|
|
|
|
|
| J Biol Chem, 1996 Aug 2, 271(31), 18705 - 10 Autoregulation of the plasmid addiction operon of bacteriophage P1; Magnuson R et al.; The P1 plasmid addiction operon increases the apparent stability of a plasmid that carries it by killing plasmid-free (cured) segregants . The operon consists of a gene encoding an endotoxin responsible for death on curing (doc), preceded by a gene encoding a relatively unstable antidote that can prevent host death (phd) . When the copy number of the operon was increased, expression of a lacZ reporter fused to the promoter of the operon decreased, indicating that expression of the operon was stabilized by an autoregulatory circuit . Transcription of the lacZ reporter was repressed about 10-fold when phd, without doc, was expressed from an exogenous promoter . DNase I footprinting showed that Phd binds a perfect 10-base pair palindromic DNA sequence and, at higher concentrations, an adjacent, imperfect palindrome . The palindromic sites are located between the -10 region of the putative promoter and the start codon of phd . Electrophoretic mobility of DNA containing the promoter region was retarded in the presence of Phd and further retarded in the presence of Phd and Doc . When doc was co-expressed with phd, repression of the lacZ fusion was enhanced more than 100-fold . Thus, both products of the addiction operon participate in its autoregulation. Mol Microbiol, 1996 Aug, 21(3), 567 - 78 Integration host factor alleviates the H-NS-mediated repression of the early promoter of bacteriophage Mu; van Ulsen P et al.; Integration host factor (IHF), which is a histone-like protein, has been shown to positively regulate transcription in two different ways . It can either help the formation of a complex between a transcription factor and RNA polymerase or it can itself activate RNA polymerase without the involvement of other transcription factors . In this study, we present a third mechanism for IHF-stimulated gene expression, by counteracting the repression by another histone-like protein, H-NS . The early (Pe) promoter of bacteriophage Mu is specifically inhibited by H-NS, both in vivo and in vitro . For this inhibition, H-NS binds to a large DNA region overlapping the Pe promoter . Binding of IHF to a binding site just upstream of Pe alleviates the H-NS-mediated repression of transcription . This same ihf site is also involved in the direct activation of Pe by IHF . In contrast to the direct activation by IHF, however, the alleviating effect of IHF appears not to be dependent on the relevant position of the ihf site on the DNA helix, and it also does not require the presence of the C-terminal domain of the alpha subunit of RNA polymerase . Footprint analysis shows that binding of IHF to the ihf site destabilizes the interaction of H-NS with the DNA, not only in the IHF-binding region but also in the DNA regions flanking the ihf site . These results suggest that IHF disrupts a higher-order nucleoprotein complex that is formed by H-NS and the DNA. Indian J Biochem Biophys, 1996 Aug, 33(4), 253 - 60 The multifaceted roles of the RNA processing enzyme ribonuclease III; Srivastava RA et al.; Ribonuclease III was initially characterized as an endoribonuclease specific for double stranded RNA . Subsequently RNase III was found to be involved in the processing and maturation of ribosomal and tRNAs . Recent studies demonstrate that RNase III also participates in the processing of small stable RNAs . A number of other biological processes in which RNase III participates are: (a), conversion of polycistronic transcript of the bacteriophage T7 early region into discrete monocistronic mRNAs, (b), controlling expression of a variety of genes by processing of gene transcripts, (c), autoregulation of its own gene and (d), regulation of mRNA stability and stimulation of translation . No single processing enzyme displays such a wide variety of roles in RNA metabolism and gene expression as RNA processing enzyme ribonuclease III . This review provides an account of the various roles of RNase III in regulating gene expression and RNA metabolism. Mol Microbiol, 1996 Aug, 21(4), 751 - 61 The CII protein of bacteriophage 186 establishes lysogeny by activating a promoter upstream of the lysogenic promoter; Neufing PJ et al.; We have shown previously that the cII gene product of the non-lambdoid temperate bacteriophage 186 is required for the establishment of lysogeny . We show here that CII, a potential helix-turn-helix DNA-binding protein, establishes lysogeny by activating a promoter (PE) which spans the apl/cII intergenic region, upstream of the lysogenic promoter, PL . The start site of the PE transcript (+1) has been mapped by primer extension and we have identified the CII binding determinants at PE by DNase I footprinting . CII binds to inverted repeat sequences separated by two turns of the helix, with binding half-sites centred at the 38 and -58 positions of PE . Oligomerisation studies with purified CII protein indicate that a CII tetramer may be the species that binds to this site . We also show that PE is subject to direct negative feedback by the CI repressor. Mol Microbiol, 1996 Aug, 21(4), 667 - 74 Three conserved consensus sequences identify the NAD-binding site of ADP-ribosylating enzymes, expressed by eukaryotes, bacteria and T-even bacteriophages; Domenighini M et al.; It has been previously reported that the three-dimensional structures of the NAD-binding and catalytic site of bacterial toxins with ADP-ribosylating activity are superimposable, and that the key amino acids for the enzymatic activity are conserved . The model includes an NAD-binding and catalytic site formed by an alpha-helix bent over a beta-strand, surrounded by two beta-strands bearing a Glu and a His, or Arg, that are required for catalysis . We show here that the model can be extended to comprise all proteins with ADP-ribosylating activity known to date, including all eukaryotic mono- and poly-ADP-ribosyltransferases, the bacterial ADP-ribosylating enzymes which do not have toxic activity, and the analogous enzymes encoded by T-even bacteriophages . We show that, in addition to the common Glu and Arg/His amino acids previously identified, the conserved motifs can be extended as follows: (i) the Arg/His motif is usually arom-His/Arg (where 'arom' is an aromatic residue); (ii) in the sequences of the CT group the beta-strand forming part of the 'scaffold' of the catalytic cavity has an arom-ph-Ser-Thr-Ser-ph consensus (where 'ph' represents a hydrophobic residue); and (iii) the motif centered in the key glutamic residue is Glu/Gin-X-Glu; while (iv) in the sequences of the DT group the NAD-binding motif is Tyr-X10-Tyr . We believe that the model proposed not only accounts for all ADP-ribosylating proteins known to date, but it is likely to fit other enzymes (currently being analysed) which possess such an activity. J Mol Cell Cardiol, 1996 Aug, 28(8), 1823 - 8 Organization of the mouse cardiac natriuretic peptide locus encoding BNP and ANP; Huang H et al.; The genes encoding the mouse atrial natriuretic peptide and B-type natriuretic peptide were previously shown to be physically linked on mouse chromosome 4 (Steinhelper ME, 1993, Structure, expression, and genomic mapping of the mouse natriuretic peptide type-B gene . Circ Res 72: 984-992) . In the present study the spatial relationship and orientation of the mouse atrial natriuretic peptide and B-type natriuretic peptide transcription units were identified and a physical map of the mouse cardiac natriuretic peptide locus was obtained . To this end, genomic clones encoding atrial natriuretic peptide and B-type natriuretic peptide were isolated from a mouse genomic library in bacteriophage P1 . Three independent clones encoding atrial natriuretic peptide were isolated and two of these also encode B-type natriuretic peptide . Both transcripts were shown to arise from the same DNA strand, with B-type natriuretic peptide encoded approximately 15 kb 5'-of atrial natriuretic peptide based on field inversion gel electrophoresis of fragments amplified with specific oligonucleotides . This finding was confirmed by isolation of subclones comprising the entire locus and by blot hybridization analysis of mouse genomic DNA . The results show that the genes encoding the two natriuretic peptides expressed predominantly in mammalian cardiac myocytes are organized in tandem on mouse chromosome 4 . This information provides a physical framework for investigating mechanisms that regulate transcription of the cardiac natriuretic peptide locus. Proteins, 1996 Aug, 25(4), 486 - 500 Structure model of a complex between the factor for inversion stimulation (FIS) and DNA: modeling protein-DNA complexes with dyad symmetry and known protein structures; Sandmann C et al.; A method is presented to predict overall conformations of protein-DNA complexes on the basis of the known three-dimensional structures of the proteins . The method is restricted to proteins with a common twofold symmetry axis, which show only minor conformational changes upon binding to DNA . The method uses a numerical finite difference solution of the linearized Poisson-Boltzmann equation and subsequent energy minimization cycles . Structural parameters-the rotation angle of the DNA relative to the protein around the common symmetry axis, the protein-DNA distance, and intermolecular hydrogen-bonding contacts-are presented for two test cases, DNA bound to CAP (catabolite gene activator protein) and to the Cro-repressor of bacteriophage 434 . The DNA curvature in the starting model of the docking procedure was chosen as a smoothed approximation of the conformation found in the X-ray structures of these complexes . The method is further used to predict the unknown structure of the complex between the factor for inversion stimulation (FIS) and DNA, which is bent upon binding to FIS . In contrast to the test cases, the unknown curvature of the starting model is derived from a calibration of electrostatic precalculations for different proteins according to crystallographically observed DNA bending . The results of the modeling are in good accordance with the experimentally observed overall structure of protein-DNA complexes for the two test cases; for FIS, they correspond to several of the experimentally proposed protein-DNA contacts. Genetics, 1996 Aug, 143(4), 1507 - 20 Repair of double-strand breaks in bacteriophage T4 by a mechanism that involves extensive DNA replication; George JW et al.; We investigated double-strand break (dsb) repair in bacteriophage T4 using a physical assay that involves a plasmid substrate with two inverted DNA segments . A dsb introduced into one repeat during a T4 infection induces efficient dsb repair using the second repeat as a template . This reaction is characterized by the following interesting features . First, the dsb induces a repair reaction that is directly coupled to extensive plasmid replication; the repaired/replicated product is in the form of long plasmid concatemers . Second, repair of the dsb site is frequently associated with exchange of flanking DNA . Third, the repair reaction is absolutely dependent on the products of genes uvsX, uvsY, 32, 46, and 59, which are also required for phage genomic recombination-dependent DNA replication . Fourth, the coupled repair/replication reaction is only partly dependent on endonuclease VII (gp49), suggesting that either another Holliday-junction-cleaving activity or an alternate resolution pathway is active during T4 infections . Because this repair reaction is directly coupled to extensive replication, it cannot be explained by the SZOSTAK et al . model . We present and discuss a model for the coupled repair/replication reaction, called the extensive chromosome replication model for dsb repair. Biol Reprod, 1996 Aug, 55(2), 370 - 8 Identification, characterization, and expression of a new prolactin-like molecule in the hamster placenta; Barnes SW et al.; In the hamster, serum total lactogenic activity increases during the latter half of gestation (Days 8-16) . On Days 10 and 12 a substantial amount of lactogenic activity cannot be attributed to prolactin (PRL) and hamster placental lactogen-II (haPL-II); therefore, the presence of a molecule similar to placental lactogen-I (PL-I), as found in the rat and mouse at midpregnancy, has been hypothesized for the hamster . The objectives of this study were to identify PRL-like molecules synthesized by the hamster placenta and to determine the temporal and cellular synthesis of identified molecules . Oligonucleotides (20-23 bp) corresponding to regions of nucleotide homology between mouse PL-I (mPL-I) and rat PL-I (rPL-I) along with midgestation hamster placental RNA were used in 3' rapid amplification of cDNA ends (RACE) methodology to generate PRL-like cDNA . A 444-bp cDNA fragment that had nucleotide sequence similarity with members of the prolactin-growth hormone (PRL-GH) gene family was generated . This cDNA fragment was utilized to screen a Day 16 hamster placental bacteriophage cDNA library, and a clone containing the entire coding region was identified and sequenced . The molecule had 77% nucleotide sequence homology with mouse proliferin-related protein (mPRP) and somewhat less homology (approximately 60%) with hamster, rat, and mouse PRL or placental lactogens (PL) . The derived amino acid sequence of the identified molecule contained a 15-residue signal sequence and a 219-residue peptide with a calculated molecular weight of 25477 . The peptide shared 58% amino acid sequence identity with mPRP . Placental expression of the PRL-like molecule during the latter half of gestation was evaluated by Northern and slot-blot analyses using the 444-bp cDNA fragment as a hybridization probe . A 1-kb transcript was detected on Days 9-15 with peak expression on Days 10 and 11 . Messenger RNA for the PRL-like molecule was localized to cytotrophoblast but not giant trophoblast cells of the placental trophospongium region . In addition, specific immunostaining using an antibody to mPRP was confined to cytotrophoblast cells. Genomics, 1996 Aug 1, 35(3), 405 - 14 Antibody expression from the core region of the human IgH locus reconstructed in transgenic mice using bacteriophage P1 clones; Wagner SD et al.; Mice carrying transgenic human immunoglobulin gene miniloci can be used for the production of human monoclonal antibodies . The human variable region (V) gene segments in these miniloci undergo productive rearrangement in mouse lymphoid tissue to yield a population of B lymphocytes expressing a repertoire of antibodies . Many of the miniloci studied to date have included only a small number of germline gene segments in an artificially compact configuration . Here we describe the use of the bacteriophage P1 cloning system to create mice carrying the core region of the human immunoglobulin heavy chain (IgH) locus . Three P1 clones carrying overlapping regions of the human IgH locus (spanning the five JH-proximal VH segments, the entire DH and JH clusters, and the C mu and C delta constant regions) were injected into mouse eggs and appear to have reconstituted the core region of the locus (> 180 kb) following homologous recombination with each other . While this translocus yielded a titer of serum immunoglobulin similar to that obtained with a smaller plasmid-based minilocus, the P1-based locus gave rise to substantially greater diversification by somatic hypermutation . Such diversification is important for obtaining high-affinity antibodies . The results show the usefulness of the P1 system in facilitating the manipulation and recreation of large transgenes. Anal Biochem, 1996 Aug 1, 239(2), 136 - 44 A comparison of electrophoretic resolution for snapshot and finish-line imaging; Sutherland JC et al.; Finish-line imaging, in which DNA or other macromolecules are detected after electrophoresis for a constant distance, usually improves resolution compared to snapshot imaging, in which molecules are electrophoresed for a constant time in an apparatus of comparable dimensions . Resolving power, which is an objective measure of the ability of different separatory methods to detect closely spaced molecular species, can be used to compare directly the performance of systems employing both snapshot and finish-line imaging {E . A . Ribeiro and J . C . Sutherland, Anal . Biochem . 210, 378-388 (1993)} . Experimentally determined values of resolving power are influenced both by the method of imaging (snapshot vs finish-line) and by instrument-specific factors that affect resolution . Previous comparisons of the resolving power obtained with finish-line and snapshot imaging involved data sets acquired by different instruments with different instrumental resolutions . To reduce the influence of instrumental effects, we constructed a scanning laser fluorometer that can measure both snapshot and finish-line images of fluorochrome-labeled DNA . Snapshot and finish-line images of a DNA sample containing HaeII restriction fragments of the DNA from bacteriophage T7, which range in length from 474 to 6514 base pairs, were obtained under otherwise identical electrophoretic conditions . Snapshot and finish-line imaging give similar resolving powers for DNA molecules up to about 1.5 kbp long . For both imaging modes, maximum resolving power was achieved for DNA molecules between 2 and 3 kbp in length . For larger DNA molecules, finish-line imaging provided higher resolving power . The ratio of the resolving power of finish-line images to that of snapshot images increased monotonically as a function of DNA length . For the longest restriction fragments studied (6514 bp), the resolving power for finish-line images exceeded that of snapshot images by about 50%. Virology, 1996 Aug 1, 222(1), 169 - 75 In vitro transcripts from cloned cDNAs of the lettuce infectious yellows closterovirus bipartite genomic RNAs are competent for replication in Nicotiana benthamiana protoplasts; Klaassen VA et al.; Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter . To assess RNA replication, Nicotiana benthamiana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derived in vitro transcripts . Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG) in vitro transcripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions . Furthermore, protoplasts inoculated with only capped in vitro transcripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2 . Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived capped in vitro transcripts . These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectious in vitro transcripts can be derived from the cloned cDNAs of a closterovirus genome. Exp Cell Res, 1996 Aug 1, 226(2), 346 - 55 Specific association of cyclin-like uracil-DNA glycosylase with the proliferating cell nuclear antigen; Muller-Weeks SJ et al.; We have previously isolated a human gene that encodes a cyclin-like protein with uracil-removing activity (UDG2) (Muller, S.J., and Caradonna, S . 1993 . J . Biol . Chem . 268, 1310-1319) . The structural and regulatory similarities shared between this uracil-DNA glycosylase and cyclins suggested that it may interact with additional proteins . Using a unique affinity purification protocol (Ugi-Sepharose) and anti-UDG2 antibodies, we have identified a physical interaction between the cyclin-like uracil-DNA glycosylase and PCNA in extracts derived from HeLa cells . Conversely, we show that anti-PCNA immunoprecipitates possess significant uracil-DNA glycosylase activity . This activity is specifically blocked by the addition of uracil-DNA glycosylase inhibitor protein (Ugi) derived from bacteriophage PBS2 . To further characterize this association, we performed in vitro mixing experiments using 35S-labeled PCNA and uracil-DNA glycosylase (UDG2) that were generated in a coupled transcription/translation system . We show that UDG2 and PCNA are coprecipitated using anti-PCNA antibodies and anti-UDG2 antibodies as well as Ugi-Sepharose . When PCNA is preincubated with synthetic peptides corresponding to amino acid residues 73-90 of UDG2, the PCNA-UDG2 association is prevented . By contrast, addition of synthetic peptides corresponding to amino acid residues 208-223 has no effect on this interaction . These findings suggest that the UDG2 domain encompassing amino acids 73-90 is directly involved in binding PCNA. Eur J Biochem, 1996 Aug 1, 239(3), 810 - 7 Membrane topology of the mannose transporter of Escherichia coli K12; Huber F et al.; The mannose transporter of the bacterial phosphotransferase system mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation . It also functions as a receptor for bacterial chemotaxis {Adler.J . & Epstein, W . (1974) Proc . Natl Acad . Sci . USA 71 . 2895-2899} and is required for infection of the cell by bacteriophage lambda where it most likely functions as a pore for penetration of phage DNA {Elliott, J . & Arber, W . (1978) Mol . & Gen . Genet . 161, 1-8} . The transporter consists of two transmembrane subunits (27-kDa IICMan and 31-kDa IIDMan) and a hydrophilic subunit (35-kDa IIABMan) . Protein fusions of IICMan and IIDMan with beta-galactosidase (LacZ) and with alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the two proteins . Protein fusions were obtained by progressively deleting the manY and manZ genes from their 3' ends and ligating them to lacZ and 'phoA that lack promotor and leader sequences . Based on the analysis of 30 IICMan-PhoA . 10 IICMan-LacZ, 12 IIDMan-PhoA, and 30 IIDMan-LacZ fusions, it is predicted that IICMan has six membrane-spanning segments with the N- and C-termini on the cytoplasmic face of the membrane . IIDMan is anchored in the membrane by a single membrane-spanning segment at the end of the C-terminus, while most of the protein (250 residues) protrudes into the cytoplasm. J Bacteriol, 1996 Aug, 178(15), 4747 - 50 Analysis of the enzymatic cleavage (beta elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: reexamination; Hanfling P et al.; The capsular K5 polysaccharide of Escherichia coli is the receptor of the capsule-specific coliphage K5, which harbors an enzyme that degrades the capsular K5 polysaccharide to a number of oligosaccharides . Analysis of the degradation products using gel permeation chromatography, the periodate-thiobarbituric acid and bicinchoninic acid reactions, and nuclear magnetic resonance spectroscopy showed that the major reaction products are hexa-, octa-, and decasaccharides with 4,5-unsaturated glucuronic acid (delta4,5GlcA) at their nonreducing end . Thus, the bacteriophage enzyme is a K5 polysaccharide lyase and not, as we had reported previously, an endo-N-acetylglucosaminidase. J Mol Biol, 1996 Jul 26, 260(4), 484 - 91 Identification of an UP element within the IHF binding site at the PL1-PL2 tandem promoter of bacteriophage lambda; Giladi H et al.; An UP element defines a supplementary promoter element located upstream of the -35 region that stimulates transcription by interacting with the C-terminal domain of the RNA polymerase alpha subunit (alpha CTD) . The alpha CTD also responds to various transcription activators, including the integration host factor protein, IHF, in the stimulation of the bacteriophage lambda PL promoter . PL consists of the tandem PL1-PL2 promoters where PL1 is stimulated and PL2 is repressed by IHF . We identified a functional UP element that binds the alpha subunit of RNA polymerase and is located in the region from -36 to -60 relative to the PL2 start site . PL2 expression requires the presence of the UP element and requires an intact alpha CTD . The UP element is nested within the DNA region protected by IHF against DNase I digestion . We used mutational analysis to identify the IHF recognition sequence which was found to be located downstream of the UP element, overlapping the -35 region of PL2 . The possible function of the complex structure of the PL promoter is discussed. J Biol Chem, 1996 Jul 26, 271(30), 17675 - 86 The RNA chain elongation rate of the lambda late mRNA is unaffected by high levels of ppGpp in the absence of amino acid starvation; Tedin K et al.; In this study, the effects of high levels of guanosine tetraphosphate (ppGpp) on the decay and RNA chain elongation kinetics of the bacteriophage lambda late transcript in Escherichia coli were examined in the absence of amino acid starvation . The accumulation, mRNA decay kinetics, and RNA chain elongation rate of the lambda late mRNA were determined after heat induction of lambdacI857 lysogens in the presence of high levels of ppGpp induced from a RelAalpha fragment-overproducing plasmid . The accumulation kinetics and elongation rate determinations of the late mRNA were made at long times after induction to allow a new steady state of transcriptional activities under conditions of elevated intracellular levels of ppGpp . The results indicate no prolonged or significant effect on either mRNA decay or the RNA chain elongation rate of the late mRNA as a result of elevated ppGpp levels . Surprisingly, the RNA chain elongation rate determinations indicate an RNA polymerase processivity of approximately 90-100 nucleotides/s for the lambda late transcript despite the presence of high levels of ppGpp . The results are discussed in terms of various models for regulation of stable and messenger RNA synthesis in E . coli. Biochemistry, 1996 Jul 23, 35(29), 9603 - 9 Raman spectroscopy of the Ff gene V protein and complexes with poly(dA): nonspecific DNA recognition and binding; Benevides JM et al.; Raman spectra of crystals and solutions of the single-stranded DNA binding protein of bacteriophage Ff (gene V protein, gVp) and of solution complexes of gVp with single-stranded poly-(deoxyadenylic acid) {poly(dA)} reveal the following: (i) The gVp secondary and tertiary structures are similar in solution and in the crystal and are dominated by beta-sheet domains, in agreement with NMR and X-ray findings . (ii) Subunit conformation and side chain environments of gVp are virtually unchanged over a wide range of salt concentration (0 < {NaCl} < 100 mM); however, the solution conformation of poly(dA) exhibits sensitivity to added salt . The perturbed Raman markers indicate subtle changes in helix backbone geometry with accompanying small differences in base stacking as the concentration of NaCl is changed . (iii) In complexes with poly(dA), neither the conformation of gVp nor its side chain environments are altered significantly in comparison to the free protein . This is the case at both high salt (nucleotide-to-subunit binding stoichiometry n = 4) and low salt (n = 3) . (iv) The Raman signature of poly(dA) undergoes small perturbations upon gVp binding, indicative of small changes in base stacking and phosphodiester backbone conformation . The present results show that the different stoichiometric binding modes of gVp to poly(dA) are accomplished without significant changes in gVp subunit structure and with only modest changes in the single-stranded poly(dA) ligand . This contrasts sharply with sequence-specific double-stranded DNA binding proteins, such as the phage lambda and D108 repressors, which undergo substantial structural changes upon DNA binding, and which also alter more dramatically the Raman fingerprints of their DNA target sites . Thus, nonspecific and specific nucleic acid recognition modes are distinguishable by Raman spectroscopy . The Raman signature of gVp also allows examination of hydrogen bonding interactions of unique side chains within the hydrophobic core (cysteine 33) and at the binding interface (tyrosine 41) . These are discussed in relation to the recently published gVp crystal structure. Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7761 - 6 A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage; Fisch I et al.; It has been suggested that recombination and shuffling between exons has been a key feature in the evolution of proteins . We propose that this strategy could also be used for the artificial evolution of proteins in bacteria . As a first step, we illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (> 10(11) members) of peptides from two different exons . Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides . The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein and selected by binding to several proteins, including beta-glucuronidase . One of the peptides selected against beta-glucuronidase was chemically synthesized and shown to inhibit the enzymatic activity (inhibition constant: 17 nM); by further exon shuffling, an improved inhibitor was isolated (inhibition constant: 7 nM) . Not only does this approach provide the means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and of the linker, it may be possible to evolve small folded peptides and proteins. Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7446 - 51 Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage; Jockusch S et al.; The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy . The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus . In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus . Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye . At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer . Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching . Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution. J Mol Biol, 1996 Jul 19, 260(3), 359 - 68 Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain; Low NM et al.; Human antibodies can now be isolated from antibody repertoires displayed on the surface of filamentous bacteriophage in a process that mimics the primary immune response . Here we have attempted to mimic the secondary response, the natural process of affinity maturation of antibodies occurring in germinal centres, by multiple cycles of random mutation and selection . Phage displaying a human antibody fragment recognising the hapten 2-phenyl-5-oxazolone were grown in a mutator strain of bacteria (Escherichia coli: mutD5) to generate a large repertoire of antibodies that should include the majority of possible single nucleotide point mutations . The repertoire of phage antibody mutants was then selected by binding to hapten . By multiple rounds of growth in the mutator strain, and increasingly stringent selection, we succeeded in isolating mutants with improved binding affinities; furthermore, the distribution of mutations and nucleotide substitution preferences strongly resembled those of somatic hypermutation . We then constructed a genealogical tree from the sequences of mutants taken at different rounds, and identified four sequentially acquired mutations that together improve the binding affinity of the antibody by a factor of 100-fold (from Kd 320 nM to 3.2 nM). J Mol Biol, 1996 Jul 19, 260(3), 332 - 46 In vitro characterization of transcription termination factor Rho from Escherichia coli rho(nusD) mutants; Washburn RS et al.; Escherichia coli nusD strains are bacteria that carry mutations in rho, the gene for transcription termination factor Rho, that block the growth of phages T4 and lambdar32 . We have identified the rho mutation in six independent nusD strains, and although five of the strains have different mutations, with one exception the mutations are in the proposed RNA-binding domain of Rho . We overexpressed, purified, and characterized the five different mutant Rho proteins . All show substantial RNA-dependent ATPase activity with several homoribopolymers or the lambda cro message as cofactor . At the lambda tR1 Rho-dependent terminator in vitro, all mutant Rho proteins show decreased termination compared with wild-type, and all also terminate within cro at a new terminator, tRE, with endpoints 5' to tR1 at 170, 200, 245 and 260 nucleotides 3' from the transcription start . The mutant Rho proteins are proposed to interfere with bacteriophage T4 growth through indirect effects on host gene expression. J Mol Biol, 1996 Jul 19, 260(3), 289 - 98 Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels; Miroux B et al.; We have investigated the over-production of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system . In all seven cases, when expression of the target membrane protein was induced, most of the BL21(DE3) host cells died . Similar effects were also observed with expression vectors for ten globular proteins . Therefore, protein over-production in this expression system is either limited or prevented by bacterial cell death . From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high saturation cell density, and produced the protein as inclusion bodies at an elevated level without toxic effect . Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3) . The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior to BL21(DE3) as a host for protein over-expression . However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3) . Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells containing the expression plasmid for subunit b of bacterial F-ATPase . In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H(+) symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced . The transcription of the gene for the OGCP and subunit b was lower in C41(DE3) and C43(DE3), respectively, than in BL21(DE3) . In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane . The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts in order to overcome other toxic effects associated with over-expression. Mol Gen Genet, 1996 Jul 19, 251(5), 526 - 31 Telomere-homologous sequences occur near the centromeres of many tomato chromosomes; Presting GG et al.; Several bacteriophage lambda clones containing interstitial telomere repeats (ITR) were isolated from a library of tomato genomic DNA by plaque hybridization with the cloned Arabidopsis thaliana telomere repeat . Restriction fragments lacking highly repetitive DNA were identified and used as probes to map 14 of the 20 lambda clones . All of these markers mapped near the centromere on eight of the twelve tomato chromosomes . The exact centromere location of chromosomes 7 and 9 has recently been determined, and all ITR clones that localize to these two chromosomes map to the marker clusters known to contain the centromere . High-resolution mapping of one of these markers showed cosegregation of the telomere repeat with the marker cluster closest to the centromere in over 9,000 meiotic products . We propose that the map location of interstitial telomere clones may reflect specific sequence interchanges between telomeric and centromeric regions and may provide an expedient means of localizing centromere positions. Biochemistry, 1996 Jul 16, 35(28), 9253 - 65 Role of adenosine 5'-triphosphate hydrolysis in the assembly of the bacteriophage T4 DNA replication holoenzyme complex; Berdis AJ et al.; Steady-state and pre-steady-state rates of ATP hydrolysis by the 44/62 accessory protein were determined to elucidate the role of ATP hydrolysis in bacteriophage T4 holoenzyme complex formation . Steady-state ATPase measurements of the 44/62 protein under various combinations of 45 protein, DNA substrate, and T4 exo- polymerase indicate that although the 44/62 protein synergistically hydrolyzes ATP in the presence of 45 protein and DNA substrate, the ATPase activity of 44/62 is diminished substantially upon the formation of the holoenzyme complex . The decrease in activity is primarily in kcat while the K(m) for ATP is changed unsubstantially by the various combinations . Data suggest that the decrease in the rate of ATP hydrolysis upon the addition of T4 exo- polymerase in the presence of 45 protein and DNA substrate is due to formation of a stable holoenzyme complex consisting of only the 45 protein and T4 exo- polymerase in a 1:1 ratio . The 44/62 protein acts catalytically to load 45 protein onto the DNA substrate and does not remain a component of the holoenzyme complex . Pre-steady-state kinetic analysis of the ATP hydrolysis reaction catalyzed by the 44/62 protein loading the 45 protein onto the DNA substrate in the absence or presence of polymerase is biphasic, in which a burst in ATP hydrolysis precedes the steady-state rate of ATP hydrolysis . An identical burst in ATP consumption is obtained under either condition, indicating that ATP hydrolysis is not required to load polymerase into the holoenzyme complex . The data suggest one turnover of ATP at each of the four ATPase active sites of the 44/62 protein per 45 protein loaded . ATP hydrolysis by the 44/62 protein under conditions of holoenzyme complex formation is the rate-limiting step in holoenzyme complex formation . The process of holoenzyme formation appears to be identical for leading and lagging strand synthesis. Nucleic Acids Res, 1996 Jul 15, 24(14), 2821 - 8 A viral genome containing an unstable aflatoxin B1-N7-guanine DNA adduct situated at a unique site; Bailey EA et al.; A problem that has hindered the study of the biological properties of certain DNA adducts, such as those that form at the N7 atoms of purines, is their extreme chemical lability . Conditions are described for the construction of a single-stranded genome containing the chemically and thermally labile 8,9-dihydro-8- (N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct, the major DNA adduct of the potent liver carcinogen aflatoxin B1 (AFB1) . A 13mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react with the exo-8,9-epoxide of AFB1 to form an oligonucleotide containing a single AFB1-N7-Gua (at the underlined guanine) . This modified 13mer was 5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome generated by annealing a 53mer uracil-containing scaffold to M13mp7L2 linearized by EcoRI . Following ligation, the scaffold was enzymatically removed with uracil DNA glycosylase and exonuclease III . The entire genome construction was complete within 3 h and was carried out at 16 degrees C, pH 6.6, conditions determined to be optimal for AFB1-N7-Gua stability . Characterization procedures indicated that the AFB1-N7-Gua genome was approximately 95% pure with a small (5%) contamination by unmodified genome . This construction scheme should be applicable to other chemically or thermally unstable DNA adducts. J Immunol, 1996 Jul 15, 157(2), 549 - 56 Regulation of the B cell response to T-dependent antigens by classical pathway complement; Fischer MB et al.; Mice deficient in complement components C3 (C3 -/-) and C4 (C4 -/-) were found to have a profound defect in their Ab response to a T-dependent Ag (bacteriophage (phi X174) . Characterization of the deficient mice demonstrated a diminished level of peanut agglutinin+ germinal centers and a failure in isotype switching despite normal B cell signaling in vitro . The nature of the defect was found to lie at the B cell level, as the T cells were primed in C3- and C4-deficient mice as well as those in wild-type mice . These results, and the finding that the defect could be partly reversed by a 10-fold increase in Ag dose, support the hypothesis that covalent attachment of complement ligands, i.e., C3b and C3d to the Ag-Ab complex, increases its immunogenicity. EMBO J, 1996 Jul 15, 15(14), 3487 - 97 Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP; Teplyakov A et al.; NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor . Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP . The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement . The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution . The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop . The second domain, consisting of five alpha-helices, forms the NMP binding pocket . A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme . The mechanism of active centre formation via domain closure is described . Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding . Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination. Genomics, 1996 Jul 15, 35(2), 299 - 307 Rapid mapping of genomic P1 clones: the mouse L-isoaspartyl/D-aspartyl methyltransferase gene; MacLaren DC et al.; We report the mapping of the gene for the murine protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1 . 77) from a 129 mouse strain . This gene encodes an enzyme present in all tissues that can catalyze the first step of a repair reaction in which age-damaged proteins containing abnormal l-isoaspartyl (or d-aspartyl) residues can be converted to forms containing normal l-aspartyl residues . We first mapped the restriction sites from a genomic P1 clone using a rapid method generally applicable to all bacteriophage P1 clones containing large DNA inserts . We show that a single pulsed-field electrophoresis blot can be used to map an entire 89-kb P1 clone insert for eight restriction endonucleases with an error of no more than 2% of the length of the fragment, or 1 kb at the middle of the insert . In this method, we combine complete restriction endonuclease digestion at rare sites within the P1 vector with partial restriction endonuclease digestion within the insert . After size separation by pulsed-field gel electrophoresis and blotting, the fragments are detected by Southern hybridization with probes to the vector . This method is potentially useful for restriction mapping other large DNA clones such as artificial chromosomes . We then determined the positions of the exons of the methyltransferase gene by restriction mapping of long PCR fragments . The previously unidentified exon 8, which encodes the -DEL C-terminus of the more acidic isozyme II, was sequenced and mapped 5 . 