical field increases in magnitude . A ratchet-based procedure is developed here to achieve continuous preparative gel electrophoresis.

Biochem Biophys Res Commun, 2001 May 25, 283(5), 1099 - 104
Bacteriophage WO and virus-like particles in Wolbachia, an endosymbiont of arthropods; Masui S et al.; Wolbachia are intracellular symbionts mainly found in arthropods, causing various sexual alterations on their hosts by unknown mechanisms . Here we report the results that strongly suggest that Wolbachia have virus-like particles of phage WO, which was previously identified as a prophage-like element in the Wolbachia genome . Wolbachia (strain wTai) infection in an insect was detected with the antibody against Wsp, an outer surface protein of Wolbachia, by fluorescence microscopy and immunoelectron-microscopy for the first time . Virus-like particles in Wolbachia were observed by electron-microscopy . The 0.22-microm filtrate of insect ovary contained DAPI-positive particles, and PCR analysis demonstrated that a phage WO DNA passed through the filter while Wolbachia DNA were eliminated, suggesting that the DAPI-positive particles were phage WO .

Biotechniques, 2001 May, 30(5), 1142 - 7
S-Gal: an autoclavable dye for color selection of cloned DNA inserts; Heuermann K et al.; Blue/white selection is the standard method for detecting a cloned DNA fragment . In the absence of an insert, uninterrupted expression of the vector-encoded alpha-complement of beta-galactosidase (beta-gal), results in the hydrolysis of X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) and the subsequent blue staining of the host colony or bacteriophage plaque expressing the carboxyterminal portion of the beta-gal gene (lacZ) . A white or clear colony or plaque indicates the presence of an insert . Because of its water insolubility, X-gal is dissolved in hazardous solvents such as dimethylformamide and then added to the medium following autoclaving . X-gal can be spread on previously plated medium, but this may result in an uneven color development . Also, incubation at 4 degrees C is frequently required for the distinction between a positive recombinant (unstained colony or plaque) and a stained negative . S-Gal (3,4-cyclohexenoesculetin-beta-D-galactopyranoside), a novel beta-gal substrate, is autoclavable and microwavable, allowing for dry-blending of the dye directly into the medium . Black S-Gal-stained colonies are visibly distinguishable from unstained colonies at an earlier time than X-gal . In addition, detection of the unstained signal over background is enhanced by 25% using S-Gal-containing medium, compared to medium containing X-gal . These characteristics offer convenience and better suitability for automated colony or plaque analyses.

Anal Chem, 2001 May 1, 73(9), 2126 - 31
MALDI-TOF mass spectrometric method for detection of hybridized DNA oligomers; Isola NR et al.; Two new approaches for nucleic acid hybridizations by MALDI-TOF mass spectrometry are described . Hybridization using genomic DNA without polymerase chain reaction was demonstrated . Total genomic DNA of bacteriophages bound to charge-modified nylon membranes was identified by the hybridization of species-specific oligonucleotide probes . lambda-Phage DNA and M13 were used for the test with good success . Since MALDI-TOF mass spectrometry can be used to measure the molecular weights of different probes, mass spectrometry can be used for the detection of hybridizations with multiple probes . We demonstrate that multiple-probe hybridization can be resolved by mass spectrometry . Six probes with different mass tag were used for hybridization on a single spot . MALDI-TOF mass spectrometry was successfully used to measure these probes simultaneously . This provides a simple nonradioactive method for multiplex hybridization analysis . It has the potential to drastically increase the speed for microarray hybridization analysis in the future.

J Mol Biol, 2001 May 18, 308(5), 1081 - 9
Stabilization of short collagen-like triple helices by protein engineering; Frank S et al.; Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase . Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs . Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain . The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli . The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation . We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability . The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling . Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition . As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability . When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased . The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide . Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions .

J Mol Biol, 2001 May 11, 308(4), 745 - 64
What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR; Goto NK et al.; Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate . Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding . To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods . The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures . The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures) . A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant . Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS) . In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees) . Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond . The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure .

J Mol Biol, 2001 May 11, 308(4), 579 - 85
The TolC protein of Escherichia coli serves as a cell-surface receptor for the newly characterized TLS bacteriophage; German GJ et al.; The TolC protein of Escherichia coli is implicated in a variety of diverse cellular functions, including antibiotic efflux and alpha-hemolysin secretion . An incidental role of TolC is to facilitate the entry of the bacteriophage TLS and colicin E1 into the bacterial cell . Despite the resolution of TolC's atomic structure, the roles of specific residues in its diverse functions are unknown . Here, we describe a genetic strategy for isolating missense tolC mutations that abolish the bacteriophage receptor activity of the TolC protein without influencing its role in antibiotic efflux . These spontaneous mutations affected two regions of the TolC protein and included base-pair substitutions, insertions, and deletions . Comparison of the TolC sequence with those of its homologues revealed two hypervariable stretches that were predicted to represent loops . Interestingly, all but one of the TolC alterations preventing phage binding were located in these two hypervariable regions, which are likely to be exposed on the cell surface . This was substantiated by the recently solved three-dimensional structure of TolC . Curiously, all the phage-resistant TolC mutants showed varying degrees of resistance to colicin E1, suggesting the involvement of overlapping regions of TolC in colicin E1 import and phage binding.The phage used in this study, TLS, was earlier reported as a strain of U3 . However, we show here that, unlike the previously reported lipopolysaccharide-specific U3 phage, this phage displays a distinctly different host range and discrete morphological features and, in addition to utilizing TolC as receptor, it requires the inner core of a lipopolysaccharide .

RNA, 2001 May, 7(5), 774 - 81
Primer-independent RNA sequencing with bacteriophage phi6 RNA polymerase and chain terminators; Makeyev EV et al.; Here we propose a new general method for directly determining RNA sequence based on the use of the RNA-dependent RNA polymerase from bacteriophage phi6 and the chain terminators (RdRP sequencing) . The following properties of the polymerase render it appropriate for this application: (1) the phi6 polymerase can replicate a number of single-stranded RNA templates in vitro . (2) In contrast to the primer-dependent DNA polymerases utilized in the sequencing procedure by Sanger et al . (Proc Natl Acad Sci USA, 1977, 74:5463-5467), it initiates nascent strand synthesis without a primer, starting the polymerization on the very 3'-terminus of the template . (3) The polymerase can incorporate chain-terminating nucleotide analogs into the nascent RNA chain to produce a set of base-specific termination products . Consequently, 3' proximal or even complete sequence of many target RNA molecules can be rapidly deduced without prior sequence information . The new technique proved useful for sequencing several synthetic ssRNA templates . Furthermore, using genomic segments of the bluetongue virus we show that RdRP sequencing can also be applied to naturally occurring dsRNA templates . This suggests possible uses of the method in the RNA virus research and diagnostics.

Protein Eng, 2001 Mar, 14(3), 189 - 93
Selection of novel ligands from a whole-molecule randomly mutated C5a library; Cain SA et al.; Novel antagonists of the proinflammatory leukocyte chemoattractant C5a have been produced from a phage display library of whole-molecule random mutants . The cDNA for the inflammatory polypeptide C5adR(74) was used as template in a PCR reaction doped with the mutagenic nucleoside triphosphates dPTP {dP: 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-{4,5-c}{1,2}oxazin-7-one} and 8-oxodGTP (8-oxodG: 8-oxo-2'-deoxyguanosine) to allow the introduction of mutations in a highly controlled manner throughout the cDNA . The resultant library of mutants was displayed on bacteriophage M13 using a jun/fos linker sequence . Functional polypeptides were isolated by several rounds of selection against the receptor for C5a expressed on the surface of CHO cells . From this selection procedure, a limited number of variants of C5adR(74) were obtained . When expressed as free polypeptide, the binding affinities of the selected C5adR(74) sequences were increased 5-fold relative to wild-type protein . Site-directed mutagenesis of the C-terminus of these variants resulted in the production of antagonists of C5adR(74) activity.

Biochemistry, 2001 May 15, 40(19), 5665 - 73
Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose; Leung AK et al.; The three-dimensional structure of the lytic transglycosylase from bacteriophage lambda, also known as bacteriophage lambda lysozyme, complexed to the hexasaccharide inhibitor, hexa-N-acetylchitohexaose, has been determined by X-ray crystallography at 2.6 A resolution . The unit cell contains two molecules of the lytic transglycosylase with two hexasaccharides bound . Each enzyme molecule is found to interact with four N-acetylglucosamine units from one hexasaccharide (subsites A-D) and two N-acetylglucosamine units from the second hexasaccharide (subsites E and F), resulting in all six subsites of the active site of this enzyme being filled . This crystallographic structure, therefore, represents the first example of a lysozyme in which all subsites are occupied, and detailed protein-oligosaccharide interactions are now available for this bacteriophage lytic transglycosylase . Examination of the active site furthermore reveals that of the two residues that have been implicated in the reaction mechanism of most other c-type lysozymes (Glu35 and Asp52 in hen egg white lysozyme), only a homologous Glu residue is present . The lambda lytic transglycosylase is therefore functionally closely related to the Escherichia coli Slt70 and Slt35 lytic transglycosylases and goose egg white lysozyme which also lack the catalytic aspartic acid.

Annu Rev Biophys Biomol Struct, 2001, 30, 87 - 104
A structural view of cre-loxp site-specific recombination; Van Duyne GD; Structural models of site-specific recombinases from the lambda integrase family of enzymes have in the last four years provided an important new perspective on the three-dimensional nature of the recombination pathway . Members of this family, which include the bacteriophage P1 Cre recombinase, bacteriophage lambda integrase, the yeast Flp recombinase, and the bacterial XerCD recombinases, exchange strands between DNA substrates in a stepwise process . One pair of strands is exchanged to form a Holliday junction intermediate, and the second pair of strands is exchanged during resolution of the junction to products . Crystal structures of reaction intermediates in the Cre-loxP site-specific recombination system, together with recent biochemical studies in the field, support a "strand swapping" model for recombination that does not require branch migration of the Holliday junction intermediate in order to test homology between recombining sites.