3 kb from the end of exon 7. J Biol Chem, 1996 Jul 12, 271(28), 16962 - 6 Recombinant protein synthesis in Chinese hamster ovary cells using a vaccinia virus/bacteriophage T7 hybrid expression system; Ramsey-Ewing A et al.; The vaccinia virus/bacteriophage T7 expression system was adapted to Chinese hamster ovary (CHO) cells . Vaccinia virus undergoes abortive infection in CHO cells, which is characterized by a sharp reduction in protein synthesis at the stage of viral intermediate gene expression . We determined that expression of a T7 promoter-regulated chloramphenicol acetyltransferase gene was at least 20 times more efficient in permissive BS-C-1 than in CHO cells . The encephalomyocarditis virus 5'-untranslated region, which confers cap-independent translatability to mRNA, stimulated recombinant protein synthesis by 10-fold in both cell lines, maintaining the advantage of the BS-C-1 cells over CHO cells . Since the cowpox virus hr gene overcomes vaccinia virus host range restriction in CHO cells, we constructed a recombinant virus that carries an intact hr gene in addition to the T7 RNA polymerase gene . With this virus, synthesis of T7 RNA polymerase was enhanced and production of a recombinant protein occurred in CHO cells at the level observed in permissive cell lines . Extension of the vaccinia virus/bacteriophage T7 expression system to CHO cells should be of wide interest, as these cells have advantages for preparation of recombinant proteins in research and biotechnology. J Biol Chem, 1996 Jul 12, 271(28), 16678 - 82 Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork; Schrock RD et al.; The gene 41 protein is the DNA helicase associated with the bacteriophage T4 DNA replication fork . This protein is a major component of the primosome, being essential for coordinated leading and lagging strand DNA synthesis . Models suggest that such DNA helicases are loaded only onto DNA at origins of replication, and that they remain with the ensuing replication fork until replication is terminated . To test this idea, we have measured the extent of processivity of the 41 protein in the context of an in vitro DNA replication system composed of eight purified proteins (the gene 43, 44/62, 45, 32, 41, 59, and 61 proteins) . After starting DNA replication in the presence of these proteins, we diluted the 41 helicase enough to prevent any association of new helicase molecules and analyzed the replication products . We measured an association half-life of 11 min, revealing that the 41 protein is processive enough to finish replicating the entire 169-kilobase T4 genome at the observed replication rate of approximately 400 nucleotides/s . This processivity of the 41 protein does not require the 59 protein, the protein that catalyzes 41 protein assembly onto 32 protein-covered single-stranded DNA . The stability we measure for the 41 protein as part of the replication fork is greater than estimated for it alone on single-stranded DNA . We suggest that the 41 protein interacts with the polymerase holoenzyme at the fork, both stabilizing the other protein components and being stabilized thereby. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7022 - 7 Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry; Cheng X et al.; The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS) . Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution . Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer . A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1 . Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry . The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data . These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS . The sensitivity and high resolution of ESI-MS should make it a useful too} in the study of protein-DNA interactions. Mutat Res, 1996 Jul 5, 368(3-4), 235 - 48 Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms; Adams SP et al.; Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA . To follow-up positive responses in in vitro tests, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA . Calf thymus DNA was used as the target for the direct detection of adducts by 32P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage lambda DNA . To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B1, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate) . Each of the nine compounds produced a positive result for one or both endpoints . Using multifraction contact-transfer TLC, we detected 32P-labeled DNA adducts produced by aflatoxin B1, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide) . Aflatoxin B1, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts . Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels . The damage to lambda DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70 degrees C . Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage . Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests. Mutat Res, 1996 Jul 5, 354(1), 113 - 27 Instability of repeated dinucleotides in bacteriophage T7 genomes; Yang Y et al.; The ligase gene of bacteriophage T7 was interrupted with an insert of synthetic DNA that included a series of dinucleotide repeats which altered the reading frame of the gene and prevented the phage from growing on a host deficient in Escherichia coli ligase . The insert was designed so that gain of an additional copy of the repeat would restore the reading frame and produce ligase positive T7 . It was found that a pair of nucleotides was gained at a frequency of 1.4 x 10(-3) and a dinucleotide was lost from this sequence at a frequency of 1 x 10(-4) . The same measurements were made using T7 with a DNA polymerase without the 3' --> 5' exonuclease function . This mutant DNA polymerase lacks proofreading edit function and is more processive than its wild-type counterpart . Phage without the edit function gained or lost a dinucleotide repeat significantly more frequently than wild-type T7 . Thus, the frequency of two base frameshifting is very high during DNA replication . Proofreading activity attenuates the frequency of two base frameshifts . Inactivation of the 3' --> 5' exonuclease of T7 DNA polymerase had essentially no effect on the frequency of deletion of one member of a pair of 10 bp tandem repeats. J Mol Biol, 1996 Jul 5, 260(1), 9 - 21 Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage; Malik P et al.; Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed . The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion . However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions . X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo . We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell . A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion . These results have important implications for the design of peptide display systems based on filamentous bacteriophage. J Mol Biol, 1996 Jul 5, 260(1), 85 - 98 Three-dimensional structure of scaffolding-containing phage p22 procapsids by electron cryo-microscopy; Thuman-Commike PA et al.; The procapsids of bacterial viruses are the products of the polymerization of coat and scaffolding subunits, as well as the precursors in DNA packaging . Electron cryo-microscopy has been used to study the three-dimensional structures of bacteriophage P22 procapsids containing wild-type and mutant scaffolding proteins . The scaffolding mutant structure has been resolved to 19 A resolution and agrees with the 22 A resolution wild-type procapsid reconstruction . Both procapsid reconstructions contain an outer icosahedral coat protein shell and an inner scaffolding protein core . The outer core protein forms a T = 7 icosahedral lattice with distinctive channels present at the centers of the pentons and hexons . In addition, the hexons display a prominent skew . Computational isolation of the skewed hexon shows the presence of a local 2-fold axis that reduces the number of unique conformations in the asymmetric unit to four at this resolution . We have classified the four unique subunits into three distinct classes, based upon the shape of the upper domain and the presence of a channel leading to the inner coat protein surface . In addition, at the inner surface of the coat protein, finger-like regions that extend towards the scaffolding protein core are present in two of the subunits . The finger-like regions suggest the presence of an ordered interaction between the inner coat protein and the scaffolding protein . However, an icosahedral scaffolding protein shell is not formed, and the innermost scaffolding protein core does not pack with icosahedral symmetry. Genet Anal, 1996 Jul, 13(2), 33 - 42 Retrofitting high molecular weight DNA cloned in P1: introduction of reporter genes, markers selectable in mammalian cells and generation of nested deletions; Chatterjee PK et al.; The bacteriophage P.1 . cloning system is proving to be quite useful for the cloning and analysis of genomic DNA inserts of up to 95 kb in size . In an effort to use that DNA directly in biological experiments we have embarked on a scheme to retrofit the P.1 . DNA using a mini-Tn10 transposon system . This transposon system is used in two ways: (i) to introduce a variety of sequence signals that are recognizable in mammalian cells, such as mammalian cell-responsive resistance markers and reporter genes, and (ii) to generate a nested set of deletions in a P.1 . clone by using a ioxP site located within the transposon . In this report we show that such transpositions into P.1 . DNA are efficient, distributed throughout the entire length of the genomic fragment and do not disrupt the DNA in any location other than the site of insertion of the transposon . The Tn10-based P.1 . transduction system described here provides a general scheme for retrofitting any large genomic DNA cloned in a P.1 . vector, thus facilitating the use of clones from the current P.1 . recombinant libraries in cellular transformation studies. Mol Microbiol, 1996 Jul, 21(1), 159 - 70 Characterization of Mycobacterium smegmatis gene that confers resistance to phages L5 and D29 when overexpressed; Barsom EK et al.; Bacteriophage infection requires a specific interaction with the outer surface of a bacterial host followed by interaction with the cell membrane and phage DNA injection . Phages of the mycobacteria encounter a cell wall that is rich in unusual lipid- and sugar-containing components which form a formidable barrier that must be passed to gain access to the membrane . We describe here a gene of Mycobacterium smegmatis that confers resistance to mycobacteriophages L5 and D29 . The phage-resistance phenotype results not from mutation but from elevated expression of a wild-type gene . It appears that the product of this multicopy phage-resistance (mpr) gene may alter the structure of the host cell wall or membrane, thereby inhibiting productive phage DNA injection. Biotechniques, 1996 Jul, 21(1), 106 - 12 NIRCA: a rapid robust method for screening for unknown point mutations; Goldrick MM et al.; We describe a method for screening for dispersed point mutations, based on the observation that RNase frequently cleaves both strands of base pair mismatches in duplex RNA targets . The mismatched substrates are generated by in vitro transcription of wild-type and mutant templates amplified by the PCR or reverse transcription (RT)-PCR; bacteriophage promoters are incorporated into the PCR primers to permit both strands of the products to be transcribed into RNA . Complementary wild-type and mutant transcripts are hybridized and treated with RNase, and the cleavage products are separated on agarose gels and detected by visualization of the ethidium-stained sample under UV light . The method is thus non-isotopic, and since the cleavage products remain double-stranded during analysis, the labor-intensive RNase inactivation steps required in the original procedure can be eliminated . Also, nonspecific background cleavage is reduced so that longer target regions (1 kb) can be screened in a single step . The Non-Isotopic RNase Cleavage Assay (NIRCA) achieved a detection rate of 88%-90% in blind studies in a Factor IX model system, and it was also used to detect unknown p53 mutations in breast tumor samples . NIRCA provides a rapid method for sensitive, non-isotopic, high-throughput genetic screening. Biotechniques, 1996 Jul, 21(1), 99 - 104 Partial CviJI digestion as an alternative approach to generate cosmid sublibraries for large-scale sequencing projects; Gingrich JC et al.; We demonstrate an alternative method for the generation of random subclones for large-scale shotgun human DNA sequencing projects . Miniprep DNA from a human cosmid clone was partially digested with CviJI, size-fractionated by agarose gel electrophoresis and cloned into bacteriophage M13 . A library consisting of 10(5) subclones of 1.1 kb average size was recovered from one gel fraction containing approximately 300 ng of partially digested DNA . DNA sequences from an initial 103 subclones demonstrate that 100 of the subclones cover 90% of the cosmid, which is close to the 92% expected if the subclones were generated at random . DNA sequences from three of the subclones did not match the cosmid sequences, establishing that miniprep DNA can be used for library construction with little concern for contamination with genomic E . coli DNA . The use of CviJI to generate random DNA fragments thus offers a simple alternative to other commonly used fragmentation methods, such as shearing or sonication, for the generation of random sublibraries for large-scale human shotgun DNA sequencing projects. Genetics, 1996 Jul, 143(3), 1081 - 90 Bacteriophage T4 mutants hypersensitive to an antitumor agent that induces topoisomerase-DNA cleavage complexes; Woodworth DL et al.; Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex . A pathway of recombination can apparently repair this DNA damage . In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4 . Substantial increases in sensitivity to the antitumor agent m-AMSA {4'-(9-acridinylamino)methanesulfon-m-anisidide} were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), alpha-gt (alpha-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (beta subunit of ribonucleotide reductase) . The role of the rnh gene in drug sensitivity was further characterized . First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA . Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase . Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage . Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage. Genetics, 1996 Jul, 143(3), 1069 - 79 Genetic analysis of the bacteriophage lambda attL nucleoprotein complex; MacWilliams MP et al.; Site-specific recombination in bacteriophage lambda involves interactions among proteins required for integration and excision of DNA molecules . We have analyzed the elements required to form an in vivo nucleoprotein complex of integrase (Int) and integration host factor (IHF) . Interaction of Int with the core (the site of strand exchange) is stabilized by the flanking arm region of attL . IHF, in addition to Int, is required for efficient Int-core binding . We used the in vivo attL binding assay to characterize several Int variants for their abilities to form stable attL complexes . Substitution of Int active site tyrosine 342 by phenylalanine had no effect on the ability of the protein to form attL complexes . Three other amino acids that are completely conserved in the integrase family of recombinases (arginine 212, histidine 308, and arginine 311) were separately substituted by glutamine, leucine, and histidine, respectively . In each case, the mutant protein was altered in its ability to form attL complexes while retaining its ability to bind to the lambda arm-type sites . We propose that, in addition to their role in catalysis, this triad of amino acids helps the Int protein to interact with the lambda core sites. J Bacteriol, 1996 Jul, 178(14), 4115 - 21 Effects of T4 phage infection and anaerobiosis upon nucleotide pools and mutagenesis in nucleoside diphosphokinase-defective Escherichia coli strains; Zhang X et al.; Bacteriophage T4 encodes nearly all of its own enzymes for synthesizing DNA and its precursors . An exception is nucleoside diphosphokinase (ndk gene product), which catalyzes the synthesis of ribonucleoside triphosphates and deoxyribonucleoside triphosphates (dNTPs) from the corresponding diphosphates . Surprisingly, an Escherichia coli ndk deletion strain grows normally and supports T4 infection . As shown elsewhere, these ndk mutant cells display both a mutator phenotype and deoxyribonucleotide pool abnormalities . However, after T4 infection, both dNTP pools and spontaneous mutation frequencies are near normal . An E . coli strain carrying deletions in ndk and pyrA and pyrF, the structural genes for both pyruvate kinases, also grows and supports T4 infection . We examined anaerobic E . coli cultures because of reports that in anaerobiosis, pyruvate kinase represents the major route for nucleoside triphosphate synthesis in the absence of nucleoside diphosphokinase . The dNTP pool imbalances and the mutator phenotype are less pronounced in the anaerobic than in the corresponding aerobic ndk mutant strains . Anaerobic dNTP pool data, which have not been reported before, reveal a disproportionate reduction in dGTP, relative to the other pools, when aerobic and anaerobic conditions are compared . The finding that mutagenesis and pool imbalances are mitigated in both anaerobic and T4-infected cultures provides strong, if circumstantial, evidence that the mutator phenotype of ndk mutant cells is a result of the dNTP imbalance . Also, the viability of these cells indicates the existence of a second enzyme system in addition to nucleoside diphosphokinase for nucleoside triphosphate synthesis. J Bacteriol, 1996 Jul, 178(14), 4077 - 83 Phage HK022 Roi protein inhibits phage lytic growth in Escherichia coli integration host factor mutants; Clerget M et al.; Temperate coliphage HK022 requires integration host factor (IHF) for lytic growth . The determinant responsible for this requirement was identified as a new gene (roi) located between genes P and Q . This gene encodes a DNA-binding protein (Roi) containing a helix-turn-helix motif . We have shown that Roi binds a site within its own gene that is closely linked to an IHF binding site . By gel retardation experiments, we have found that IHF binding stabilizes the interaction of Roi with its gene . We have isolated three independent phage mutants that are able to grow on an IHF- host . They carry different mutations scattered in the roi gene and specifying single amino-acid changes . The interactions of all three Roi mutant proteins with the Roi binding site differed from that of the wild type . Roi displays strong similarities, in its C-terminal half, to two putative DNA-binding proteins of bacteriophage P1: Ant1 and KilA . The mode of action of the Roi protein and the possibility that IHF is modulating the expression and/or the action of Roi are discussed. J Bacteriol, 1996 Jul, 178(14), 4031 - 8 Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein; Vianney A et al.; The TolQ, TolR, TolA, TolB, and Pal proteins appear to function in maintaining the integrity of the outer membrane, as well as facilitating the uptake of the group A colicins and the DNA of the infecting filamentous bacteriophages . Sequence data showed that these genes are clustered in a 6-kb segment of DNA with the gene order orf1 tolQ tolR tolA tolB pal orf2 (a newly identified open reading frame encoding a 29-kD9 protein) . Like those containing orf1, bacteria containing an insertion mutation in this gene showed no obvious phenotype . Analysis of beta-galactosidase activity from fusion constructs in which the lac operon was fused to various genes in the cluster showed that the genes in this region constitute two separate operons: orf1 tolQRA and tolB pal orf2 . In the orf1 tolQRA operon, translation of MR was dependent on translation of the upstream tolQ region . Consistent with this result, no functional ribosome-binding site for TolR synthesis was detected. J Bacteriol, 1996 Jul, 178(14), 4004 - 11 Integration of bacteriophage Mx8 into the Myxococcus xanthus chromosome causes a structural alteration at the C-terminal region of the IntP protein; Tojo N et al.; Mx8 is a generalized transducing phage that infects Myxococcus xanthus cells . This phage is lysogenized in M . xanthus cells by the integration of its DNA into the host chromosome through site-specific recombination . Here, we characterize the mechanism of Mx8 integration into the M . xanthus chromosome . The Mx8 attachment site, attP, the M . xanthus chromosome attachment site, attB, and two phage-host junctions, attL and attR, were cloned and sequenced . Sequence alignments of attP, attB, attL, and attR sites revealed a 29-bp segment that is absolutely conserved in all four sequences . The intP gene of Mx8 was found to encode a basic protein that has 533 amino acids and that carries two domains conserved in site-specific recombinases of the integrase family . Surprisingly, the attP site was located within the coding sequence of the intP gene . Hence, the integration of Mx8 into the M . xanthus chromosome results in the conversion of the intP gene to a new gene designated intR . As a result of this conversion, the 112-residue C-terminal sequence of the intP protein is replaced with a 13-residue sequence . A 3-base deletion within the C-terminal region had no effect on Mx8 integration into the chromosome, while a frameshift mutation with the addition of 1 base at the same site blocked integration activity . This result indicates that the C-terminal region is required for the enzymatic function of the intP product. Mol Biol Evol, 1996 Jul, 13(6), 749 - 57 Relative apparent synapomorphy analysis (RASA) . I: The statistical measurement of phylogenetic signal; Lyons-Weiler J et al.; We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA) . RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices . The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase . Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal . A first advantage is computational efficiency . A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power . The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied . RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates. Kaohsiung J Med Sci, 1996 Jul, 12(7), 381 - 7 Bacteriophage T4 expression-packaging-processing vector that encapsidates HIV-type I GP120-V3 fusion protein inside the head; Hong YR et al.; Bacteriophage T4 as an expression-packaging-processing vector has recently been developed by using the T4 non-essential capsid scaffold protein IPIII(1-3) . The IPIII gene was expressed at high level in E . coli from plasmid and was truncated at its C-terminus to permit construction of gene fusion in three different reading frames of EP-16 vector . Regulated higher-expression PPL reading frame vectors were also constructed . Infection of the plasmid-containing bacteria with bacteriophage mutants deleted for the IPIII gene showed that viable phage encapsidated and proteolytically processed the IPIII fusion proteins . An IPIII gene fused to human immunodeficiency virus type I (HIV-1) gp120 loop3 domain (V3) IPIII-V3 fusion gene products has been constructed and was packaged and processed within viable phage particle . The packaged fusion protein Gp120-V3 may be used for production of vaccine in the future. J Inorg Biochem, 1996 Jul, 63(1), 1 - 7 Metal-ion discrimination by phage T7; Whang T et al.; The discovery of new metal-selective complexing agents may be facilitated by applying an in vitro selection strategy . Such a strategy was recently devised to identify and enrich populations of bacteriophage that rely on Mg(II)-, Zn(II)-, or Au(III)-selective stabilization for survival in the presence of denaturing urea . The potential for extension of the strategy to other metal ions is investigated here . The kinetics of phage inactivation in 5 M urea was measured for a spectrum of metals . At a concentration of 1 mM, Mg(II), Ca(II), Co(II), and Ni(II) were found to be the most stabilizing, followed by Cd(II), Cu(II), and Zn(II), respectively . K(I) had virtually no effect . In contrast, Al(III) and Au(III) significantly destabilized the phage, even at concentrations of 0.25 mM. J Virol, 1996 Jul, 70(7), 4444 - 50 Transcription of a vaccinia virus late promoter template: requirement for the product of the A2L intermediate-stage gene; Passarelli AL et al.; Evidence is presented that a 26-kDa protein encoded by the vaccinia virus A2L open reading frame, originally shown to be one of three intermediate-stage genes that together can transactivate late-stage gene expression in transfection assays (J . G . Keck, C . J . Baldick, and B . Moss, Cell 61:801-809, 1990), is required for in vitro transcription of a template with a late promoter . The critical step in this analysis was the preparation of an extract containing all the required factors except for the A2L protein . This extract was prepared from cells infected with a recombinant vaccinia virus expressing the bacteriophage T7 RNA polymerase in the presence of the DNA synthesis inhibitor cytosine arabinoside and transfected with plasmids containing the two other known transactivator genes, A1L and G8R, under T7 promoter control . Reaction mixtures made with extracts of these cells had background levels of late transcription activity, unless they were supplemented with extracts of cells transfected with the A2L gene . Active transcription mixtures were also made by mixing extracts from three sets of cells, each transfected with a gene (A1L, A2L, or G8R) encoding a separate factor, indicating the absence of any requirement for their coexpression . To minimize the possibility that the A2L protein functions indirectly by activating another viral or cellular protein, this gene was expressed in insect cells by using a baculovirus vector . The partially purified recombinant protein complemented the activity of A2L-deficient cell extracts . Recombinant A1L, A2L, and G8R proteins, all produced in insect cells, together complemented extracts from mammalian cells containing only viral early proteins, concordant with previous in vivo transfection data. EMBO J, 1996 Jul 1, 15(13), 3458 - 65 Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase; Biebricher CK et al.; A large variety of different RNA species that are replicated by DNA-dependent RNA polymerase from bacteriophage T7 have been generated by incubating high concentrations of this enzyme with substrate for extended time periods . The products differed from sample to sample in molecular weight and sequence, their chain lengths ranging from 60 to 120 . The mechanism of autocatalytic amplification of RNA by T7 RNA polymerase proved to be analogous to that observed with viral RNA-dependent RNA polymerases (replicases): only single-stranded templates are accepted and complementary replica strands are synthesized . With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template . The plus strands, present at 90% of the replicating RNA species, were found to have GG residues at both termini . Consensus sequences were not found among the sequences of the replicating RNA species . The secondary structures of all species sequenced turned out to be hairpins . The RNA species were specifically replicated by T7 RNA polymerase; they were not accepted as templates by the RNA polymerases from Escherichia coli or bacteriophage SP6 or by Qbeta replicase; T3 RNA polymerase was partially active . Template-free production of RNA was completely suppressed by addition of DNA to the incubation mixture . When both DNA and RNA templates were present, transcription and replication competed, but T7 RNA polymerase preferred DNA as a template . No replicating RNA species were detected in vivo in cells expressing T7 RNA polymerase. Virology, 1996 Jul 1, 221(1), 67 - 77 Modulation of bacteriophage T4 capsid size; Haynes JA et al.; Bacteriophage T4 capsid assembly requires the vertex protein (gp24) . Mutations that bypass this requirement are found in gene 23, which produces the major capsid protein (gp23) . The latter were used to study the role of gp24 in head length control . We found that gp24 is no longer present in the capsids of several gp24 bypass mutants . We measured the capsid lengths of several of these bypass mutants, because gp24 had been reported to be implicated in head length control . One bypass mutant (reported in 1977) produced 40-60% short headed ("petite") phage in the presence of wild-type amounts of gp24 . The bypass mutations, when combined with amber mutations in gene 24, produced normal size heads in either suppressor or nonsuppressor host bacteria . When several known bypass mutations were back-crossed with wild-type phage, one-third of the byp/24wt mutants isolated produced large amounts of petite phage, indicating that the ability to produce petite phage is a general property of the bypass mutations . Sequencing several of these bypass mutants showed that those that produced petite phage contained at least one additional missense mutation in gene 23 . This suggests that gp24 itself has no direct role in head length regulation, but that in the presence of bypass 24 mutations and certain easily acquired gene 23 mutations (called trb) the gp23-gp24 interactions can modulate head length. J Biol Chem, 1996 Jun 28, 271(26), 15307 - 10 Identification of a second functional glutaredoxin encoded by the bacteriophage T4 genome; Gvakharia BO et al.; Thioredoxins and glutaredoxins are small ubiquitous redox proteins that were discovered as hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis . Some organisms encode more than one redox protein . In this study, we demonstrate that an open reading frame in the bacteriophage T4 genome, reported earlier and designated as Y55.7 (Tomaschewski, J., and Ruger, W . (1987) Nucleic Acids Res . 15, 3632-3633), encodes a second functional redox protein . Gene y55.7 was cloned and expressed in Escherichia coli . Purified Y55.7 protein had glutathione-dependent thioltransferase and dehydroascorbate reductase activities indicative of a functional glutaredoxin . The protein is expressed at all stages of the T4 infection cycle and can serve as a hydrogen donor for the phage ribonucleotide reductase in in vitro experiments. Gene, 1996 Jun 26, 172(2), 295 - 8 Recombinant rabbit Fab with binding activity to type-1 plasminogen activator inhibitor derived from a phage-display library against human alpha-granules; Lang IM et al.; The display of panels of antibody (Ab) fragments on the surface of filamentous bacteriophage offers a way of making Ab with defined binding specificities . Because rabbit Ab are routinely utilized as immunologic probes in a variety of biological techniques, the aim of this study was to design and utilize primers for the amplification of mRNAs encoding rabbit kappa light and gamma heavy chains for the construction of an Ab library from this species . Using the polymerase chain reaction, a diverse Ab library with a repertoire of 2 x 10(7) clones was derived from the spleen and bone marrow of a rabbit that had been immunized with purified human platelet alpha-granules . From this library, specific clones were isolated after three rounds of affinity selection with binding activity to type-1 plasminogen activator inhibitor, a trace protein contained in platelet alpha-granules . These data indicate that recombinant phage-displayed Ab libraries obtained after immunization with complex biological antigens can be employed for the isolation of rabbit monoclonal Fab against specific antigens contained in the biological sample. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6676 - 81 Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment; Li SC et al.; Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins . Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma . We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment . To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro) . Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol) . The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents . Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media . Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water . The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels . The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane. J Mol Biol, 1996 Jun 21, 259(4), 622 - 31 Bacteriophage T4 strand transfer protein UvsX tolerates symmetric and asymmetric heterologies in short double-stranded oligonucleotides; Birkenkamp K et al.; The UvsX protein of bacteriophage T4 catalyzes strand transfer from double-stranded DNA to homologous single-stranded DNA to generate both paranemic and plectonemic joints . We demonstrate here that UvsX mediates strand transfer efficiently from synthetic double-stranded donor oligonucleotides of 30 to 117 bp in length to circular single-stranded recipient M13mp19 DNA . Recovery of a diagnostic BamHI-restriction site, activated in the recipient after strand transfer, demonstrates that recipient and donated strands are perfectly base-paired after the exchange reaction has taken place . The transfer reaction progresses with greatest efficiency using donor DNA with a 3' overhang . Use of donor DNA having recessed 3' ends or blunt ends reduces the transfer efficiency by half . Single-stranded heterologies, centrally located in either strand of the donor DNA and forming either heteroduplex loops or a bulge in the donor are transferred with 80 to 100% efficiency . Also, a centrally located C/C-mismatch in the donor does not affect the transfer efficiency . Double-stranded heterologies are tolerated by the UvsX-catalyzed reaction but have different effects on the transfer efficiencies, depending on length and location in the molecule . A heterology of 24 bp located at the proximal end (start of transfer), the distal end (termination of transfer) and at each end of the donor molecule results in transfer efficiencies of 100%, 50% and 50 to 60%, respectively . Strand transfer efficiency is markedly reduced to about 15% if the 24 bp heterology is at a central location . However, insertion of a 4 bp heterology at this position yields a transfer efficiency of about 30% . Also, large double-stranded heterologies of 187 bp at the proximal end or 590 bp at the distal end of control-donor DNAs derived from plasmid digests did not impair the transfer activity of UvsX . This result differs from published results obtained with strand transfer reactions with large distally located heterologies catalyzed by RecA of Escherichia coli in vitro. J Biol Chem, 1996 Jun 21, 271(25), 14840 - 8 Structure-function analysis of the zinc finger region of the DnaJ molecular chaperone; Banecki B et al.; DnaJ is a molecular chaperone, which not only binds to its various protein substrates, but can also activate the DnaK cochaperone to bind to its various protein substrates as well . DnaJ is a modular protein, which contains a putative zinc finger motif of unknown function . Quantitation of the released Zn(II) ions, upon challenge with p-hydroxymercuriphenylsulfonic acid, and by atomic absorption showed that two Zn(II) ions interact with each monomer of DnaJ . Following the release of Zn(II) ions, the free cysteine residues probably form disulfide bridge(s), which contribute to overcoming the destabilizing effect of losing Zn(II) . Supporting this view, infrared and circular dichroism studies show that the DnaJ secondary structure is largely unaffected by the release of Zn(II) . Moreover, infrared spectra recorded at different temperatures, as well as scanning calorimetry, show that the Zn(II) ions help to stabilize DnaJ's tertiary structure . An internal 57-amino acid deletion of the cysteine-reach region did not noticeably affect the affinity of this mutant protein, DnaJDelta144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity . However, the DnaJDelta144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex . Additionally, the DnaJDelta144-200 mutant protein alone was unimpaired in its ability to interact with its final sigma32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and lambdaP substrates . Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage lambda growth in a DnaJDelta144-200 mutant background. Biochemistry, 1996 Jun 18, 35(24), 8045 - 57 Iterative optimization of high-affinity proteases inhibitors using phage display . 1 . Plasmin; Markland W et al.; We generated a series of libraries having variants of the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1, also known as tissue-factor pathway inhibitor-I) displayed on bacteriophage M13 as pIII-fusions . We varied LACI-DI iteratively in two regions: the P1 region (positions 10-21) and the "second loop", (positions 31-39), which together form one end of the domain . Display-phage library Lib#1 allows 31 200 amino-acid sequences in P1 region (residues 13, 16-19) . Preliminary, we screened Lib#1 against human plasmin (PLA, EC 3.4.21.7) immobolized on agarose to enrich for phage displaying variants with PLA affinity . We introduced a 1600-fold increase in second-loop diversity (residues 31, 32, 34, 39) into the population of selectants from Lib#1, yielding Lib#2 . Lib#2 (allowing approximately 50 million amino-acid sequences) was screened against PLA-agarose to isolate highest affinity binders . Protein EPI-P211, derived from the best isolate of Lib#2, inhibits PLA with Ki = 2 nM (at least 500-fold better than LACI-D1) and with high specificity . We used amino-acid sequences of PLA-binding selectants to design a PLA-biased library (Lib#3) which we screened against PLA . The protein EPI-P302 (derived from the best binder obtained from Lib#3) has Ki for PLA inhibition of 87 pM, which is 25-fold better than the first-round best binder and > or = 12 500-fold better than LACI-D1 . EPI-P302 also shows very high specificity for PLA vs other human proteases and is resistant to inactivation by oxidants and extremes of temperature or pH . Thus, one can use selectants from one library to design target-tailored combinatorial libraries and obtain quite stable, highly specific, very high-affinity binding molecules while maintaining an essentially human framework. Biochemistry, 1996 Jun 18, 35(24), 7675 - 83 A quadruple mutant T4 RNA hairpin with the same structure as the wild-type translational repressor; Mirmira SR et al.; The solution structure of a 16-nucleotide RNA hairpin, 5'-GCCUAG{CAAC}CUGGGC (loop bases in square brackets), has been determined by proton, phosphorus, and carbon (natural abundance) nuclear magnetic resonance (NMR) spectroscopy . This RNA tetraloop hairpin varies in four loop nucleotides from the wild-type T4 RNA hairpin (with eight loop nucleotides) involved in the translational repression of bacteriophage T4 DNA polymerase . Despite the differences in their sequence and proposed secondary structures, these two hairpins bind T4 DNA polymerase with equal affinity . The NMR spectra of the mutant hairpin indicate that its stem is extended in comparison to that of the wild-type hairpin by the formation of two additional Watson-Crick base pairs . The NMR data provide a precisely defined structure for the mutant hairpin with an average root mean square deviation of approximately 0.7 A for all 16 residues in the molecule . The structure of the mutant loop is very similar to that determined previously for the wild-type hairpin . The three loop bases that are conserved between the mutant and wild-type hairpins point out in solution with the groups capable of hydrogen bond formation exposed to the solution . This is exactly what was seen for the wild-type hairpin . Also, unusual, long-range NOEs, loop hydrogen bonds, and even the position at which the loop bends are common features between the two loops . This explains how two different hairpins, by adopting similar three-dimensional structures, have the same affinity for the DNA polymerase. Biochemistry, 1996 Jun 18, 35(24), 7664 - 74 NMR structure of a bacteriophage T4 RNA hairpin involved in translational repression; Mirmira SR et al.; A high-resolution structure of a 16-nucleotide bacteriophage T4 RNA hairpin, 5'-GCCU{AAUAACUC}GGGC (loop bases in square brackets), has been determined in solution by proton, phosphorus, and carbon (natural abundance) NMR spectroscopy . This RNA hairpin is known to play a crucial role in the translational repression of bacteriophage T4 DNA polymerase . Ultraviolet absorbance melting curves indicate that the structure formed is unimolecular . The NMR spectra indicate that a single conformation consistent with a hairpin structure is formed . Strong imino-imino NOEs confirm the formation of the G.U base pair at the stem-loop junction . There is no evidence that A5 is protonated (at pH 6.0) and involved in an A+.C pair . However, the NMR data indicate that the stem is extended beyond the G.U pair and that A-form stacking continues for three nucleotides on the 5' side and one nucleotide on the 3' side . Structure calculations using restraints obtained from NMR data give a precisely defined structure with an average root mean square deviation (RMSD) of approximately 1.2 A for the entire molecule . The assignment of all the protons and most of the 31P resonances in the loop yielded a large number of distance and torsion angle restraints for these nucleotides . These helped obtain a well-defined loop with an average RMSD of 1.1 A for the loop nucleotides of 11 converged structures. Nucleic Acids Res, 1996 Jun 15, 24(12), 2352 - 9 Complementation of RNA binding site mutations in MS2 coat protein heterodimers; Peabody DS et al.; The coat protein of bacteriophage MS2 functions as a symmetric dimer to bind an asymmetric RNA hairpin . This implies the existence of two equivalent RNA binding sites related to one another by a 2-fold symmetry axis . In this view the symmetric binding site defined by mutations conferring the repressor-defective phenotype is a composite picture of these two asymmetric sites . In order to determine whether the RNA ligand interacts with amino acid residues on both subunits of the dimer and in the hope of constructing a functional map of the RNA binding site, we performed heterodimer complementation experiments . Taking advantage of the physical proximity of their N- and C-termini, the two subunits of the dimer were genetically fused, producing a duplicated coat protein which folds normally and allows the construction of the functional equivalent of obligatory heterodimers containing all possible pairwise combinations of the repressor-defective mutations . The restoration of repressor function in certain heterodimers shows that a single RNA molecule interacts with both subunits of the dimer and allows the construction of a functional map of the binding site. Nucleic Acids Res, 1996 Jun 15, 24(12), 2281 - 7 Characterization of proteolytic fragments of bacteriophage T7 DNA ligase; Doherty AJ et al.; Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion . These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively . The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion . The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of {alpha-32P}ATP, confirming that it contains the active site lysine residue . In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding . It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA . We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA. Eur J Biochem, 1996 Jun 15, 238(3), 706 - 13 The solution structure of the synthetic circular peptide CGVSRQGKPYC . NMR studies of the folding of a synthetic model for the DNA-binding loop of the ssDNA-binding protein encoded by gene V of phage M13; Rietman BH et al.; The cyclic disulfide peptide CGVSRQGKPYC was prepared to obtain a constrained analogue of residues 17-27 of the DNA-binding loop of the gene-V-encoded sDNA-binding protein of filamentous bacteriophage M13 . Amino acid sequences very similar to that of the beta-loop have been found in various phage-encoded ssDNA-binding proteins, and it has been proposed that such a loop may occur as a common motif in this class of proteins . The conformation, in aqueous solution, of the synthetic gene-V-protein binding-loop analogue has been investigated by means of two-dimensional-1H-NMR techniques . Subsequent structure calculations show that the molecule forms a beta-loop that includes a turn formed by three residues . This structure, very unusually for a cyclic disulfide peptide, is highly similar to that of the analogous part of the binding loop of the native protein . Comparison with experiments on other cyclic disulfide peptides indicates that the formation, of the beta-sheet (beta-hairpin) secondary structure is essentially governed by the amino acid composition of the 11-residue sequence . The disulfide bridge in the 11-residue sequence is essential for conformational stability, as indicated by the finding that the open peptide analogue that encompasses residues Ser17-Ser27 does not adopt a detectable secondary structure in water . The bridge replaces the role of the loop formed by residues 49-58 in the protein, which act as a scaffold to hold the N-terminal and C-terminal ends of the DNA-binding loop together. J Mol Biol, 1996 Jun 14, 259(3), 542 - 59 Thermodynamic and structural compensation in "size-switch" core repacking variants of bacteriophage T4 lysozyme; Baldwin E et al.; Previous analysis of randomly generated multiple mutations within the core of bacteriophage T4 lysozyme suggested that the "large-to-small" substitution Leu121 to Ala (L121A) and the spatially adjacent "small-to-large" substitution Ala129 to Met (A129M) might be mutually compensating . To test this hypothesis, the individual variants L121A and A129M were generated, as well as the double "size-switch" mutant L121A/A129M . To make the interchange symmetrical, the combination of L121A with A129L to give L121A/A129L was also constructed . The single mutations were all destabilizing . Somewhat surprisingly, the small-to-large substitutions, which increase hydrophobic stabilization but can also introduce strain, were less deleterious than the large-to-small replacements . Both Ala129 --> Leu and Ala129 --> Met offset the destabilization of L121A by about 50% . Also, in contrast to typical Leu --> Ala core substitutions, which destabilize by 2 to 5 kcal/mol, Leu121 --> Ala slightly stabilized A129L and A129M . Crystal structure analysis showed that a combination of side-chain and backbone adjustments partially accommodated changes in side-chain volume, but only to a limited degree . For example, the cavity that was created by the Leu121 to Ala replacement actually became larger in L121A/A129L . The results demonstrate that the destabilization associated with a change in volume of one core residue can be specifically compensated by an offsetting volume change in an adjacent residue . It appears, however, that complete compensation is unlikely because it is difficult to reconstitute an equivalent set of interactions . The relatively slow evolution of core relative to surface residues appears, therefore, to be due to two factors . First, a mutation in a single core residue that results in a substantial change in size will normally lead to a significant loss in stability . Such mutations will presumably be selected against . Second, if a change in bulk does occur in a buried residue, it cannot normally be fully compensated by a mutation of an adjacent residue . Thus, the most probable response will tend to be reversion to the parent protein. J Biol Chem, 1996 Jun 14, 271(24), 14074 - 81 Stoichiometry and DNA unwinding by the bacteriophage T4 41:59 helicase; Raney KD et al.; The bacteriophage T4 41 protein is a replicative helicase that forms a hexamer in the presence of ATP and associates with the T4 59 protein . The stoichiometry of the 41:59 helicase complex and its mechanism for DNA unwinding have been investigated using steady-state and single-turnover kinetics . A partial duplex DNA fork containing two regions of single-stranded DNA (ssDNA) of 30 nucleotides each, and 30 base pairs served as the substrate . 