Curr Opin Mol Ther, 2001 Apr, 3(2), 159 - 69
Phage as gene delivery vectors; Monaci P et al.; Bacteriophage can be considered as a natural system to efficiently condense and package DNA . They tolerate many different types of mutations, including those that lead to the display of polypeptides as a fusion to any of the structural proteins comprising the phage particle . In addition, they are a powerful biological system for generating and screening mutants with the desired functional properties . It has also been shown that phage vectors can be engineered for receptor-mediated gene transfer to mammalian cells . The attractive features offered by this system have paved the way for various attempts to develop phage as a vector for gene therapy applications.

Genetics, 2001 May, 158(1), 177 - 86
Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations; Hadjimarcou MI et al.; We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta . Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure . The mutant DNA pol delta has a serine substitution in place of glycine at position 447 . DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays . An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene . Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts . Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.

Genetics, 2001 May, 158(1), 19 - 28
Repair of topoisomerase-mediated DNA damage in bacteriophage T4; Stohr BA et al.; Type II topoisomerase inhibitors are used to treat both tumors and bacterial infections . These inhibitors stabilize covalent DNA-topoisomerase cleavage complexes that ultimately cause lethal DNA damage . A functional recombinational repair apparatus decreases sensitivity to these drugs, suggesting that topoisomerase-mediated DNA damage is amenable to such repair . Using a bacteriophage T4 model system, we have developed a novel in vivo plasmid-based assay that allows physical analysis of the repair products from one particular topoisomerase cleavage site . We show that the antitumor agent 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) stabilizes the T4 type II topoisomerase at the strong topoisomerase cleavage site on the plasmid, thereby stimulating recombinational repair . The resulting m-AMSA-dependent repair products do not form in the absence of functional topoisomerase and appear at lower drug concentrations with a drug-hypersensitive topoisomerase mutant . The appearance of repair products requires that the plasmid contain a T4 origin of replication . Finally, genetic analyses demonstrate that repair product formation is absolutely dependent on genes 32 and 46, largely dependent on genes uvsX and uvsY, and only partly dependent on gene 49 . Very similar genetic requirements are observed for repair of endonuclease-generated double-strand breaks, suggesting mechanistic similarity between the two repair pathways.

Genetics, 2001 May, 158(1), 7 - 17
Recognition and specific degradation of bacteriophage T4 mRNAs; Ueno H et al.; Gene 61.5 of bacteriophage T4 has a unique role in gene expression . When this gene is mutated, mRNAs of many late genes are rapidly degraded, resulting in late-gene silencing . Here, we characterize an extragenic suppressor, ssf5, of a gene 61.5 mutation . ssf5 was found to be an amber mutation in motA, which encodes a transcription activator for T4 middle genes . When this gene is mutated, both degradation and specific cleavage of late-gene mRNA is induced after a delay, as exemplified by soc mRNA . Consequently, partial late-gene expression occurs . In an ssf5 genetic background, a gene 61.5 mutation exhibits a novel phenotype: in contrast to late-gene mRNA, middle-gene mRNA is stabilized and the expression of middle genes is prolonged . This is attributable to an activity of gene 61.5 specific for degradation of middle-gene mRNA . The degradation of middle-gene mRNA in the presence of a normal gene 61.5 appears in parallel with the degradation of late-gene mRNA in its absence . This observation suggests that the mRNA-degrading activity that silences late genes in cells infected with a gene 61.5 mutant is targeted to middle-gene mRNA when gene 61.5 is wild type . These results and the results obtained in the presence of a normal motA gene suggest that gene 61.5 protein functions to discriminate mRNAs for degradation in a stage-dependent manner.

Cancer Gene Ther, 2001 Mar, 8(3), 176 - 84
Kinetic characterization of ribozymes directed against the cisplatin resistance-associated ABC transporter cMOAT/MRP2/ABCC2; Materna V et al.; The enhanced expression of the human ABC transporter, cMOAT (MRP2/ABCC2), is associated with resistance of tumor cells against platinum-containing compounds, such as cisplatin . Therefore, cMOAT represents an interesting candidate factor for modulation of antineoplastic drug resistance . Two different hammerhead ribozymes, which exhibit high catalytic cleavage activities towards specific RNA sequences encoding cMOAT, were designed . Cleavage sites of these ribozymes are the GUC sites in codons 704 and 708 of the open reading frame in the cMOAT-specific mRNA molecule . Hammerhead ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence . cMOAT-encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using RNA prepared from the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS overexpressing the cMOAT-encoding transcript . In a cell-free system, both anti-cMOAT ribozymes cleaved their substrate in a highly efficient manner at a physiologic pH and temperature . The cleavage reaction was dependent on time and ribozyme:substrate ratio for determining specific kinetic parameters.

Biochemistry, 2001 Feb 20, 40(7), 2267 - 75
Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA; Mou TC et al.; The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides . Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions {Mou, T . C., Gray, C . W., and Gray, D . M . (1999) Biophys . J . 76, 1537-1551} . We found that the DNA backbone can also dramatically affect the binding affinity . The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl . CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers . The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site . Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences . The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence . These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.

Biochemistry, 2001 Feb 27, 40(8), 2606 - 13
Stability studies of FhuA, a two-domain outer membrane protein from Escherichia coli; Bonhivers M et al.; FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics . Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it . This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein . To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork . Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively . Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C . Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome . FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.

J Mol Biol, 2001 Apr 27, 308(2), 147 - 63
The two active-site tyrosine residues of the a protein play non-equivalent roles during initiation of rolling circle replication of bacteriophage p2; Odegrip R et al.; The A protein of bacteriophage P2 initiates rolling circle DNA replication by a single-stranded cut at the origin . Two well-conserved tyrosine residues, interspaced by three amino acid residues, are required for the cleavage-joining activity of the protein . The functional relationship between these tyrosine residues was investigated by site-directed mutagenesis . We found that the two tyrosine residues located in the presumed catalytic site of P2 A play non-equivalent functional roles . Tyrosine residue 454 is superior in nicking single-stranded DNA compared to tyrosine residue 450, while both could promote joining at equal efficiency . Specific peptide-oligonucleotide adducts after cleavage reaction and protease digestion could be observed for both tyrosine residues . We propose that tyrosine 454 initiates replication and that tyrosine 450 is able to cleave the DNA only when tyrosine 454 is covalently joined to DNA, thereby reinitiating replication . Also, the involvement of divalent cations in the catalytic activity of P2 A was investigated . While the cleavage reaction was strongly discriminating between different divalent cations, primarily prefering magnesium, the joining reaction showed the same efficiency independently of what divalent cation was provided . This phenomenon could reflect conformational changes of the protein upon binding to DNA . Finally, we found that a large part of the C terminus but not the N terminus is dispensable for initiation of replication both in vivo and in vitro .

Science, 2001 Apr 27, 292(5517), 744 - 8
Virus maturation involving large subunit rotations and local refolding; Conway JF et al.; Large-scale conformational changes transform viral precursors into infectious virions . The structure of bacteriophage HK97 capsid, Head-II, was recently solved by crystallography, revealing a catenated cross-linked topology . We have visualized its precursor, Prohead-II, by cryoelectron microscopy and modeled the conformational change by appropriately adapting Head-II . Rigid-body rotations ( approximately 40 degrees) cause switching to an entirely different set of interactions; in addition, two motifs undergo refolding . These changes stabilize the capsid by increasing the surface area buried at interfaces and bringing the cross-link-forming residues, initially approximately 40 angstroms apart, close together . The inner surface of Prohead-II is negatively charged, suggesting that the transition is triggered electrostatically by DNA packaging.

J Bacteriol, 2001 May, 183(10), 3176 - 83
Functional and mutational analysis of p19, a DNA transfer protein with muramidase activity; Bayer M et al.; Protein P19 encoded by the conjugative resistance plasmid R1 has been identified as being one member of a large family of muramidases encoded by bacteriophages and by type III and type IV secretion systems . We carried out a mutational analysis to investigate the function of protein P19 and used in vivo complementation assays to test those of several P19 mutants . The results indicated that conserved residues present in the presumed catalytic center of P19 are absolutely essential for its function in conjugation of plasmid R1 and infection by the RNA phage R17 . Overexpression of protein P19 in an early growth phase resulted in a massive lysis of Escherichia coli cells in liquid culture, as indicated by a rapid and distinct decrease in cell culture densities after induction . Change of the proposed catalytic glutamate at position 44 to glutamine completely abolished this effect . P19-induced cell lysis was directly shown by transmission and scanning electron microscopy . Typically, P19-overexpressing cells showed bulges protruding from the cell surfaces . Our interpretation is that these protrusions arose from a localized and spatially confined disruption of the bacterial cell wall . To our knowledge such an effect has not previously been documented for any member of the lytic transglycosylase family . From the data presented here, we conclude that protein P19 possesses the proposed localized peptidoglycan-hydrolyzing activity . This activity would be a prerequisite for efficient penetration of the cell envelope by the DNA translocation complex encoded by the conjugative plasmid.

Virus Genes, 2001 Mar, 22(2), 127 - 32
Bacteriophage lambda cIII gene product has an additional function apart from inhibition of cII degradation; Latala B et al.; For lysogenization of Escherichia coli cells by bacteriophage lambda, functions of three lambda genes called c are necessary . The cI gene codes for a repressor that blocks activities of lytic promoters . However, early after infection, expression of cI is dependent on the function of the cII gene, coding for a specific transcriptional activator . The cII protein is unstable in E . coli cells due to FtsH-mediated proteolysis . The cIII gene product is an inhibitor of the FtsH protease . Here we demonstrate that cIII may have another function apart from inhibition of cII degradation . We found that overexpression of the cII gene results in impaired lysogenization by phage lambda, however simultaneous overexpression of the cIII gene abolished this negative effect on lysogenization . Analysis of cII-mediated transcriptional activation of certain promoters at different levels of cII and cIII proteins in cells confirmed that observed effects cannot be explained assuming that the only role of cIII is inhibition of FtsH-mediated degradation of cII . We propose that cIII has an additional role apart from its well-known function in indirect stabilization of cII . Apparently, cIII influences not only cII level but also activity of this transcriptional stimulator, especially at its high concentrations.