59 was found to increase the steady-state unwinding rate of the substrate by 200-fold over the rate of 41 alone . Maximum unwinding occurred when 59 and 41 were equimolar, revealing a 1:1 stoichiometry for the complex . Varying 41 while holding 59 constant resulted in sigmoidal kinetics suggesting strong cooperativity for formation of the 41 hexamer and providing a lower limit for hexamer assembly of 65 nM . Substrates were prepared that contained a biotin-streptavidin block in either the leading or lagging strand of the duplex region of the substrate . The first order rate constant for unwinding was reduced only when the block was placed in the lagging strand of the DNA fork, indicating that the helicase interacts primarily with the lagging DNA strand. Genes Cells, 1996 Jun, 1(6), 569 - 79 Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense; Kato A et al.; BACKGROUND: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae . These viruses possess a single stranded negative sense RNA as the genome . Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering . However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+)RNA . Starting with genomic negative sense (-)RNA has been unsuccessful . Furthermore, the recovery efficiency has often been extremely low . RESULTS: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus-driven bacteriophage T7 polymerase . Our system was able to recover the virus from cDNA directing not only (+)RNA but also (-)RNA . Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (-)RNA . This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors . In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter-specific nucleotides to the 5' ends . An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage-activation site of the viral fusion glycoprotein . CONCLUSION: We have established methods which greatly improve the recovery of Sendai virus from cDNA . There is essentially no absolute obstacle to recovery of the virus from the (-)RNA template . Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template. J Biomol Struct Dyn, 1996 Jun, 13(6), 945 - 51 Inhibition of restriction endonuclease activity by DNA binding fluorochromes; Meng X et al.; Activity of type II restriction endonuclease is affected by many common factors including buffer composition and sequences flanking the recognition site (Brabec et al., Eur.J . Biochem . 216, 183, 1993) . The successful development of Optical Mapping (Schwartz et al., Science, 262, 110, 1993; Meng et al., Nature Genet . 9, 432, 1995; Wang and Schwartz, PNAS, 1995 Cai et al., PNAS, 92, 5164, 1995) relied on optimization of light microscope-based imaging of fluorescently labeled DNA molecules during restriction endonuclease digestion . Little was known about the effects of commonly used DNA-fluorochromes on restriction endonuclease activity . Thus, we developed an enzyme activity assay using lambda bacteriophage DNA or adenovirus-2 DNA to evaluate the effects of five DNA binding fluorochromes (4'-6-daimidine-2-phenylindole (DAPI), ethidium bromide (EtdBr), ethidium bromide homodimer (EthD-1), bis-benzimide (H33258) and benzothiazolium-4-quinolinium dimer (TOTO-1)) on the enzymatic activities of eleven type II restriction endonucleases (Asc I, Csp I, Dra I, EcoR I, Hha I, Hind III, Not I, Rsr II, Sfi I, SgrA I and Sma I) . We found that the minor groove binding fluorochrome, DAPI, did not measurably inhibit activity of this group, with the exception of Dra I . Similarly, another minor groove binding fluorochrome H33258 inhibited Dra I and Not I (slightly) . The three intercalating fluorochromes EtdBr, EthD-1 and TOTO-1, however, variably inhibited the other enzymes . Since Beta-mercaptoethanol (Beta-ME) is used to discourage photodamage of stained DNA molecules, we also assessed its effect on restriction endonuclease activity . Interestingly, Dra I, Hind III, Sfi I and Sma I retained full activities at high concentration of Beta-ME (5%), but Asc I, Csp I, Not I, Rsr II and SgrA I showed varying sensitivities to the Beta-ME . Isoschizomers Csp I and Rsr II behaved differently to both fluorochromes and Beta-ME . The results presented here should provide a basis for further development of new Optical Mapping-based techniques requiring fluorescence labeling of other actively imaged enzymatic reactions. Genome Res, 1996 Jun, 6(6), 525 - 37 Generation of a transcriptional map for a 700-kb region surrounding the polycystic kidney disease type 1 (PKD1) and tuberous sclerosis type 2 (TSC2) disease genes on human chromosome 16p3.3; Burn TC et al.; A 700-kb region of DNA in human chromosome 16p13.3 has been shown to contain the polycystic kidney disease 1 (PKD1) and the tuberous sclerosis type 2 (TSC2) disease genes . An estimated 20 genes are present in this region of chromosome 16 . We have initiated studies to identify transcribed sequences in this region using a bacteriophage P1 contig containing 700 kb of DNA surrounding the PKD1 and TSC2 genes . We have isolated 96 unique exon traps from this interval, with 23 of the trapped exons containing sequences from five genes known to be in the region . Thirty exon traps have been mapped to additional transcription units based on data base homologies, Northern analysis, or their presence in cDNA or reverse transcriptase (RT)-PCR products . We have mapped the human RNPS gene to the cloned interval . We have obtained cDNAs or RT-PCR products from eight novel genes, with sequences from seven of these genes having homology to sequences in the data bases . Two of the newly identified genes represent human homologs for rat and murine genes identified previously . We have isolated three exon traps with homology to sequences in the data bases but have been unable to confirm the presence of these exon traps in expressed sequences . In addition, we have isolated 43 exon traps that do not map to our existing cDNAs or PCR products and have no homology to sequences in the data bases . In this report we present a transcriptional map for the 700 kb of DNA surrounding the PKD1 and TSC2 genes. J Biochem (Tokyo), 1996 Jun, 119(6), 1062 - 9 The virion proteins encoded by bacteriophage phi K and its host-range mutant phi KhT: host-range determination and DNA binding properties; Kodaira K et al.; The microvirid phage phi K, specific for Escherichia coli K12, contains a circular single-stranded (SS) DNA in the icosahedral virion, which comprises four phage gene products, F (capsid), G (major spike), H (minor spike), and J (core) . phi KhT, a host-range mutant of phi K, can grow on E . coli C and B, besides K12, and is more thermosensitive than the parental phage phi K . Sequencing analysis revealed that the genome of phi K and phi KhT consists of 6,089 nucleotides (nt), and codes for eleven genes, whose sequences are similar to those of alpha 3, phi X174, and G4 infective to strain C . In phi KhT, two nt had changed: one is in the gene G, resulting in replacement of the 75th codon Ala with Ser, and the other is at 67th codon of the gene H: Val to Ala . Chemically synthesized gene J protein composed of 23 amino acids (aa) binds to phi K SS DNA more tightly than and preferentially over the host E . coli SS-DNA-binding protein (SSB) . These results indicate that the two spike proteins G and H are involved in the determination of phi K host-range, and support a model in which the gene J protein functions in packaging the viral SS DNA into the virion vesicle. Mol Microbiol, 1996 Jun, 20(6), 1145 - 54 Role of recombinational repair in sensitivity to an antitumour agent that inhibits bacteriophage T4 type II DNA topoisomerase; Neece SH et al.; The bacteriophage T4-encoded type II DNA topoisomerase is the major target for the antitumour agent m-AMSA (4'-(9-acridinylamino)methanesulphonm-ansidide) in phage-infected bacterial cells . Inhibition of the purified enzyme by m-AMSA results in formation of a cleavage complex that contains the enzyme covalently attached to DNA on both sides of a double-strand break . In this article, we provide evidence that this cleavage complex is responsible for inhibition of phage growth and that recombinational repair can reduce sensitivity to the antitumour agent, presumably by eliminating the complex (or some derivative thereof) . First, topoisomerase-deficient mutants were shown to be resistant to m-AMSA, indicating that m-AMSA inhibits growth by inducing the cleavage complex rather than by inhibiting enzyme activity . Second, mutations in several phage genes that encode recombination proteins (uvsX, uvsY, 46 and 59) increased the sensitivity of phage T4 to m-AMSA, strongly suggesting that recombination participates in the repair of topoisomerase-mediated damage . Third, m-AMSA stimulated recombination in phage-infected bacterial cells, as would be expected from the recombinational repair of DNA damage . Finally, m-AMSA induced the production of cleavage complexes involving the T4 topoisomerase within phage-infected cells. Antiviral Res, 1996 Jun, 31(1-2), 79 - 86 Anti-vaccinia virus effect of M13 bacteriophage DNA; Mori K et al.; Single-stranded DNA derived from M13 phage was evaluated for antiviral activity in mice infected with vaccinia virus . M13 DNA at a dose as low as 16.7 mg/kg was effective in reducing the number of tail lesions caused by vaccinia virus by more than 90% . A single administration of M13 DNA 1 day before infection was sufficient to reduce significantly the number of tail lesions caused by vaccinia virus . Denatured eukaryotic nucleic acids such as calf thymus DNA and human placenta DNA were not effective . A mixture of nucleotides prepared according to the nucleotides composition of M13 DNA was also ineffective . Within 4 h after the administration of M13 DNA, the serum interferon (IFN, predominantly type beta) titer rose from undetectable levels to as much as approximately 700 IU/ml . IFN was detectable for up to 12 h after the administration of M13 DNA . IFN titers as high as 1050 IU/ml were detected in vitro when M13 DNA was added to spleen cultures . We conclude that at least part of the antiviral activity of M13 DNA can be explained on the basis of IFN induction. Nucleic Acids Res, 1996 Jun 1, 24(11), 2190 - 1 Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers; Lench NJ et al.; We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats . The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers . The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones . We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3. Nucleic Acids Res, 1996 Jun 1, 24(11), 2166 - 75 Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase; Belanger KG et al.; The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels . Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails . A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail . After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid . DNA samples were generated from infections with either wild-type or primase-deletion mutant phage . The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis . Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection . We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent. Gene, 1996 Jun 1, 171(2), 299 - 300 The cDNA sequence and infectious transcripts of peanut stripe virus; Flasinski S et al.; A full-length cDNA clone of the blotch isolate of the peanut stripe potyvirus (PStV) RNA genome was constructed downstream from the bacteriophage SP6 RNA polymerase promoter . The full-length PStV cDNA clone (PStVSF9) was sequenced and compared to the previously published sequence of PStV-B . In vitro-synthesized PStV transcripts capped with m7GpppG were infectious in Nicotiana benthamiana plants, and the progeny virus was aphid transmissible . To confirm the origin of infection, a mutant PStV (PStVDAE), with a Gly14 to Glu mutation in the coat protein-encoding gene, was constructed . Transcripts from PStVDAE produced symptoms indistinguishable from native or PStVSF9 virus, but was not transmitted by aphids. J Bacteriol, 1996 Jun, 178(12), 3447 - 56 Topology of the membrane protein LamB by epitope tagging and a comparison with the X-ray model; Newton SM et al.; We previously developed a genetic approach to study, with a single antibody, the topology of the outer membrane protein LamB, an Escherichia coli porin with specificity towards maltodextrins and a receptor for bacteriophage lambda . Our initial procedure consisted of inserting at random the same reporter epitope (the C3 neutralization epitope from poliovirus) into permissive sites of LamB (i.e., sites which tolerate insertions without deleterious effects on the protein activities or the cell) . A specific monoclonal antibody was then used to examine the position of the inserted epitope with respect to the protein and the membrane . In the present work, we set up a site-directed procedure to insert the C3 epitope at new sites in order to distinguish between two-dimensional folding models . This allowed us to identify two new surface loops of LamB and to predict another periplasmic exposed region . The results obtained by random and directed epitope tagging are analyzed in light of the recently published X-ray structure of the LamB protein . Study of 23 hybrid LamB-C3 proteins led to the direct identification of five of the nine external loops (L4, L5, L6, L7, and L9) and led to the prediction of four periplasmic loops (I1, I4, I5, and I8) of LamB . Nine of the hybrid proteins did not lead to topological conclusions, and none led to the wrong predictions or conclusions . The comparison indicates that parts of models based on secondary structure predictions alone are not reliable and points to the importance of experimental data in the establishment of outer membrane protein topological models . The advantages and limitations of genetic foreign epitope insertion for the study of integral membrane proteins are discussed. Gene, 1996 May 24, 171(1), 49 - 51 New vectors for peptide display on the surface of filamentous bacteriophage; Malik P et al.; We have modified the genome of the filamentous bacteriophage fd and also constructed a number of new vectors for the purpose of displaying peptides on the surface of the virion . These vectors facilitate the directional cloning of DNA encoding a peptide of interest at or near the N terminus of the major coat protein, the product of the bacteriophage gene VIII, and the construction of hybrid capsids in which the modified coat protein is interspersed with wild-type coat protein subunits. J Biol Chem, 1996 May 24, 271(21), 12241 - 6 Equilibrium binding studies of recombinant anti-single-stranded DNA Fab . Role of heavy chain complementarity-determining regions; Komissarov AA et al.; We previously isolated nucleic acid-binding antibody fragments (Fab) from bacteriophage display libraries representing the immunoglobulin repertoire of automimune mice to expedite the analysis of antibody-DNA recognition . In the present study, the binding properties of one such anti-DNA Fab, high affinity single-stranded (ss) DNA-binding Fab (DNA-1), were defined using equilibrium gel filtration and fluorescence titration . Results demonstrated that DNA-1 had a marked preference for oligo(dT) (100 nM dissociation constant) and required oligo(dT) >5 nucleotides in length . A detailed analysis of the involvement of the individual heavy chain (H) complementarity-determining regions (CDR) ensued using previously constructed HCDR transplantation mutants between DNA-1 and low affinity ssDNA-binding Fab (D5), a Fab that binds poorly to DNA (Calcutt, M . J . Komissarov, A . A., Marchbank, M . T., and Deutscher, S . L . (1996) Gene (Amst.) 168, 9-14) . Circular dichroism studies indicated that the wild type and mutant Fab studied were of similar overall secondary structure and may contain similar combining site shapes . The conversion of D5 to a high affinity oligo(dT)-binding Fab occurred only in the presence of DNA-1 HCDR3 . Results with site-specific mutants in HCDR1 further suggested a role of residue 33 in interaction with nucleic acid . The results of these studies are compared with previously published data on DNA-antibody recognition. J Mol Biol, 1996 May 24, 258(5), 839 - 50 A discontinuous headful packaging model for packaging less than headful length DNA molecules by bacteriophage T4; Leffers G et al.; Bacteriophage T4 and other double-stranded DNA-containing bacteriophages package DNA by the classical headful packaging mechanism . In this mechanism, the packaging machinery cuts a DNA concatemer and packages a single unit length genome within the viral capsid . The length of the packaged DNA molecule is determined by the size of the viral capsid . Surprisingly, during large DNA cloning experiments, we observed that the in vitro phage T4 packaging system can package and transduce DNA molecules that are much smaller than the T4 headful size . We analyzed this phenomenon by using defined plasmid DNAs as substrates for in vitro packaging . The data showed that phage T4 can successfully package and transduce 4 to 29 kb plasmid DNA molecules . When two plasmid DNAs with different antibiotic markers were added to the packaging reaction mixture, transductants that are resistant to both the antibiotics were obtained, suggesting that both the plasmid DNAs are packaged within the same head . Analysis of the transducing particles by equilibrium CsCl density-gradient centrifugation showed that the particles have the same density as the wild-type phage . That the less than headful length molecules were not converted to T4 headful length prior to packaging was established by a number of independent approaches . Finally, unit length plasmid DNA molecules of appropriate size were isolated from the in vitro packaged particles . Based on these data, we propose a discontinuous headful packaging model for packaging less than headful length molecules . In this model, the packaging machinery packages the first available less than headful length DNA molecule and generates a partially full head . The partially full head then reinitiates packaging on a second DNA molecule . This process continues until the head is filled with DNA. J Mol Biol, 1996 May 24, 258(5), 747 - 62 Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage; Bonnycastle LL et al.; The structural requirements for peptide binding to an antibody may be elucidated by probing it with a variety of peptides having different constraints . To this end, we have constructed and screened a panel of peptide libraries displayed by filamentous bacteriophage . The peptides in most of the libraries have the potential for constraint by fixed Cys residues, which have been placed at different sites within a randomized amino acid sequence of varying length . When taken together, the binding data obtained from screening the panel with a given antibody allow one to determine the types of constraints that promote binding, as well as the residues that are critical for binding . We describe the construction of 11, pVIII-displayed, peptide libraries, whose sizes range from 150 million to 10 billion clones . The libraries were screened with a number of polyclonal and monoclonal antibodies against peptides, proteins and carbohydrates . Cross-reactivity with peptides was always found for antibodies produced against peptides, linear epitopes on folded proteins and, surprisingly, carbohydrates, whereas antibodies against discontinuous epitopes on proteins were found less frequently . The implications of these results are discussed in terms of the structural basis for cross-reactivity with peptides. Biochemistry, 1996 May 21, 35(20), 6276 - 82 Mutational analysis of the catalytic subunit of muscle protein phosphatase-1; Zhang J et al.; A mutational analysis of rabbit skeletal muscle protein phosphatase-1 was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli . The selection of the sites to be mutated was based on sequence alignments which showed the existence of a number of invariant residues when eukaroytic Ser/Thr protein phosphatases were compared with bacteriophage phosphatases and adenosinetetraphosphatase {Barton et al . (1995) Eur . J . Biochem . 220, 225-237} . In other studies, it had been shown that PP1 is a metalloprotein {Chu et al . (1996) J . Biol . Chem . 271, 2574-2577}, and in this study, we have largely focused on invariant histidine and aspartate residues which may be involved in metal binding . The residues which were mutated were H66, H125, H173, H248, D64, D71, D92, D95, N124, and R96E . The results showed that mutation of H66, H248, D64, and D92 resulted in severe loss of catalytic function . Mutation of D95, N124, and R96 also led to loss of function, while attempts to mutate H125 and H173 led to production of insoluble, inactive proteins . The results of the mutational analysis are consistent with the involvement of conserved His and Asp residues in metal binding, and are discussed in the context of the recently described crystal structure of PP1 {Goldberg et al . (1995) Nature, 376, 745-753}, which reveals that PP1 possesses a bimetallic center at the active site . The behavior of the D95, R96, and N124 mutants supports a catalytic mechanism involving nucleophilic attack by a hydroxide ion with H125 functioning as a proton donor to the leaving alcohol group. J Biol Chem, 1996 May 17, 271(20), 12095 - 102 Function of the htrB high temperature requirement gene of Escherchia coli in the acylation of lipid A: HtrB catalyzed incorporation of laurate; Clementz T et al.; By assaying lysates of Escherichia coli generated with the hybrid lambda bacteriophages of an ordered library (Kohara, Y., Akiyama, K., and Isono, K . (1987) Cell 50, 495-508), we identified two clones (lambda232 and lambda233) capable of overexpressing the lauroyl transferase that functions after 3-deoxy-D-manno-octulosonic acid (Kdo) addition in lipid A biosynthesis (Brozek, K . A., and Raetz, C . R . H . (1990) J . Biol . Chem . 265, 15410-15417) . The E . coli DNA inserts in lambda232 and lambda233 suggested that a known gene (htrB) required for rapid growth above 33 degrees C might encode the lauroyl transferase . Using the intermediate (Kdo)2-lipid IVA as the laurate acceptor, extracts of strains with transposon insertions in htrB were found to contain no lauroyl transferase activity . Cells harboring hybrid htrB+ plasmids overproduced transferase activity 100-200-fold . The overproduced transferase was solubilized with a non-ionic detergent and purified further by DEAE-Sepharose chromatography . With lauroyl acyl carrier protein as the donor, the purified enzyme rapidly incorporated one laurate residue into (Kdo)2-lipid IVA . The rate of laurate incorporation was reduced by several orders of magnitude when either one or both Kdos were absent in the acceptor . With a matched set of acyl-acyl carrier proteins, the enzyme incorporated laurate 3-8 times faster than decanoate or myristate, respectively . Transfer of palmitate, palmitoleate, or R-3-hydroxymyristate was very slow . Taken together with previous studies, our findings indicate that htrB encodes a key, late functioning acyltransferase of lipid A biosynthesis. J Biol Chem, 1996 May 17, 271(20), 11963 - 70 Transgenic mice that overexpress mouse apolipoprotein B . Evidence that the DNA sequences controlling intestinal expression of the apolipoprotein B gene are distant from the structural gene; McCormick SP et al.; An 87-kilobase (kb) P1 bacteriophage clone (p649) spanning the mouse apolipoprotein (apo) B gene was used to generate transgenic mice that express high levels of mouse apoB . Plasma levels of apoB, low density lipoprotein cholesterol, and low density lipoprotein triglycerides were increased, and high density lipoprotein cholesterol levels were decreased in the transgenic mice, compared with nontransgenic littermate controls . Although p649 contained 33 kb of 5'-flanking sequences and 11 kb of 3'-flanking sequences, the tissue pattern of transgene expression was different from that of the endogenous apoB gene . RNA slot blots and RNase protection analysis indicated that the transgene was expressed in the liver but not in the intestine, whereas the endogenous apoB gene was expressed in both tissues . To confirm the absence of transgene expression in the intestine, the mouse apoB transgenic mice were mated with the apoB knockout mice, and transgenic mice that were homozygous for the apoB knockout mutation were obtained . Because of the absence of transgene expression in the intestine, those mice lacked all intestinal apoB synthesis, resulting in a marked accumulation of fats within the intestinal villus enterocytes . The current studies, along with prior studies of human apoB transgenic animals, strongly suggest that the DNA sequence element(s) controlling intestinal expression of the apoB gene is located many kilobases from the structural gene. Cell, 1996 May 17, 85(4), 607 - 15 Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7; Subramanya HS et al.; The crystal structure of the ATP-dependent DNA ligase from bacteriophage T7 has been solved at 2.6 A resolution . The protein comprises two domains with a deep cleft running between them . The structure of a complex with ATP reveals that the nucleotide binding pocket is situated on the larger N-terminal domain, at the base of the cleft between the two domains of the enzyme . Comparison of the overall domain structure with that of DNA methyltransferases, coupled with other evidence, suggests that DNA also binds in this cleft . Since this structure is the first of the nucleotidyltransferase superfamily, which includes the eukaryotic mRNA capping enzymes, the relationship between the structure of DNA ligase and that of other nucleotidyltransferases is also discussed. Structure, 1996 May 15, 4(5), 543 - 54 The crystal structure of bacteriophage Q beta at 3.5 A resolution; Golmohammadi R et al.; BACKGROUND: The capsid protein subunits of small RNA bacteriophages form a T = 3 particle upon assembly and RNA encapsidation . Dimers of the capsid protein repress translation of the replicase gene product by binding to the ribosome binding site and this interaction is believed to initiate RNA encapsidation . We have determined the crystal structure of phage Q beta with the aim of clarifying which factors are the most important for particle assembly and RNA interaction in the small phages . RESULTS: The crystal structure of bacteriophage Q beta determined at 3.5 A resolution shows that the capsid is stabilized by disulfide bonds on each side of the flexible loops that are situated around the fivefold and quasi-sixfold axes . As in other small RNA phages, the protein capsid is constructed from subunits which associate into dimers . A contiguous ten-stranded antiparallel beta sheet facing the RNA is formed in the dimer . The disulfide bonds lock the constituent dimers of the capsid covalently in the T = 3 lattice . CONCLUSIONS: The unusual stability of the Q beta particle is due to the tight dimer interactions and the disulfide bonds linking each dimer covalently to the rest of the capsid . A comparison with the structure of the related phage MS2 shows that although the fold of the Q beta coat protein is very similar, the details of the protein-protein interactions are completely different . The most conserved region of the protein is at the surface, which, in MS2, is involved in RNA binding. Virology, 1996 May 15, 219(2), 443 - 52 Bacteriophage P4 capsid-size determination and its relationship to P2 helper interference; Nilssen O et al.; The sid (size determination) gene product of phage P4 is known to be involved in capsid-size determination . Moreover, the capsid-size determination function interferes with the lytic development of its helper P2, presumably because the helper genome is too large to be packaged into P4-size capsids . In order to study P4-specified helper interference, we cloned the sid gene for expression during phage infection . Even though gpSid restores the capsid-size determination function of a sid defective P4 mutant, we find that gpSid alone is not sufficient to establish full interference of helper P2 phage production . Complete helper interference requires some P4 function in addition to gpSid . Complementation tests show that none of the known P4 genes display this property . We propose that P4 encodes a yet-unidentified function that in concert with gpSid establishes full P2 helper interference at the level of capsid-size determination. Virology, 1996 May 15, 219(2), 432 - 42 Mutational analysis of the bacteriophage P4 capsid-size-determining gene; Nilssen O et al.; Satellite phage P4 (11,624 bp) depends on the morphopoietic genes (capsid, tail) and lysis genes of its helper phage P2 (33.5 kb) for its lytic development . In the morphopoietic process, P4 redirects the assembly pathway of large, P2 size, capsids (diameter = 60 nm) to yield smaller, P4 size, capsids (diameter = 45 nm), 1/3 in volume of that of its helper . The P4-specified capsid size determination is dependent on the function of the 27-kDa gpSid . To study the capsid size-determining function, we carried out a mutational analysis of the P4 sid gene . Use of a P4-derived genome of 29.1 kb (P461), which can be packaged only into large, P2 size, capsids allowed us to select P4 Sid- mutants . By DNA sequencing we characterized 25 P4 Sid- mutants, of which 10 contain base pair substitutions and 15 contain deletions . Both types of mutations are clustered in separate locations within the sid gene . Our results suggest that the Sid polypeptide contains three distinct functional domains. Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 5019 - 24 Epitope-specific tolerance induction with an engineered immunoglobulin; Zambidis ET et al.; Isologous and heterologous immunoglobulins have been shown to be extremely effective as tolerogenic carriers for nearly 30 years . The efficacy of these proteins is due in part to their long half-life in vivo, as well as their ability to crosslink surface IgM with Fc receptors . The concept of using IgG as a carrier molecule to induce unresponsiveness in the adult immune system has been exploited for simple haptens, such as nucleosides, as well as for peptides . To further evaluate the in vivo potential of these molecules for inducing tolerance to a defined epitope, we have engineered a fusion protein of mouse IgG1 with the immunodominant epitope 12-26 from bacteriophage lambda cI repressor protein . This 15-mer, which contains both a B-cell and T-cell epitope, has been fused in-frame to the N terminus of a mouse heavy chain IgG1 construct, thus creating a "genetic hapten-carrier" system . We describe a novel in vitro and in vivo experimental system for studying the feasibility of engineered tolerogens, consisting of a recombinant flagellin challenge antigen and a murine IgG1 tolerogen, both expressing the lambda repressor epitope 12-26 . Herein, we show that peptide-grafted IgG molecules injected i.v., or expressed by transfected, autologous B cells, can efficiently modulate the cellular and humoral immune responses to immunodominant epitopes . This model displays the feasibility of "tailor-designing" immune responses to whole antigens by selecting epitopes for either tolerance or immunity. FEBS Lett, 1996 May 13, 386(1), 72 - 4 Determination of binding site of anti-tumour necrosis factor-alpha monoclonal antibody using hybrid and mutant proteins; Shingarova LN et al.; In order to map the immunogenic epitope for the monoclonal antibody E7H2 on the human tumour necrosis factor (hTNF-alpha) molecule, a number of chimeric proteins were developed by in-frame joining segments of the human genes encoding TNF-alpha and lymphotoxin (TNF-beta) as well as by coupling appropriate coding regions for human and mouse TNF-alpha . High level expression of these chimeric genes was achieved in Escherichia coli by placing the coding sequences under control of either E . coli trp-promoter or a tandem of bacteriophage T7 constitutive promoters A2 and A3 . As revealed by Western blot analysis with monoclonal antibody E7H2 directed against human TNF-alpha, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the N-terminal part of the TNF-alpha molecule. J Mol Biol, 1996 May 10, 258(3), 433 - 46 cis-acting elements within an RNA coliphage genome: fold as you please, but fold you must!! Arora R, Priano C, Jacobson AB, Mills DR. Using an in vivo complementation system, we conducted a mutational analysis of the bacteriophage Q beta readthrough cistron . In the Q beta cDNA-containing plasmid, pQ beta m100, we constructed six defined Q beta deletion cDNA genomes, each missing between 86 and 447 nucleotides from within the readthrough cistron . These deletion plasmids were introduced into host cells that are constitutively supplied with Q beta readthrough protein from the plasmid pQ beta RT . Under these conditions, all six deletion genomes spontaneously generated phage particles, each exhibiting a characteristic plaque phenotype and virus forming potential . Isolated readthrough-defective phage particles were subsequently used to infect host cells that carried helper readthrough protein . Passaged viruses yielded both larger plaques and higher titers, compared with those of the parent phages . Sequence analysis revealed that the genomes of the passaged viruses had deleted additional regions of readthrough RNA sequence . We discuss the possibilities that (1) the disruption of a well-defined structural domain in Q beta RNA was selectively disadvantageous to phage infection, and that (2) the evolved viral populations were selected by virtue of their ability to restore critical integrity of short and/or long-range nucleotide interactions within this region of Q beta RNA. J Biol Chem, 1996 May 10, 271(19), 11236 - 46 A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein; Karzai AW et al.; The DnaK and DnaJ heat shock proteins function as the primary Hsp70 and Hsp40 homologues, respectively, of Escherichia coli . Intensive studies of various Hsp70 and DnaJ-like proteins over the past decade have led to the suggestion that interactions between specific pairs of these two types of proteins permit them to serve as molecular chaperones in a diverse array of protein metabolic events, including protein folding, protein trafficking, and assembly and disassembly of multisubunit protein complexes . To further our understanding of the nature of Hsp70-DnaJ interactions, we have sought to define the minimal sequence elements of DnaJ required for stimulation of the intrinsic ATPase activity of DnaK . As judged by proteolysis sensitivity, DnaJ is composed of three separate regions, a 9-kDa NH2-terminal domain, a 30-kDa COOH-terminal domain, and a protease-sensitive glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids that serves as a flexible linker between the two domains . The stable 9-kDa proteolytic fragment was identified as the highly conserved J-region found in all DnaJ homologues . Using this structural information as a guide, we constructed, expressed, purified, and characterized several mutant DnaJ proteins that contained either NH2-terminal or COOH-terminal deletions . At variance with current models of DnaJ action, DnaJ1-75, a polypeptide containing an intact J-region, was found to be incapable of stimulating ATP hydrolysis by DnaK protein . We found, instead, that two sequence elements of DnaJ, the J-region and the G/F-rich linker segment, are each required for activation of DnaK-mediated ATP hydrolysis and for minimal DnaJ function in the initiation of bacteriophage lambda DNA replication . Further analysis indicated that maximal activation of ATP hydrolysis by DnaK requires two independent but simultaneous protein-protein interactions: (i) interaction of DnaK with the J-region of DnaJ and (ii) binding of a peptide or polypeptide to the polypeptide-binding site associated with the COOH-terminal domain of DnaK . This dual signaling process required for activation of DnaK function has mechanistic implications for those protein metabolic events, such as polypeptide translocation into the endoplasmic reticulum in eukaryotic cells, that are dependent on interactions between Hsp70-like and DnaJ-like proteins. J Biol Chem, 1996 May 10, 271(19), 11156 - 62 T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex; Wheeler LJ et al.; After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase . At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex . The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized . Several specific protein-protein contacts within the complex are described in this paper . We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands . Elsewhere we have described interactions involving three T4 enzymes found in the complex . In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E . coli nucleoside diphosphokinase, . All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32 . At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins . Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase . Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini. J Biol Chem, 1996 May 10, 271(19), 11083 - 9 Bacteriophage T7 DNA ligase . Overexpression, purification, crystallization, and characterization; Doherty AJ et al.; The bacteriophage T7 DNA ligase gene was amplified using polymerase chain reaction-based methods and cloned into a T7 promoter-based expression vector . The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of protein per liter of bacterial culture . An initial physical and biochemical characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt-ended DNA fragments . Inhibition of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated . The enzyme has been crystallized from methoxypolyethylene glycol . The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 A . The unit cell dimensions are a = 66.1 A, b = 87.6 A, and c = 78.6 A, with one monomer in the asymmetric unit (Vm = 2.77 A3/Da) . This is the first member of the DNA ligase family of enzymes to be crystallized. J Mol Biol, 1996 May 3, 258(2), 299 - 307 Purification and characterization of T7 head-tail connectors expressed from the cloned gene; Cerritelli ME et al.; Cloned gene 8, which specifies the protein of the head-tail connector of bacteriophage T7, was expressed in Escherichia coli . Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates . Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography perhaps because of dissociation of monomer-connector complexes or disassembly of unstable connectors . Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography but not before . Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography . The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis . Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay . No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA. J Mol Biol, 1996 May 3, 258(2), 286 - 98 Assembly of T7 capsids from independently expressed and purified head protein and scaffolding protein; Cerritelli ME et al.; Prohead-like capsid shells containing the scaffolding and head proteins of bacteriophage T7 were isolated after both proteins were expressed from the cloned genes in the same cell . When the head-tail connector protein was also expressed, the isolated capsids contained neither connector nor scaffolding protein and resembled mature phage capsids rather than proheads . However, only a small fraction of the head protein was converted to stable capsid structures in either case . Purified scaffolding protein (expressed individually from the cloned gene) appeared to be a monomer in solution; purified head protein appeared to be a tetramer . The purified proteins reacted in the presence of polyethylene glycol or dextran to produce prohead-like capsid shells and also polycapsids consisting primarily of head protein, similar to the polycapsids observed after infection by T7 mutants lacking connector or core proteins . Neither capsids nor polycapsids were produced in the absence of scaffolding protein . Polycapsids were usually the predominant product even when scaffolding protein was in excess, and a small fraction of scaffolding protein catalyzed the conversion of an excess of head protein to polycapsids . Our results suggest that the first step in the natural pathway to prohead formation is the assembly of incomplete prohead shells, which are normally closed by insertion of a connector-core complex . In the absence of a functional connector-core complex, incomplete capsid shells apparently react further to form polycapsids or completely closed capsid shells. Cell, 1996 May 3, 85(3), 435 - 45 Three-site synapsis during Mu DNA transposition: a critical intermediate preceding engagement of the active site; Watson MA et al.; The chemical steps of bacteriophage Mu DNA transposition take place within a higher order nucleoprotein structure . We describe a novel intermediate that precedes the previously characterized transpososomes and directly demonstrates the interaction of a distant enhancer element with recombination regions . The transpositional enhancer interacts with the Mu left and right ends to form a three-site synaptic (LER) complex . Under normal reaction conditions, the LER complex is rapidly converted into the more stable Mu transpososomes . However, mutation of the Mu terminal nucleotides results in accumulation of the LER and a failure to form the type 0 transpososome . During the transition from LER to type 0, the Mu DNA termini and the active site of the transposase engage in a catalytically competent conformation. J Struct Biol, 1996 May-Jun, 116(3), 390 - 8 The interaction of DNA with bacteriophage phi 29 connector: a study by AFM and TEM; Valle M et al.; The connector of bacteriophage phi 29 is involved in DNA packaging during viral morphogenesis and we have studied its in vitro binding to DNA using either linear or circular DNA . The protein-DNA complexes have been analyzed by transmission electron microscopy (TEM) and by atomic force microscopy (AFM) of samples directly deposited on mica . TEM showed the presence of a specific binding due to the interaction of the protein with the free ends of the DNA . The study of these samples by AFM showed two major types of morphologies: The interaction of the connector with circular DNA revealed that the strands of DNA that enter and exit the protein complex form an angle with a mean value of 132 degrees . Nevertheless, when the connector was incubated with linear DNA (and later circularized), there was an additional bend angle of about 168 degrees . Further morphological analysis of the latter samples by AFM revealed a structure of the protein-DNA complex consistent with the DNA traversing the connector, probably through the inner channel . On the other hand, images from the samples obtained by incubation of the connector with circular DNa were consistent with an interaction of the DNA with the outer side of the connector. Chem Biol, 1996 May, 3(5), 393 - 403 Elucidation of the metal-binding properties of the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase by lanthanide(III) luminescence spectroscopy; Frey MW et al.; BACKGROUND: The exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I has a double binding site for the two essential divalent metal ions in the presence of the nucleotide monophosphate dTMP . RESULTS: The luminescence spectroscopy observed upon binding of Eu3+ to the exonuclease active site of T4 DNA polymerase was interpreted relative to the binding of Eu3+ or Tb3+ observed with KF . Both wild-type enzymes tightly bind a single Ln3+ ion but in two isomeric forms . The single mutants of KF (D424A) and T4 (D219A) also bind a single Eu3+ ion tightly, but the alignment of the coordinating ligands is altered . The KF double mutant (D355A, E357A) exhibits a markedly altered and weakened binding site (Kd = 20-26 microM) . Eu3+ serves as a competitive inhibitor of Mg2+-induced polymerase and exonuclease activity, validating its use as a probe for these active sites . CONCLUSIONS: Ln3+ luminescence spectroscopy is established as a sensitive way to determine the consequences of exonuclease binding-site mutations and to examine binding site similarities and differences among DNA polymerases from different sources . The binding sites of KF and T4 DNA polymerase are shown to be quite similar. Mol Microbiol, 1996 May, 20(4), 685 - 92 Phage presentation; Hill HR et al.; There has recently been great interest in the use of the filamentous bacteriophage fd as a vehicle for the display of peptides and proteins . Phage libraries displaying random peptides up to 38 amino acids in length can be used (i) to select for ligands able to bind specific target molecules; (ii) to mimic non-proteinaceous ligands; and (iii) as a tool to map epitopes recognized by antibodies . The display of proteins or their functional domains provides a system for the analysis of structure-function relationships, and the potential to generate proteins with altered binding characteristics or novel catalytic properties . The display of short immunogenic determinants on fusion phage may provide a basis for the development of novel peptide vaccines, whilst the expression of libraries of antibody fragments may provide a method to by-pass hybridoma technology in the generation of monoclonal antibodies. Mol Med, 1996 May, 2(3), 349 - 57 Conserved structure and adjacent location of the thrombin receptor and protease-activated receptor 2 genes define a protease-activated receptor gene cluster; Kahn M et al.; BACKGROUND: Thrombin is a serine protease that elicits a variety of cellular responses . Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism . Recently, a second protease-activated receptor was discovered and dubbed PAR2 . PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain . Also like the thrombin receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of receptor cleavage . Thus, functionally, the thrombin receptor and PAR2 constitute a fledgling receptor family that shares a novel proteolytic activation mechanism . To further explore the relatedness of the two known protease-activated receptors and to examine the possibility that a protease-activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin receptor and PAR2 genes . MATERIALS AND METHODS: The genomic structures of the two protease-activated receptor genes were determined by analysis of lambda phage, P1 bacteriophage, and bacterial artificial chromosome (BAC) genomic clones . Chromosomal location was determined with fluorescent in situ hybridization (FISH) on metaphase chromosomes, and the relative distance separating the two genes was evaluated both by means of two-color FISH and analysis of YACs and BACs containing both genes . RESULTS: Analysis of genomic clones revealed that the two protease-activated receptor genes share a two-exon genomic structure in which the first exon encodes 5'-untranslated sequence and signal peptide, and the second exon encodes the mature receptor protein and 3'-untranslated sequence . The two receptor genes also share a common locus with the two human genes located at 5q13 and the two mouse genes at 13D2, a syntenic region of the mouse genome . These techniques also suggest that the physical distance separating these two genes is less than 100 kb . CONCLUSIONS: The fact that the thrombin receptor and PAR2 genes share an identical structure and are located within approximately 100 kb of each other in the genome demonstrates that these genes arose from a gene duplication event . These results define a new protease-activated receptor gene cluster in which new family members may be found. Mol Microbiol, 1996 May, 20(3), 519 - 28 The effects on Escherichia coli of expression of the cloned bacteriophage T4 nucleoid disruption (ndd) gene; Bouet JY et al.; Immediately after T4 bacteriophage infection, the Escherichia coli nucleoid undergoes rapid delocalization . The ndd gene of T4 is responsible for this nuclear disruption phenomenon . We have cloned two alleles of this gene and studied the effects of their expression on E . coli cells . We have shown that the Ndd protein (i) is able to reproduce the disruption of the nucleoid characteristic of T4 infection, (ii) is highly toxic and results in a logarithmic decrease in cell viability, and (iii) inhibits genomic DNA replication by blocking progression of replication forks . Induction of Ndd does not result in degradation of genomic DNA and does not significantly alter the general processes of transcription and translation during the entire period of exponential cell death . These results support the notion that the target of Ndd is some aspect of the nucleoid architecture. Plant Physiol, 1996 May, 111(1), 293 - 9 Purification and properties of Arabidopsis thaliana COR (cold-regulated) gene polypeptides COR15am and COR6.6 expressed in Escherichia coli; Gilmour SJ et al.; Arabidopsis thaliana cold-regulated genes COR15a and COR6.6 encode 15- and 6.6-kD polypeptides, respectively . The COR15a polypeptide is known to be targeted to chloroplasts and, during import, to be processed to a 9.4-kD polypeptide designated COR15am . The COR6.6 polypeptide is thought to be located in the cytosol . The coding sequences for COR15am and COR6.6 were fused to the bacteriophage T7 promoter and expressed in Escherichia coli . The recombinant polypeptides COR15amr and COR6.6r were purified to near homogeneity using a combination of ammonium sulfate fractionation, ion-exchange chromatography, and adsorption chromatography on hydroxyapatite . COR15amr and the major species of COR15am in chloroplasts co-migrated on both two-dimensional O'Farrell gels and nondenaturing polyacrylamide gels . These data corroborate the site of COR15a processing and indicate no difference in charge or quaternary structure between COR15amr and the major species of COR15am in plants . In contrast, the migration patterns of COR6.6r and COR6.6 on two-dimensional gels suggest that a considerable portion of the COR6.6 population in plants is modified . In the accompanying papers (M.S . Webb, S.J . Gilmour, M.F . Thomashow, P.L . Steponkus {1996} Plant Physiology 111: 301-312; M . Uemura, S.J . Gilmour, M.F . Thomashow, P.L . Steponkus {1996} Plant Physiology 111: 313-327), the effects of COR15amr and COR6.6r on the cryostability and lyotropic phase behavior of liposomes are examined. Nucleic Acids Res, 1996 May 1, 24(9), 1625 - 31 Photolysis of N-hydroxpyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA; Epe B et al.; N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on irradiation with visible light, and two related compounds, viz . N-hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were tested for their potency to induce DNA damage in L1210 mouse leukemia cells and in isolated DNA from bacteriophage PM2 . DNA single-strand breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) were quantified . Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl radicals, i.e . single-strand breaks and the various endonuclease-sensitive modifications were formed in the same ratios as after exposure to established hydroxyl radical sources . In contrast, HAT plus light gave rise to a completely different DNA damage profile, namely that characteristic for singlet oxygen . Experiments with various scavengers (t-butanol, catalase, superoxide dismutase) and in D2O as solvent confirmed that hydroxyl radicals are directly responsible for the DNA damage caused by photoexcited 2-HPT and 4-HPT, while the damage by HAT plus light is mediated by singlet oxygen and type I reactions . The type of DNA damage characteristic of hydroxyl radicals was also observed in L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2 at 0 degrees C . t-Butanol (2%) inhibited the cellular DNA damage by approximately 50% . A dose of 2-HPT plus light that generated single-strand breaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell survival . No DNA damage and cytotoxicity was observed after treatment with 2-HPT in the dark . We propose that 2-HTP and 4-HTP may serve as new agents to study the consequences of DNA damage induced by hydroxyl radicals in cells . In addition, the data provide direct evidence that hydroxyl radicals are ultimately responsible for the genotoxic effects caused by H2O2 in the dark. J Eukaryot Microbiol, 1996 May-Jun, 43(3), 171 - 6 An arrayed bacteriophage P1 genomic library of Pneumocystis carinii; Metcheva IS et al.; We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system . The library consists of approximately 4800 independent clones with an average insert size of approximately 55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device . Screening of the library for unique P . carinii sequences detected an average of 4-5 positive clones for each, consistent with a several-fold coverage of the approximately 10-mbp P . carinii genome . Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts . Thus, this arrayed, large insert library of P . carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen. J Bacteriol, 1996 May, 178(10), 2902 - 10 Characterization of the primary immunity region of the Escherichia coli linear plasmid prophage N15; Lobocka MB et al.; N15 is the only bacteriophage of Escherichia coli known to lysogenize as a linear plasmid . Clear-plaque mutations lie in at least two regions of the 46-kb genome . We have cloned, sequenced, and characterized the primary immunity region, immB . This region contains a gene, cB, whose product shows homology to lambdoid phage repressors . The cB3 mutation confers thermoinducibility on N15 lysogens, consistent with CB being the primary repressor of N15 . Downstream of cB lies the locus of N15 plasmid replication . Upstream of cB lies an operon predicted to encode two products: one homologous to the late repressor of P22 (Cro), the other homologous to the late antiterminator of phi 82 (Q) . The Q-like protein is essential for phage development . We show that CB protein regulates the expression of genes that flank the cB gene by binding to DNA at symmetric 16-bp sites . Three sites are clustered upstream of cB and overlap a predicted promoter of the cro and Q-like genes as well as two predicted promoters of cB itself . Two sites downstream of cB overlap a predicted promoter of a plasmid replication gene, repA, consistent with the higher copy number of the mutant, N15cB3 . The leader region of repA contains terminators in both orientations and a putative promoter . The organization of these regulatory elements suggests that N15 plasmid replication is controlled not only by CB but also by an antisense RNA and by a balance between termination and antitermination. J Virol, 1996 May, 70(5), 3169 - 75 Branched structures in the intracellular DNA of herpes simplex virus type 1; Severini A et al.; Herpes simplex virus type 1 (HSV-1) replication produces large intracellular DNA molecules that appear to be in a head-to-tail concatemeric arrangement . We have previously suggested (A . Severini, A.R . Morgan, D.R . Tovell, and D.L.J . Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure . We now provide direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication . On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates) . Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures . Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis . Branched structures were also visualized by electron microscopy . Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions . We had previously shown that a restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis . We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient . Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules . The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4 . Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions. J Virol, 1996 May, 70(5), 2797 - 808 Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis; Wolffe EJ et al.; We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins . Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin . To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome . Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside . Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures . Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited . We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis. Virology, 1996 May 1, 219(1), 190 - 4 Preliminary crystallographic studies of bacteriophage T4 fibritin confirm a trimeric coiled-coil structure; Strelkov SV et al.; Fibritin, a 52-kDa product of gene wac of bacteriophage T4, forms fibrous "whiskers" that connect to the phage tail and facilitate the later stages of phage assembly . Preliminary experiments suggest that fibritin is a trimer, and its predominant central part has a parallel alpha-helical coiled-coil structure . To investigate the oligomerization and function of fibritin, we have designed and studied two related deletion mutants, denoted M and E, that consist of its last 75 and 120 amino acids, respectively . Both proteins contain part of the coiled-coil region and the 29 amino acid carboxy-terminal domain essential for the trimerization of fibritin . The proteins are expressed as a soluble product in an Escherichia coli system . We have obtained crystals of fibritins M and E . Complete native X-ray diffraction data sets have been collected to 1.85 and 2.7 A resolution, respectively . The crystals have space group P3 with a=44.3 A, c=91.3 A (fibritin M) and R32 with a=41.2 A, b=358.7 A (fibritin E) in the hexagonal setting . Symmetry and packing considerations show that fibritin is a triple coiled coil. Radiat Res, 1996 May, 145(5), 619 - 23 Protein-protein interactions between the Escherichia coli single-stranded DNA-binding protein and exonuclease I; Sandigursky M et al.; It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E . coli single-stranded DNA-binding protein (Ssb) . This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease . We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb . When the E . coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity . We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites . These results suggest that Ssb may be required for efficient base-excision repair in bacteria. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4096 - 101 Identification of the promoter of the mouse obese gene; de la Brousse FC et al.; Primer extension and RACE (rapid amplification of cDNA ends) assays were used to identify and sequence the 5' terminus of mouse ob mRNA . This sequence was used to obtain a recombinant bacteriophage containing the first exon of the encoding gene . DNA sequence analysis of the region immediately upstream of the first exon of the mouse ob gene revealed DNA sequences corresponding to presumptive cis-regulatory elements . A canonical TATA box was observed 30-34 base pairs upstream from the start site of transcription and a putative binding site for members of the C/EBP family of transcription factors was identified immediately upstream from the TATA box . Nuclear extracts prepared from primary adipocytes contained a DNA binding activity capable of avid and specific interaction with the putative C/EBP response element; antibodies to C/EBP alpha neutralized the DNA binding activity present in adipocyte nuclear extracts . When linked to a firefly luciferase reporter and transfected into primary adipocytes, the presumptive promoter of the mouse ob gene facilitated luciferase expression . When transfected into HepG2 cells, which lack C/EBP alpha, the mouse ob promoter was only weakly active . Supplementation of C/EBP alpha by cotransfection with a C/EBP alpha expression vector markedly stimulated luciferase expression . Finally, an ob promoter variant mutated at the C/EBP response element was inactive in both primary adipocytes and HepG2 cells . These observations provide evidence for identification of a functional promoter capable of directing expression of the mouse ob gene. Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 3932 - 6 Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene; Wang Y et al.; Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in development and disease . The cre-loxP system from bacteriophage P1 has been used in transgenic animals to induce site-specific DNA recombination leading to gene activation or deletion . To regulate the recombination in a spatiotemporally controlled manner, we constructed a recombinant adenoviral vector, Adv/cre, that contained the cre recombinase gene under regulation of the herpes simplex virus thymidine kinase promoter . The efficacy and target specificity of this vector in mediating loxP-dependent recombination were analyzed in mice that had been genetically engineered to contain loxP sites in their genome . After intravenous injection of the Adv/cre vector into adult animals, the liver and spleen showed the highest infectivity of the adenovirus as well as the highest levels of recombination, whereas other tissues such as kidney, lung, and heart had lower levels of infection and recombination . Only trace levels of recombination were detected in the brain . However, when the Adv/cre vector was injected directly into specific regions of the adult brain, including the cerebral cortex, hippocampus, and cerebellum, recombination was detectable at the injection site . Furthermore, when the Adv/cre vector was injected into the forebrains of neonatal mice, the rearranged toxP locus from recombination could be detected in the injected regions for at least 8 weeks . Taken together, these results demonstrate that the Adv/cre vector expressing a functional cre protein is capable of mediating loxP-dependent recombination in various tissues and the recombined gene locus may in some cases be maintained for an extended period . The use of the adenovirus vector expressing cre combined with localized delivery to specific tissues may provide an efficient means to achieve conditional gene expression or knockout with precise spatiotemporal control. J Virol Methods, 1996 Apr 26, 58(1-2), 41 - 51 A replication-deficient adenovirus enhances liposome-mediated nucleic acid transfer into a stable cell line expressing T7 RNA polymerase; Honda M et al.; Liposome-mediated transfer of nucleic acids into a cell line expressing bacteriophage T7 RNA polymerase was enhanced by addition of a replication-deficient adenovirus (Ad5-259A) to transfection mixtures . Increasing quantities of Ad5-259A resulted in a dose-related (up to 30-fold) enhancement of reporter gene activity expressed in BT7-H cells transfected with plasmid DNA containing the reporter sequence fused to the internal ribosome entry site of encephalomyocarditis virus . Similarly, Ad5-259A enhanced reporter gene expression 7-fold following transfection of DNA containing the reporter sequence under transcriptional control of the Rous sarcoma virus long terminal repeat . Addition of Ad5-295A to transfection mixtures increased the proportion of cells staining positively for reporter gene activity, from 2 to 25% when the reporter was expressed via the T7 polymerase and from 20 to 50% when the reporter was under the control of a eucaryotic promoter . Thus, Ad5-259A enhanced reporter protein activities expressed by cytoplasmic T7-directed transcription and cap-independent initiation of translation, or nuclear transcription and cap-dependent translation . Transfection enhancement was blocked by neutralizing antibody to Ad5, and is most likely related to the endosome-disrupting activities of the virus . Adenovirus enhancement of liposome-mediated transfection provides a useful method for efficient nucleic acid transfer into eucaryotic cells. Biochemistry, 1996 Apr 23, 35(16), 5145 - 57 Structure and dynamics of bacteriophage IKe major coat protein in MPG micelles by solution NMR; Williams KA et al.; The structure and dynamics of the 53-residue filamentous bacteriophage IKe major coat protein in fully protonated myristoyllysophosphatidylglycerol (MPG) micelles were characterized using multinuclear solution NMR spectroscopy . Detergent-solubilized coat protein {sequence: see text} mimics the membrane-bound "assembly intermediate" form of the coat protein which occurs during part of the phage life cycle . NMR studies of the IKe coat protein show that the coat protein is largely alpha-helical, exhibiting a long amphipathic surface . helix (Asn 4 to Ser 26) and a shorter "micelle-spanning" C-terminal helix which begins at TRP 29 and continues at least to Phe 48 . Pro 30 likely occurs in the first turn of the C-terminal helix, where it is ideally situated given the hydrogen bonding and steric restrictions imposed by this residue . The similarity of 15N relaxation values (T1, T2, and NOE and 500 MHz and T2 at 600 MHz) among much of the N-terminal helix and all of the TM helix indicates that the N-terminal helix is as closely associated with the micelle as the TM helix . The description of the protein in the micelle is supported by the observation of NOEs between lysolipid protons and protein amide protons between asn 8 and Ser 50 . The N-terminal and TM helices exhibit substantial mobility on the microsecond to second time scale, which likely reflects changes in the orientation between the two helices . The overall findings serve to clarify the role of individual residues in the context of a TM alpha-helix and provide an understanding of the secondary structure, dynamics, and aqueous and micellar environments of the coat protein. J Biol Chem, 1996 Apr 19, 271(16), 9739 - 45 Interactions between the repressor and the early operator region of bacteriophage Mu; Rousseau P et al.; The repressor of bacteriophage Mu, c, binds to three operator sites, O1, O2, and O3, overlapping two divergent promoters, which regulate the lytic and lysogenic pathways . Its binding to this operator region generates several complexes, which were analyzed by DNase I protection experiments . We demonstrate that c first binds to two 11-base pair partially repeated sequences in O2 that could represent "core" binding sites for the repressor . This initial interaction serves as an organizer of a more complex nucleoprotein structure in which O2, O1, and O3 become successively occupied . The quaternary structure of the repressor was also investigated . Size exclusion chromatography and protein-protein crosslinking experiments with chemicals that possess linking arms of various lengths indicate that the repressor oligomerizes in solution . A model is proposed describing the successive interactions of c with the operator sites O2, O1, and O3 leading to the elaboration of a higher order structure in which the early lytic functions are repressed. Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3565 - 9 Mimotope/anti-mimotope probing of structural relationships in platelet glycoprotein Ib alpha; Miller JL et al.; A bacteriophage library displaying random decapeptides was used to characterize the binding preference of C-34, a monoclonal antibody originally raised against platelet-type von Willebrand disease platelets heterozygous for the mutation 23OWKQ (G --> V)233V234 in the alpha chain of glycoprotein Ib (GPIb alpha) . Three rounds of biopanning C-34 against the library resulted in striking convergence upon the sequence WNWRYREYV . Since no portion of this sequence corresponds to a recognizable peptide sequence within human platelet GPIb alpha, it may be considered a "mimotope" of the naturally occurring C-34 epitope, presumably bearing similarity to it in three-dimensional structure . Synthetic AWNWRYREYV peptide preincubated with C-34 fully neutralized the ability of C-34 to inhibit platelet aggregation, with an IC50 of approximately 6 microg/ml . When biotinylated AWNWRYREYV was subsequently bioparmed against the original decapeptide library, the sole clone demonstrating inhibitory activity above background level in a functional platelet assay displayed the sequence RHVAWWRQGV, and chemically synthesized peptide fully inhibited ristocetin-induced aggregation, with an IC50 of 200-400 microg/ml . Synthesized RHVAWWKQGV peptide exerted only slight inhibition, whereas RHVAWWKQVV peptide showed potent inhibitory activity . Moreover, whereas synthesized wild-type 228YVWKQGVDVK237 GPIb alpha peptide was virtually without inhibitory activity, the 228YVWKQ(G -->V) 233VDVK237 peptide fully inhibited ristocetin-induced aggregation, with an IC50 of approximately 400 microg/ml . These studies raise the possibility of an intramolecular association of peptide regions within GPIb alpha that may play a role in the regulation of von Willebrand factor-dependent platelet aggregation. Nucleic Acids Res, 1996 Apr 15, 24(8), 1582 - 4 Improved method for selecting RNA-binding activities in vivo; Fouts DE et al.; RNA challenge phages are modified versions of bacteriophage P22 that allow one to select directly for a specific RNA-protein interaction in vivo . The original construction method for generating a bacteriophage that encodes a specific RNA target requires two homologous recombination reactions between plasmids and phages in bacteria . An improved method is described that enables one to readily construct RNA challenge phages through a single homologous recombination reaction in vivo . We have applied the new method to construct a derivative of P22R17, an RNA challenge phage that undergoes lysogenic development in bacterial cells that express the bacteriophage R17/MS2 coat protein. EMBO J, 1996 Apr 15, 15(8), 2010 - 9 Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange; Kong D et al.; Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange . Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions . The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein . In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA . Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate . The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein. EMBO J, 1996 Apr 15, 15(8), 1850 - 6 FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5; Bonhivers M et al.; The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(-)ferrichrome and is the receptor of phage T5 and phi 80 . The purified protein inserted into planar lipid bilayers showed no channel activity . Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels . The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the 'gating loop' (delta 322-355) had been removed . binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo . These data suggest that binding of T5 to loop 322-355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA . Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening. J Mol Biol, 1996 Apr 12, 257(4), 745 - 55 Identification of a phage-coded DNA-binding protein that regulates transcription from late promoters in bacteriophage P4; Polo S et al.; The genetic element P4 can propagate as a temperate phage or as a multicopy plasmid in its host Escherichia coli . Late in the lytic cycle and in the plasmid condition, transcription of the P4 essential genes depends on the activation of the late promoters P(LL) and P(sid), which control the transcription of the left and right operons, respectively . Both P4 late promoters are positively regulated by the product of the P4 delta gene, which is transcribed from P(sid) . We have identified a new P4 gene, vis, that appears to play a relevant role in P4 late transcription control . vis is the first gene downstream of P(LL) and codes for a basic 88 amino acid protein with a potential helix-turn-helix motif . Expression of the cloned vis gene suppresses all the phenotypic traits exhibited by P4 vir1, a mutant that carries a promoter-up mutation in the late promoter P(LL) . By Northern hybridization analysis we showed that vis negatively regulates transcription from P(LL) and enhances transcription from P(sid) . Thus, vis auto-regulates its expression by repressing its own promoter and enhancing transcription of delta, which is required for P(LL) activation . The vis gene was fused with the glutathione S-transferase gene and the GST-Vis fusion protein was partially purified . By gel retardation assays and DNA footprinting we demonstrated that GST-Vis binds to a 32 bp long region immediately downstream of P(LL) . We also showed, by gel retardation, that GST-Vis binds to the P sid region . A sequence present in both P(LL) and P(sid) regions may represent the Vis binding consensus sequence . The dual role of Vis on the control of P4 late transcription may be required for a regulated expression of the replication functions when P4 propagates in the plasmid state. J Mol Biol, 1996 Apr 5, 257(3), 561 - 73 Holliday junction resolvases encoded by homologous rusA genes in Escherichia coli K-12 and phage 82; Mahdi AA et al.; The RusA protein of Escherichia coli is an endonuclease that can resolve Holliday intermediates and correct the defects in genetic recombination and DNA repair associated with inactivation of RuvAB or RuvC . The structure of the rusA gene, its organisation in the genome, and its interaction with the Ruv and RecG proteins have been investigated . Recombinant plasmids carrying rusA were identified by their ability to make ruv mutants resistant to UV light . The gene was located to an open reading frame encoding a polypeptide of 120 amino acids . It forms the fifth gene in an operon containing a chain of short, interlinked open reading frames . A similar arrangement was found in the genome of the lambdoid bacteriophage, 82 . The two rusA genes show 95% sequence identity . The E . coli operon forms part of the defective lambdoid prophage, DLP12, and is probably derived from a phage related to 82 and PA-2 . rusA appears to be very poorly expressed in E . coli, but can be activated by insertion of IS2 or IS10 upstream of the coding sequence to promote transcription . These insertions arise spontaneously in ruv strains as suppressors of the mutant phenotype . Deletion of rusA from the chromosome of either wild-type or ruv mutant strains has no obvious effect on recombination or sensitivity to UV light . Multicopy plasmids expressing RusA alone make ruvA, ruvB, and ruvC mutants resistant to UV light . Suppression depends critically on RecG. Biochemistry, 1996 Apr 2, 35(13), 4187 - 98 Structure of the autoregulatory pseudoknot within the gene 32 messenger RNA of bacteriophages T2 and T6: a model for a possible family of structurally related RNA pseudoknots; Du Z et al.; A 36-nucleotide RNA with a sequence corresponding to the 5' end region of the gene 32 mRNA of bacteriophages T2 and T6 was analyzed by one- and two-dimensional NMR methods . NMR results provide clear evidence that the RNA is folded into a pseudoknot structure with two coaxial stems connected by two loops, in a classic pseudoknot topology . The pseudoknot is unusual in that one of the loops consists of only one nucleotide, which spans the major groove of a seven base pair helical stem . Imino proton resonances indicate the hydrogen bonding pattern within the pseudoknot, and two-dimensional NOE spectra provide information that describes many of the structural features . The temperature dependence of the UV absorption and imino proton exchange rates provides insight into the stability of the pseudoknot . A three-dimensional model of the pseudoknot that is consistent with our NMR data is presented, and features that may be important for stabilizing the pseudoknot structure are discussed . A substantial number of other putative RNA pseudoknots described in the literature have sequences and topologies that appear to be related to the T2 and T6 pseudoknots . We propose that these RNAs may be members of a family of pseudoknots related by a similar structural motif, which we refer to as "common pseudoknot motif 1" or CPK1 . The bacteriophage T2/T6 pseudoknot can be considered a structural model for the CPK1 family . The common features of the CPK1 pseudoknots are a stem 2 with six or seven base pairs, a loop 1 consisting of a single adenosine, and a variable length stem 1 and loop 2 . The first "dangling" nucleotide at the 3' end of the molecule probably stabilizes stem 2 . The CPK1 family includes several of the retroviral pseudoknots associated with mRNA frameshifting and readthrough . The work presented here describes the first detailed NMR analysis of an RNA pseudoknot with an entirely natural nucleotide sequence. Biochemistry, 1996 Apr 2, 35(13), 4176 - 86 Thermodynamics of folding of the RNA pseudoknot of the T4 gene 32 autoregulatory messenger RNA; Qiu H et al.; Nucleotides U(-67) to C(-40) at the extreme 5' end of the gene 32 mRNA in bacteriophage T4 have been shown to fold into an RNA pseudoknot proposed to be important for translational autoregulation . The thermal denaturation of three in vitro transcribed RNAs corresponding to the pseudoknot region has been investigated as a function of Mg2+ concentration to begin to elucidate the determinants of the structure and stability of this conformation . T4-35 is a 35-nucleotide RNA containing a 5' G followed by the natural T4 sequence starting with the mature 5' end of the mRNA, nucleotides A(-71) to C(-38) . A 32-nucleotide RNA, termed T4-32, contains the native sequence form U(-67) to C(40) with 5'GC and 5'CA single-stranded regions appended to the 5' and 3' ends of the core sequence, respectively . T4-28 contains only the 28 core nucleotides, and the predicted closing U(-67)-A(-52) base pair in stem 1 has been replaced with a phylogenetically allowed G(-67)-C(-52) base pair . Ribonuclease mapping of T4-32 and imino proton NMR experiments of T4-35 show that both sequences adopt a pseudoknotted conformation . At pH 6.9 and 50 mM NaCl, T4-35 and T4-32 RNAs are characterized by a single major melting transition over a wide range of {Mg2+} (0-6 mM) . The delta H degree of unfolding for T4-35 and T4-32 shows a large dependence on Mg2+ concentration; the maximum delta H degree occurs at about 2.0 mM Mg2+ with further addition of Mg2+ simply increasing the tm . Investigation of the {Mg2+} dependence of the tm suggests that a net of one Mg2+ ion is released upon denaturation of T4-35 and T4-32 RNAs . Over the entire {Mg2+} range, the delta G degree (37 degrees C) for the folding of T4-35 is consistently 1-1.5 kcal mol(-1) more negative than T4-32 due to a higher stabilization enthalpy for the natural sequence molecule . In contrast to this behavior, T4-28 gives consistently higher tm's but less negative enthalpies and is destabilized (at 37 degrees C) by about 0.5-1.5 kcal mol(-1) relative to T4-32 and by about 2-3 kcal mol(-1) relative to T4-35, depending upon cation concentration . (1)H NMR experiments suggest that, even in the presence of 4.0 mM Mg2+, T4-28 RNA does not adopt a stable pseudoknotted conformation . These data show that the stability of the pseudoknot in the gene 32 mRNA encoded by the 28-nucleotide core sequence is significantly influenced by the number and nature of the immediately adjacent "single-stranded" 5' and/or 3' nucleotides appended to the core structure . These findings are discussed within the context of the structural model for the evolutionarily related phage T2 and T6 gene 32 mRNA pseudoknots presented in the following paper {Du, Z., Giedroc, D . P., & Hoffman, D . W . (1996) Biochemistry 35, 4187-4198}. Microb Drug Resist, 1996 Spring, 2(1), 147 - 53 Bacteriophage lambda lysis gene product modified and inserted into Escherichia coli outer membrane: Rz1 lipoprotein; Taylor A et al.; Lysis proteins of bacteriophage lambda were localized in different parts of the host envelope: S in the inner membrane,36 Rz in the membrane adhesion sites,14 and Rz1 in the outer membrane . The R gene product, the transglycosylase destroying bacterial murein, is a soluble protein . Computer-assisted analysis of the Rz1 protein amino acids sequence revealed that its N-terminal part contained the site 15VVVG {symbol: see text} C20, which could be recognizable for the SPase II and cleaved leaving lipid modified C20 as the N-terminal amino acid of the mature protein . Microsequencing of the Rz1 protein isolated from the expression products of E . coli {pSB54} carrying the Rz1 gene showed that the N-terminal part of the protein was cleaved as predicted . Lipid labeling with {3H}palmitate confirmed the expectation that Rz1 was a lipoprotein . E . coli {pSB54} treated with globomycin accumulated prolipoprotein, the Rz1 precursor, which was detectable by the anti-Rz1 serum on electropherograms as the 6.5-kDa protein, larger than mature protein . Physiological function of the Rz1 protein remains to be discovered, but as a first hint we noticed that it evokes increase of the fraction of adhesion sites of outer and inner membranes when overproduced from pSB54 . The same effect was observed in induced