Plasmid, 2001 Mar, 45(2), 122 - 6
Complete nucleotide sequence of the mycoplasma virus P1 genome; Tu AH et al.; Mycoplasma virus P1 is one of only four viruses isolated from the genus Mycoplasma . The host for P1, Mycoplasma pulmonis, possesses complex, phase-variable restriction and modification enzymes and the Vsa family of phase-variable surface proteins . The ability of P1 virus to infect host cells is influenced by these phase-variable systems, rendering P1 a valuable tool for assessing host properties . The double-stranded P1 DNA genome was sequenced (11,660 bp) and 11 ORFs were identified . The predicted P1 DNA polymerase is similar to that of phages that are known to have terminal protein (TP) attached to the 5' end of their genome, consistent with previous studies indicating that P1 DNA has covalently attached TP . Most of the other predicted P1 proteins have little sequence similarity to known proteins, and P1 virus is unrelated to the other mycoplasma virus, MAV1, for which the genome sequence is known . One of the predicted P1 proteins, the ORF 8 gene product, contains a repetitive collagen-like motif characteristic of some bacteriophage tail fiber proteins and is a candidate for interacting with the Vsa proteins .

Gene Ther, 2001 Apr, 8(8), 649 - 53
Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes; Lacy-Hulbert A et al.; Sustained expression of recombinant proteins is a critical factor for the effectiveness of numerous applications in the biomedical sciences including the treatment of human disease by gene therapy, the large scale production of therapeutic proteins, as well as the investigation of gene function by transgenesis or cell type specific mutagenesis . Although much attention has been paid to the optimisation of regulatory sequences such as promoters, untranslated regions and polyadenylation signals, effective and sustained expression of recombinant genes in vivo is often difficult to achieve . Here we report that the creation of artificial exons, by insertion of two short heterologous introns into open reading frames, is not only compatible with functional expression, but also leads to a 30-fold enhancement of mRNA production for both green fluorescent protein and the bacteriophage P1-derived Cre recombinase . The levels of green fluorescence were increased five-fold in cell lines and sustained long-term expression at increased levels was observed in rat brain after transduction with a herpes simplex virus-based vector . The data presented identify a means by which the expression of recombinant genes can be enhanced considerably, in addition to and independently from the surrounding regulatory sequences . The method should help obtain sustained and effective expression of recombinant proteins in vivo.

Plasmid, 2001 Jan, 45(1), 1 - 17
The plasmid status of satellite bacteriophage P4; Briani F et al.; P4 is a natural phasmid (phage-plasmid) that exploits different modes of propagation in its host Escherichia coli . Extracellularly, P4 is a virion, with a tailed icosahedral head, which encapsidates the 11.6-kb-long double-stranded DNA genome . After infection of the E . coli host, P4 DNA can integrate into the bacterial chromosome and be maintained in a repressed state (lysogeny) . Alternatively, P4 can replicate as a free DNA molecule; this leads to either the lytic cycle or the plasmid state, depending on the presence or absence of the genome of a helper phage P2 in the E . coli host . As a phage, P4 is thus a satellite of P2 phage, depending on the helper genes for all the morphogenetic functions, whereas for all its episomal functions (integration and immunity, multicopy plasmid replication) P4 is completely autonomous from the helper . Replication of P4 DNA depends on its alpha protein, a multifunctional polypeptide that exhibits primase and helicase activity and binds specifically the P4 origin . Replication starts from a unique point, ori1, and proceeds bidirectionally in a straight theta-type mode . P4 negatively regulates the plasmid copy number at several levels . An unusual mechanism of copy number control is based on protein-protein interaction: the P4-encoded Cnr protein interacts with the alpha gene product, inhibiting its replication potential . Furthermore, expression of the replication genes cnr and alpha is regulated in a complex way that involves modulation of promoter activity by positive and negative factors and multiple mechanisms of transcription elongation-termination control . Thus, the relatively small P4 genome encodes mostly regulatory functions, required for its propagation both as an episomal element and as a temperate satellite phage . Plasmids that, like P4, propagate horizontally via a specific transduction mechanism have also been found in the Archaea . The presence of P4-like prophages or cryptic prophages often associated with accessory bacterial functions attests to the contribution of satellite phages to bacterial evolution .

Appl Environ Microbiol, 2001 May, 67(5), 2062 - 9
Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296; Morgan JA et al.; Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae . From one of these strains (X . nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity . Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E . coli (50% lethal concentration {LC(50)} of 2 to 6 microg of total protein/g of diet) . The complete sequence of one cosmid (cHRIM1) was obtained . On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity) . Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P . brassicae . One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity . The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E . coli clone . Although multiple genes may be needed for full activity, E . coli cells expressing the xptA1 gene from the bacteriophage lambda P(L) promoter were shown to have insecticidal activity (LC(50) of 112 microg of total protein/g of diet) . This is contrary to the toxin genes identified in P . luminescens, which were not insecticidal when expressed individually in E . coli . High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E . coli clone expressing xptA1 . The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X . nematophilus PMFI296.

Appl Environ Microbiol, 2001 May, 67(5), 2037 - 43
Brachyspira (Serpulina) hyodysenteriae gyrB mutants and interstrain transfer of coumermycin A(1) resistance; Stanton TB et al.; To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a lambdaZAPII library of strain B204 genomic DNA and sequenced . The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes . B . hyodysenteriae coumermycin A(1)-resistant (Cn(r)) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 microg of coumermycin A(1)/ml . The coumermycin A(1) MICs were 25 to 100 microg/ml for the resistant strains and 0.1 to 0.25 microg/ml for strain B204 . Four Cn(r) strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly(78) to Ser (two strains), Gly(78) to Cys, and Thr(166) to Ala . When Cn(r) strain 435A (Gly(78) to Ser) and Cm(r) Km(r) strain SH (DeltaflaA1::cat Deltanox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56 degrees C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A(1), and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain . Seven Cn(r) Km(r) Cm(r) strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH . Cn(r) Km(r) Cm(r) cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added . These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B . hyodysenteriae cells . Gene transfer readily occurs between B . hyodysenteriae cells in broth culture, a finding with practical importance . VSH-1 is the likely mechanism for gene transfer.

Genes Cells, 2001 Apr, 6(4), 291 - 301
Sequence-specific termination by T7 RNA polymerase requires formation of paused conformation prior to the point of RNA release; Song H et al.; BACKGROUND: The sequence-specific, hairpin-independent termination signal for the bacteriophage RNA polymerases in Escherichia coli rrnB t1 terminator consists of two modules . The upstream module includes the conserved sequence and the downstream one is U-rich . RESULTS: Elongation complexes of T7 RNA polymerase paused 2 bp before reaching the termination site at a 500 microM concentration of NTP . At 5-50 microM NTP, however, they paused and terminated there or resumed elongation beyond the termination site . Only at higher concentrations of NTP (500 microM), the pause complex proceeded slowly to and became incompetent at the termination site . At 4 bp or more before the termination site, the unprotected single-stranded region of transcription bubble shrank at the trailing edge to 4-5 bp from approximately 10 bp, resulting from duplex formation of the conserved sequence . The pause and bubble collapse were not observed with an inactive mutant of the termination signal . CONCLUSION: Sequence-specific termination requires the slow elongation mode of paused conformation, working only at high concentrations of NTP for a few bp prior to the RNA release site . The collapse of bubble that was observed several base pairs before the termination site and/or the resulting duplex might subsequently lead to the paused conformation of T7 elongation complexes.

Biochemistry, 2001 May 1, 40(17), 5200 - 7
Bacteriophage T7 RNA polymerase transcription elongation is inhibited by site-specific, stereospecific benzo{c}phenanthrene diol epoxide DNA lesions; Roth RB et al.; Benzo{c}phenanthrene diol epoxide (B{c}PhDE), the ultimate carcinogenic metabolite of the environmental pollutant benzo{c}phenanthrene, reacts with DNA primarily at the exocyclic amino groups of purines, forming B{c}PhDE-DNA adducts that differ in their stereochemical configurations and their effect on biological processes such as transcription . To determine the effect of these stereoisomers on RNA synthesis, in vitro T7 RNA polymerase transcription assays were performed using DNA templates modified on the transcribed strand by either a site-specific (+)-trans- or (-)-trans-anti-B{c}PhDE-N(6)-dA lesion located within the sequence 5'-CTCTCACTTCC-3' . The results show that both (-)-trans-anti-B{c}PhDE-N(6)-dA and (+)-trans-anti-B{c}PhDE-N(6)-dA block RNA synthesis . Furthermore, both B{c}PhDE-dA stereoisomeric adducts lead to lower levels of initiation of transcription relative to that observed using an unmodified DNA template . In contrast to these results, placement of the adduct on the nontranscribed strand within the template does not impede transcription elongation . In addition to the assessment of the effect of the lesions on transcription elongation, the resulting transcripts were characterized in terms of their base composition . A high level of base misincorporation is detected at the 3'-ends of truncated transcripts, with guanosine being most frequently incorporated opposite the modified nucleotide rather than the expected uridine . This result supports the notion that translocation past a modified base in a DNA template relies in part on correct base incorporation, and suggests that stalling of RNA polymerases at damaged sites in DNA may well be dependent on both the presence of the lesion and the base which is incorporated opposite the modified nucleotide.