E . coli (lambda) just before the lysis onset, however, one should be cautious in interpreting the results obtained in conditions of the overproduction of the Rz1 lipoprotein. Appl Environ Microbiol, 1996 Apr, 62(4), 1336 - 41 Effects of sunlight on bacteriophage viability and structure; Wommack KE et al.; Current estimates of viral abundance in natural waters rely on direct counts of virus-like particles (VLPs), using either transmission or epifluorescence microscopy . Direct counts of VLPs, while useful in studies of viral ecology, do not indicate whether the observed VLPs are capable of infection and/or replication . Rapid decay in bacteriophage viability under environmental conditions has been observed . However, it has not been firmly established whether there is a corresponding degradation of the virus particles . To address this question, viable and direct counts were carried out employing two Chesapeake Bay bacteriophages in experimental microcosms incubated for 56 h at two depths in the York River estuary . Viruses incubated in situ in microcosms at the surface yielded decay rates in full sunlight of 0.11 and 0.06 h-1 for CB 38 phi and CB 7 phi, respectively . The number of infective particles in microcosms in the dark and at a depth of 1 m was not significantly different from laboratory controls, with decay rates averaging 0.052 h-1 for CB 38 phi and 0.037 h-1 for CB 7 phi . Direct counts of bacteriophages decreased in teh estuarine microcosms, albeit only at a rate of 0.028 h-1, and were independent of treatment . Destruction of virus particles is concluded to be a process separate from loss of infectivity . It is also concluded that strong sunlight affects the viability of bacteriophages in surface waters, with the result that direct counts of VLPs overestimate the number of bacteriophage capable of both infection and replication . However, in deeper waters, where solar radiation is not a significant factor, direct counts should more accurately estimate numbers of viable bacteriophage. J Clin Microbiol, 1996 Apr, 34(4), 959 - 61 Molecular epidemiology of Escherichia coli O157:H7 by pulsed-field gel electrophoresis and comparison with that by bacteriophage typing; Krause U et al.; One hundred twenty-four Escherichia coli O157:H7 isolates were characterized by pulse-field gel electrophoresis, bacteriophage typing, and PCR of verotoxin genes . Diversity indices obtained--0.786 for phage types and 0.987 for pulsed-field gel electrophoresis types--demonstrated that phage typing falls below the critical value (0.9) required for confident interpretation of results. Bioorg Khim, 1996 Apr, 22(4), 256 - 63 {Organization of the gene for the beta-subunit of human photoreceptor cyclic GMP phosphodiesterase}; Suslova VA et al.; Two recombinant bacteriophage lambda clones encoding a 27.8-kb fragment of the human phosphodiesterase beta-subunit gene were isolated from a human genomic library . The nucleotide sequences of 19 exons (from the 4th to 22nd), 18 introns, and the 3'-flanking region were determined . The analysis of the nucleotide sequence of the phosphodiesterase beta-subunit gene revealed four Alu repeats and four minisatellite regions. Mol Immunol, 1996 Apr, 33(6), 493 - 502 A novel and efficient route for the isolation of antibodies that recognise T cell receptor V alpha(s); Popov S et al.; Studies of the T cell repertoire have been hindered by the lack of antibodies that recognise V region families, particularly for V alpha regions . In this report, single chain Fv (scFv) fragments have been isolated that recognise both recombinant V alpha(s) and native V alpha(s) on the surface of T cells . Mice have been immunised with purified soluble T cell receptors (TCRs) and antibody heavy and light chain variable domain (VH and VL, respectively) genes isolated from splenocytes using the polymerase chain reaction (PCR) . The VH and VL genes have been assembled as scFv gene libraries and a bacteriophage display system used to isolate scFvs that recognise a soluble V alpha . Five scFvs have been purified and characterised in detail using enzyme-linked immunosorbent assays (ELISAs) and flow cytometry . Three of these five scFvs recognise native V alpha(s) on the surface of T cell hybridomas . This method therefore offers a rapid route to the generation of scFvs that recognise native TCRs and can readily be extended to the production of anti-human TCR antibodies for use in therapy and diagnosis. Acta Paediatr Jpn, 1996 Apr, 38(2), 182 - 8 Adenovirus vector technology: an efficient method for constructing recombinant adenovirus and on/off switching of gene expression; Kanegae Y et al.; An efficient method of constructing recombinant adenoviruses (Ad) has been established . The expression unit to be introduced into recombinant Ad was first inserted into the unique SwaI site of the full-length Ad genome cloned in a cassette cosmid . The cassette bearing the expression unit was then cotransfected to 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I . The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the restriction digestion drastically reduced regeneration of the parent virus . This method may facilitate the application of recombinant Ad and should be useful for further improvement of Ad vectors . Also a recombinant adenovirus expressing Cre recombinase derived from bacteriophage P1 was constructed . To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed . Co-infection experiments together with the Cre-expressing and the reporter recombinant Ad showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1 cells . These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in future gene therapy. J Parasitol, 1996 Apr, 82(2), 250 - 4 Cysticercosis: identification and cloning of protective recombinant antigens; Manoutcharian K et al.; We describe the cloning and the evaluation of the protective capacity of 5 recombinant antigens expressed during the cysticercus stage of both Taenia crassiceps and Taenia solium . A cDNA library was constructed in bacteriophage lambda ZAP using mRNA isolated from larvae of T . crassiceps of the ORF strain . The recombinant phage library was screened with polyclonal antibodies against 56- and 74-kDa protective antigen fractions . This screening identified 13 recombinant clones, 5 of which were also strongly recognized by pooled sera from pigs experimentally infected with T . solium . The native antigens are proteins of 56 (clones KETcl, 4, 7) and 74 and 78 kDa (clones KETc11, 12) of T . crassiceps cysticerci . Vaccination experiments using these 5 recombinant clones against murine cysticercosis point to the relevance of KETcl, 4, 7, and 12 in host protection, whereas immunization with the clone KETc11 does not modify the parasite load in females and facilitates the parasitosis in males . We report here the DNA and the deduced amino acid sequence (100 amino acids) of the first protective antigen (KETc7) of potential interest for T . solium pig cysticercosis prevention. Mutat Res, 1996 Mar 26, 351(1), 19 - 31 Tobacco plants expressing T4 endonuclease V show enhanced sensitivity to ultraviolet light and DNA alkylating agents; Lapointe G et al.; DNA repair processes and UV-filtering pigments protect organisms from the cytotoxicity of UV light and endow plants with a high degree of natural UV resistance . In an attempt to further enhance this UV resistance we have constructed transgenic tobacco lines that express a DNA repair enzyme encoded by the bacteriophage T4 denV gene . The denV gene encodes endonuclease V, an enzyme which initiates base excision repair of cyclobutane pyrimidine dimers . Its presence is expected to provide transgenotes with a repair pathway complementary to, but likely distinct from, the repair pathways found in tobacco . The denV gene, flanked by a CaMV 35S promoter and poly(A) addition site, was introduced into tobacco and mature plants regenerated . The transgenotes expressed high levels of a UV-specific endonuclease and no such activity was found in control plants . Curiously, assays which detected several different biological endpoints showed that the denV+ transgenotes were also hypersensitive to UV-C light . This hypersensitivity segregated with the denV gene and was not caused by altered concentrations of UV-filtering pigments . Moreover, the denV+ transgenotes were also hypersensitive to high levels of baseless lesions that would be generated by a transgenically expressed beta-eliminating lyase such as endonuclease V. J Mol Biol, 1996 Mar 22, 257(1), 102 - 15 Structure, interactions and dynamics of PRD1 virus II . Organization of the viral membrane and DNA; Tuma R et al.; Structure, dynamics and stability of the membrane and double-stranded (ds) DNA genome packaged within the native PRD1 virion have been probed by laser Raman spectroscopy . The Raman signature of PRD1 is complex, but exhibits distinctive marker bands diagnostic of the internal lipid bilayer and dsDNA . The Raman markers demonstrate, respectively, a liquid crystalline lipid phase(L(alpha)) and B DNA conformation throughout the temperature range (5 degrees to 50 degrees) of virion stability . Despite the absence of large scale lipid phase transitions or DNA melting, small temperature-dependent changes in the Raman markers of lipid and DNA are detected, indicating coupling between their structures . Minor deviations of DNA from the canonical B form are imposed by the membrane . The Raman markers indicate further that base stacking and phosphate group interactions of the packaged PRD1 genome differ from those of unpackaged PRD1 DNA . Specific Raman band perturbations are proposed as indicators of DNA-membrane interaction . Hydrogen-deuterium exchange kinetics of packaged and unpackaged PRD1 DNA are indistinguishable, demonstrating that base imino and amino protons are not affected significantly by either the condensation or membrane enclosure associated with DNA packaging . This contrasts with the significant acceleration of base exchanges detected in the packaged DNA of bacteriophage P22, which lacks a viral membrane . The distinctive H-->2H exchange profile of the PRD1 genome, the absence of packaging-induced acceleration of exchange kinetics and the apparent direct interaction between DNA and phospholipids suggest a specific role for the viral membrane in PRD1 assembly . We propose a "membrane-surface-catalyzed" model for dsDNA condensation and organization within the PRD1 virion. J Theor Biol, 1996 Mar 21, 179(2), 161 - 72 Evolution of the genetic switch in temperate bacteriophage . I . Basic theory; Mittler JE; While the molecular mechanisms underlying lysogeny and induction in bacteriophage have been intensely studied, relatively little has been done to relate these findings to their presumed selective functions . To explore the ecological basis for these traits, I have used a resource-based model for competition between bacteriophage with different probabilities of lysogeny and different spontaneous induction rates . In any given habitat the fitness of a phage will depend on the inputs of sensitive cells and nutrient resources . In equable environments (modeled here using chemostats with constant inputs of nutrients and sensitive cells), bacteriophage with low probabilities of lysogeny and low induction rates can always invade when rare and will generally be good competitors . In variable environments (chemostats with seasonal inputs), bacteriophage with higher probabilities of lysogeny and higher induction rates are favored . In both equable and variable environments, the ability of a phage to invade when rare will depend on the properties of the resident phage, and it is possible for phages with divergent parameter values to coexist . The modeling suggests that bacteriophage that have evolved moderately low induction and lysogeny rates will be able to "hedge their bets" against environmental change without sacrificing the ability to compete well in a constant environment . Implications of this theory for understanding the molecular basis of gene regulation in temperate bacteriophage and other viruses are discussed. Eur J Biochem, 1996 Mar 15, 236(3), 887 - 94 The characterisation of the 5' regulatory region of a temperature-induced myosin-heavy-chain gene associated with myotomal muscle growth in the carp; Gauvry L et al.; We have isolated and characterised the 5' region of a member of the carp myosin heavy chain gene family . Expression of this gene has previously been shown to be induced by an increase in environmental temperature and is restricted to the small-diameter white myotomal muscle fibres which are associated with growth . The whole isoform gene, including potential regulatory sequence 5' to the transcription start site and the 3' untranslated region was cloned in a lambda2001 bacteriophage vector . Studies of the structure of the 5'-end of the gene revealed high amino acid sequence similarity with translated exons 3-7 of mammalian myosin heavy chain genes indicating identical exon/intron boundaries . The overall length of the gene was however only about one half of that in mammals and birds due to shorter introns . The region 5' to the transcription unit was sequenced and revealed the presence of putative TATA and CCAAAT boxes . In order to study the regulation of expression, a series of endonuclease-generated fragments from the 5' flanking sequence were spliced to chloramphenicol acetyltransferase reporter vectors and used in cell transfection assays or direct gene injection into carp skeletal muscle . The 5' flanking region, which contains a consensus sequence known as an E-box (CANNTG) and a MEF2 binding site, was shown to improve the expression of the reporter gene in fish acclimated at 18 degrees C or 28 degrees C . Unlike the coding region, there was little similarity between the 5'-upstream sequence (promoter region) when compared with sequences flanking the 5'-end of the other myosin heavy chain genes in mammals or chicken. EMBO J, 1996 Mar 15, 15(6), 1421 - 33 Defining the enzyme binding domain of a ribonuclease III processing signal . Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants; Li H et al.; Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose-phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III . Two RNase III mutants were employed, which bind substrate in vitro similarly as wild-type enzyme, but lack detectable phosphodiesterase activity . Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage . The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal . One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1{WC-L} RNA, undergoes coordinate double cleavage . The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites . Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern . The footprinting and interference patterns indicate that (i) RNase III contacts the sugar-phosphate backbone; (ii) the RNase III-substrate interaction spans two turns of the A-form helix; and (iii) divalent metal ion does not play an essential role in binding specificity . These results rationalize the conserved two-turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity . In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1{WC-L} RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop. J Biol Chem, 1996 Mar 15, 271(11), 6537 - 44 Cloning and characterization of the Neurospora crassa cyt-5 gene . A nuclear-coded mitochondrial RNA polymerase with a polyglutamine repeat; Chen B et al.; The Neurospora crassa mutants, cyt-5-1 and cyt-5-4 have a cytochrome b- and aa3-deficient phenotype, suggesting that they result from a deficiency in a nuclear-coded component of the mitochondrial gene expression apparatus (Bertrand, H., Nargang, F . E., Colllins, R . A., and Zagozeski, C . A . (1977) Mol . Gen . Genet . 153,247-257) . The complementing wild-type gene has been cloned and and shown to encode a protein with significant sequence similarity to Saccharomyces cerevisiae mitochondrial RNA polymerase and bacteriophage RNA polymerases . There are remarkable differences between the N . crassa protein and its yeast homologue, including a region of very little homology near the N termini of the two gene products . The cyt-5 gene encodes a stretch of polyglutamine in this region of uniquesequence . In addition, an acidic insertion (86 amino acids, of which 24 are Asp or Glu and 10 are Arg or Lys) is present near the C terminus of the cyt-5 gene product . Transcript levels of the cytochrome b and cytochrome oxidase subunit III genes are severely reduced in cyt-5 mutants, suggesting a likely mechanism for the cytochrome-deficient phenotype . In contrast, mitochondrial rRNAs accumulate to nearly normal levels in cyt-5 mutants . However, mitochondrial rRNA levels are not indicative of the rate of transcription of the corresponding genes, since crude lysates of mitochondria from cyt-5 mutants exhibit greatly reduced transcriptional activity with a 19 S rRNA promoter . The cyt-5 gene is flanked by at least one gene whose product also may be involved in mitochondrial function. Biochemistry, 1996 Mar 12, 35(10), 3341 - 50 Cooperativity mutants of bacteriophage lambda cI repressor: temperature dependence of self-assembly; Burz DS et al.; Analytical ultracentrifugation was used to study higher order self-assembly of lambda cI repressors, including eight mutants whose monomer to dimer reactions were recently characterized {Burz et al . (1994) Biochemistry 33, 8399} . Six of the mutants were found to remain dimeric up to 50 microM total protein; the remaining mutants (EK102 and PT158) were found to undergo higher order oligomerization, as does wild-type cI . For these three repressors, we determined the stoichiometries and energetics of higher order assembly over the temperature range 5-40 degrees C . Weak dimerization exhibited by two other mutants, SN228 and SR228, was also evaluated by sedimentation equilibrium over this same temperature range . The end-state for higher order assembly of wild-type cI was determined to be octameric, in agreement with Senear et al . {(1993) Biochemistry 32, 6179-6189} . The assembly free energies resolved by the sedimentation analysis program NONLIN {Johnson, M . L., et al . (1981) Biophys . J . 36, 575-588} leads to the prediction that tetramers may contribute significantly to the intermediate populations during assembly . This analysis of the species populations is in accord with recent conclusions from fluorescence anisotropy studies {Banik et al . (1993) J . Biol . Chem . 268, 3938} . It was found that two of the mutant repressors (EK102 and PTI58) assemble into octamers, but with differing possible intermediates . PT158 satisfies the stoichiometry 8M1 <--> 2M4 <--> M8, while the EK102 data conforms to a 4M2 <--> 2M4 <--> M8 model, similar to WT (both the EK102 and WT data could also be described by a dimer-octamer model with no intermediates) . Of the six repressors found in this study to remain dimeric, three exhibit non-cooperative DNA binding (GD147, KN192, YH210), two express intermediate cooperativity (EK188, SR228), and one is fully cooperative (SN228) . The three octamerizing repressors are fully cooperative {Burz & Ackers (1994) Biochemistry 33, 8399}, suggesting a correlation between their ability to form higher order assemblies and to engage in cooperative DNA binding . Linear van't Hoff plots were obtained for overall assembly of wild-type and EK102 dimers, while that of PT158 monomers was curved, indicating a negative heat capacity change . The van't Hoff analyses of dimerization constants for SN228 and SR228 were distinctly different from each other and also from that of wild type; such differences might be related to the disparate cooperative behavior found previously for these mutants (Burz & Ackers, 1994). J Mol Biol, 1996 Mar 8, 256(4), 736 - 50 Atomic structure of the degraded procapsid particle of the bacteriophage G4: induced structural changes in the presence of calcium ions and functional implications; McKenna R et al.; Bacteriophage G4 and phiX174 are members of the Microviridae family . The degree of similarity of the structural proteins ranges from 66% identity of the F protein to 40% identity of the G protein . The atomic structure of the phiX174 virion had previously been determined by X-ray crystallography . Bacteriophage G4 procapsids, consisting of the structural proteins F, G, D, B, H, and small traces of J but no DNA, were set up for crystallization . However, the resultant crystals were of degraded procapsid particles, which had lost the assembly scaffolding proteins D and B, resulting in particles that resembled empty virions . The structure of the degraded G4 procapsid has been determined to 3.0 angstrom resolution . The particles crystallized in the hexagonal space group P6(3)22 with unit cell dimensions a=b=414.2(5) angstrom and c=263.0(3) angstrom . The diffraction data were collected at the Cornell High Energy Synchrotron Source (CHESS) on film and image plates using oscillation photography . Packing considerations indicated there were two particles per unit cell . A self-rotation function confirmed that the particles were positioned on 32 point group special positions in the unit cell . Initial phases were calculated to 6 angstrom resolution, based on the known phiX174 virion model . Phase information was then extended in steps to 3.0 angstrom resolution by molecular replacement electron density modification and particle envelope generation . The resulting electron density map was readily interpretable in terms of the F and G polypeptides, as occur in the mature capsid of phiX174 . In a few regions of the electron density map there were inconsistencies between the density and the published amino acid sequence . Redetermining the amino acid sequence confirmed that the density was correct . The r.m.s . deviation between the Calpha backbone of the mature capsid of phiX174 and the degraded G4 procapsid was 0.36 angstrom for the F protein and 1.38 angstrom for the G protein . This is consistent with the greater conservation of the F protein compared to the G protein sequences among members of the Microviridae family . Functionally important features between phiX174 and G4 had greater conservation . Calcium ions (Ca2+) were shown to bind to G4 at a general site located near the icosahedral 3-fold axis on the F protein capsid, equivalent to sites found previously in phiX174 . Binding of Ca2+ also caused the ordering of the conserved region of the DNA binding protein J, which was present in the degraded procapsid particle in the absence of DNA. Biophys Chem, 1996 Mar 7, 59(1-2), 41 - 59 Mechanism of the long tail-fiber deployment of bacteriophages T-even and its role in adsorption, infection and sedimentation; Kellenberger E et al.; Models for the tail-fiber deployment of T-even bacteriophages have been experimentally tested by correlating sedimentation constants, adsorption rates, protease inactivation kinetics, and fiber configurations of individual phages observed by electron microscopy . Neither the collective nor the individualistic model, i.e . coordinated fiber retraction and expansion or oscillation of fibers independently of each other, respectively, could satisfactorily account for the results presented . We propose a new intermediary model, in which the base-plate determines a collective behaviour by fixing the hinge angle, around which individual fibers oscillate freely . The bidisperse, so-called dual sedimentation was shown to occur mainly with nascent high-concentration phage stocks in potassium glutamate containing media . Indeed, when mature intracellular phages are released in 0.5 M potassium glutamate--a condition simulating the intracellular environment--only the fast form appears . Upon storage in the cold or release into 0.5 M chloride, both forms appear . Results confirming that the sedimentation constants of the fast and slow form roughly correspond to those of the monodisperse sedimentation, characteristic of the extreme pH values, i.e . 5 and 8, do not allow to conclude that fiber configuration is the only cause of the bidisperse sedimentation. Genes Cells, 1996 Mar, 1(3), 317 - 23 A one-hybrid system for detecting RNA-protein interactions; Wilhelm JE et al.; BACKGROUND: mRNA translation, stability, and localization are controlled by regulatory proteins that bind to specific RNA motifs . Since biochemical isolation of such proteins has often proven to be difficult, a genetic system for studying RNA-protein interactions would be of great utility in the identification of novel RNA binding proteins and in understanding how these proteins recognize particular RNA sequences . The bacteriophage lambda gene product N protein is a sequence specific RNA binding protein that when bound to its target sequence allows RNA polymerase to ignore transcription termination signals . The fact that the binding of N protein to RNA is directly coupled to gene expression suggests that N protein could be used to develop a general system for studying RNA-protein interactions . RESULTS: Our results show that fusion of the RNA binding protein, R17, to N causes antitermination in a beta-galactosidase reporter construct that has the R17 binding site substituted for the normal N target sequence . This system can both detect low affinity interactions as well as discriminate between binding events with equilibrium dissociation constants from 10(-5) to 10(-8)M . The differences in antitermination activity with various mutant binding sites can be reliably detected by colony colour on X-Gal plates as well as by liquid culture assay . CONCLUSIONS: We have demonstrated that N protein can cause antitermination through a heterologous RNA-protein interaction and that the system is capable of detecting RNA-protein interactions of differing affinities . This approach may also be useful in screening libraries for proteins that bind to novel RNA regulatory elements . Our results are also consistent with a model of N protein function in which binding to the nascent transcript increases the effective concentration of N in the vicinity of RNA polymerase leading to antitermination. Appl Environ Microbiol, 1996 Mar, 62(3), 994 - 7 Natural variability and diurnal fluctuations within the bacteriophage population of the rumen; Swain RA et al.; To investigate the impact of nutritional and environmental factors on bacteriophage activity in the rumen, it is first valuable to determine the extent of natural variations and fluctuations in phage populations from different animal species, and from animals located together and separately, and variation in animals over time . Differences in phage populations between sheep on different diets, between sheep and goats, and within the rumen over time were investigated by using pulsed-field gel electrophoresis and comparing total phage DNA in ruminal fluid . It was found that no two individuals had similar DNA banding patterns, even when similarly fed and penned together, indicating there is considerable individual diversity in phage populations between animals . Despite these individual differences, the quantities, but not the banding patterns, of phage DNA were similar for animals within groups but varied between groups, suggesting that nutritional factors may influence overall phage activity in the rumen . In sheep fed once daily, a distinct diurnal variation in the phage population was observed . Two hours postfeeding, total phage DNA dropped to its lowest level . The phage population then increased, reaching a maximal level 8 to 10 h postfeeding before declining over the next 4 h to reach a stable concentration for the rest of the cycle . The general trend in phage DNA concentration appeared similar to previously recorded diurnal fluctuations in ruminal bacterial populations in cattle fed once daily. Genome Res, 1996 Mar, 6(3), 218 - 25 Construction of a 750-kb bacterial clone contig and restriction map in the region of human chromosome 21 containing the progressive myoclonus epilepsy gene; Stone NE et al.; The gene responsible for progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is located on human chromosome 21q22.3 in a region defined by recombination breakpoints and linkage disequilibrium . As part of an effort to clone the EPM1 gene on the basis of its chromosomal location, we have constructed a 753-kb bacterial clone contig that encompasses the region containing the gene . Because DNA markers from the region did not identify intact yeast artificial chromosome (YAC) clones after screening several libraries, we built the contig from cosmid clones and used bacterial artificial chromosome (BAC) and bacteriophage P1 clones to fill gaps . In addition to constructing the clone contig, we determined the locations of the EcoRI, SacII, EagI, and NotI restriction sites in the clones, resulting in a high-resolution restriction map of the region . Most of the contig is represented by a level of redundancy that allows the orders of most restriction sites to be determined, provides multiple data points supporting the clone orders and orientations, and allows a set of clones with a minimum degree of overlap to be chosen for efficient additional analysis . The clone and restriction maps are in excellent agreement with maps generated of the region by other methods . These ordered bacterial clones and the mapping information obtained from them provide valuable reagents for isolating candidate genes for EPM1, as well as for determining the nucleotide sequence of a 750 kb region of the human genome. Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 94 - 101 Efficient production of Thermus protease aqualysin I in Escherichia coli: effects of cloned gene structure and two-stage culture; Sakamoto S et al.; The DNA sequence encoding Thermus protease aqualysin I was inserted downstream from a bacteriophage T7 promoter in an expression vector . In the T7 expression system, using a strain lacking an F' episome, aqualysin I was produced in soluble form without chemical induction . The deletions of part (30 amino acid residues) or all (105 residues) of the C-terminal pro-sequence from the C terminus significantly affected both cellular growth and the production of the enzyme . Complete deletion adversely affected both . In contrast, the 30-residue deletion markedly improved productivity by approximately four times compared to non-deletion, and shortened the time needed for the activation of a precursor to active enzyme . The concentration of inducer isopropyl beta-D-thiogalactopyrano-side (IPTG) was varied to examine its effects, and it was found that a low concentration of IPTG improved aqualysin I production . To avoid the inhibitory effects of acetic acid accumulation in the culture medium, the use of other carbon sources besides glucose was examined . When cells were cultivated with glycerol, the acetic acid level remained relatively low, and both good cellular growth and a high level of production were attained . The aqualysin I productivity for a fed-batch culture using two carbon sources, glucose and glycerol, reached more than 150 kU/ml enzymatically active aqualysin I. Photochem Photobiol, 1996 Mar, 63(3), 281 - 5 Induction of lambda prophage by 213 nm laser radiation: a quantitative comparison with 193 nm excimer radiation using image analysis; Matchette LS et al.; We compared the DNA damage produced by radiation from two UV laser wavelengths, 213 nm and 193 nm, with that produced by noncoherent 254 nm radiation . Following irradiation of Escherichia coli BR339, a bacteriophage lambda lysogen containing the lacZ gene, pro-phage induction was measured by assaying for beta-galactosidase . Because of the limited penetration by UV laser wavelengths an agar overlay of the lysogen was used as the irradiation target . Irradiation of 254 nm was performed in buffer suspension followed by transfer of 5 microL spots onto assay plants . Computer image analysis was used to monitor the rate of product formation, observed as an increase in optical density of the irradiated zones on assay plates . We found that the rate of product formation was a more reproducible unit of comparison than the optical density present at the end of the reaction . Although the rate of product formation was not linearly related to enzyme concentration, the data could be fit to a simple logarithmic function . Using this method, we concluded that the DNA damaging ability of 213 nm radiation was 10 times more efficient than 193 nm radiation and about 100 times less efficient than 254 nm noncoherent radiation. Biotechnol Prog, 1996 Mar-Apr, 12(2), 196 - 200 Synthesis rates of cellular proteins involved in translation and protein folding are strongly altered in response to overproduction of basic fibroblast growth factor by recombinant Escherichia coli; Rinas U; The cellular response to temperature-induced production of human basic fibroblast growth (bFGF) factor by recombinant E . coli (bacteriophage lambda PRPL promoter/cI857 repressor expression system) was studied by one- and two-dimensional gel electrophoresis . Temperature shift from 30 to 42 degrees C caused the induction of heat-shock protein synthesis and the repression of synthesis of ribosomal proteins and the protein folding catalyst trigger factor . Compared to control cells, carrying the expression vector without structural bFGF gene cells, producing the heterologous protein exhibited a stronger increase in the synthesis rate of heat-shock proteins ClpB (HtpM), DnaK, HtpG, GroEL, GrpE, and IbpB (HtpE) in response to temperature upshift . Unexpectedly, formation of the chaperone heat-shock protein GroES was not detected after temperature shift to 42 degrees C in cells producing bFGF . In addition to amplified heat-shock protein formation, the syntheses of ribosomal proteins and of the protein folding catalyst trigger factor were more severely repressed after temperature upshift in cells producing bFGF . In conclusion, the normal cellular stress response caused by the high inducing temperature was strongly amplified by heterologous protein synthesis . In particular, syntheses of proteins involved in translation and protein folding were affected by the overproduction of the heterologous protein. Genetics, 1996 Mar, 142(3), 661 - 72 C-terminal deletions can suppress temperature-sensitive mutations and change dominance in the phage Mu repressor; Vogel JL et al.; Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity . Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q) and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts) . Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup degrees hosts . Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190 . The Sts phenotype relates to the repressor size: in Sup degrees hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts . The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179-V196), binds Mu operator DNA more stably at 42 degrees in vitro compared to its full-length counterpart (cts62 repressor) . In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor. Biophys J, 1996 Mar, 70(3), 1514 - 20 Vertical dimension of hydrated biological samples in tapping mode scanning force microscopy; Schabert FA et al.; The vertical dimensions of the well-characterized test samples tobacco mosaic virus, T4 bacteriophage polyhead, purple membrane, and hexagonally packed intermediate (HPI) layer were investigated by tapping mode scanning force microscopy (SFM) in solution . Purple membrane and HPI layer were imaged in both contact mode and tapping mode SFM for direct comparison . All vertical dimensions match the known heights . The practical implications of the absence of frictional forces in tapping mode are discussed. Biokhimiia, 1996 Mar, 61(3), 532 - 45 {Expression of HIV-1 epitopes included in particles formed by human hepatitis B virus nucleocapsid protein}; Isaguliants MG et al.; Hybrids of the core protein of hepatitis B virus (HBcAg) have been designed which carry N-terminal insertions of B- and T-cell epitopes of HIV-1 an immunodominant B-epitope from gp41, a T-cell epitope from p34 pol, and a cluster of B- and T-cell epitopes from p17 gag . The hybrids have been synthesized using two expression systems-one based on the thermoinducible PR promoter of bacteriophage lambda and the other one based on phi 10 promoter of bacteriophage T7 with 3-5% and 7-14% yields, respectively . The hybrids have dual HBV and HIV-1 immunospecificity and are assembled into particles similar to those formed by the protein carrier HBcAg . Sandwich ELISA and immune electron microscopy revealed that HIV-1 epitopes are exposed on the surface of the particles. Curr Microbiol, 1996 Mar, 32(3), 162 - 7 Promotion of bacteriophage induction and recombination by the cloned Bordetella pertussis recA gene is copy-number dependent; Kuhl SA; Favre and coworkers (Favre et al., Biochimie 73:235-244, 1991) previously reported that the Bordetella pertussis recA gene present at high copy number could promote a low frequency of recombination, but not bacteriophage induction in Escherichia coli RecA- mutants . Reexamination of these phenotypes demonstrated that, in contrast to the previous study, when this gene is present at high copy number, it can stimulate a 2- to 4-log frequency of bacteriophage induction in the presence of mitomycin C, but no appreciable spontaneous induction . The cloned gene, whether it was present in high or low copy number, also promoted a low frequency of intrachromosomal recombination of two duplicated genes in Escherichia coli . These results suggest that a high concentration of the B . pertussis RecA protein is required to promote high-frequency mitomycin C-stimulated bacteriophage induction, but it facilitates intrachromosomal recombination at a very low frequency in E . coli RecA- mutants . The ability of the B . pertussis RecA protein to promote mitomycin C induction and its inability to appreciably stimulate spontaneous induction of bacteriophage suggest that this protein possesses a unique phenotype compared with other RecA proteins. Plasmid, 1996 Mar, 35(2), 108 - 20 Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector; Wilkens K et al.; We report on the construction of promoter probe vector pKWIII, useful in cloning and analyzing strong promoters for Escherichia coli RNA polymerase . Also T4 early promoters that proved to be difficult to clone with other vectors could be tested . The promoter activities obtained with this convenient and nonradioactive system largely correspond to those determined by pulse-labeling of transcripts in the same system . Results with well-characterized control promoters are in good agreement with values given by other authors . We present relative activities of several early promoters of phage T4 and compare these to promoter activities of other phages . Sorting the T4 promoters according to strength suggests the importance of distinct sequence elements to promoter functioning . They are centered around positions -52, -42, and -15. Vet Parasitol, 1996 Mar, 62(1-2), 9 - 25 Use of genomic DNA probes for the diagnosis of acute sarcocystosis in experimentally infected cattle; Ndiritu W et al.; Two clones of 1.4 and 4.33 kilobase pairs (kbp) DNA inserts, were selected from a Sarcocystis cruzi sporozoite genomic library constructed in bacteriophage lambda gt10 . These clones strongly hybridized with sporozoite and merozoite DNA and were evaluated as probes for detection of merozoite DNA in clinical samples . Of five calves in the experiment, four were each orally dosed with approximately 200,000 S . cruzi sporocysts; one calf served as non-infected control . Subsequently, blood was collected from the calves twice weekly for 3.5 months and fractionated into buffy coats, polymorphonuclear cells, and plasma . Total cellular DNA extracted from these fractions was dot blotted on nylon membranes and hybridized with the probes radiolabeled with {alpha-32P}dATP . The probes detected merozoites on Day 22 post infection in the buffy coats and intermittently from Day 25-39 in the granulocyte fraction . Parasitemia (i.e . merozoites in blood) was also detected by indirect fluorescent antibody technique (IFAT) and direct microscopy, Diagnosis of sarcocystosis in cattle using genomic DNA probes by dot blot hybridization provides an alternative method of detecting parasitemia that is more rigorous than the other two tests (IFAT, direct microscopy) which rely on morphology of the merozoite and visualization by the examiner . As probes detected merozoite DNA in the granulocyte fraction, polymorphonuclear cells may be involved in the pathogenesis of S.cruzi; however this hypothesis requires further study. J Bacteriol, 1996 Mar, 178(5), 1484 - 6 Proteins responsible for lysogenic conversion caused by coliphages N15 and phi80 are highly homologous; Vostrov AA et al.; Lysogenic conversion caused by lambdoid bacteriophage phi80 and that caused by coliphage N15 have similar characteristics, suggesting that similarities in their cor genes and Cor proteins are responsible for this effect . Here we present the nucleotide sequence of the N15 cor gene . The N15 cor gene homolog was found in the phi80 cor region, but in the opposite direction of that of the open reading frame to which the phi80 cor gene had previously been assigned (M . Matsumoto, N . Ichikawa, S . Tanaka, T . Morita, and A . Matsushiro, Jpn . J . Genet . 