Protein Sci, 2001 May, 10(5), 979 - 87
Constrained modeling of spin-labeled major coat protein mutants from M13 bacteriophage in a phospholipid bilayer; Bashtovyy D et al.; The family of three-dimensional molecular structures of the major coat protein from the M13 bacteriophage, which was determined in detergent micelles by NMR methods, has been analyzed by constrained geometry optimization in a phospholipid environment . A single-layer solvation shell of dioleoyl phosphatidylcholine lipids was built around the protein, after replacing single residues by cysteines with a covalently attached maleimide spin label . Both the residues substituted and the phospholipid were chosen for comparison with site-directed spin labeling EPR measurements of distance and local mobility made previously on membranous assemblies of the M13 coat protein purified from viable mutants . The main criteria for identifying promising candidate structures, out of the 300 single-residue mutant models generated for the membranous state, were 1) lack of steric conflicts with the phospholipid bilayer, 2) good match of the positions of spin-labeled residues along the membrane normal with EPR measurements, and 3) a good match between the sequence profiles of local rotational freedom and a structural restriction parameter for the spin-labeled residues obtained from the model . A single subclass of structure has been identified that best satisfies these criteria simultaneously . The model presented here is useful for the interpretation of future experimental data on membranous M13 coat protein systems . It is also a good starting point for full-scale molecular dynamics simulations and for the design of further site-specific spectroscopic experiments.

Res Microbiol, 2001 Mar, 152(2), 187 - 91
Improving the display of proteins on filamentous phage; Jestin JL et al.; In phage display technology, polypeptides are displayed on the surface of filamentous bacteriophage by genetic fusion to a coat protein . However, the fraction of phage particles bearing the fusion protein can be low . Here we found that we could improve the display of a protein (Stoffel fragment of Taq polymerase fused to the p3 protein of the phage) by mutation of the signal sequence and use of helper phage with a protease-cleavable coat protein . Over multiple rounds of infection, proteolysis and binding to an anti-Taq antibody, we were able to select strongly for display of the fusion protein (> 50-fold), and for mutations in the translation initiation region and in the signal sequence of the fusion . This suggests a general means of improving the display of proteins on phage.

Cancer Immunol Immunother, 2001 Mar, 50(1), 51 - 9
In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody; Roovers RC et al.; Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours . The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting . We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages . Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro . One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells . The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding . It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 x 10(-2) s-1 . To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (gamma 1) hinge region and CH3 domain . This "minibody" was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis . These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting.

Virology, 2001 Apr 25, 283(1), 49 - 58
Capsid size determination in the P2-P4 bacteriophage system: suppression of sir mutations in P2's capsid gene N by supersid mutations in P4's external scaffold gene sid; Kim KJ et al.; The sid gene of the P2-dependent phage P4 provides an external scaffold so P2 N gene encoded protomers assemble as T = 4 capsids rather than as P2's T = 7 capsids . Mutations (sir) in the middle of N interfere with Sid's function . We describe a new P4 mutant class, nms ("supersid") mutations, which direct also P2 sir to provide small capsids . Three different nms mutations were located near the sid end, commingled with sid(-) mutations . Suppression of sir by nms is not allele-specific . Our results favor this interpretation of capsid size control: (i) sir mutations reduce pN protomer flexibility and thereby interfere with the generation of T = 4 compatible hexons; (ii) the C-termini of Sid molecules link up when forming the scaffold; nms mutations strengthen these Sid-Sid contacts and thus allow the scaffold to force even sir-type protomers to form T = 4 compatible hexons . Some related findings concern suppression of N ts mutations by P4 .

J Biol Chem, 2001 Jun 29, 276(26), 23268 - 74 Epub 2001 Apr 17.
Effects of 3' terminus modifications on mRNA functional decay during in vitro protein synthesis; Lee K et al.; The pcnB gene, which encodes the principal poly(A) polymerase of Escherichia coli, promotes 3'-polyadenylation and chemical decay of mRNA . However, there is no evidence that pcnB-mediated mRNA destabilization decreases protein synthesis, suggesting that polyadenylation may enhance translational efficiency . Using in vitro translation by E . coli cell extracts and toeprinting analysis of transcripts encoded by the chloramphenicol acetyltransferase (CAT) and beta-galactosidase genes to investigate this notion, we found no effect of poly(A) tails on protein synthesis . However, we observed that 3'-polyguanylation delayed the chemical decay of CAT mRNA and, even more dramatically, increased the ability of CAT mRNA to produce enzymatically active full-length protein in 30 S E . coli cell fractions . This resulted from interference with the primary mechanism for inactivation of CAT transcript function in cell extracts, which occurred by 3'-exonucleolytic degradation rather than endonucleolytic fragmentation by RNase E . Using bacteriophage T7 RNA polymerase to install poly(G) tails on mRNAs transcribed from polymerase chain reaction-generated DNA templates, we observed sharply increased synthesis of active proteins in vitro in coupled transcription/translation reactions . The ability of poly(G) tails to functionally stabilize transcripts from polymerase chain reaction-generated templates allows proteins encoded by translational open reading frames on genomic DNA or cDNA to be synthesized directly and efficiently in vitro.

J Biol Chem, 2001 Jul 6, 276(27), 25236 - 42 Epub 2001 Apr 17.
Identification and mapping of protein-protein interactions between gp32 and gp59 by cross-linking; Ishmael FT et al.; The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination . Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA requires gp59 to remove gp32 and replace it with gp41 . Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of gp32 and Cys-42 of gp59 . Since Cys-166 lies in the DNA binding core domain of gp32, this interaction may affect the association of gp32 with DNA . In the presence of gp32 or DNA, gp59 is capable of forming a multimer consisting of at least five gp59 subunits . Kinetics studies suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that gp59 is capable of forming a hexamer (Raney, K . D., Carver, T . E., and Benkovic, S . J . (1996) J . Biol . Chem . 271, 14074-14081) . The C-terminal A-domain of gp32 is needed for gp59 oligomer formation . Cross-linking has established that gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species . The results support a model in which gp59 binds to gp32 on a replication fork, destabilizing the gp32-single-stranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at the replication fork.

Anal Chem, 2001 Mar 15, 73(6), 1277 - 85
Identification of bacteriophage MS2 coat protein from E . coli lysates via ion trap collisional activation of intact protein ions; Cargile BJ et al.; Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E . coli lysates . Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching . The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues . The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion . Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server . These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein . These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap.

Biol Pharm Bull, 2001 Apr, 24(4), 418 - 21
Involvement of reactive oxygen species in hemoglobin oxidation and virus inactivation by 1,9-dimethylmethylene blue phototreatment; Hirayama J et al.; The participation of reactive oxygen species (ROS) in virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment in stroma-free hemoglobin (SFH) was investigated with the use of scavengers, quenchers and enhancer . Virus (R17 bacteriophage) photoinactivation by either activated monomer or dimer DMMB was suppressed by sodium azide (singlet oxygen quencher) and promoted by the substitution of H2O for deuterium oxide (D2O), which is known to prolong the lifespan of singlet oxygen . There was no or little effect of mannitol (hydroxyl radical scavenger) and superoxide dismutase (superoxide scavenger) on the photoinactivation . Similar experiments were conducted to investigate the mechanism of methemoglobin (Met-Hb) formation by the activated monomer of DMMB . There was little effect of the singlet oxygen quencher, histidine, or the enhancer, D2O, on Met-Hb formation . However, rutin, which inhibits not only singlet oxygen but also other ROS, and mannitol supressed the formation of Met-Hb by activated monomer . The addition of superoxide dismutase (SOD) did not inhibit the formation . In contrast to the activity of the DMMB monomer, that of the dimer was inhibited by histidine and enhanced by D2O . The addition of neither mannitol nor SOD affected Met-Hb formation by activated dimer . These results collectively suggest that virus photoinactivation by the activated monomer and dimer of DMMB as well as Met-Hb formation by the activated dimer proceed via a singlet oxygen mediated pathway . In contrast, singlet oxygen may play a less important role in Met-Hb formation by the activated monomer.

J Mol Biol, 2001 Apr 20, 308(1), 9 - 14
The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold; Maxwell KL et al.; Protein W (gpW) from bacteriophage lambda is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis . Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA . Thus, gpW is an intriguing subject for detailed structural studies . We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two alpha-helices and a single two-stranded beta-sheet arranged around a well-packed hydrophobic core . The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution .

Mol Microbiol, 2001 Apr, 40(1), 1 - 8
No syringes please, ejection of phage T7 DNA from the virion is enzyme driven; Molineux IJ; Development of a sensitive assay that measures the rate of cellular internalization of an infecting bacteriophage T7 genome has led to surprising observations on the initiation of infection . Proteins ejected from the phage virion probably function as an extensible tail to form a channel across the cell envelope . This channel is subsequently used for translocating the phage genome into the cell . One of these ejected proteins also controls the amount of DNA that enters the cell, rendering subsequent internalization of the remainder of the genome dependent on transcription . Mutations affecting this protein allow the entire phage genome to enter a cell by the transcription-independent process . This process exhibits pseudo-zero-order reaction kinetics and a temperature dependence of translocation rate that are not expected if DNA ejection from a phage capsid were caused by a physical process . The temperature dependence of transcription-independent T7 DNA translocation rate is similar to those of enzyme-catalysed reactions . Current data suggest a highly speculative model, in which two of the proteins ejected from the phage head establish a molecular motor that ratchets the phage genome into the cell.

Mol Biochem Parasitol, 2001 Apr 6, 113(2), 261 - 9
Identification and characterization of a Plasmodium falciparum RNA polymerase gene with similarity to mitochondrial RNA polymerases; Li J et al.; Nearly all mitochondrial RNA polymerase genes identified to date are encoded in the nucleus and have similarities to T3 and T7 bacteriophage RNA polymerases . Some chloroplast genes are also transcribed by T3/T7 phage-like RNA polymerases, raising the possibility that the apicomplexan parasites, which have both a mitochondrion and a plastid, might have two such genes . As part of an investigation of Plasmodium falciparum organelle transcription, we initiated a search for T3/T7 bacteriophage-like RNA polymerase genes . We employed degenerate primers based on highly conserved plant, animal and fungal mitochondrial RNA polymerase sequences to amplify corresponding P . falciparum sequences by polymerase chain reaction (PCR) . Less well-conserved flanking sequences were obtained by inverse PCR . The resulting sequence predicts a 1503 amino acid open reading frame with similarity to other T3/T7 phage-like RNA polymerases . Essential amino acids that have been identified in T7 mutant analyses are conserved in the P . falciparum RNA polymerase gene . Comparison of the sequence with preliminary data from the P . falciparum genome sequencing project revealed strain heterogeneity within two regions of the gene . The amino-terminal predicted amino acid sequence of the RNA polymerase gene has similarities to mitochondrial targeting sequences . Taken together, these points suggest that we have identified the P . falciparum mitochondrial RNA polymerase gene.

Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4328 - 33 Epub 2001 Apr 03.
Translocation pathway of protein substrates in ClpAP protease; Ishikawa T et al.; Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases . These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion . In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings . We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA . In complexes stabilized by ATPgammaS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA . On ATP addition, RepA is translocated through approximately 150 A into the digestion chamber inside ClpP . Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer . When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites . These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.

J Virol, 2001 May, 75(9), 4176 - 83
Genetic targeting of an adenovirus vector via replacement of the fiber protein with the phage T4 fibritin; Krasnykh V et al.; The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues . This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue . Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand . Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand . Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules . Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner.

Biochim Biophys Acta, 2001 Apr 2, 1511(2), 309 - 16
Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes; Houbiers MC et al.; Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane after phage infection of Escherichia coli . The way of insertion of this small protein (32 amino acids) into membranes is still unknown . Here we show that the protein is able to insert in monolayers . The limiting surface pressure of 35 mN/m for 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol lipid systems indicates that this spontaneous insertion can also occur in vivo . By carboxyfluorescein leakage experiments of vesicles it is demonstrated that protein monomers, or at least small aggregates, are more effective in releasing carboxyfluorescein than highly aggregated protein . The final orientation of the protein in the bilayer after insertion was addressed by proteinase K digestion, thereby making use of the unique C-terminal location of the antigenic binding site . After insertion the C-terminus is still available for the enzymatic digestion, while the N-terminus is not . This leads to the overall conclusion that the protein is able to insert spontaneously into membranes without the need of any machinery or transmembrane gradient, with the positively charged C-terminus remaining on the outside . The orientation after insertion of gene 9 protein is in agreement with the 'positive inside rule'.

Biochim Biophys Acta, 2001 Apr 2, 1511(2), 224 - 35
Conformation and orientation of the gene 9 minor coat protein of bacteriophage M13 in phospholipid bilayers; Houbiers MC et al.; The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in model membrane systems, which varied in lipid head group and lipid acyl chain composition . By using FTIR spectroscopy and subsequent band analysis a quantitative analysis of the secondary structure of the protein was obtained . The secondary structure of the gene 9 protein predominantly consists of alpha-helical (67%) and turn (33%) structures . The turn structure is likely to be located C-terminally where it has a function in recognizing the phage DNA during bacteriophage assembly . Attenuated total reflection FTIR spectroscopy was used to determine the orientation of gene 9 protein in the membrane, revealing that the alpha-helical domain is mainly transmembrane . The conformational and orientational measurements result in two models for the gene 9 protein in the membrane: a single transmembrane helix model and a two-helix model consisting of a 15 amino acid long transmembrane helix and a 10 amino acid long helix oriented parallel to the membrane plane . Potential structural consequences for both models are discussed.

J Mol Biol, 2001 Apr 6, 307(4), 1145 - 58
The active site of the junction-resolving enzyme T7 endonuclease I; Declais AC et al.; Endonuclease I is a junction-resolving enzyme encoded by bacteriophage T7, that selectively binds and cleaves four-way DNA junctions . We have recently solved the structure of this dimeric enzyme at atomic resolution, and identified the probable catalytic residues . The putative active site comprises the side-chains of three acidic amino acids (Glu20, Asp55 and Glu65) together with a lysine residue (Lys67), and shares strong similarities with a number of type II restriction enzymes . However, it differs from a typical restriction enzyme as the proposed catalytic residues in both active sites are contributed by both polypeptides of the dimer . Mutagenesis experiments confirm the importance of all the proposed active site residues . We have carried out in vitro complementation experiments using heterodimers formed from mutants in different active site residues, showing that Glu20 is located on a different monomer from the remaining amino acid residues comprising the active site . These experiments confirm that the helix-exchanged architecture of the enzyme creates a mixed active site in solution . Such a composite active site structure should result in unilateral cleavage by the complemented heterodimer; this has been confirmed by the use of a cruciform substrate . Based upon analogy with closely similar restriction enzyme active sites and our mutagenesis experiments, we propose a two-metal ion mechanism for the hydrolytic cleavage of DNA junctions .

Biochemistry, 2001 Apr 10, 40(14), 4459 - 77
Molecular mechanisms of the functional coupling of the helicase (gp41) and polymerase (gp43) of bacteriophage T4 within the DNA replication fork; Delagoutte E et al.; Processive strand-displacement DNA synthesis with the T4 replication system requires functional "coupling" between the DNA polymerase (gp43) and the helicase (gp41) . To define the physical basis of this functional coupling, we have used analytical ultracentrifugation to show that gp43 is a monomeric species at physiological protein concentrations and that gp41 and gp43 do not physically interact in the absence of DNA, suggesting that the functional coupling between gp41 and gp43 depends significantly on interactions modulated by the replication fork DNA . Results from strand-displacement DNA synthesis show that a minimal gp41-gp43 replication complex can perform strand-displacement synthesis at approximately 90 nts/s in a solution containing poly(ethylene glycol) to drive helicase loading . In contrast, neither the Klenow fragment of Escherichia coli DNA polymerase I nor the T7 DNA polymerase, both of which are nonprocessive polymerases, can carry out strand-displacement DNA synthesis with gp41, suggesting that the functional helicase-polymerase coupling may require the homologous system . However, we show that a heterologous helicase-polymerase pair can work if the polymerase is processive . Strand-displacement DNA synthesis using the gp41 helicase with the T4 DNA polymerase holoenzyme or the phage T7 DNA polymerase-thioredoxin complex, both of which are processive, proceeds at the rate of approximately 250 nts/s . However, replication fork assembly is less efficient with the heterologous helicase-polymerase pair . Therefore, a processive (homologous or heterologous) "trailing" DNA polymerase is sufficient to improve gp41 processivity and unwinding activity in the elongation stage of the helicase reaction, and specific T4 helicase-polymerase coupling becomes significant only in the assembly (or initiation) stage.

Biochemistry, 2001 Apr 10, 40(14), 4293 - 302
Solution structure of the transcriptional activation domain of the bacteriophage T4 protein, MotA; Li N et al.; Bacteriophage T4 encodes a transcription factor, MotA, that binds to the -30 region of middle-mode promoters and activates transcription by host RNA polymerase . The crystal structure of the N-terminal domain of MotA (MotNF) revealed a six-helix domain in which the two C-terminal alpha-helices mediate the formation of a dimer via a coiled-coil motif and hydrophobic interactions . This structure suggested that full-length MotA binds DNA as a dimer, but subsequent biochemical results have shown that a monomeric form of MotA binds DNA . In this study, gel filtration chromatography, dynamic light scattering, and NMR-based diffusion measurements show conclusively that MotNF is a monomer, and not a dimer, in solution . In addition, we have determined the monomeric solution structure of MotNF using NMR spectroscopy, and have compared this with the dimer structure observed in crystals . The core of the protein assumes the same helical conformation in solution and in crystals, but important differences are observed at the extreme C-terminus . In solution, helix alpha5 is followed by five disordered residues that probably link the N-terminal and C-terminal domains of MotA . In crystals, helix alpha5 forms the dimer interface and is followed by a short sixth helix that further stabilizes the dimer configuration . The solution structure of MotNF supports the conclusion that MotA functions as a monomer, and suggests that the existence of the sixth helix in crystals is a consequence of crystal packing . Our work highlights the importance of investigating protein structures in both crystals and solution to fully understand biomolecular structure and to accurately deduce relationships between structure and function.

Mol Cell Biol, 2001 Apr, 21(8), 2706 - 15
UvsW protein regulates bacteriophage T4 origin-dependent replication by unwinding R-loops; Dudas KC et al.; The UvsW protein of bacteriophage T4 is involved in many aspects of phage DNA metabolism, including repair, recombination, and recombination-dependent replication . UvsW has also been implicated in the repression of origin-dependent replication at late times of infection, when UvsW is normally synthesized . Two well-characterized T4 origins, ori(uvsY) and ori(34), are believed to initiate replication through an R-loop mechanism . Here we provide both in vivo and in vitro evidence that UvsW is an RNA-DNA helicase that catalyzes the dissociation of RNA from origin R-loops . Two-dimensional gel analyses show that the replicative intermediates formed at ori(uvsY) persist longer in a uvsW mutant infection than in a wild-type infection . In addition, the inappropriate early expression of UvsW protein results in the loss of these replicative intermediates . Using a synthetic origin R-loop, we also demonstrate that purified UvsW functions as a helicase that efficiently dissociates RNA from R-loops . These and previous results from a number of studies provide strong evidence that UvsW is a molecular switch that allows T4 replication to progress from a mode that initiates from R-loops at origins to a mode that initiates from D-loops formed by recombination proteins.

J Biol Chem, 2001 Jun 15, 276(24), 21250 - 6 Epub 2001 Mar 29.
A single amino acid substitution within a coiled-coil motif changes the assembly of a 53-amino acid protein from two-dimensional sheets to filamentous structures; Bravo A et al.; The bacteriophage phi29 replication protein p1 self-interacts in vitro, generating highly ordered structures . Specifically, the 53-amino acid protein p1DeltaN33, which retains the sequence of p1 spanning amino acids Met(34) to Lys(85), assembles into two-dimensional protofilament sheets . The region of protein p1 located between residues Glu(38) and Asn(65) presumably forms an alpha-helical coiled-coil structure . Here we have examined the role of this coiled-coil sequence in the formation of protofilament sheets . Using sedimentation assays and negative-stain electron microscopy analysis, we demonstrate that residues Leu(46), Met(53), and Leu(60), but not Leu(39), are essential for p1DeltaN33 assembly into sheets . Remarkably, replacement of Leu(46) by Val shifts the pathway of molecular assembly, leading to the formation of filamentous polymers approximately 10 nm in diameter . These results show, for the first time, that a short coiled-coil motif can mediate protein assembly into protofilament sheet structures.