60:475-483, 1985). J Virol, 1996 Mar, 70(3), 1792 - 8 Transfer of single gene-containing long terminal repeats into the genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP site; Choulika A et al.; Retroviral vectors contain viral cis-acting elements to achieve the packaging, reverse transcription, integration, and expression of the retroviral genomic nucleic acid sequence . However, these elements are not useful in the integrated provirus and can be the cause of problems . We have developed a vector which eliminates the majority of these viral elements . This vector, a long terminal repeat (LTR) enhancer-deleted vectors, exploits the Cre-lox recombination system of the P1 bacteriophage . The Cre-lox system is neutral for eukaryotic cells . The 32-nucleotide loxP site is inserted within the U3 of the 3' LTR along with with the gene to be transduced (in place of the viral enhancers) . Following the LTR-mediated loxP duplication, the LTRs can be recombined by the Cre enzyme . The structure of the resulting provirus in the host genome corresponds to a single LTR (deleted of the viral enhancers) carrying a single copy of the gene to be transduced . If the Cre expression unit is furnished after the integration of a loxP-containing virus, the efficiency of the recombination is not absolute . If the Cre expression unit is inserted between the two LTRs, only single LTR proviral structures are found following infection by the retroviral vector. J Bacteriol, 1996 Mar, 178(6), 1585 - 92 Regulatory factors acting at the bacteriophage Mu middle promoter; Kahmeyer-Gabbe M et al.; Lytic development of bacteriophage Mu proceeds through three phases of transcription: early, middle, and late . Initiation of middle transcription from Pm requires the phage-encoded activator, Mor . An examination of the sequences surrounding the promoter revealed possible binding sites for Mu proteins A and c, as well as for Escherichia coli integration host factor . Promoter fragments containing 5' and 3' deletions were fused to the lacZ reporter gene and assayed for activity after induction of a Mu prophage or a plasmid-borne mor gene . Sequences upstream of position -62 and downstream of +10 were dispensable for promoter activity . In DNase I footprinting with both crude extract and purified protein, Mor protected Pm sequences from position -56 to -33 . Mutations disrupting the dyad symmetry of the terminator of early transcription overlapping the Mor binding site did not reduce promoter activity, suggesting that the symmetry per se is not required for Mor binding or Pm activation . Purified Mu lysogenic repressor (c) also bound to Pm, overlapping the Mor binding site . Production of large amounts of repressor in vivo reduced Mor-dependent promoter activity nearly 10-fold . Promoters with mutations in the repressor binding site showed a reduction in this repressor-mediated inhibition of Pm activity. RNA, 1996 Mar, 2(3), 289 - 96 RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation; Lapham J et al.; Large quantities of RNA for study by NMR and X-ray crystallography can be produced by transcription reactions in vitro using T7 bacteriophage RNA polymerase . A limitation on producing RNA with this polymerase has been the strong dependence of the yield of the transcription reaction on the sequence at the 5' end of the RNA produced . We report a procedure for obtaining large quantities of enzymatically synthesized RNA from T7 RNA polymerase that has no dependence on the 5' end sequence of the target RNA . Ribonuclease H has been shown previously (Inoue H, Hayase Y, Iwai S, Ohtsuka E, 1987, FEBS Lett 215:327-330) to cleave RNA site specifically using 2'-O-methyl RNA/DNA chimeras to direct the cleavage site . We show that 2'-O-methyl RNA nucleotides on the 5'-side of the DNA nucleotides in the chimera are not essential for site-specific cleavage . This allowed us to design the method such that the same 2'-O-methyl chimera may be used to process any RNA sequence . We have adapted this reaction to the cleavage of NMR-scale quantities of RNA at high yield . RNA is synthesized using T7 RNA polymerase with a 15-nt high-yielding leader sequence at the 5' end, and then this sequence is cleaved off with the RNase H cleavage reaction . The cleaved RNA has 3'-hydroxyl and 5'-phosphate ends, so that the products can be used directly as substrates for ligation by T4 DNA ligase . We show that the cleavage reaction occurs efficiently in solution and on a solid streptavidin/agarose matrix . We report an example in which we are able to improve transcription yield by more than five-fold using this technique in the synthesis of a 15N isotopically labeled hairpin found in the Crithidia fasciculata spliced leader RNA . We are able to obtain a 0.5-mM NMR sample from this inherently poorly transcribing sequence, while minimizing the amount of isotopically labeled rNTPs used to produce it . The NMR spectroscopic results are consistent with the predicted RNA secondary structure. J Mol Biol, 1996 Mar 1, 256(3), 517 - 32 Computer-aided discrimination between active and inactive mutants of the N-terminal domain of the bacteriophage lambda repressor; Kombo DC et al.; Binding of the N-terminal domain of the lambda repressor to DNA is coupled to dimerization . Hydrophobic interactions between helix-5 and helix-5' drive the packing at the dimer interface . We have carried out computations of the conformational energy of packing of the fifth helices (and of the helix-4-loop-helix-5 portions) of variants of the lambda repressor operator binding domain, using an ECEPP/3-based packing algorithm . Here, we report the results for 26 mutants chosen among those that hve been characterized experimentally . We find that the relative orientation of the fifth helices for active mutants is very similar to the wild-type . The fifth helices of the inactive mutants have a significantly different relative orientation . This result illustrates that a unique specific orientation pattern of helix-5 relative to helix-5' is required for dimerization-coupled DNA binding activity . This finding is further supported by computational studies of the whole N-terminal domain of ten variants that showed that the active mutants, including the wild-type protein, have similar values of the number of contacts between the two monomers in the dimer, involving two amino acid residues of the fifth helices (positions 84 and 87 in each monomer) . A decrease in the number of such contacts abolishes DNA-binding activity . Furthermore, all active mutants have their "DNA-recognition helices", numbers 3 and 3' positioned so that they can fit in the DNA operator like those of the wild-type protein, while some inactive mutants exhibit a substantial change in the relative orientation of their recognition helices. Nucleic Acids Res, 1996 Mar 1, 24(5), 835 - 42 Cleavage by RNase P of gene N mRNA reduces bacteriophage lambda burst size; Li Y et al.; RNase P, an enzyme essential for tRNA biosynthesis, can be directed to cleave any RNA when the target RNA is in a complex with a short, complementary oligonucleotide called an external guide sequence (EGS) . RNase P from Escherichia coli can cleave phage lambda N mRNA in vitro or in vivo when the mRNA is in a complex with an EGS . The EGS can either be separate from or covalently linked to M1 RNA, the catalytic RNA subunit of RNase P . The requirement for Mg2+ in the reaction in vitro is lower when the EGS is covalently linked to M1 RNA . Substrates made of DNA can also be cleaved by RNase P in vitro in complexes with RNA EGSs . When either kind of EGS construct is used in vivo, burst size of phage lambda is reduced by > or = 40% . Reduction in burst size depends on efficient expression of the EGS constructs . The product of phage lambda gene N appears to function in a stoichiometric fashion. Virology, 1996 Mar 1, 217(1), 332 - 7 DNA sequences responsible for specificity of DNA packaging and phage growth interference of bacteriophages T3 and T7; Tsuchida SI et al.; T3 and T7 phages package recombinant plasmids carrying DNA necessary for DNA packaging (the pac sequences) of T3 and T7, respectively . Packaging is specific between T3 and T7 . The pac sequence has a bipartite structure, consisting of target sequences for processing of concatemeric DNA (pac C) and its left side flanking sequence containing a promoter for phage RNA polymerase (pac B) . To determine the sequences responsible for the specificity of plasmid DNA packaging, plasmids chimeric for the pac B and pac C sequences of T3 and T7 were constructed . Analysis of packaging of the chimeric plasmid DNAs showed that pac B is responsible for the packaging specificity of T3 and T7 DNAs . Plasmids carrying the genetic right end of T3 and T7 DNA interfered with the growth of T3 and T7 phages, respectively . Interference was specific between T3 and T7 . pac B and sequences between pac B and pac C, but not pac C, were responsible for the interference . The specificity of interference was determined by pac B and sequences responsible for interference were partially defined. Virology, 1996 Mar 1, 217(1), 200 - 10 Bacteriophage Mu head assembly; Grimaud R; The protein composition of defective particles produced by various bacteriophage Mu head-gene mutants was analyzed by SDS-PAGE . An abundant 20-kDa protein was detected in only one type of defective head . This protein exhibits properties of a scaffolding protein . A 50-kDa structural protein present in most defective heads was shown to be produced by cleavage of the C-terminus of the 64-kDa polypeptide encoded by gene H . Cleavage occurs during head assembly at a site which, according to earlier results, might separate two different functional domains in gpH . A fraction of the gpH molecules produced upon Mu induction sediment in a 25 S complex, suggesting that gpH participates in the formation of an early intermediate of Mu head assembly . Characteristics of gpH suggest that it may be the Mu portal protein. DNA Res, 1996 Feb 29, 3(1), 37 - 42 An improved cosmid vector for the nested deletion method using the bacteriophage T3 DNA packaging system; Kawarabayasi Y et al.; We constructed a new cosmid vector suitable for the previously developed nested deletion method which used the in vitro DNA packaging system of bacteriophage T3 . The first step of this method is linearization of a cosmid clone to be packaged, and we previously introduced cleavage at the cos site using lambda-Terminase, but optimization of the reaction conditions was required for complete digestion because of its instability . In the newly constructed vector, pAT5, the sites of 4 different restriction enzymes, Sse8387I, Asc I, Fse I and Pme I, each of which recognizes an 8-bp sequence (8-base cutter) were introduced in the vicinity of the cos site . In addition, the species of restriction sites for cloning were increased to broaden its application . The cosmid clone constructed by this new vector could be linearized at one of the 8-base cutter sites which are assumed to rarely occur in the genome, and followed by in vitro packaging, nested deletion clones were successfully prepared. Biochemistry, 1996 Feb 27, 35(8), 2796 - 803 Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi; Hwang Y et al.; Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively . Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine . By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP . Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3{32P} ATP in the presence of UV light . Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively . ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM) . The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity . The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase . ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA . In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA. J Mol Biol, 1996 Feb 23, 256(2), 330 - 9 Crystal structures of MS2 capsids with mutations in the subunit FG loop; Stonehouse NJ et al.; The loop between the F and G beta strands (FG loop) of the bacteriophage MS2 coat protein subunit forms inter-subunit contacts around the 5-fold and 3-fold (quasi 6-fold) axes of the T=3 protein shell . In capsids, the loop is found in two very different conformations, one in B subunits, which form the 5-fold contact, and one in A and C subunits, which form the quasi 6-fold contact . One proline residue, Pro78, is strictly conserved in the coat protein of all related bacteriophages, and in the case of MS2 this proline residue is preceded by a cis peptide bond in the B subunit . In order to probe the role of the FG loop in capsid assembly, we have determined the crystal structures of two MS2 capsids, formed by coat proteins with mutations at two positions in the FG loop, P78N or E76D . These mutants show conformational changes in the FG loops that explain the reduced temperature stability of the capsids . The P78N mutant has a normal trans peptide bond at position 78. J Mol Biol, 1996 Feb 23, 256(2), 235 - 48 Characterization of pre-transcription complexes made at a bacteriophage T4 middle promoter: involvement of the T4 MotA activator and the T4 AsiA protein, a sigma 70 binding protein, in the formation of the open complex; Hinton DM et al.; Bacteriophage T4 middle promoters have excellent matches to the -10 consensus sequence for the sigma 70 subunit of Escherichia coli RNA polymerase, but a binding site for the T4 transcriptional activator MotA replaces the sigma 70 -35 consensus . E . Coli RNA polymerase transcribes from middle promoters with or without the activator . In contrast, transcription by T4-modified E . coli RNA polymerase, which is present during T4 infection, requires NotA . We show that transcription by unmodified polymerase from the T4 middle promoter P uvsx is independent of the specific sequences within the -35 region, and the Dnase I footprint obtained with unmodified polymerase and P uvsx resembles those seen previously with E . coli extended -10" promoters . In contrast, although T4-modified polymerase alond binds P uvsx, promoter unwinding and detection of a Dnase I footprint requires MotA . This footprint is significantly different from that obtained with unmodified polymerase, starting upstream of around position -20 . Previous work has indicated that the T4 AsiA protein, which binds tightly to sigma 70, is the phage modification required for MotA activation . We show that in the presence of AsiA, MotA, and otherwise unmodified polymerase, Dnase I protection of P uvsx is now similar to that obtained with the fully modified polymerase and MotA up to around position -40 . However, protection upstream of -40 is still similar to that seen with unmodified polymerase . Our results support the idea that MotA-dependent activation requires AsiA binding to sigma 70 to achieve specific protein-DNA contacts within the -20 to -40 region of a middle promoter. Biochemistry, 1996 Feb 20, 35(7), 2349 - 56 In vitro selection of RNA specifically cleaved by bacteriophage T4 RegB endonuclease; Jayasena VK et al.; T4 RegB endonuclease specifically cleaves at -GGAG- sites in several early T4 messages, rendering them nonfunctional . Not all -GGAG- sites are processed equally by RegB; those found at the Shine-Dalgarno sequences and in intercistronic regions are processed with higher efficiency than the -GGAG- sites located in coding regions . The low activity of RegB observed in vitro is enhanced by 1-2 orders of magnitude by the Escherichia coli ribosomal protein S1 . We have used SELEX (systematic evolution of ligands by exponential enrichment) on a combinatorial RNA library to obtain molecules that are specifically cleaved by T4 RegB endonuclease in the presence of S1 . The sequences obtained contain the required -GGAG- tetranucleotide and were unusually enriched in adenosine and cytosine nucleotides . No consensus structure or sequence motif other than -GGAG- was conserved among the selected molecules . The majority of the RNAs are entirely dependent on S1 for RegB-catalyzed cleavage; however, a few RNAs are found to be S1 independent but are cleaved by RegB with much lower rates. Biochemistry, 1996 Feb 20, 35(7), 2218 - 28 Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases; Hingorani MM et al.; The equilibrium nucleotide binding and oligomerization of bacteriophage T7 gene 4 helicases have been investigated using thymidine 5'-triphosphate (dTTP), deoxythymidine 5'-(beta, gamma-methylenetriphosphate)(dTMP-PCP), thymidine 5'-diphosphate (dTDP), adenosine 5'-triphosphate (ATP), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) . In the presence of nucleotide ligands, T7 helicases self-assemble into hexamers with six potential nucleotide binding sites that are nonequivalent both in the absence and in the presence of single-stranded DNA . All nucleotides tested bind with high affinity to three sites (K(d) = 5 x 10(-6) M, dTTP; 6 x 10(-7) M, dTMP-PCP; 4 x 10(-6) M, dTDP; 3 x 10(-5) M, ATP; 2 x 10(-6) M, ATP gamma S), while binding to the remaining sites is undetectable . Interestingly, nucleotide binding to the high-affinity sites exhibits positive cooperativity which is sensitive to protein concentration . This effect is a result of ligand binding-linked oligomerization wherein helicase oligomer equilibrium changes as a function of both nucleotide and protein concentration . A study of DNA binding shows that 1-2 NTPs bound per hexamer are sufficient for stoichiometric interaction between the helicase and DNA . Thus, the ring-shaped helicase hexamers assemble around DNA with one, two, or three NTPs bound to each hexamer . This study also examines the preferred use of dTTP for T7 helicase-catalyzed DNA unwinding by comparison with ATP, the more commonly used nucleotide ligand . ATP binds to the helicase with 6-fold weaker affinity than dTTP and promotes hexamerization as well as DNA binding . Nevertheless, DNA unwinding with ATP is at least 100-fold slower than with dTTP . Thus, the difference in ATP and dTTP utilization probably lies in a highly specific step in the coupling of NTP hydrolysis to DNA unwinding. Proc Natl Acad Sci U S A, 1996 Feb 20, 93(4), 1694 - 8 Sequence scanning: A method for rapid sequence acquisition from large-fragment DNA clones; Nurminsky DI et al.; A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes . The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size range 8-12 kb . A number of subclones, typically 96 or 192, are chosen at random, and the ends of the inserts are sequenced using vector-specific primers . Then long-range spectrum PCR is used to order and orient the clones . This combination of shotgun and directed sequencing results in a high-resolution physical map suitable for the identification of coding regions or for comparison of sequence organization among genomes . Computer simulations indicate that, for a target clone of 100 kb, the scanning of 192 subclones with sequencing reads as short as 350 bp results in an approximate ratio of 1:2:1 of regions of double-stranded sequence, single-stranded sequence, and gaps . Longer sequencing reads tip the ratio strongly toward increased double-stranded sequence. Proc Natl Acad Sci U S A, 1996 Feb 20, 93(4), 1535 - 9 Mutational properties of the primary aflatoxin B1-DNA adduct; Bailey EA et al.; The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo . An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13 . Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4% . The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin . The G --> T mutations of AFB1-N7-Gua, unlike those (if the AFB1-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern matching that in intact cells treated with the toxin . It is concluded that the AFB1-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells . While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine . In contrast, the apurinic site-containing genome gave rise only to targeted mutations . The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face of the guanine residue . These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1. Mutat Res, 1996 Feb 19, 350(1), 9 - 16 Selection of bacteriophage T4 antimutator DNA polymerases: a link between proofreading and sensitivity to phosphonoacetic acid; Reha-Krantz LJ et al.; During DNA replication, DNA polymerases alternate between DNA synthesis and proofreading the newly synthesized DNA . In order to understand the molecular details of how DNA polymerases determine the balance between polymerase and proofreading activities, it would be useful to have mutants which switch between the two activities either more or less frequently . Antimutator DNA polymerases switch more frequently and thus have more opportunity for proofreading . We have observed that mutant DNA polymerases which proofread less frequently have a mutator phenotype and are inhibited by the pyrophosphate analogue phosphonoacetic acid . Sensitivity to phosphonoacetic acid can be used to isolate second-site suppressor mutations . These suppressor mutations encode amino acid substitutions which produce antimutator DNA polymerases. Genomics, 1996 Feb 15, 32(1), 91 - 6 The generation and regional localization of 303 new chromosome 5 sequence-tagged sites; Grady DL et al.; With the ultimate goal of creating a sequence-ready physical map of all of chromosome 5, 303 new human chromosome 5-specific STS markers have been systematically generated and regionally ordered . Chromosome 5 DNA prepared from flow-sorted chromosomes was digested with restriction enzymes BamHI and HindIII and cloned in bacteriophage M13mp18 . Random clones were sequenced, and appropriate PCR deoxyoligomers were synthesized . An acceptable sequence-tagged site (STS)-PCR assay yielded the appropriate size amplification product from both total human DNA and hybrid cell line DNA containing only human chromosome 5 . Each STS has been regionally localized by breakpoint analysis using a set of hybrid cell panels consisting of natural deletions or translocations of human chromosome 5 . This hybrid cell panel was able to localize the STSs to 1 of 51 bins on the short arm and 1 of 15 bins on the long arm . The STS markers appear to be randomly distributed along the length of this 194-Mb chromosome . The current overall density of these markers (approximately 1 STS/640 kb), combined with the numerous PCR-based physical and genetic markers generated by other groups, will provide sufficient "nucleation points" for YAC contig assembly and verification in any region of human chromosome 5. EMBO J, 1996 Feb 15, 15(4), 935 - 44 ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis; Kruklitis R et al.; During transposition bacteriophage Mu transposase (MuA) catalyzes the transfer of a DNA strand at each Mu end to target DNA and then remains tightly bound to the Mu ends . Initiation of Mu DNA replication on the resulting strand transfer complex (STC1) requires specific host replication proteins and host factors from two partially purified enzyme fractions designated Mu replication factors alpha and beta (MRFalpha and beta) . Escherichia coli ClpX protein, a molecular chaperone, is a component required for MRFalpha activity, which removes MuA from DNA for the establishment of a Mu replication fork . ClpX protein alters the conformation of DNA-bound MuA and converts STC1 to a less stable form (STC2) . One or more additional components of MRFalpha (MRFalpha2) displace MuA from STC2 to form a nucleoprotein complex (STC3), that requires the specific replication proteins and MRFbeta for Mu DNA synthesis . MuA present in STC2 is essential for its conversion to STC3 . If MuA is removed from STC2, Mu DNA synthesis no longer requires MRFalpha2, MRFbeta and the specific replication proteins . These results indicate that ClpX protein activates MuA in STC1 so that it can recruit crucial host factors needed to initiate Mu DNA synthesis by specific replication enzymes. Virology, 1996 Feb 15, 216(2), 389 - 96 Immunity specificity determinants in the P4-like retronphage phi R73; Sabbattini P et al.; Retronphage phi R73 exhibits extensive sequence homology to the satellite bacteriophage P4 . Bacteriophage P4 superinfection immunity is elicited by a small RNA (CI RNA) that causes premature transcription termination within the operon coding for the P4 replication functions . This control is exerted via interaction of the CI RNA with two complementary target sites on the untranslated leader RNA of the replication operon . We found that phi R73 is endowed with a similar immunity system but is heteroimmune to P4 . The heteroimmunity is due to six base differences in the CI RNA and to compensatory base substitutions in the target sequences . The sequence differences in the CI RNA are all located in single-stranded regions, which appear to play a predominant role in the interaction with the target sites . Analysis of phage carrying a hybrid immunity system indicates that, although two target sequences are required for the establishment of lysogeny, a single site is sufficient to make a phage sensitive to the prophage immunity. Biochim Biophys Acta, 1996 Feb 8, 1292(2), 324 - 34 T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer; Latham KA et al.; Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution {Nickell and Lloyd (1991) Biochemistry 30, 8638}, although the enzyme has only been crystallized in the absence of substrate as a monomer {Morikawa et al . (1992) Science 256, 523} . In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution . In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen . To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue . These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes . The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer. Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1352 - 6 Extending the chemistry that supports genetic information transfer in vivo: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA, and methylphosphonate DNA; Thaler DS et al.; DNA and RNA are the polynucleotides known to carry genetic information in life . Chemical variants of DNA and RNA backbones have been used in structure-function and biosynthesis studies in vitro, and in antisense pharmacology, where their properties of nuclease resistance and enhanced cellular uptake are important . This study addressed the question of whether the base(s) attached to artificial backbones encodes genetic information that can be transferred in vivo . Oligonucleotides containing chemical variants of DNA or RNA were used as primers for site-specific mutagenesis of bacteriophage f1 . Progeny phage were scored both genetically and physically for the inheritance of information originally encoded by bases attached to the nonstandard backbones . Four artificial backbone chemistries were tested: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA and methylphosphonate DNA . All four were found capable of faithful information transfer from their attached bases when one or three artificial positions were flanked by normal DNA . Among oligonucleotides composed entirely of nonstandard backbones, only phosphorothioate DNA supported genetic information transfer in vivo. Gene, 1996 Feb 2, 168(1), 9 - 14 Analysis of a nucleic-acid-binding antibody fragment: Construction and characterization of heavy-chain complementarity-determining region switch variants; Calcutt MJ et al.; The display of antibody (AB) fragments (Fab) on the surface of filamentous bacteriophage (phage) and selection of phage that interact with a particular antigen (Ag) has enabled the isolation of Fab that bind nucleic acids . Nucleic acid (NA) binding Ab occur in vivo in connective tissue disease patients and certain inbred strains of mice and are thought to be pathogenic . Although there is ample data concerning the amino acid (aa) sequence of murine monoclonal Ab (mAb) reactive with DNA, significantly less is known about how autoAb interact with NA . The complementarity-determining regions (CDR) contained in the Fab contribute to most Ag binding, especially through heavy (H)-chain CDR 3 . We have examined the role of individual H-chain CDR of a previously isolated recombinant single-stranded DNA-binding Fab (DNA-1) in nucleic acid interaction using a combination of H-chain CDR switching and solution-binding experiments . The three H-chain CDR of DNA-1 Fab were independently switched with the H-chain CDR of a Fab (D5) with very similar sequence and framework (FR) that binds DNA poorly in order to create all possible H-chain CDR combinations . The chimeric Fab genes were bacterially expressed, and their products were purified and analyzed . Results indicated that the H-chain CDR 3 of DNA-1 Fab, in the context of the remainder of the H-chain of D5 Fab, restored binding to oligo(dT)15 to 60% of DNA-1 levels, whereas H-chain CDR 1 and 3 of DNA-1 with CDR 2 of D5 Fab restored binding to 100% A combination of H-chain CDR 2 and 3 of DNA-1 Fab with H-chain CDR 1 of D5, unexpectedly resulted in the ability of the chimeric Fab to bind RNA preferentially over DNA . These studies demonstrate the importance of both H-chain CDR 1 and 3 in DNA recognition and further suggest that the specificity of the type of NA recognized by a particular Fab can be drastically altered by exchanging CDR. Gene, 1996 Feb 2, 168(1), 1 - 8 The Rz1 gene product of bacteriophage lambda is a lipoprotein localized in the outer membrane of Escherichia coli; Kedzierska S et al.; The Rz1 gene of bacteriophage lambda is located within the Rz1 lysis gene . It codes for the 6.5-kDa prolipoprotein (Rz1) which undergoes N-terminal signal sequence cleavage and post-translational lipid modification of the N-terminal Cys of the mature protein . Globomycin, the antibiotic which inhibits bacterial signal peptidase II, specific for prolipoproteins containing diacylglyceryl cysteine {Hayashi and Wu, J . Bioenerg . Biomembr . 22 (1990) 451-471} inhibits the N-terminal sequence cleavage of the Rz1 precursor . The mature protein is rich in Pro, which constitutes 25% of its amino acids (aa) . Using a computer-predicted, synthetic, 15-aa antigenic determinant of Rz1 polyclonal anti-Rz{46-60} antibodies, were obtained, and employed to localize Rz1 in bacterial fractions . In induced Escherichia coli lambda lysogens Rz1 was found almost exclusively in the outer membrane (OM) . In a strain overproducing Rz1 from the pSB54 plasmid, it was distributed in all the fractions, OM, fraction A and inner membrane (IM) . Expression of Rz1 from the pSB54 caused enlargement of fraction A, corresponding to the adhesion sites of OM and IM . Such an enlargement was previously observed in induced lambda lysogens, shortly before the onset of lysis. Mol Divers, 1996 Feb, 1(2), 79 - 86 Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody; De Ciechi PA et al.; Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet . These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4 . After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteine-constrained peptides . DNA sequencing of the enriched phage revealed two distinct consensus sequences: HXG(A/Y)XH and DVCXXGGPGC . Phage expressing these consensus sequences bound to L307.4 but not to an isotype matched antibody, indicating that binding was antibody specific . Synthetic peptides corresponding to both motifs inhibited phage binding to L307.4, indicating that the gene III protein is not required for peptide binding . In addition, the cyclized forms of synthetic peptides containing the DVCXXGGPGC motif were capable of inhibiting L307.4 binding to soluble B7-1/Fc fusion . Moreover, phage expressing only the HXG(A/Y)XH consensus sequence were inhibited from binding to L307.4 by the presence of chelating agents . These results indicate that the framework within which the peptide is presented on the surface of the phage may allow the identification of unique peptide motifs with distinct binding characteristics . These peptide motifs could be used for the design of peptidomimetics with therapeutic applications if they inhibit the binding of B7-1 to its T cell receptors. Microbiology, 1996 Feb, 142 ( Pt 2), 261 - 7 A gene cloning system for 'Streptomyces toyocaensis'; Matsushima P et al.; We explored different methods of introducing DNA into 'Streptomyces toyocaensis' and Streptomyces virginiae to construct stable recombinant strains . Plasmid pIJ702 isolated from Streptomyces lividans transformed protoplasts of 'S . toyocaensis' at a frequency of 7 x 10(3) transformants (mu g DNA)-1 . pIJ702 prepared from 'S . toyocaensis' transformed 'S . toyocaensis' protoplasts at a frequency of 1 center dot 5 x 10(5) (mu g DNA)-1, suggesting that 'S . toyocaensis' expresses restriction and modification . Plasmid pRHB126 was transduced by bacteriophage FP43 into 'S . toyocaensis' at a frequency of 1.2 x 10(-6) (p.f.u)-1 . Plasmids pOJ436 and pRHB304 were introduced into 'S . toyocaensis' by conjugation from Escherichia coli S17-1 at frequencies of about 2 x 10(-4) and 1 x 10(-4) per recipient, respectively . Analysis of several exconjugants indicated that pOJ436 and pRHB304 inserted into a unique phiC31 attB site and that some of the insertions had minimal deleterious effects on glycopeptide A47934 production . The results indicate that 'S . toyocaensis' is a suitable host for gene cloning, whereas S . virginiae does not appear to be. J Mol Evol, 1996 Feb, 42(2), 91 - 6 Horizontal gene transfer contributes to the wide distribution and evolution of type II restriction-modification systems; Jeltsch A et al.; Restriction modification (RM) systems serve to protect bacteria against bacteriophages . They comprise a restriction endonuclease activity that specifically cleaves DNA and a corresponding methyltransferase activity that specifically methylates the DNA, thereby protecting it from cleavage . Such systems are very common in bacteria . To find out whether the widespread distribution of RM systems is due to horizontal gene transfer, we have compared the codon usages of 29 type II RM systems with the average codon usage of their respective bacterial hosts . Pronounced deviations in codon usage were found in six cases: EcoRI, EcoRV, KpnI, SinI, SmaI, and TthHB81 . They are interpreted as evidence for horizontal gene transfer in these cases . As the methodology is expected to detect only one-fourth to one-third of all horizontal gene transfer events, this result implies that horizontal gene transfer had a considerable influence on the distribution and evolution of RM systems . In all of these six cases the codon usage deviations of the restriction enzyme genes are much more pronounced than those of the methyltransferase genes . This result suggests that in these cases horizontal gene transfer had occurred sequentially with the gene for the methyltransferase being first acquired by the cell . This can be explained by the fact that an active restriction endonuclease is highly toxic in cells whose DNA is not protected from cleavage by a corresponding methyltransferase. Biophys J, 1996 Feb, 70(2), 917 - 23 Structural prediction of A- and B-DNA duplexes based on coordinates of the phosphorus atoms; Tung CS et al.; The sequence-dependent structure of DNA double helices was studied extensively during the past 10 years . How the backbone structure correlates with the base structure in a duplex conformation is still an important yet open question . Using a set of reduced coordinates and a least-squares fitting procedure, we have developed a method to predict structures for B-DNA duplexes based on coordinates of the phosphorus atoms . This method can be used to predict all-atom structures for both bent and straight molecules . We estimated the accuracies of the predictions by studying a set of 10 oligonucleotides with their structures available from the Protein Data Bank . We used this method to construct a modeled structure for the bacteriophage lambda cro operator for which the phosphorus coordinates were known from 3.5-angstrum resolution crystal data (4CRO). Virus Res, 1996 Feb, 40(2), 135 - 40 Infectious in vitro transcripts from cloned cDNA of the potato A potyvirus; Puurand U et al.; A full-length cDNA copy of potato virus A RNA was constructed downstream from the bacteriophage T7 RNA polymerase promoter . A single extra guanosine not present in PVA RNA was added to the 5'-end of the cDNA . The capped in vitro transcripts from cloned cDNA were infectious in the mesophyll protoplasts and intact plants of Nicotiana tabacum . PVA coat protein accumulated in transcript-inoculated tobacco protoplasts and infected systematically intact plants producing high PVA titres according to the ELISA and immunoblot assay . The infected tobacco plants produced similar mild mosaic symptoms in the systematically infected leaves, irrespective of whether the RNA transcripts or parental virus were used as inoculum; the virus particles were also morphologically similar according to immunosorbent electron microscopy. Mol Immunol, 1996 Feb, 33(3), 279 - 85 Selection of phage displayed antibodies based on kinetic constants; Duenas M et al.; The display of antibody fragments on the surface of filamentous bacteriophages and the selection of binders from antibody libraries have provided powerful tools to generate human antibodies . We reported recently a new concept (SAP system) for the selection of specific phages by linking antigenic recognition and phage replication, using a soluble fusion protein containing the antigen and a fragment of the M13 coat protein 3 . In this investigation, a model library has been composed using six different antibody fragments which were characterized individually regarding their kass, kdiss and Ka . All Fab fragments were specific for a 15 amino acid region of the V3 loop of gp120 (HIV-1) . We demonstrated that the SAP system could discriminate between the kinetic parameters of each clone, using different selection strategies . Phages expressing high affinity clones were selected preferentially using low doses of antigen but clones of lower affinity also could be selected by increasing the antigen concentration or using a preselection procedure . Phages expressing antibody fragment with high association or low dissociation rate constants were retrieved by utilizing short contact times between antigen and antibody or antigen-chase conditions. J Gen Virol, 1996 Feb, 77 ( Pt 2 ), 247 - 55 Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C; Gosert R et al.; The RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins . So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified . A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase . A HAV-specific 27.5 kDa expression product was identified as peptide 2B . The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells . The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836-837 (Gln-Ala) . Furthermore, heterologous expression in the same system of regions P1-P2 and of the protease 3C (3Cpro) gene, showed that P1-P2 polyprotein is not cleaved autocatalytically but by 3Cpro . Hence, 3Cpro is effective in cleaving the polyprotein 2A-2B junction. Carcinogenesis, 1996 Feb, 17(2), 327 - 31 Influence of the antioxidant N-acetylcysteine and its metabolites on damage induced by bleomycin in PM2 bacteriophage DNA; Cloos J et al.; Bleomycin is considered to be a useful model compound for studying environmental carcinogenesis, due to its broad spectrum of DNA damaging properties . In addition, bleomycin is a useful antitumor drug because of its cytotoxic properties . To investigate the influence of the antioxidant N-acetylcysteine and its metabolites glutathione and cysteine on bleomycin-induced DNA damage and more importantly to gain insight into the biological relevance of such damage, PM2 DNA was exposed to Cu(2+)-bleomycin in the presence and absence of the thiols N-acetylcysteine, glutathione and cysteine . It was found that the presence of these thiols led to a considerable enhancement of bleomycin-induced single- and double-strand breaks and a concomitant decrease in the biological activity of PM2 DNA in a dose-dependent way . A similar observation was made when ascorbic acid was used . Bleomycin showed no DNA damaging activity when PM2 DNA was pretreated with the strong Fe ion chelator desferal and its activity was strongly inhibited by the addition of Cu2+ ions or under hypoxic (N2) conditions . Cu(2+)-bleomycin under our conditions is not active by itself, but most probably after binding to DNA exchanges Cu2+ for Fe3+ bound to DNA . Fe(3+)-bleomycin is then reduced to Fe(2+)-bleomycin, a process potentiated by the added antioxidants, and subsequently activated by O2 . The contribution to biological inactivation of bleomycin alone or in the presence of ascorbic acid is only approximately 15% . The contribution to lethality in the presence of thiols is higher . These results indicate that ascorbic acid only enhances the DNA damaging properties of bleomycin, whereas the thiol compounds in addition influence the type of DNA damage . The remainder of the biological inactivation is probably caused by double damage, such as single-strand breaks with closely opposed alkali-labile sites or base damage. Virology, 1996 Feb 1, 216(1), 20 - 5 Isolation of a mutant bacteriophage T7 deleted in nonessential genetic elements, gene 19.5 and m; Kim SH et al.; Half of the 55 potential genes of bacteriophage T7 appear to be dispensable . One of the major obstacles in the study of these nonessential genes is the difficulty in obtaining mutants . During a study of genes involved in the packaging of bacteriophage T7, we hypothesized that some nonessential genes may be required for optimal growth . Mutant phages lacking such nonessential genes may form plaques but grow slowly . One gene located at the extreme right end of the linear T7 genome, gene 19.5 with no known mutants, and a genetic element m responsible for a unique hairpin end, were studied . Mutant T7 phages deleted in gene 19.5 and m (T7 delta 19.5-M) were generated in vivo by homologous recombination with a recombinant plasmid . This phage produces small plaques and the production of progeny phage particles per infected cell was reduced fourfold . Investigation of the intracellular DNA after infection with T7 delta 19.5-M showed the persistence of Escherichia coli DNA as well as delayed conversion of concatemers to unit-length T7 DNA . The inefficiency of concatemer processing confirmed the proposed function of the M-hairpin in duplication of the concatemer junction . Since it is not likely that the M-hairpin influences the degradation of host DNA, we propose that the gene 19.5 product is partly responsible for the degradation of E . coli chromosomal DNA. Virology, 1996 Feb 1, 216(1), 158 - 64 Functional characterization of the P2 A initiator protein and its DNA cleavage site; Liu Y et al.; The A protein of bacteriophage P2 initiates DNA replication by a single-stranded cut at the origin, and the DNA replication proceeds unidirectionally by a modified rolling circle type of replication . The P2 A protein belongs to a family of proteins involved in the initiation of rolling circle DNA replication, and the prototype for this family is the well-characterized A protein of phage phi X174 . One of the common motifs of this family contains two conserved tyrosine residues, which have been shown to be able to alternate in catalyzing the cleavage as well as joining reactions in the phi X174 A protein . We investigated the role of the conserved tyrosine residues in P2 A protein by in vitro mutagenesis . Only one of the two conserved tyrosine residues was found to be involved in the cleavage reaction . The tyrosine residue dispensable for cleavage and ligation is, however, required at some other stage of the P2 growth cycle, since viable recombinants containing this mutation could not be obtained . The sequence requirements for cleavage of the target site were analyzed with a set of oligonucleotides having single base alterations in the nick region, and the results indicate that only five core nucleotides need to be conserved for efficient cleavage. Nucleic Acids Res, 1996 Feb 1, 24(3), 450 - 7 Transcription activation by the bacteriophage Mu Mor protein: analysis of promoter mutations in Pm identifies a new region required for promoter function; Artsimovitch I et al.; Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase holoenzyme and a Mu-encoded protein, Mor . Consistent with these requirements, the middle promoter, Pm, has a recognizable -10 region but lacks a -35 region . Mutagenesis of this promoter (from -70 to +10) was performed using mutagenic oligonucleotide-directed PCR . The resulting fragments were cloned into a promoter-lacZfusion vector and analyzed for promoter activity by assaying beta-galactosidase production . Single point mutations with a Down phenotype were clustered in three regions: the -10 region, the Mor footprint region and the spacer between them . Gel retardation experiments with purified Mor protein and promoter mutants demonstrated that sequences important for Mor binding are located within the Mor footprint region and lead us to propose the existence of a dyad symmetry element involved in Mor binding . In agreement with this prediction, glutaraldehyde crosslinking of Mor in solution generated a species with the size of a dimer . These experiments also identified an unusual group of mutations located in the spacer region adjacent to the Mor footprint . These mutations alter promoter activity without affecting Mor binding . A circular permutation assay revealed that Mor does not introduce a significant bend upon binding to its target sequence. EMBO J, 1996 Feb 1, 15(3), 684 - 93 Recombination associated with replication of malarial mitochondrial DNA; Preiser PR et al.; Mitochondrial DNA of the malarial parasite Plasmodium falciparum comprises approximately 20 copies per cell of a 6 kb genome, arranged mainly as polydisperse linear concatemers . In synchronous blood cultures, initiation of mtDNA replication coincides with the start of the 4-5 doublings in nuclear DNA that mark the reproductive phase of the erythrocytic cycle . We show that mtDNA replication coincides with a recombination process reminiscent of the replication mechanism used by certain bacteriophages and plasmids . The few circular forms of mtDNA which are also present do not replicate by a theta mechanism, but are themselves the product of recombination, and we propose they undergo rolling circle activity to generate the linear concatemers. EMBO J, 1996 Feb 1, 15(3), 665 - 74 The downstream box: an efficient and independent translation initiation signal in Escherichia coli; Sprengart ML et al.; The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon . Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role . Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene . In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region . The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence . Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region . We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation. EMBO J, 1996 Feb 1, 15(3), 569 - 80 Probes of chromatin accessibility in the Drosophila bithorax complex respond differently to Polycomb-mediated repression; McCall K et al.; The Polycomb group (PcG) of genes are required for maintenance of the repressed state of the homeotic genes in Drosophila . There are similarities between the PcG repression and mating-type silencing in yeast or heterochromatic position effect in Drosophila, which suggest that PcG repression may involve a highly compacted chromatin structure . To test for such a structure, heterologous DNA- binding proteins were used as probes for DNA accessibility in Drosophila embryos . Binding sites for the yeast transcriptional activator GAL4 and for bacteriophage T7 RNA polymerase were inserted into the bithorax (bx) regulatory region of the endogenous Ultrabithorax (Ubx) gene, which is regulated by the PcG . Ubiquitously expressed GAL4 protein directs transcription through its binding sites only in the posterior segments where the bx region is active . The block to GAL4 activation in the more anterior segments is dependent on Polycomb (Pc) function . In contrast, T7 RNA polymerase can transcribe from its target promoter in all segments of the embryo . Thus, Pc-mediated repression blocks activated polymerase II transcription, but does not simply exclude all proteins. J Bacteriol, 1996 Feb, 178(4), 1099 - 104 Three functions of bacteriophage P1 involved in cell lysis; Schmidt C et al.; Amber and deletion mutants were used to assign functions in cell lysis to three late genes of bacteriophage P1 . Two of these genes, lydA and lydB of the dar operon, are 330 and 444 bp in length, respectively, with the stop codon of lydA overlapping the start codon of lydB . The third, gene 17, is 558 bp in length and is located in an otherwise uncharacterized operon . A search with the predicted amino acid sequence of LydA for secondary motifs revealed a holin protein-like structure . Comparison of the deduced amino acid sequence of gene 17 with sequences of proteins in the SwissProt database revealed homologies with the proteins of the T4 lysozyme family . The sequence of lydB is novel and exhibited no known extended homology . To study the effect of gp17, LydA, and LydB in vivo, their genes were cloned in a single operon under the control of the inducible T7 promoter, resulting in plasmid pAW1440 . A second plasmid, pAW1442, is identical to pAW1440 but has lydB deleted . Induction of the T7 promoter resulted in a rapid lysis of cells harboring pAW1442 . In contrast, cells harboring pAW1440 revealed only a small decrease in optical density at 600 nm compared with cells harboring vector alone . The rapid lysis phenotype in the absence of active LydB suggests that this novel protein might be an antagonist of the holin LydA. J Virol, 1996 Feb, 70(2), 1041 - 9 Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells; Schultz DE et al.; Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells (S . P . Day, P . Murphy, E . A . Brown, and S . M . Lemon, J . Virol . 66: 6533-6540, 1992) . To determine whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, respectively . Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs containing bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT . In both cell types, transcripts containing the 5'NTR of HM175/P16 expressed CAT at levels that were 50- to 100-fold lower than transcripts containing the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES . However, in BT7-H cells, transcripts containing the 5'NTR of wild-type virus . This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16 . These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates . These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is determined in part by differences in cellular translation initiation factors. Infect Immun, 1996 Feb, 64(2), 495 - 502 Regulation of the Shiga-like toxin II operon in Escherichia coli; Muhldorfer I et al.; Investigations of the regulation of the bacteriophage-encoded Shiga-like toxin II (SLT-II) in Escherichia coli demonstrated that bacteriophages exhibit a regulatory impact on toxin production by two mechanisms . Firstly, replication of the toxin-converting bacteriophages brings about an increase in toxin production due to concomitant multiplication of toxin gene copies . Secondly, an influence of a phage-encoded regulatory molecule was demonstrated by using low-copy-number plasmid pADR-28, carrying a translational gene fusion between the promoter and proximal portion of slt-IIA and the structural gene for bacterial alkaline phosphatase (phoA) . PhoA activity, reflecting the slt-II promoter activity, was significantly enhanced in E . coli strains which and been lysogenized with an SLT-I or SLT-II-converting bacteriophage (H-19B or 933W, respectively) or bacteriophage lambda . Both mechanisms are dependent on bacteriophage induction and hence are recA dependent . Moreover, the study revealed that the DNA-binding protein H-NS has a regulatory impact on both bacteriophage-mediated SLT-II synthesis and the activity of the slt-II promoter of plasmid pADR-28 . While a slight impact of growth temperature on SLT-II expression was observed, no impact of either osmolarity, pH, oxygen tension, acetates, iron level, or utilized carbon source could be demonstrated. J Biotechnol, 1996 Jan 26, 44(1-3), 129 - 37 Design of immunogens as components of a new generation of molecular vaccines; Loktev VB et al.; Three new approaches to design effective immunogens are considered . At first, we derived an expression vector from bacteriophage M13 allowing the exposure of short peptides on the virion surface . EIA demonstrates that antibodies against a recombinant phage carrying the antigenic determinant of the HIV-1 gag protein reacted with the 17-kDa core protein of the virus and also with its polyprotein precursor p55 in immunoblotting . In another approach, we chose the hepatitis B core antigen (HBcAg) particle as a vehicle for the presentation of foreign antigenic determinants to the immune system . Chimerical particles of HBcAg containing epitope of the VEE virus were obtained . A vector system for insertion of foreign antigenic determinants and production of both hybrid and wild HBcAg proteins were also obtained . The third approach relies on construction of immunogens from different T- and B-cell epitopes of the HIV-1 . We suggested to construct HIV-1 vaccines in a form of the TBI (T- and B-cell epitopes containing Immunogen) with a predetermined tertiary structure, namely, a four-alpha-helix bundle . The gene of the TBI protein consisting of nine HIV-1 epitopes was synthesized and expressed in Escherichia coli cells . Mice immunized with TBI showed humoral and cellular immune responses to HIV-1 . Anti-TBI antibodies displayed HIV-1 neutralizing activity . These new approaches offer promise in the development of new effective vaccines. J Mol Biol, 1996 Jan 26, 255(3), 412 - 24 Intron-encoded endonuclease I-TevII binds across the minor groove and induces two distinct conformational changes in its DNA substrate; Loizos N et al.; I-TevII is the homing endonuclease encoded by the sunY intron of bacteriophage T4 . The enzyme cleaves an intronless sunY gene near the exon I-exon II junction, thereby initiating intron homing into its cognate intronless allele . Specifically, I-TevII cleaves its DNA target 13 to 15 nucleotides (nt) downstream of the sunY intron insertion site, generating 2-nl 3'-OH extensions . Here, we present evidence that I-TevII makes predominantly minor groove contacts in two regions of its recognition sequence, as does I-TevI, the other homing endonuclease encoded by phage T4 . Following cleavage, I-TevII was shown to remain bound to one of its DNA products, suggesting possible additional roles for the endonuclease in the mobility process . Interestingly, two distinct conformational changes were detected by gel analysis in the DNA substrate following binding by I-TevII, one occurring in the absence of Mg2+, the second being dependent on the presence of Mg2+ . The Mg(2+)-induced distortion accompanies a nick in one strand, and may serve to bring the cleavage site on the other strand into proximity with the catalytic domain of the protein. Cell, 1996 Jan 26, 84(2), 211 - 21 Beyond homing: competition between intron endonucleases confers a selective advantage on flanking genetic markers; Goodrich-Blair H et al.; The closely related B . subtilis bacteriophages SPO1 and SP82 have similar introns inserted into a conserved domain of their DNA polymerase genes . These introns encode endonucleases with unique properties . Other intron-encoded "homing" endonucleases cleave both strands of intronless DNA; subsequent repair results in unidirectional gene conversion to the intron-containing allele . In contrast, the enzymes described here cleave one strand on both intron-containing and intronless targets at different distances from their common intron insertion site . Most surprisingly, each enzyme prefers DNA of the heterologous phage . The SP82-encoded endonuclease is responsible for exclusion of the SPO1 intron and flanking genetic markers from the progeny of mixed infections, a novel selective advantage imparted by an intron to the genome in which it resides. Biochemistry, 1996 Jan 23, 35(3), 735 - 42 High-resolution structure of an engineered Cro monomer shows changes in conformation relative to the native dimer; Albright RA et al.; A rationally designed, genetically engineered, monomeric form of the Cro protein from bacteriophage lambda has been crystallized and its structure determined by isomorphous replacement and refined to a resolution of 1.54 A . The structure confirms the rationale of the design but, at the same time, reveals 1-2 A shifts throughout the monomer structure relative to the previously determined structure of the dimeric wild-type protein . These changes include a 1.6 A main-chain shift in part of the beta-sheet region of the molecule relative to the alpha-helical region and a 1.1 A shift of a buried phenylalanine within the core as well as a correlated 2.2 A shift in a solvent-exposed beta-hairpin . The conformational adjustments appear to reflect an inherent flexibility of the protein that is associated with its DNA-binding function. Biochemistry, 1996 Jan 23, 35(3), 1084 - 92 Dual role of the 44/62 protein as a matchmaker protein and DNA polymerase chaperone during assembly of the bacteriophage T4 holoenzyme complex; Kaboord BF et al.; Processive DNA synthesis in the bacteriophage T4 system requires the formation of a holoenzyme complex composed of the T4 DNA polymerase and the 44/62 and 45 accessory proteins . While ATP hydrolysis by the 44/62 protein is essential for holoenzyme formation, the role of the sliding clamp or processivity factor is attributed to the 45 protein . Beyond the need for ATP hydrolysis, the exact role of the 44/62 protein in complex assembly has not been clearly defined . In this paper, we have investigated the kinetics of complex assembly in the presence of both saturating and substoichiometric concentrations of the 44/62 protein . Under saturating conditions, complex assembly is 100% efficient, with all of the polymerase bound in a processive complex . Under conditions of limiting 44/62 protein, the 44/62 protein can act catalytically to assemble the 45 protein and polymerase into a productive complex . However, kinetic simulations indicate that a significant fraction of polymerase is sequestered in a nonproductive complex with the 45 protein . Thus, a second role for the 44/62 protein during complex assembly is that of a chaperone protein to ensure productive pol.45.DNA complex formation . We have also investigated the stability of the 45 protein on the DNA . The off rate of 0.003 s-1 for the 45 protein closely parallels that of the holoenzyme complex . Therefore, disassembly of the complex appears to involve the coordinated dissociation of both the 45 protein and the polymerase from the DNA. Mutat Res, 1996 Jan 17, 349(1), 21 - 32 Deletion during recombination in bacteriophage T7; Yang Y et al.; The ligase gene of bacteriophage T7 was interrupted with inserts made from synthetic DNA . A pair of inserts were designed so that each insert contained one copy of an identical 17 bp sequence (repeat) positioned such that intermolecular recombination between the 17 bp homologies on separate genomes could delete enough of the insert to produce a functional ligase gene . The frequency of deletion by intermolecular recombination was compared to deletion frequency when repeated copies of the identical 17 bp sequence were present on the same genome separated by 39 bp . When the 17 bp homologous sequences were on different genomes the formation of ligase positive phage was about 7% to 13% of the deletion frequency measured with both repeats on the same genome . A second set of inserts contained the same 17 bp sequence present in the first pair of inserts . The sequence of this second set of inserts was such that when both 17 bp repeats were present on the same genome there was no separation between the repeats . With the second set of inserts (no separation) deletion by intermolecular recombination was about two orders of magnitude higher than what was measured with the first set of inserts where 39 bp of nonhomologous sequence intervened between the 17 bp repeats and the normal T7 genome . These data are interpreted to suggest that in T7 misalignment between repeated sequences during intermolecular recombination may play a role in deletion mutagenesis. EMBO J, 1996 Jan 15, 15(2), 437 - 44 Bacteriophage Mu repressor as a target for the Escherichia coli ATP-dependent Clp Protease; Laachouch JE et al.; Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP-dependent ClpXP protease . We further investigated the determinants of the mutant repressor's sensitivity to Clp . We show the crucial importance of a C-terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein . The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F . CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon . In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy- dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease. Mol Gen Genet, 1996 Jan 15, 250(1), 129 - 34 Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations; Hobman JL et al.; The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system . Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity . Several mutant merT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Gly14Arg, Gly15Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter. Cell, 1996 Jan 12, 84(1), 147 - 54 E . coli SSB activates N4 virion RNA polymerase promoters by stabilizing a DNA hairpin required for promoter recognition; Glucksmann-Kuis MA et al.; Bacteriophage N4 virion RNA polymerase transcription of double-stranded promoter-containing DNAs requires supercoiled template and E . coli single-stranded DNA-binding protein (EcoSSB); other single-stranded DNA-binding proteins cannot substitute . The DNA determinants of virion RNA polymerase binding at the promoter comprise a small template-strand hairpin . The requirement for EcoSSB is surprising, since single-stranded DNA-binding proteins destabilize hairpin structures . DNA footprinting of EcoSSB on wild-type and mutant promoters indicates that EcoSSB stabilizes the template-strand hairpin owing to the hairpin-loop sequences . Other single-stranded DNA-binding proteins destabilize the promoter hairpin, explaining the specificity of EcoSSB activation . We conclude that EcoSSB activates transcription by providing the appropriate DNA structure for polymerase binding . The existence of small hairpins stable to single-stranded protein binding suggests a novel mechanism that provides structural determinants for specific recognition in single-stranded DNA transactions by an otherwise nonspecific DNA-binding protein. Biochemistry, 1996 Jan 9, 35(1), 144 - 52 Asp537 and Asp812 in bacteriophage T7 RNA polymerase as metal ion-binding sites studied by EPR, flow-dialysis, and transcription; Woody AY et al.; Asp537 and Asp812 are essential in the catalytic mechanism of T7 RNA polymerase . The mutants D537N and D812N have no detectable activity whereas the mutants D537E and D812E have significantly reduced activity relative to the wild-type . The hypothesis that these two amino acids act as metal-binding ligands has been tested using EPR with Mn2+ as the metal ion . Mn2+ is able to substitute for Mg2+ in transcription by T7 RNAP on templates containing the T7 promoter . Mg2+ and Mn2+ compete for binding sites, with the former having lower affinity . Mn2+ binding to the wild-type enzyme and the mutants D537N, D812N, D537E, D812E, and Y649F was measured over the concentration range of 25 microM to 1.5 mM . The data were analyzed by nonlinear least-squares fits to the binding isotherms, and the analysis gave approximately two Mn(2+)-binding sites in all cases and a Kd for the wild-type of approximately 340 microM . The Kd value for the mutant Y639F, in which Asp537 and Asp 812 are not mutated, is comparable to the value for the wild-type . Mn2+ binding to the double mutants, D537N/D812N and D537E/D812E, appears to be nonspecific . The Kd values of the Asp-->Asn mutants are only 2-5 times larger than the value for the wild-type, in contrast to the drastic diminution of enzymatic activity in the mutants . The geometry of metal binding to these Asp residues may be crucial in determining the catalytic competence . Mn2+ binding to the wild-type enzyme in the presence of nucleotides, measured by flow dialysis, is characterized by two Mn(2+)-binding sites with a Kd value of ca . 150 microM . The similarity in values of Kd with and without nucleotide suggests that nucleotides do not have a drastic effect on Mn2+ binding . Our results indicate that monodentate carboxylate oxygens of both conserved Asp residues bridge the two metal ions. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 342 - 6 Bacteriophage lambda N protein alone can induce transcription antitermination in vitro; Rees WA et al.; Specific and processive antitermination by bacteriophage lambda N protein in vivo and in vitro requires the participation of a large number of Escherichia coli proteins (Nus factors), as well as an RNA hairpin (boxB) within the nut site of the nascent transcript . In this study we show that efficient, though nonprocessive, antitermination can be induced by large concentrations of N alone, even in the absence of a nut site . By adding back individual components of the system, we also show that N with nut+ nascent RNA is much more effective in antitermination than is N alone . This effect is abolished if N is competed away from the nut+ RNA by adding, in trans, an excess of boxB RNA . The addition of NusA makes antitermination by the N-nut+ complex yet more effective . This NusA-dependent increase in antitermination is lost when delta nut transcripts are used . These results suggest the formation of a specific boxB RNA-N-NusA complex within the transcription complex . By assuming an equilibrium model, we estimate a binding constant of 5 x 10(6) M-1 for the interaction of N alone with the transcription complex . This value can be used to estimate a characteristic dissociation time of N from the complex that is comparable to the dwell time of the complex at an average template position, thus explaining the nonprocessivity of the antitermination effect induced by N alone . On this basis, the effective dissociation rate of N should be approximately 1000-fold slower from the minimally processive (100-600 bp) N-NusA-nut+ transcription complex and approximately 10(5)-fold slower from the maximally processive (thousands of base pairs) complex containing all of the components of the in vivo N-dependent antitermination system. Chin J Biotechnol, 1996, 12(4), 207 - 13 T7-promoter-based Escherichia coli expression system induced with bacteriophage M13HEP; Chen C et al.; The bacteriophage M13HEP was constructed by cloning the T7 RNA polymerase gene into phage M13mp18 RF DNA to express T7 RNA polymerase under the control of the lac promoter . Through M13HEP phage infection, T7 RNA polymerase could be introduced into an expression strain and heterologous genes under the control of the T7 promoter can be induced to express . Using this phage M13HEP induction system, many heterologous genes, especially some genes whose products are toxic to the host strain, were successfully expressed . By transferring F' pilli from E . coli XL1-blue to E . coli HMS174, a new E . coli strain HMS174F' was obtained to make the construction, expression, and single-stranded DNA rescue of the T7 expression plasmid be conveniently performed in the same strain. Biochimie, 1996, 78(10), 856 - 61 Mammalian cell binding and transfection mediated by surface-modified bacteriophage lambda; Dunn IS; Bacteriophage lambda virions whose tail tube major subunit (V) proteins are modified with a cyclizable Arg-Gly-Asp (RGD) peptide are able to promote the binding of certain mammalian cells to a solid surface . This effect was shown to be specific by peptide competition experiments, and control phage lacking the RGD peptide showed no significant cellular interaction . RGD-modified but not control phage bearing a reporter gene could transfect COS cells at a significant frequency . Phage-mediated transfection therefore benefits when the efficiency of only one step in the multi-stage uptake process is improved. Hum Antibodies Hybridomas, 1996, 7(3), 106 - 12 Short synthetic CDR-peptides forming the antibody combining site of the monoclonal antibody against RNA bacteriophage fr neutralize the phage activity; Steinbergs J et al.; The construction of a mouse hybridoma FR52 secreting neutralizing monoclonal antibody specific for RNA bacteriophages fr, MS2 and GA is reported . The genes encoding the variable domains of the monoclonal antibody FR52 heavy and light chains were cloned and sequenced and the corresponding complementarity determining region (CDR) peptides were chemically synthesized . The CDR-peptides were tested for their ability to neutralize the activity of RNA phage fr and related RNA phages MS2 and GA . The CDR-derived peptides H2, L2 and L3 interacted with the fr phage particles and neutralized fr phage activity . Two of these peptides--H2 and L3 also had the ability to neutralize partly the activity of related bacteriophage MS2, but L1 and especially L3 neutralize the activity of the RNA phage GA . These results provide an excellent system for further antibody-antigen interaction studies and raise the possibility that simple CDR-peptides may serve as a new class of anti-viral molecules. DNA Seq, 1996, 6(6), 347 - 50 Identification of the bacteriophage T5 dUTPase by protein sequence comparisons; Kaliman AV; It is shown by protein sequence comparisons that a 148 amino acid open reading frame (ORF 148) located at 67% of the bacteriophage T5 genome encodes a protein with strong similarity to known dUTPases . This protein contains five characteristic amino acid sequence motifs that are common to the dUTPase gene family . A similarity in size and high degree of sequence identity strongly suggest that the protein encoded by the ORF 148 of bacteriophage T5 is dUTPase. Chem Res Toxicol, 1996 Jan-Feb, 9(1), 179 - 87 Benzo{a}pyrene-DNA adducts inhibit the DNA helicase activity of the bacteriophage T7 gene 4 protein; Yong Y et al.; The gene 4 protein of bacteriophage T7 provides the essential helicase and primase activities for the replication of the T7 genome . In addition, it also displays a DNA-dependent deoxyribonucleoside triphosphatase activity, the preferred substrate of which is dTTP . Previous investigations have demonstrated that the translocation of the gene 4 protein along single-stranded DNA is blocked by the presence of benzo{a}pyrene-DNA adducts and that the gene 4 protein is likely to be sequestered at the sites of these adducts . In the present study, we directly show that the helicase activity of the gene 4 protein is also profoundly inhibited by the benzo{a}pyrene-DNA adducts . The inhibitory effects of these adducts are strand-specific in that they block the DNA helicase activity of the gene 4 protein only when they are located in the DNA strand where the gene 4 protein translocates when it unwinds double-stranded DNA . Consistent with the hypothesis that the gene 4 protein is sequestered at the adduct site, we also show that the complexes formed by the gene 4 protein and benzo{a}pyrene-modified DNA are far more stable than those formed by the gene 4 protein and unmodified DNA. Rapid Commun Mass Spectrom, 1996, 10(12), 1475 - 8 Analyzing sequencing reactions from bacteriophage M13 by matrix-assisted laser desorption/ionization mass spectrometry; Mouradian S et al.; The current demand for improved DNA sequencing methodologies posed by the Human Genome Project has spurred the investigation of alternatives to gel electrophoresis . Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has great potential for the rapid analysis of DNA fragments . Mock Sanger sequencing mixtures have been successfully analyzed by MALDI by pooling synthesized oligonucleotides corresponding to the M13 bacteriophage sequence . More recently, analyses of Sanger sequencing fragments enzymatically generated from synthetic templates of 45 or 50 bases were reported . In the present study, these feasibility demonstrations are extended to show MALDI sequencing from the M13 bacteriophage DNA template commonly used in actual Sanger sequencing . The results show sequence determination for extension products up to 35 bases in length . Different desalting and purification procedures were investigated and it was found that salt could be efficiently reduced by removal of the template in a post-reaction step . Work in progress to stabilize DNA by chemical modification, employed in conjunction with the methods described here, should enable significant extension of the length of readable sequence. J Biochem Toxicol, 1996, 11(2), 67 - 71 DNA breakage by uric acid and Cu(II): binding of uric acid to DNA and biological activity of the reaction; Shamsi FA et al.; Uric acid is present in human plasma in relatively high concentrations and is considered to be a natural physiological antioxidant . We have earlier shown that in the presence of Cu(II) and molecular oxygen, uric acid causes strand breakage in DNA . In this article, we show that uric acid fluorescence is quenched by addition of DNA, indicating the formation of uric acid-DNA complex . Uric acid-Cu(II)-mediated DNA strand scission is capable of bacteriophage inactivation and such inactivation is mediated through reduction of Cu(II) to Cu(I) and the generation of oxygen-derived radicals . It is indicated that the DNA breakage is repaired in E . coli and involves the repair of DNA polymerase. Virus Genes, 1996, 12(1), 5 - 14 Intracellular membrane proliferation in E . coli induced by foot-and-mouth disease virus 3A gene products; Weber S et al.; During picornavirus infection replication of genomic RNA occurs in membrane-associated ribonucleoprotein complexes . These replication complexes contain different nonstructural viral proteins with mostly unknown function . To examine the function of nonstructural picornaviral proteins in more detail, cDNA of foot-and-mouth-disease virus (FMDV) strain O1 Lausanne was cloned into lambda ZAP II, and different parts of the P3-coding sequence were expressed in E . coli by the T7 polymerase system . Expression products constituted (a) fusion proteins composed of N-terminal leader peptide of bacteriophage T7 phi 10 protein fused to FMDV P3-sequences of different lengths, (b) translation products of authentic P3-region genes, and (c) carboxy-terminally truncated 3A proteins . Expression products were characterized by NaDodSO4-polyacrylamide gel electrophoresis, immunoblotting, as well as electron and immunoelectron microscopy . We show here that in the T7 polymerase system a high level of expression of 3A-containing peptides is achieved in E . coli . Remarkably, the expression of 3A-derived proteins induced a dramatic intracellular membrane proliferation in E . coli cells, similar to the vesicle induction observed in FMDV-infected cells . By immunoelectron microscopy, 3A-reactive material was found associated with these membranes . We hypothesize that the FMDV 3A protein is instrumental in eliciting intracellular membrane proliferation in infected cells as a prerequisite for viral RNA replication. J Protein Chem, 1996 Jan, 15(1), 77 - 86 Effects on protein structure and function of replacing tryptophan with 5-hydroxytryptophan: single-tryptophan mutants of the N-terminal domain of the bacteriophage lambda repressor; Kombo DC et al.; Conformational energy computations have been carried out on the N-acetyl-N'-methylamide of 5-hydroxytryptophan (5OH-Trp) using ECEPP/3 . As observed with tryptophan (Trp), the most preferred conformation about the C alpha-C beta bond of the side chain is g+ or t . This preference is reduced to only the t conformational state when 5-hydroxyTrp is in the middle of a right-handed poly(L-alanine) alpha-helix . A similar result has been obtained with Trp {Piela et al . (1987), Biopolymers 1987, 1273-1286} . These results suggest that replacement of Trp by its analog 5-hydroxyTrp may be tolerated in an alpha-helix . To test this hypothesis, we have replaced Trp by 5OH-Trp in the fifth helices of two functionally active mutants of the N-terminal domain of the bacteriophage lambda repressor . Computations on the packing of these helices have shown that no significant structural changes results from the replacement of Trp by 5OH-Trp . The DNA-binding activity of these mutants, as assessed indirectly through geometrical parameters, is also unaltered. Immunogenetics, 1996, 44(5), 340 - 50 Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1; Kelly JM et al.; Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes . We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene . The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases . Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16 . Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA . The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene . A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines . The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines . Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1 . The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells . Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1. EXS, 1996, 75, 185 - 222 Lysozyme: a model enzyme in protein crystallography; Strynadka NC et al.; The review concentrates on the crystal structure results from several protein crystallography laboratories on three different lysozymes, the type-c lysozymes such as hen egg-white lysozyme (HEWL), the type-g lysozyme, such as goose egg-white lysozyme (GEWL), and the lysozyme from T4 bacteriophage (T4L) . The crystallographic studies on HEWL in several different crystal forms have shown that the lysozyme molecule is relatively rigid with the residues of the active site Glu35 and Asp52 adopting almost identical conformations in all structures and species variants . The NMR results also confirm the presence of a similar conformation of HEWL in solution . All three enzymes, HEWL, GEWL and T4L are composed of two domains, one that is predominantly alpha-helical and a smaller domain that is mainly beta-sheet in nature . The general acid/general base residue in each lysozyme (Glu35 in HEWL, Glu73 in GEWL and Glu11 in T4L) is contributed by the larger alpha-helical domain . The beta-sheet domains of HEWL and T4L contribute an aspartate to their respective active sites, which is likely involved in electrostatic stabilization of the oxycarbonium ion intermediate of the site D sugar on the hydrolytic pathway of oligosaccharides . There is no analogous aspartate carboxylate group in GEWL although minor conformational changes could position one or other of Asp86 or Asp97 for such a stabilization role . The binding of substrate analogues, transition state mimics and oligosaccharide products of hydrolysis to HEWL, GEWL and T4L have contributed greatly to our understanding of sugar binding to proteins . The observed subtle conformational differences of the free vs bound forms of these enzymes are best described by a narrowing of the active site clefts in the presence of the inhibitors . Details of the binding interactions of those residues lining the oligosaccharide binding clefts of the three-enzymes HEWL, GEWL and T4L with the sugar residues in sites A, B, C and D are presented and discussed . Oligosaccharides of (GlcNAc)n and alternating MurNAc-GlcNAc-MurNAc have been bound to these three enzymes and the structures determined at high resolution . These binding studies have contributed greatly to our understanding of the catalytic mechanism of the lysozyme glycosidase activity . The currently accepted view of this mechanism is presented and discussed in this review. EXS, 1996, 75, 35 - 64 Phage lysozymes; Fastrez J; Bacteriophage genomes encode lysozymes whose role is to favour the release of virions by lysis of the host cells or to facilitate infection . In this review, the evolutionary relationships between the phage lysozymes are described . They are grouped into several classes: the V-, the G-, the lambda- and the CH-type lysozymes . The results of structure determinations and of enzymological studies indicate that the enzymes belonging to the first two classes, and possibly the third, share common structural elements with C-type lysozymes (eg . hen egg white lysozyme) . The proteins of the fourth class, on the other hand, are structurally similar to the S . erythraeus lysozyme . Several phage lysozymes feature a modular construction: besides the catalytic domain, they contain additional domains or repeated motifs presumed to be important for binding to the bacterial walls and for efficient catalysis . The mechanism of action of these enzymes is described and the role of the important amino acid residues is discussed on the basis of sequence comparisons and of mutational studies . The effects of mutations affecting the structure and of multiple mutations are also discussed, particularly in the case of the T4 lysozyme: from these studies, proteins appear to be quite tolerant of potentially disturbing modifications. J Clin Microbiol, 1996 Jan, 34(1), 87 - 93 Molecular typing by random amplification of polymorphic DNA and M13 southern hybridization of related paired isolates of Aspergillus fumigatus; Anderson MJ et al.; Three forms of DNA-based typing procedures for Aspergillus fumigatus isolates have been developed over the last five years . The procedures are random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) detection, and Southern hybridizations with various repetitive sequence-based probes . Using two of these procedures, we compared 16 selected isolates, grouped into eight pairs on the basis of epidemiology or previously assigned RFLP types . RAPD with four primers (R108, RC08, 2, and 4), including three previously used with A . fumigatus, showed that one primer, R108, gave the best discrimination (8 types) . Southern hybridization of total genomic DNA digested with HindIII and probed with the total bacteriophage M13 genome resulted in the highest overall level of discrimination . Combination of the RAPD and Southern hybridization with the previously assigned RFLP types discriminated 10 isolates of 16 . Isolates closely linked epidemiologically could not be distinguished from each other . In addition, three pairs of isolates previously unlinked by epidemiology had the same overall types . Two pairs were obtained from the same hospital within 2 years of each other, whereas the third pair were isolated from California and Germany . A full understanding of the epidemiology and ecology of A . fumigatus requires multiple discriminatory typing procedures. Methods Enzymol, 1996, 267, 68 - 82 Affinity maturation of proteins displayed on surface of M13 bacteriophage as major coat protein fusions; Roberts BL et al.; This chapter described the preparation and fractionation of libraries of M13 phage displaying proteins as fusions to the major coat protein . High titer (10(13) pfu/ml) phage libraries can readily be generated using a single vector and the level of display surpasses that of gene III fusion phage . Since the synthetic VIII fusion gene can be customized, this system should provide the flexibility required to construct phage libraries displaying a variety of different peptides and proteins and to select variants possessing the highest affinity for target molecules of a diverse chemical nature. Arch Virol, 1996, 141(5), 885 - 900 Complete nucleotide sequence and synthesis of infectious in vitro transcripts from a full-length cDNA clone of a rakkyo strain of tobacco mosaic virus; Chen J et al.; The complete nucleotide sequence of the genome of a rakkyo strain of tobacco mosaic virus (TMV-R), which exhibits distinct host range differences from the common strain of TMV, was determined . The overall nucleotide sequence homology with TMV-U1 (a common strain of TMV) is 94.2% . The amino acid sequence homologies of the four encoded proteins (180K, 130K, 30K, coat protein) are from 95.9% to 98.0% compared with TMV-U1 . To facilitate the analysis of the novel host range of TMV-R, a full-length clone of the genome containing a bacteriophage T7 RNA polymerase promoter was assembled from two cDNA clones and designated pRF3 . In vitro transcripts derived from pRF3 were highly infectious . The infections of RF3, wild-type TMV-R, and U3/12-4 (derived from pU3/12-4, an infectious clone of TMV-U1) were compared on Nicotiana tabacum cv . Bright Yellow (BY) plants . No systemic mosaic symptoms were observed on plants inoculated with RF3 and TMV-R, while BY plants inoculated with U3/12-4 developed distinct mosaic symptoms on the upper leaves 8-9 days post-inoculation . The green fluorescent protein (GFP) gene was introduced into pRF3 and pU3/12-4 by replacing the coat protein gene to get two GFP expressing chimeric virus clones: pR-GFP or pU1-GFP . Transcripts from pU1-GFP produced strong fluorescence when inoculated onto BY leaves, while those from pR-GFP produced only very faint fluorescence. Mutagenesis, 1996 Jan, 11(1), 111 - 8 Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations; Dolle ME et al.; To study in vivo somatic mutations a C57BL/6 transgenic mouse model was constructed harboring multiple chromosomally integrated copies of the plasmid pUR288, which carried the lacZ reporter gene as the mutational target . We previously demonstrated that lacZ-containing plasmids could be rescued from their integrated state efficient enough to detect mutations in lacZ by positive selection . The smaller size of the plasmid vector, as compared with our earlier transgenic mouse model based on bacteriophage lambda vectors, should offer considerable advantages in terms of rescue efficiency and sensitivity to large size alterations in the lacZ gene . To evaluate the plasmid-based mouse model for its suitability to detect in vivo mutations, we determined mutant frequencies in different organs of untreated and ethyl nitrosourea (ENU)-treated animals using a new, improved protocol . The rescue efficiencies