Curr Opin Microbiol, 2001 Apr, 4(2), 138 - 44
Transcriptional regulation at a distance in bacteria; Xu H et al.; Transcriptional enhancers are cis-acting DNA elements that are binding sites for regulatory proteins and function at large distances from promoter elements to stimulate transcription . Once thought to be unique to eukaryotes, enhancer-like elements have been discovered in a wide variety of bacteria . The regulatory proteins that bind to these bacterial enhancers must contact RNA polymerase to activate transcription . In principle, interactions between bacterial enhancer-binding proteins and RNA polymerase can occur by either DNA looping or tracking of the enhancer-binding protein along the DNA . Paradigms for each of these methods are found in bacterial systems . Activators of sigma(54)-RNA polymerase holoenzyme contact polymerase by DNA looping, while bacteriophage T4 gp45 functions as a sliding clamp that tracks along DNA until it engages RNA polymerase . Significant advances have been made over the last few years towards understanding the mechanisms by which bacterial enhancer-binding proteins activate transcription, but important aspects of these mechanisms are still poorly defined.

Comb Chem High Throughput Screen, 2001 Apr, 4(2), 193 - 205
High-throughput screening of surface displayed gene products; Walter G et al.; With the human genome project approaching completion, there is a growing interest in functional analysis of gene products . The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes . As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules . Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence . High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling . This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput . Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands . Binding activity is confirmed by magnetic particle ELISA in the microtitre format . The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable . Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.

Comb Chem High Throughput Screen, 2001 Apr, 4(2), 121 - 33
Alternative bacteriophage display systems; Castagnoli L et al.; Filamentous phage has been extensively used to implement various aspects of phage display technology . The success of these organisms as vectors to present foreign peptides and to link them to their coding sequences is a consequence of their structural and biological characteristics . Some of these properties, however, represent a limitation when one attempts to display proteins that cannot be efficiently exported through the bacterial membrane or do not fold properly in the periplasm . Thus, the desirability of developing alternative display systems was recognised recently and led to the development of a different class of display vectors that assemble their capsid in the cytoplasm and are released via cell lysis . This review describes and compares the properties of these alternative display systems.

J Biol Chem, 2001 Jun 15, 276(24), 21809 - 20 Epub 2001 Mar 28.
A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization; Kato M et al.; The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them . Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities . The primase is also required for the utilization of RNA primers by T7 DNA polymerase . It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis . In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity . Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates . The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis . These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.

J Biol Chem, 2001 Jun 8, 276(23), 20175 - 81 Epub 2001 Feb 27.
Biophysical characterization of the DNA binding domain of gpNu1, a viral DNA packaging protein; Bain DL et al.; Terminase enzymes are common to double-stranded DNA viruses . These enzymes "package" the viral genome into a pre-formed capsid . Terminase from bacteriophage lambda is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits . We have described the expression and biochemical characterization of gpNu1DeltaK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C . E . (1999) Biochemistry 38, 465-477) . Here we present a biophysical characterization of this construct . Thermally induced loss of secondary and tertiary structures is fully reversible . Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative . NMR and limited proteolysis data suggest that approximately 30 amino acids of gpNu1DeltaK100 are solvent-exposed and highly flexible . We therefore constructed gpNu1DeltaE68, a protein consisting of the N-terminal 68 residues of gpNu1 . gpNu1DeltaE68 is a dimer with no evidence of dissociation or further aggregation . Thermally induced unfolding of gpNu1DeltaE68 is reversible, with concomitant loss of both secondary and tertiary structure . The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled . The data suggest that gpNu1DeltaE68 represents the minimal DNA binding domain of gpNu1 . We further suggest that the C-terminal approximately 30 residues in gpNu1DeltaK100 adopt a pseudo-stable alpha-helix that extends from the folded core of the protein . A model describing the role of this helix in the assembly of the packaging apparatus is discussed.

J Biol Chem, 2001 May 4, 276(18), 14933 - 8 Epub 2001 Feb 13.
ADP-dependent DNA strand exchange by the mutant {P67G/E68A} RecA protein; Nayak S et al.; We have prepared a mutant RecA protein in which proline 67 and glutamic acid 68 in the NTP binding site were replaced by a glycine and alanine residue, respectively . The {P67G/E68A}RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of ATP and is able to promote the standard ATP-dependent three-strand exchange reaction between a circular bacteriophage phiX174 (phiX) single-stranded DNA molecule and a homologous linear phiX double-stranded (ds) DNA molecule (5.4 kilobase pairs) . The strand exchange activity differs from that of the wild type RecA protein, however, in that it is (i) completely inhibited by an ATP regeneration system, and (ii) strongly stimulated by the addition of high concentrations of ADP to the reaction solution . These results indicate that the strand exchange activity of the {P67G/E68A}RecA protein is dependent on the presence of both ATP and ADP . The ADP dependence of the reaction is reduced or eliminated when (i) a shorter linear phiX dsDNA fragment (1.1 kilobase pairs) is substituted for the full-length linear phiX dsDNA substrate, or (ii) the Mg(2+) concentration is reduced to a level just sufficient to complex the ATP present in the reaction solution . These results indicate that it is the branch migration phase (and not the initial pairing step) of the {P67G/E68A}RecA protein-promoted strand exchange reaction that is dependent on ADP . It is likely that the {P67G/E68A}RecA mutation has revealed a requirement for ADP that also exists (but is not as readily apparent) in the strand exchange reaction of the wild type RecA protein.

J Biol Chem, 2001 Jun 8, 276(23), 19836 - 44 Epub 2001 Mar 06.
Intrinsic DNA distortion of the bacteriophage Mu momP1 promoter is a negative regulator of its transcription . A novel mode of regulation of toxic gene expression; Basak S et al.; The momP1 promoter of the bacteriophage Mu mom operon is an example of a weak promoter . It contains a 19-base pair suboptimal spacer between the -35 (ACCACA) and -10 (TAGAAT) hexamers . Escherichia coli RNA polymerase is unable to bind to momP1 on its own . DNA distortion caused by the presence of a run of six T nucleotides overlapping the 5' end of the -10 element might prevent RNA polymerase from binding to momP1 . To investigate the influence of the T(6) run on momP1 expression, defined substitution mutations were introduced by site-directed mutagenesis . In vitro probing experiments with copper phenanthroline ((OP)(2)Cu) and DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared with the wild type, the mutants showed an increase (variable) in momP1 promoter activity in vivo . Promoter strength analyses were in agreement with the ability of these mutants to form open complexes as well as to produce momP1-specific transcripts . No significant role is attributed to the overlapping and divergently organized promoter, momP2, in the expression of momP1 activity, as determined by promoter disruption analysis . These data support the view that an intrinsic DNA distortion in the spacer region of momP1 acts in cis as a negative element in mom operon transcription . This is a novel mechanism of regulation of toxic gene expression.

J Biol Chem, 2001 Apr 27, 276(17), 13695 - 700 Epub 2001 Jan 25.
Revisiting the lysogenization control of bacteriophage lambda . Identification and characterization of a new host component, HflD; Kihara A et al.; Upon infection to the Escherichia coli cell, the genome of bacteriophage lambda either replicates to form new progenies (lytic growth) or integrates into the host chromosome (lysogenization) . The lambda CII protein is a key determinant in the lysis-lysogeny decision . It is a short-lived transcription activator for the lambda genes essential for lysogeny establishment . In this study, we isolated a new class of hfl (high frequency lysogenization) mutants of E . coli, using a new selection for enhancement of CII-stimulated transcription . The gene affected was termed hflD, which encodes a protein of 213 amino acids . An hflD-disrupted mutant indeed showed an Hfl phenotype, indicating that HflD acts to down-regulate lysogenization . HflD is associated peripherally with the cytoplasmic membrane . Its interaction with CII was demonstrated in vitro using purified proteins as well as in vivo using the bacterial two-hybrid system . Pulse-chase examinations demonstrated that the HflD function is required for the rapid in vivo degradation of CII, although it interfered with FtsH-mediated CII proteolysis in an in vitro reaction system using detergent-solubilized components . We suggest that HflD is a factor that sequesters CII from the target promoters and recruits it to the membrane where the FtsH protease is localized.

J Biol Chem, 2001 Apr 27, 276(17), 14075 - 82 Epub 2001 Jan 25.
Peculiar 2-aminopurine fluorescence monitors the dynamics of open complex formation by bacteriophage T7 RNA polymerase; Bandwar RP et al.; The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA polymerase was investigated using 2-aminopurine (2-AP) modified promoters . 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases . All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner . The 2-AP at -4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA polymerase . Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at -4 from the guanine at -5, during open complex formation . The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA polymerase active site upon promoter melting for template directed RNA synthesis . Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA polymerase binds promoter double-stranded DNA by a three-step mechanism . The initial collision complex or a closed complex, ED(c) is formed with a K(d) of 1.8 microm . This complex isomerizes to an open complex, ED(o1), in an energetically unfavorable reaction with an equilibrium constant of 0.12 . The ED(o1) further isomerizes to a more stable open complex, ED(o2), with a rate constant around 300 s(-1) . Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s(-1).

J Biol Chem, 2001 May 18, 276(20), 17324 - 31 Epub 2001 Feb 08.
The role of leucine 191 of Escherichia coli uracil DNA glycosylase in the formation of a highly stable complex with the substrate mimic, ugi, and in uracil excision from the synthetic substrates; Handa P et al.; Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, initiates the uracil excision repair pathway . Ugi, a bacteriophage-encoded peptide, potently inhibits UDGs by serving as a remarkable substrate mimic . Structure determination of UDGs has identified regions important for the exquisite specificity in the detection and removal of uracils from DNA and in their interaction with Ugi . In this study, we carried out mutational analysis of the Escherichia coli UDG at Leu191 within the 187HPSPLS192 motif (DNA intercalation loop) . We show that with the decrease in side chain length at position 191, the stability of the UDG-Ugi complexes regresses . Further, while the L191V and L191F mutants were as efficient as the wild type protein, the L191A and L191G mutants retained only 10 and 1% of the enzymatic activity, respectively . Importantly, however, substitution of Leu191 with smaller side chains had no effect on the relative efficiencies of uracil excision from the single-stranded and a corresponding double-stranded substrate . Our results suggest that leucine within the HPSPLS motif is crucial for the uracil excision activity of UDG, and it contributes to the formation of a physiologically irreversible complex with Ugi . We also envisage a role for Leu191 in stabilizing the productive enzyme-substrate complex.

J Biol Chem, 2001 Jun 8, 276(23), 19691 - 8 Epub 2001 Feb 27.
Evidence for a functional monomeric form of the bacteriophage T4 DdA helicase . Dda does not form stable oligomeric structures; Morris PD et al.; The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA . In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated . Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species . The specific activity for ssDNA stimulated ATPase activity was independent of Dda concentration . Dda was mutated to produce an ATPase-deficient protein (K38A Dda) by altering a residue within a conserved, nucleotide binding loop . The helicase activity of K38A Dda was inactivated, although DNA binding properties were similar to Dda . In the presence of limiting DNA substrate, the rate of unwinding by Dda was not changed; however, the amplitude of product formation was reduced in the presence of increasing concentrations of K38A Dda . The reduction was between that expected for a monomeric or dimeric helicase based on simple competition for substrate binding . When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no reduction in the observed rate for strand separation . Taken together, these results indicate that oligomerization of Dda is not required for DNA unwinding.

J Biol Chem, 2001 Apr 27, 276(17), 14271 - 8 Epub 2001 Jan 18.
Foreign DNA integration . Genome-wide perturbations of methylation and transcription in the recipient genomes; Muller K et al.; In hamster cells transgenic for the DNA of adenovirus type 12 (Ad12) or for the DNA of bacteriophage lambda, the patterns of DNA methylation in specific cellular genes or DNA segments remote from the site of transgene insertion were altered . In the present report, a wide scope of cellular DNA segments and genes was analyzed . The technique of methylation-sensitive representational difference analysis (MS-RDA) was based on a subtractive hybridization protocol after selecting against DNA segments that were heavily methylated and hence rarely cleaved by the methylation-sensitive endonuclease HpaII . The MS-RDA protocol led to the isolation of several cellular DNA segments that were indeed more heavily methylated in lambda DNA-transgenic hamster cell lines . By applying the suppressive subtractive hybridization technique to cDNA preparations from nontransgenic and Ad12-transformed or lambda DNA-transgenic hamster cells, several cellular genes with altered transcription patterns were cloned from Ad12-transformed or lambda DNA-transgenic hamster cells . Many of the DNA segments with altered methylation, which were isolated by a newly developed methylation-sensitive amplicon subtraction protocol, and cDNA fragments derived from genes with altered transcription patterns were identified by their nucleotide sequences . In control experiments, no differences in gene expression or DNA methylation patterns were detectable among individual nontransgenic BHK21 cell clones . In one mouse line transgenic for the DNA of bacteriophage lambda, hypermethylation was observed in the imprinted Igf2r gene in DNA from heart muscle . Two mouse lines transgenic for an adenovirus promoter-indicator gene construct showed hypomethylation in the interleukin 10 and Igf2r loci . We conclude that the insertion of foreign DNA into an established mammalian genome can lead to alterations in cellular DNA methylation and transcription patterns . It is conceivable that the genes and DNA segments affected by these alterations depend on the site(s) of foreign DNA insertion.

Biochimie, 2001 Feb, 83(2), 261 - 7
The GemA protein of phage Mu and the GyrB gyrase subunit of Escherichia coli: the search for targets and interactions leading to the reversion of Mu-induced mutations; Abbes C et al.; The mutant bacteriophage Mugem2(Ts), known to synchronize the division of infected cells, to relax DNA supercoiling and, as prophage, to give rise to precisely excised revertants, has been thought to overexpress the gemA-mor operon, and genetic evidence suggests that the B subunit of DNA gyrase (GyrB) is the target of action of GemA . In two different double hybrid tests presented here, we find no evidence of GemA-GyrB protein-protein interaction . We do observe a GemA-GemA interaction, however, indicating that GemA can dimerize . In lacZ::Mu lysogens, overexpression of the gemA-mor operon from a plasmid, under control of the L-arabinose inducible p(araBAD) promoter, does not permit the recovery of Lac(+) revertants . These observations suggest that GyrB is not the direct target of GemA action and that the various phenotypes of Mugem2(Ts) are not caused by overexpression of the gemA-mor operon.

J Bacteriol, 2001 Apr, 183(8), 2527 - 34
Cytoplasmic RNA Polymerase in Escherichia coli; Shepherd N et al.; To obtain an estimate for the concentration of free functional RNA polymerase in the bacterial cytoplasm, the content of RNA polymerase beta and beta' subunits in DNA-free minicells from the minicell-producing Escherichia coli strain chi925 was determined . In bacteria grown in Luria-Bertani medium at 2.5 doublings/h, 1.0% of the total protein was RNA polymerase . The concentration of cytoplasmic RNA polymerase beta and beta' subunits in minicells produced by this strain corresponded to about 17% (or 2.5 microM) of the value found in whole cells . Literature data suggest that a similar portion of cytoplasmic RNA polymerase subunits is in RNA polymerase assembly intermediates and imply that free functional RNA polymerase can form a small percentage of the total functional enzyme in the cell . On infection with bacteriophage T7, 20% of the minicells produced progeny phage, whereas infection in 80% of the cells was abortive . RNA polymerase subunits in lysozyme-freeze-thaw lysates of minicells were associated with minicell envelopes and were without detectable activity in an in vitro transcription assay . Together, these results suggest that most functional RNA polymerase is associated with the DNA and that little if any segregates into DNA-free minicells.

J Magn Reson, 2001 Mar, 149(1), 154 - 8
Characterization of the cholesteric phase of filamentous bacteriophage fd for molecular alignment; Barrientos LG et al.; Residual dipolar couplings arise from small degrees of alignment of molecules in a magnetic field and have proven to provide valuable structural information . Colloidal suspensions of rod-shaped viruses and bacteriophages constitute a frequently employed medium for imparting such alignment onto biomolecules . The stability and behavior of the liquid crystalline phases with respect to solution conditions such as pH, ionic strength, and temperature vary, and characterization should benefit practical applications as well as theoretical understanding . In this Communication we describe the pH dependence of the cholesteric liquid crystalline phase of the filamentous bacteriophage fd and demonstrate that the alignment tensor of the solute protein is modulated by pH . We also report the interesting observation that the relative sign of the residual dipolar coupling changes at low pH values . In addition, we demonstrate that the degree of alignment inversely scales with the lengths of the phage particles for phages with identical mass and charge per unit length .

Protein Sci, 2001 Jan, 10(1), 17 - 23
Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus; Matsumiya S et al.; The proliferating cell nuclear antigen (PCNA) is now recognized as one of the key proteins in DNA metabolic events because of its direct interactions with many proteins involved in important cellular processes . We have determined the crystal structure of PCNA from a hyperthermophilic archaeon, Pyrococcus furiosus (pfuPCNA), at 2.1 A resolution . pfuPCNA forms a toroidal, ring-shaped structure consisting of homotrimeric molecules, which is also observed in the PCNA crystals from human and yeast . The overall structure of pfuPCNA is highly conserved with other PCNA proteins, as well as with the bacterial ss clamp and the bacteriophage gp45 . This result shows that the three-dimensional structure of the sliding clamp is conserved in the three domains of life . pfuPCNA has two remarkable features compared with the human and yeast PCNA molecules: it has more ion pairs and fewer intermolecular main chain hydrogen bonds . The former may contribute to the thermal stability of pfuPCNA, and the latter may be the cause of the stimulatory effect of pfuPCNA on the DNA synthesizing activity of P . furiosus DNA polymerases in the absence of the clamp loader replication factor C in vitro.

Nucleic Acids Res . 2001 Apr 1;29(7):E40.
Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus; Nakano M et al.; A recombinant adenovirus (rAd) expressing Cre recombinase derived from bacteriophage P1 has already been extensively used for the conditional gene activation and inactivation strategies in mammalian systems . In this study, we generated AxCAFLP, a rAd expressing FLP recombinase derived from Saccharomyces cerevisiae and carried out quantitative comparisons with Cre-expressing rAd in both in vitro and in cultured cells to provide another efficient gene regulation system in mammalian cells . In the in vitro experiments, the relative recombination efficiency of FLP expressed in 293 cells infected with FLP-expressing rAd was approximately one-thirtieth that of Cre even at 30 degrees C, the optimum temperature for FLP activity, and was approximately one-ninetieth at 37 degrees C . Co-infection experiments in HeLa cells using a target rAd conditionally expressing LacZ under the control of FLP showed that an FLP-expressing rAd, infected at a multiplicity of infection (MOI) of 5, was able to activate the transgene in almost 100% of HeLa cells whereas the Cre-expressing rAd was sufficient at an MOI of 0.2 . Since an MOI of 5 is ordinarily used in rAd experiments, these results showed that the FLP-expressing rAd is useful for gene activation strategies and is probably applicable to a sequential gene regulation system in combination with Cre-expressing rAd in mammalian cells.

Nucleic Acids Res, 2001 Apr 1, 29(7), 1484 - 90
Methylation by a mutant T2 DNA {N(6)-adenine} methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage; Minko I et al.; Properties of a mutant bacteriophage T2 DNA {N:(6)-adenine} methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage . Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites . Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes . In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient . Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences . The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.

J Virol, 2001 Apr, 75(8), 3851 - 8
Hepatitis B virus X protein acts as a tumor promoter in development of diethylnitrosamine-induced preneoplastic lesions; Madden CR et al.; Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma . Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens . Although the mechanism for this cofactor role remains unknown, the ability of HBx to inhibit DNA repair and to influence cell cycle progression suggests two possible pathways . To investigate these possibilities in vivo, we treated double-transgenic mice that both express HBx (ATX mice) and possess a bacteriophage lambda transgene with the hepatocarcinogen diethylnitrosamine (DEN) . Histological examination of liver tissue confirmed that DEN-treated ATX mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates . Treatment of mice with DEN resulted in a six- to eightfold increase in the mutation frequency (MF), as measured by a functional analysis of the lambda transgene . HBx expression was confirmed by immunoprecipitation and Western blotting and was associated with a modest 23% increase in the MF . Importantly, the extent of hepatocellular proliferation in 14-day-old mice, as measured by the detection of proliferating cell nuclear antigen and by the incorporation of 5-bromo-2'-deoxyuridine, was determined to be approximately twofold higher in ATX livers than in wild-type livers . These results are consistent with a model in which HBx expression contributes to the development of DEN-mediated carcinogenesis by promoting the proliferation of altered hepatocytes rather than by directly interfering with the repair of DNA lesions.

DNA Res, 2001 Feb 28, 8(1), 11 - 22
Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12; Hayashi T et al.; Escherichia coli O157:H7 is a major food-borne infectious pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome . Here we report the complete chromosome sequence of an O157:H7 strain isolated from the Sakai outbreak, and the results of genomic comparison with a benign laboratory strain, K-12 MG1655 . The chromosome is 5.5 Mb in size, 859 Kb larger than that of K-12 . We identified a 4.1-Mb sequence highly conserved between the two strains, which may represent the fundamental backbone of the E . coli chromosome . The remaining 1.4-Mb sequence comprises of O157:H7-specific sequences, most of which are horizontally transferred foreign DNAs . The predominant roles of bacteriophages in the emergence of O157:H7 is evident by the presence of 24 prophages and prophage-like elements that occupy more than half of the O157:H7-specific sequences . The O157:H7 chromosome encodes 1632 proteins and 20 tRNAs that are not present in K-12 . Among these, at least 131 proteins are assumed to have virulence-related functions . Genome-wide codon usage analysis suggested that the O157:H7-specific tRNAs are involved in the efficient expression of the strain-specific genes . A complete set of the genes specific to O157:H7 presented here sheds new insight into the pathogenicity and the physiology of O157:H7, and will open a way to fully understand the molecular mechanisms underlying the O157:H7 infection.

Biochemistry (Mosc), 2001 Feb, 66(2), 141 - 6
Expression and properties of bacteriophage T4 gene product 11; Kurochkina LP et al.; A plasmid vector for expression of bacteriophage T4 gene product 11 (gp11) in E . coli cells has been constructed . Gp11 is a baseplate protein that connects short tail fibers providing irreversible adsorption of the virus on a cell . A method based on chromatography on hydroxyapatite has been developed for purification of recombinant gp11 . The protein is active in an in vitro complementation assay and transforms defective phage particles lacking gp11 into infective ones . Gel filtration data suggest that the biologically active protein is a trimer . According to CD spectroscopy and sequence analysis data, the polypeptide chain of gp11 contains not less than 20% alpha-helical segments, about 30% beta-structure, and belongs to the class of alpha/beta structural proteins.

Gene, 2001 Mar 7, 265(1-2), 25 - 36
Genetic analysis of the T4 holin: timing and topology; Ramanculov E et al.; The t protein of bacteriophage T4 shares with other holins the ability to cause the formation of a lethal membrane lesion which allows the phage endolysin to attack the peptidoglycan . Moreover, T, like other holins, acts in a saltatory manner at a precisely programmed time in the vegetative cycle . Unlike other holins, however, T has the unique ability to postpone its lethal function in response to a secondary infection by a T-even phage during the vegetative cycle . A signal transduction system that responds to the secondary infection is thought to be encoded by some of the numerous r genes, defined by mutations that abolish this lysis-inhibition (LIN) response . The primary structure of T differs from two main structural patterns found in more than 30 orthologous groups of holins . Genetic approaches were taken to probe the t sequence for features involved in membrane localization, functional timing and LIN regulation . Gene fusion analysis indicates that T has a single TMD near the N-terminus, with the bulk of the protein residing in the periplasm . Mapping and phenotypic analysis of deletion and point mutations in t indicates that the periplasmic domain of T is the major determinant of the timing mechanism and is involved in the LIN response.

Mol Microbiol, 2001 Mar, 39(5), 1259 - 71
Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila; Luneberg E et al.; We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila . In this study, the molecular mechanism for phase variation was investigated . We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation . Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units . Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin . In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence . Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function . As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant . The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype . Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes . Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

Mol Microbiol, 2001 Feb, 39(4), 823 - 34
The X philes: structure-specific endonucleases that resolve Holliday junctions; Sharples GJ; Genetic recombination is a critical cellular process that promotes evolutionary diversity, facilitates DNA repair and underpins genome duplication . It entails the reciprocal exchange of single strands between homologous DNA duplexes to form a four-way branched intermediate commonly referred to as the Holliday junction . DNA molecules interlinked in this way have to be separated in order to allow normal chromosome transmission at cell division . This resolution reaction is mediated by structure-specific endonucleases that catalyse dual-strand incision across the point of strand cross-over . Holliday junctions can also arise at stalled replication forks by reversing the direction of fork progression and annealing of nascent strands . Resolution of junctions in this instance generates a DNA break and thus serves to initiate rather than terminate recombination . Junction resolvases are generally small, homodimeric endonucleases with a high specificity for branched DNA . They use a metal-binding pocket to co-ordinate an activated water molecule for phosphodiester bond hydrolysis . In addition, most junction endonucleases modulate the structure of the junction upon binding, and some display a preference for cleavage at specific nucleotide target sequences . Holliday junction resolvases with distinct properties have been characterized from bacteriophages (T4 endo VII, T7 endo I, RusA and Rap), Bacteria (RuvC), Archaea (Hjc and Hje), yeast (CCE1) and poxviruses (A22R) . Recent studies have brought about a reappraisal of the origins of junction-specific endonucleases with the discovery that RuvC, CCE1 and A22R share a common catalytic core.

J Environ Monit, 2000 Aug, 2(4), 372 - 4
Isolation of coliphages specific to enterotoxigenic E . coli (ETEC); Jothikumar N et al.; Bacteriophages specific to Enterotoxigenic E . coli (ETEC) are reported for the first time . Out of 15 isolated phages only 10 were specific to strains of ETEC . All ten phages of dsDNA could be grouped into three different genotypes based on their RAPD patterns observed and it is likellly that they belong to only 3 different strains . The three phages yielded clear plaques on 10 strains of ETEC within 4-6 h at 37 degrees C.

J Biomol Struct Dyn, 2001 Feb, 18(4), 557 - 67
Papain does not cleave operator-bound lambda repressor: structural characterization of the carboxy terminal domain and the hinge; Ghosh K et al.; The circular dichroism spectra of three different purified carboxy terminal fragments 93-236, 112-236 and 132-236 of the bacteriophage lambda cI repressor have been measured and compared with those of the intact repressor and the amino terminal fragment 1-92 . All three carboxy terminal fragments contain mostly beta-strands and loops, a minor helix content increasing with the size of the fragment, showing that the 93-131 region previously called a hinge is structured . Fourier transformed infrared spectra also showed that fragment 93-236 contains alpha-helices, alpha-sheets and turns but fragment 132-236 contains no detectable alpha-helix, only beta-sheets and turns . Papain is known to cleave the lambda repressor, but it is shown here that it cannot cleave the operator-bound repressor dimer . For the 132-236 fragment, both the wt and the SN228 mutant previously shown to be dimerization defective in the intact, gave similar dimerization properties as investigated by HPLC at 2 to 100 microM protein concentration, with a KD of 13.2 microM and 19.1 microM respectively . The papain cleavage for wt and SN228 proceed at equal rates for the first cleavage at 92-93; however, the subsequent cleavages are faster for SN228 . The three Cys residues in the 132-236 fragment were found to be unreactive upon incubation with DTNB, indicating the thiol sulfur atoms are buried in the repressor carboxy terminal domain . Denaturation of the 132-236 fragment studied by tryptophan fluorescence shows two transitions centered at 1.5 M and 4.5 M of urea.

J Bacteriol, 2001 Apr, 183(7), 2289 - 97
Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations; Cicero MP et al.; Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the sigma(70) subunit . A motA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex . Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five sigma(70) mutants (Y571C, Y571H, D570N, L595P, and S604P) . All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements . The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant sigma(70) proteins were purified . All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay . The sigma(70) substitutions affected the 4.2 region, which binds the -35 sequence of E . coli promoters . In the presence of E . coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E . coli promoters . This defect could not be explained solely by a disruption in -35 recognition since similar results were obtained with extended -10 promoters . The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis . The L595P mutant, which was the most defective for in vitro transcription, failed to support E . coli growth.

Methods, 2001 Mar, 23(3), 287 - 93
Selection of RNA-binding peptides using mRNA-peptide fusions; Barrick JE et al.; We have been working to apply in vitro selection to isolate novel RNA-binding peptides . To do this, we use mRNA-protein fusions, peptides covalently attached to their own mRNA . Here, we report selection protocols developed using the arginine-rich domain of bacteriophage lambda-N protein and its binding target, the boxB RNA . Systematic investigation of possible paths for a selection round has allowed us to design a reliable and efficient protocol to enrich RNA-binding peptides from nonfunctional members of a complex mixture . The protocols we have developed should greatly facilitate the isolation of new molecules using the fusion system.

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