obtained were as high as 200,000/micrograms genomic DNA; millions of transformants could be obtained in one single experiment . The average spontaneous mutant frequency in four different organs of 4- to 8-week-old mice ranged from 4.41 to 6.82 x 10(-5), compared with a mutant frequency of the same plasmid grown in Escherichia coli of approximately 1 x 10(-5) or less . Single treatments with 100 and 250 mg ENU/kg body wt resulted in a 7- and 14-fold increase, respectively, in spleen mutant frequency at 14 days after i.p . administration of the alkylating agent . Restriction enzyme analysis showed that a considerable portion of spontaneous mutants were size changes varying from approximately 100 to 3000 bp . Some mutant plasmids contained mouse genomic sequences, which is indicative of large genetic rearrangement events involving the 3' flanking regions of the transgene cluster . Among the ENU-induced mutants, size changes comprised only a minor fraction of the total, which is in keeping with the known ENU mutation spectra in vitro and in vivo . The high rescue efficiency of this plasmid-based model, in combination with its sensitivity to a broad spectrum of mutations, including large deletions, makes it very suitable as a general in vivo mutagenicity test system. Cytogenet Cell Genet, 1996, 73(1-2), 77 - 8 The 21q22.1 STS marker, VN02 (EST00541 cDNA), is part of the 3' sequence of the human Na+/myo-inositol cotransporter (SLC5A3) gene; Berry GT et al.; The human osmoregulatory Na+/myo-inositol cotransporter gene (SLC5A3) was recently cloned and localized to the region of 21q22 . Fine mapping of this gene was accomplished by identifying YAC clones that contain SLC5A3 and utilizing known STS markers for 21q22.1 and 21q22.2 sub-bands that map to the positive YAC clones . Two bacteriophage P1 clones containing the SLC5A3 gene gave a positive PCR product when screened with the 21q22.1 marker VN02, an expressed sequence tag (EST00541) . Through DNA sequence analysis, it was determined that this STS marker if part of the 3' untranslated region of the SLC5A3 gene. Arch Virol, 1996, 141(2), 367 - 79 Restriction endonuclease mapping and molecular cloning of the human herpesvirus 6 variant B strain Z29 genome; Lindquester GJ et al.; Human herpesvirus 6(HHV-6) variants A and B differ in cell tropism, reactivity with monoclonal antibodies, restriction endonuclease profiles, and epidemiology . Nonetheless, comparative nucleotide and amino acid sequences from several genes indicate that the viruses are very highly conserved genetically, The B variant is the major etiologic agent of exanthem subitum and is frequently isolated from children with febrile illness; no disease has been etiologically associated with HHV-6A . One HHV-6A strain has been cloned and sequenced, but similar information and reagents are not available for HHV-6B . We report here the determination of maps of the restriction endonuclease cleavage sites for BamHI, C1aI, HindIII, KpnI, and Sa1I, and the cloning in plasmids and bacteriophages of fragments representing over 95% of the HHV-6B strain Z29 {HHV-6B(Z29)} genome . Hybridization experiments and orientation of several blocks of nucleotide sequence information onto the genomic map indicate that HHV-6A and HHV-6B genomes are colinear. FASEB J, 1996 Jan, 10(1), 5 - 9 A quantitative assessment of the role of the chaperonin proteins in protein folding in vivo; Lorimer GH; In vitro the chaperonin proteins, GroEL and GroES, facilitate the folding of some other proteins under conditions where that process does not occur spontaneously . Using values drawn from a number of such in vitro studies, together with the known rates of in vivo protein synthesis by Escherichia coli and the known quantities of GroEL and GroES in E . coli, an assessment of the general role of these proteins in protein folding in vivo has been made . Three specific cases are examined, where compelling evidence points to the involvement of the chaperonins; the in vivo folding of the bacteriophage coat protein during the burst phase of phage morphogenesis and of Rubisco during chloroplast development and during expression of recombinant Rubisco in E . coli . In each case the maximum in vitro rates are nearly sufficient to account for the observed in vivo rates of formation of the native protein . However, in general, there appears to be sufficient GroEL and GroES to facilitate the folding of no more than 5% of all of the proteins within E . coli. FASEB J, 1996 Jan, 10(1), 35 - 41 Structural and genetic analysis of the folding and function of T4 lysozyme; Matthews BW; The combination of directed mutagenesis with high-resolution structure analysis has made it possible to systematically address fundamental questions of protein folding and stability . Here we briefly review some recent results in this area based on studies of the lysozyme of bacteriophage T4 . Extended segments of the polypeptide chain can be substituted with alanine, suggesting that about 50%, or perhaps less, of the overall amino acid sequence protein is necessary to define the 3-dimensional structure of the protein . It is the internal residues that seem to be most important for folding and stability (although not necessarily for function) . Substitutions within the core of the protein of large nonpolar side chains with smaller ones have been used to better understand the nature of hydrophobic stabilization . Mutants that produce the largest cavities within the protein tend to be most destabilizing, allowing the energy cost of cavity formation to be estimated . Small, nonpolar ligands bind within such cavities and restore some stability to the protein . Analogous, nonpolar ligands do not bind, however, providing evidence that water molecules do not bind with high occupancy within nonpolar cavities . In a further series of studies it has been possible to re-engineer the active site region of T4 lysozyme to change the catalytic mechanism of the enzyme. FASEB J, 1996 Jan, 10(1), 159 - 63 Second-site reversion of a structural defect in bacteriophage T4 lysozyme; Bouvier SE et al.; A plasmid-borne gene encoding bacteriophage T4 lysozyme with a structural mutation, Tyr161-Ala, was mutagenized by by the use of polymerase chain reaction . The mutagenized gene was inserted into a specialized bacteriophage lambda cloning vector that must acquire a functional lysozyme gene in order to form plaques . Functional variants of the mutant lysozyme were selected . Three compensatory second-site revertants were obtained: Thr152-Met, Lys43-Ile, and Thr151-Ala . The effects of these mutations are interpreted in light of previous structural and genetic studies of T4 lysozyme. Carcinogenesis, 1996 Jan, 17(1), 5 - 11 Biological consequences of DNA damage introduced in bacteriophage PM2 DNA by hydrogen peroxide-mediated free radical reactions; Gille JJ et al.; In order to study the biological consequences of DNA damage induced by H2O2-mediated free radical reactions, DNA from bacteriophage PM2 was exposed to H2O2, Fe(3+)-citrate and ascorbate either alone or in combination . Induction of DNA lesions was determined as well as the biological activity of the phage DNA . Exposure to H2O2 alone resulted in max . 0.2 single-strand breaks per molecule; in the presence of Fe(3+)-citrate, the yield was approximately 4-fold higher . Under both conditions no double-strand breaks could be detected and the biological activity was not diminished . This indicates that low levels of single-strand breaks as generated by H2O2/Fe(3+)-citrate do not inactivate PM2 DNA . Exposure to ascorbate in the presence Fe(3+)-citrate resulted in extensive induction of single-strand breaks . However, at ascorbate concentration where approximately 3 single-strand breaks per molecule were induced, again no double-strand breaks could be detected and the biological activity of the DNA was not diminished . At 5 mM ascorbate, single-strand breaks were above the detection limit . Under these conditions, 0.02 double-strand breaks were induced and the biological activity was reduced to 50% . The contribution of double-strand breaks to biological inactivation was calculated to be approximately 3% . When PM2 DNA was exposed to H2O2 in the presence of ascorbate/Fe(3+)-citrate, a typical biphasic dose-effect relationship was observed both for the induction of double-strand breaks and biological inactivation, suggesting that one or more reactive species sensitive to H2O2 play a critical role . The .OH scavenger t-butanol appeared to be relatively inefficient in protecting PM2 DNA, which may indicate that other reactive species than .OH are involved . Our data suggest that other reactive species than .OH, such as the ferryl ion, are involved in H2O2-mediated DNA damage induction and biological inactivation. J Gen Virol, 1996 Jan, 77 ( Pt 1), 123 - 7 Detection of the ORF3 polypeptide of feline calicivirus in infected cells and evidence for its expression from a single, functionally bicistronic, subgenomic mRNA; Herbert TP et al.; Feline calicivirus (FCV) is a small positive-stranded RNA virus within the family Caliciviridae . Its genome is 7690 nucleotides in length and encodes three open reading frames (ORFs) . The smallest, ORF3, is located at the extreme 3' end of the genome and can potentially encode a polypeptide of approximately 12 kDa . In this paper, we report the identification of an ORF3-encoded polypeptide in FCV-infected cells using an antiserum raised against a bacterially-expressed bacteriophage T7 gene 10-ORF3 fusion protein . Although a small mRNA of 0-5 kb, which could potentially encode ORF3, has been described, reports on the number and size of FCV subgenomic RNAs have varied considerably . To clarify the situation, RNAs from FCV-infected cells were labelled in vivo using {32P}orthophosphate, an approach which provided definitive data . Only two RNA species were detected, the genomic RNA and a subgenomic mRNA of 2.4 kb . The 5' end of the subgenomic mRNA was mapped to position 5227 on the genomic RNA using RNA sequencing and primer extension methods . RNA isolated from FCV-infected cells in which no subgenomic RNA smaller than 2.4 kb was detectable directed the synthesis in rabbit reticulocyte lysate of the ORF3-encoded polypeptide . Furthermore, a synthetic RNA copy of the 2-4 kb subgenomic mRNA of FCV, containing both ORF2 and ORF3 polypeptides in the in vitro translation system . These data strongly suggest that ORF3 is expressed from the 2-4 kb subgenomic RNA and that this RNA is functionally bicistronic . The possible mechanisms by which ORF3 is expressed are discussed. Blood, 1996 Jan 1, 87(1), 292 - 8 Tumor-specific rearrangements of the immunoglobulin heavy-chain gene in B-cell non-Hodgkin's lymphoma detected by in situ hybridization; Ueda Y et al.; We have recently described the potential use of fluorescence in situ hybridization (FISH) to detect tumor-specific rearrangements of the immunoglobulin heavy-chain (IgH) gene in interphase nuclei . Using yeast artificial chromosome (YAC) clone Y6 containing variable region (VH) gene and bacteriophage clones Ig gamma, we analyzed 70 patients with B-cell non-Hodgkin's lymphoma (NHL) and compared the results with those obtained by the conventional G-banding method . Tumor-specific rearrangements of the IgH gene equivalent to 14q32 translocations were defined as separate signals of VH and Ig gamma genes or those of Ig gamma genes and referred to split signals . Twenty-nine patients (41.4%) showed split signals . Among these, 13 did not show 14q32 translocations by G-banding: three with other chromosomal abnormalities, one with normal karyotype, and nine with no analyzable metaphases . The partner sites of 14q32 translocations were identified in 17 patients by FISH: t(3;14)(q27;q32) including a complex variant was observed in nine patients, t(14;18)(q32;q21) in four, t(8;14)(q24;q32) in three, t(14;19)(q32;q13) in one, and t(11;14)(q13;q32) in one . Six of nine patients with t(3;14) or its variant and one of three with t(8;14) were diagnosed as having respective translocations only by FISH . Translocation t(3;14) was found most commonly, and was correlated histologically with diffuse lymphoma with large-cell components . These results indicate that interphase FISH with IgH gene probes promises to be a rapid and reliable method for use in the diagnosis of B-cell NHL. Nat Genet, 1996 Jan, 12(1), 72 - 7 A protein linkage map of Escherichia coli bacteriophage T7; Bartel PL et al.; Genome sequencing projects are predicting large numbers of novel proteins, whose interactions with other proteins must mediate the function of cellular processes . To analyse these networks, we used the yeast two-hybrid system on a genome-wide scale to identify 25 interactions among the proteins of Escherichia coli bacteriophage T7 . Among these is a set of six interactions connecting proteins that function in DNA replication and DNA packaging . Remarkably, two genes, arranged such that one entirely overlaps the other and uses a different reading frame, encode interacting proteins . Several of the interactions reflect intramolecular associations of different domains of the same polypeptide, suggesting that the two-hybrid assay may be useful in the analysis of protein folding . This global approach to protein-protein interactions may be applicable to the analysis of more complex genomes whose sequences are becoming available. J Virol, 1996 Jan, 70(1), 55 - 61 Complete inhibition of virion assembly in vivo with mutant procapsid RNA essential for phage phi 29 DNA packaging; Trottier M et al.; A highly efficient method for the inhibition of bacteriophage phi 29 assembly was developed with the use of mutant forms of the viral procapsid (or packaging) RNA (pRNA) indispensable for phi 29 DNA packaging . Phage phi 29 assembly was severely reduced in vitro in the presence of mutant pRNA and completely blocked in vivo when the host cell expressed mutant pRNA . Addition of 45% mutant pRNA resulted in a reduction of infectious virion production by 4 orders of magnitude, indicating that factors involved in viral assembly can be targets for efficient and specific antiviral treatment . The mechanism leading to the high efficiency of inhibition was attributed to two pivotal features . First, the pRNA contains two separate, essential functional domains, one for procapsid binding and the other for a DNA-packaging role other than procapsid binding . Mutation of the DNA-packaging domain resulted in a pRNA with no DNA-packaging activity but intact procapsid binding competence . Second, multiple copies of the pRNA were involved in the packaging of one genome . This higher-order dependence of pRNA in viral replication concomitantly resulted in its higher-order inhibitory effect . This finding suggested that the collective DNA-packaging activity of multiple copies of pRNA could be disrupted by the incorporation of perhaps an individual mutant pRNA into the group . Although this mutant pRNA could not be used for the inhibition of the replication of other viruses directly, the principle of using molecules with two functional domains and multiple-copy involvement as targets for antiviral agents could be applied to certain viral structural proteins, enzymes, and other factors or RNAs involved in the viral life cycle . This principle also implies a strategy for gene therapy, intracellular immunization, or construction of transgenic plants resistant to viral infection. Gene, 1995 Dec 29, 167(1-2), 49 - 52 Identification of functional interaction sites on proteins using bacteriophage-displayed random epitope libraries; van Zonneveld AJ et al.; We describe a phage-display-based method to identify epitopes or interaction sites on proteins . DNA encoding the protein of interest is partially degraded with DNase I to generate random fragments of 50-200 bp . These fragments are then cloned into a phagemid vector that has been modified to allow the expression of the random fragments and the construction of a (bacterio)phage-displayed random epitope library . Phages displaying functional epitopes can be selected from these libraries by affinity selection or panning . To test this method we have constructed a random-epitope library for human plasminogen-activator inhibitor 1 and used this library to map the epitope of a monoclonal antibody (mAb) directed against this protein . By alignment of the selected overlapping epitope-containing fragments, we were able to locate the epitope of the mAb on a stretch of 39 amino acids spanning from E128 to V166 . The approach may also be applied to more complex systems than single-protein genes, such as viral genomes or complete cDNA libraries. J Biol Chem, 1995 Dec 22, 270(51), 30428 - 33 Interference of PR-bound RNA polymerase with open complex formation at PRM is relieved by a 10-base pair deletion between the two promoters; Mita BC et al.; Bacteriophage lambda promoters PR and PRM direct RNA synthesis in divergent orientations from start sites 82 base pairs apart . We had previously determined that the presence on the same DNA fragment of a wild type PR promoter interfered with the utilization of the PRM promoter . The results reported here concern the effects of changing the distance between the start sites by insertion or deletion of 5 or 10 base pairs . Three different techniques (run-off transcription, gel mobility shift, and permanganate probing) were employed to monitor complex formation at PRM . Unexpectedly we find that deletion of 10 base pairs between the start sites abolishes the interference, whereas insertion of 10 base pairs does not . Deletion of 5 base pairs, however, essentially prevents joint complex formation at PR and PRM . These findings suggest several ways in which for the wild type separation of the two promoters the utilization of PRM could be affected by an RNA polymerase at PR . In addition to direct steric interference, these include the obstruction of access to DNA sites necessary for optimal contact with the RNA polymerase. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12250 - 4 Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: stability of lacZ mRNA inversely correlates with polymerase speed; Makarova OV et al.; When in Escherichia coli the host RNA polymerase is replaced by the 8-fold faster bacteriophage T7 enzyme for transcription of the lacZ gene, the beta-galactosidase yield per transcript drops as a result of transcript destabilization . We have measured the beta-galactosidase yield per transcript from T7 RNA polymerase mutants that exhibit a reduced elongation speed in vitro . Aside from very slow mutants that were not sufficiently processive to transcribe the lacZ gene, the lower the polymerase speed, the higher the beta-galactosidase yield per transcript . In particular, a mutant which was 2.7-fold slower than the wild-type enzyme yielded 3.4- to 4.6-fold more beta-galactosidase per transcript . These differences in yield vanished in the presence of the rne-50 mutation and therefore reflect the unequal sensitivity of the transcripts to RNase E . We propose that the instability of the T7 RNA polymerase transcripts stems from the unmasking of an RNase E-sensitive site(s) between the polymerase and the leading ribosome: the faster the polymerase, the longer the lag between the synthesis of this site(s) and its shielding by ribosomes, and the lower the transcript stability. EMBO J, 1995 Dec 15, 14(24), 6078 - 86 DNA packaging orders the membrane of bacteriophage PRD1; Butcher SJ et al.; Bacteriophage PRD1 contains a linear dsDNA genome enclosed by a lipid membrane lying within a protein coat . Determination of the structure of the detergent-treated particle to 2 nm by cryo-electron microscopy and three-dimensional reconstruction has defined the position of the major coat protein P3 . The coat contains 240 copies of trimeric P3 packed into positions of local 6-fold symmetry on a T = 25 lattice . The three-dimensional structures of the PRD1 virion and a DNA packaging mutant to a resolution of 2.8 nm have revealed specific interactions between the coat and the underlying membrane . The membrane is clearly visible as two leaflets separated by 2 nm and spanned by transmembrane density . The size of the coat does not change upon DNA packaging . Instead, the number of interactions seen between the protein shell and the membrane and the order of the membrane components increase . Thus the membrane of PRD1 plays a role in assembly which is akin to that played by the nucleocapsid in other membrane viruses. J Mol Biol, 1995 Dec 15, 254(5), 808 - 14 Kinetics of RNA polymerase initiation and pausing at the lambda late gene promoter in vivo; Kainz M et al.; We have measured the kinetics of transcription initiation and pausing by Escherichia coli RNA polymerase (RNAP) at the bacteriophage lambda late promoter, pR's, in growing cells . RNAP initiating transcription from pR' pauses after transcribing 16 or 17 nucleotides, and escape from this pause could in theory be the rate-limiting step in promoter function . We tested this hypothesis by analyzing pausing and non-pausing variants of both the pR' promoter segment and a more active mutant version of pR'; we measured reporter gene expression and used KMnO4 footprinting to measure directly occupancy of the promoter and pause sites in growing cells . We find that RNAP paused at +16/+17 does not limit expression of pR' . However, RNAP paused at +16/+17 does limit expression from the more active promoter by impeding formation of open complex . Therefore, the activity of the late gene regulatory protein Q to suppress the early pause, in addition to its antitermination activity, is unlikely to be important in phage gene expression. Biochim Biophys Acta, 1995 Dec 14, 1245(3), 430 - 8 Structure of genes for sperm-specific nuclear basic protein (SP4) in Xenopus laevis; Mita K et al.; Nuclear basic proteins in sperm of Xenopus laevis consist of 6 sperm-specific proteins (SPs1-6) in addition to somatic core histones . Using a cDNA for SP4 as a probe, we cloned genomic DNA containing SP4 genes from a genomic library constructed from recombinant lambda bacteriophage containing 12.0 kbp-EcoRI digests of J-strain X . laevis liver DNA . Construction of restriction maps based on Southern blot analysis revealed the existence of a total of five SP4 genes which are arranged in a tandemly repeated array forming a cluster of simple multigenes per haploid genome, over a range of 18 kbp . Among these genes, the one located at the most upstream position differed from others in possessing a single base substitution which gave rise to a replacement of one out of 78 amino acid residues . The DNA containing the second to the fourth SP4 genes, arranged at about 3 kbp intervals each, was totally sequenced for 10,165 bp . Each gene was found to contain one intron, typical TATA and CCAAT boxes in the 5'-flanking region, and a polyadenylation signal in the 3'-flanking region . Comparative sequence analyses revealed three regions of extensive homology within the upstream non-coding region among three genes, suggesting a possible relevance to their expression at a particular phase of spermatogenesis and/or in testis. Biochem Biophys Res Commun, 1995 Dec 14, 217(2), 393 - 401 Efficient regulation of gene expression by adenovirus vector-mediated delivery of the CRE recombinase; Sakai K et al.; We have constructed an E1-defective adenovirus (Ad) vector designated AdCAG-Cre containing the Cre recombinase gene derived from bacteriophage P1 under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid (CAG) promoter . We examined the Cre-loxP-based recombination by this Ad vector in C2C12 cells bearing a reporter gene construct CAG-CAT-Z, which directs expression of the E . coli lacZ gene upon Cre-mediated excision of the CAT gene located between the CAG promoter and the lacZ gene . Nearly 100% of these cells were shown to start to produce beta-galactosidase after infection with the AdCAG-Cre vector at MOI 100 . On the basis of this result, we discuss the possible use of the AdCAG-Cre vector to manipulate the gene expression in mammalian cells. Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11859 - 63 Analysis of RNA-binding proteins by in vitro genetic selection: identification of an amino acid residue important for locking U1A onto its RNA target; Laird-Offringa IA et al.; An in vitro genetic system was developed as a rapid means for studying the specificity determinants of RNA-binding proteins . This system was used to investigate the origin of the RNA-binding specificity of the mammalian spliceosomal protein U1A . The U1A domain responsible for binding to U1 small nuclear RNA was locally mutagenized and displayed as a combinatorial library on filamentous bacteriophage . Affinity selection identified four U1A residues in the mutagenized region that are important for specific binding to U1 hairpin II . One of these residues (Leu-49) disproportionately affects the rates of binding and release and appears to play a critical role in locking the protein onto the RNA . Interestingly, a protein variant that binds more tightly than U1A emerged during the selection, showing that the affinity of U1A for U1 RNA has not been optimized during evolution. J Gen Virol, 1995 Dec, 76 ( Pt 12), 3089 - 98 Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system; Abrams CC et al.; cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleave this molecule to 1AB, 1C and 1D, were constructed . These cassettes were introduced into vaccinia virus (VV) transfer vectors . Attempts to isolate recombinant VVs constitutively expressing these cassettes were unsuccessful . However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated . Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins . Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed . Electron microscopy revealed empty capsid-like particles with diameters of about 30 nm . Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles . Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins . However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles. Indian J Biochem Biophys, 1995 Dec, 32(6), 356 - 60 Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions; Shivashankar Y et al.; Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor . These proteolytic events are brought about both by host cell signalase and a virally encoded protease . The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene . In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST) . The fusion constructs were driven by the strong bacteriophage T7 promoter . Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins . Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix. Genetika, 1995 Dec, 31(12), 1630 - 6 {Growth of radiation resistance in bacteriophages as a result of intensifying expression of the stress system in host cells}; Verbenko VN et al.; By means of polyacrylamide gel electrophoresis (PAGE) of proteins from radiation-resistant Gamr mutants of Escherichia coli, it was shown that induction and elimination of RecA protein in these mutants are kinetically more rapid than in wild type cells, and heat-shock proteins (HSP) are hyperproduced even at a normal temperature (32 degrees C) . gamma-and UV-irradiated bacteriophages were used to study the results of simultaneous enhanced expression of two stress repair systems . Radiation-resistant mutants are similar to wild type cells in their ability to reactivate phages lambda CI, phi 80 vir, and T 4D inactivated by gamma-rays and UV-light . W-reactivation of gamma-irradiated phages lambda and 80 vir is respectively 1.5 and 1.2 times higher in Gamr cells in which maximal w-reactivation was observed at wide range of doses (from 300 to 2000 Gy) whereas in wild type cells the peak of W-reactivation was registered at doses of 400 to 450 Gy . The phage lambda gamma-, irradiated upon adsorption on the cells of a radiation-resistant mutant, was two times more resistant to gamma-rays (DMF = 2 at LD10) than when irradiated upon adsorption on wild type cells . Postirradiation degradation of the phage lambda DNA, when irradiated within Gamr cells, was significantly lower than in wild type cells, and preirradiation of the cells decreased phage DNA degradation (12% in Gamr cells and 30% in wild-type cells) . The role of an increased HSP level and expression of SOS-regulon in radiation resistance and possible interaction of stress systems in bacterial cells are discussed. Biophys J, 1995 Dec, 69(6), 2649 - 60 Video light microscopy of 670-kb DNA in a hanging drop: shape of the envelope of DNA; Serwer P et al.; Although its conformation has not been observed directly, double-stranded DNA in solution is usually assumed to be randomly coiled at the level of the DNA double helix . By video light microscopy of ethidium-stained DNA at equilibrium in a nonturbulent hanging drop, in the present study, the 670 kb linear bacteriophage G DNA is found to form a flexible filament that has on average 17 double helical segments across its width . This flexible filament 1) has both asymmetry and dimensions expected of a random coil and 2) has ends that move according to the statistics expected of a random walk . After unraveling the flexible filament-associated DNA double helix near the surface of a hanging drop, recompaction occurs without perceptible rotation of the DNA . Both conformational change and intermolecular tangling of the DNA are observed when G DNA undergoes nondiffusive motion in a hanging drop . The characteristics of the G DNA flexible filament are explained by the assumption that the flexible filament is a random coil of double helical segments that are unperturbed by motion of the suspending medium. Biophys J, 1995 Dec, 69(6), 2256 - 67 Rotary DNA motors; Doering C et al.; Many molecular motors move unidirectionally along a DNA strand powered by nucleotide hydrolysis . These motors are multimeric ATPases with more than one hydrolysis site . We present here a model for how these motors generate the requisite force to process along their DNA track . This novel mechanism for force generation is based on a fluctuating electrostatic field driven by nucleotide hydrolysis . We apply the principle to explain the motion of certain DNA helicases and the portal protein, the motor that bacteriophages use to pump the genome into their capsids . The motor can reverse its direction without reversing the polarity of its electrostatic field, that is, without major structural modifications of the protein . We also show that the motor can be driven by an ion gradient; thus the mechanism may apply as well to the bacterial flagellar motor and to ATP synthase. RNA, 1995 Dec, 1(10), 1041 - 50 Confirmation of the helical structure of the 5'/3' termini of the essential DNA packaging pRNA of phage phi 29; Zhang C et al.; Bacteriophage phi 29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during viral assembly . An intriguing feature of phi 29 is the presence of a 120-base virus-encoded RNA (pRNA) that is indispensable for DNA packaging . Phylogenetic comparison of similar RNAs in numerous phages has revealed that the secondary structure of the pRNA is well conserved . Computer analysis predicts the presence of an extensive segment of helix with three single-base bulges generated by the pairing of the 5' and 3' ends . The desire to understand the role played by the pRNA in DNA packaging has led to a mutational analysis of the 5'-/3'-terminal region, which is believed to be important in DNA translocation . Deletion of 3 bases from the 3' end of the RNA, shortening the pRNA from 120 to 117 bases, was tolerated without loss of activity, but additional deletion of the base 117 resulted in 100-fold less activity, and a 115-base pRNA was virtually nonfunctional . Additionally, the three unpaired one-base bulges within the helical stretches of the paired proximate ends were nonessential for pRNA activity, as demonstrated by deletion of the bulge individually . An extensive series of helix disruptions by single- and multiple-base substitution almost invariably led to the loss of DNA packaging activity . Additional mutations that restored predicted base pairings rescued pRNA activity . This second site suppression confirmed that the 5'- and 3'-end region was paired and was indeed a helical stretch . The secondary structure was of greater importance than the primary sequence, with the exception of the requirement of an adenine at either the third or fourth position . The specific requirement of an adenine in phi 29 pRNA at this position, as well as conservation of this position in other phage pRNAs, implicates that this base may play a special role in either the DNA-packaging reaction or the maintenance of the pRNA tertiary structure. FEMS Microbiol Lett, 1995 Dec 1, 134(1), 97 - 101 Mitomycin C induction of bacteriophages from Serpulina hyodysenteriae and Serpulina innocens; Humphrey SB et al.; A prophage was induced from cells of the pathogenic spirochaete Serpulina hyodysenteriae using mitomycin C . Five to seven hours after mitomycin C was added (8 micrograms/ml, final concentration) to S . hyodysenteriae B204 cultures in BHIS broth (OD620 = 0.9) cell lysis was detected as a decrease in culture optical density . Bacteriophage particles attached to whole cells and to cell debris were detected by electron microscopic analysis of negatively stained (2% PTA, pH 7.0) bacteria harvested by centrifugation from mitomycin C treated cultures . The phage particles consisted of a head (45 nm diameter) and a tail (64 nm x 9 nm) . Bacteria from untreated cultures lacked phages detectable by electron microscopy . The appearance of bacteriophage particles in mitomycin C treated cultures correlated with the appearance of extrachromosomal DNA, 7-8 kb in size as estimated by agarose gel electrophoresis, in DNA preparations from treated S . hyodysenteriae cells . When cultures of other S . hyodysenteriae strains (B78, B169, A-1, B8044, B6933, Ack300/8, R-1) and S . innocens 4/71 in BHIS were treated with mitomycin C (8-15 micrograms/ml, final concentration), phages similar in morphology and size to the S . hyodysenteriae B204 were induced. Immunology, 1995 Dec, 86(4), 636 - 45 Impaired protein catabolism in Trypanosoma cruzi-infected macrophages: possible involvement in antigen presentation; Plasman N et al.; The effect of Trypanosoma cruzi infection on the ability of mature and immature murine peritoneal macrophage (MPM) subpopulations to catabolize the bacteriophage lambda repressor cI protein (cI) has been investigated . The capacity of infected MPM to present the cI and to stimulate various CD4+, I-Ad- or I-Ed-restricted T-cell hybridomas specific for cI was also assessed . Our results show that the radioiodinated cI uptake and catabolism decreased sharply after infection of MPM with T . cruzi . A cI presentation deficiency appeared in mature and immature MPM infected with T . cruzi trypomastigotes . The ability of infected MPM to bind immunogenic cI (12-26) peptides to the plasma membrane Ia molecules was also altered, especially in immature MPM, as shown with paraformaldehyde prefixed MPM, suggesting that these MPM only have a few functional Ia molecules on their membrane . The reduced capacity of cI presentation to the I-Ed-restricted B26.1 hybridomas by infected MPM subpopulations was comparable to that of the I-Ad-restricted B24.4 and B26.2 T cells . The percentage of major histocompatibility complex (MHC) class II-positive MPM was also reduced after T . cruzi infection . The percentage of positive interleukin-2 receptor (IL-2R) MPM was sharply lowered in infected cells, even with a pre- or a post-interferon-gamma (IFN-gamma) activation . Finally, inhibition of prostaglandin with indomethacin, or of nitric oxide with N-monomethyl-L-arginine, or of tumour necrosis factor-alpha (TNF-alpha) with specific monoclonal antibodies did not restore the cI presentation capacities of the MPM subpopulations . Taken together, these results suggest that T . cruzi infection induces a reduced capacity for macrophages to take up and catabolize antigen, resulting in a deficient antigen processing and presentation of the derived immunogenic peptides to specific CD4+ T-helper type-1 cell hybridomas . The decreased cI presenting capacity was a function of the cell's burden and maturity. Chromosoma, 1995 Dec, 104(4), 242 - 51 Molecular characterization and cytogenetic analysis of highly repeated DNAs of lake trout, Salvelinus namaycush; Reed KM et al.; The chromosomes of lake trout (Salvelinus namaycush) contain a considerable amount of heterochromatin located at the centromeres and/or telomeres of several chromosomes, including a sex-specific block located distally on the X chromosome . In order to investigate further the repetitive DNAs of lake trout, genomic DNA from a female was size fractionated (<600 bp) with the restriction endonuclease AluI and fragments were cloned into the bacteriophage M13 . A total of 42 clones were isolated . Relative copy number of individual inserts within the lake trout genome was estimated by Southern analysis . Twelve clones were determined to be highly repetitive and were chosen for further investigation . Inserts of these clones contained sequences similar to the AluI/RsaI, EcoRI/DraI, DraI/BstEII, and MboI/BglII families reported from Arctic char (Salvelinus alpinus) . The chromosomal location of several of these fragments was determined in lake trout by fluorescence in situ hybridization (FISH) . Two related AluI/RsaI sequences (Type A, approximately 140 bp, and Type B,approximately 120 bp) showed differential hybridization . Type A hybridized to the centromeres of all metacentric as well as several acrocentric chromosomes . Type B hybridized to the centromeres of most acrocentric chromosomes . A sequence with homology to the EcoRI/DraI family hybridized to the centromeres of several acrocentric chromosomes . Sequences with partial similarity to the DraI/BstEII family hybridized to the major rDNA sites (nucleolar organizer regions, NORs) and several minor telomeric sites . The interstitial and telomeric heterochromatin of lake trout, including that of the X chromosome, appears to comprise sequences belonging to the MboI/BglII family. Cancer Gene Ther, 1995 Dec, 2(4), 281 - 9 A novel nonviral cytoplasmic gene expression system and its implications in cancer gene therapy; Chen X et al.; We recently have developed a unique cytoplasmic transient gene expression system based on cotransfection of target cells with bacteriophage T7 RNA polymerase (RNAP) and plasmid DNA vectors containing a T7 autogene . Because this T7 system is self-initiating, self-maintaining, and requires no cellular factors for transcription, it is therefore likely to function in any mammalian cell with any gene both in vitro and, more importantly, in vivo . In this study we demonstrate that the T7 DNA vector and T7 RNAP could be efficiently codelivered to cultured cells by lipofection . Different target genes were expressed by the T7 system in a wide variety of mammalian cells including several tumor cell lines . Gene expression could be detected in more than 30% of the cells of some tumor cell lines transiently transfected by the T7 vector . Average activity of the reporter enzyme (luciferase) expressed by a transfected cell was relatively constant regardless of the cell line used . When a T7-luciferase vector was directly injected into various tissues of mice without the use of liposomes, luciferase activity could be found in the injected liver, muscle, brain and tail connective tissues . The luciferase levels expressed by the T7 system were found to be up to 200-fold higher, depending upon the injected tissues, than levels achieved with a traditional nuclear gene expression vector . Direct tumor injection with a T7-beta-galactosidase (beta-gal) construct resulted in beta-gal gene expression in tumor cells near the injection sites . In addition, direct injection of the T7 system in mice did not generate detectable quantities of antibodies against the T7 RNAP . These results suggest that this gene expression system may be useful in many different medical applications such as cancer gene therapies and DNA vaccination, where transient but rapid and efficient gene expression is required. J Steroid Biochem Mol Biol, 1995 Dec, 55(5-6), 473 - 9 Analysis of the human gene encoding the kidney isozyme of 11 beta-hydroxysteroid dehydrogenase; Agarwal AK et al.; 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the conversion of cortisol to cortisone . This activity may be deficient in the syndrome of apparent mineralocorticoid excess (AME) . 11 beta-HSD L (Type I), isolated from liver, is widely expressed and utilizes NADP+ as a cofactor . The gene for 11 beta-HSD L was found to be normal in patients of AME . A second isoform, 11 beta-HSD K (Type II), isolated from kidney, is more tissue specific in expression and utilizes NAD+ as a cofactor . The cDNA clone encoding 11 beta-HSD K was isolated from sheep kidney . The cDNA is 1.8 kb in length and encodes a protein of 404 amino acid residues with a predicted M(r) 43,953 . The recombinant enzyme functions as an NAD(+)-dependent 11 beta-dehydrogenase with very high affinity for steroids, but it has no detectable reductase activity . It is 37% identical in amino acid sequence to an NAD(+)-dependent isozyme of 17 beta-hydroxysteroid dehydrogenase . It is expressed at high levels in the kidney, placenta, adrenal and at lower levels in colon, stomach, heart and skin . The human 11 beta-HSD K gene consists of five exons spread over 6 kb . The nucleotide binding domain lies in the first and the second exon, and the catalytic domain in the fourth exon . The promoter for 11 beta-HSD K gene lacks a TATA box and has a high GC base content, suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences . Fluorescent in situ hybridization of metaphase chromosomes with a positive bacteriophage P1 genomic 11 beta-HSD K clone localized the gene to chromosome 16q22 . In contrast, the 11 beta-HSD L gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical . Different transcriptional start sites are utilized in kidney and placenta. Curr Microbiol, 1995 Dec, 31(6), 336 - 9 Isolation and characterization of a temperate bacteriophage from a ruminal acetogen; Jiang WH et al.; Nine acetogenic bacterial cultures recently isolated from the bovine rumen were tested for phage susceptibility by plaque formation . Both clear plaques and plaques with turbid centers were occasionally seen, but could not be used repeatedly to lyse pure cultures of acetogens, suggesting the possibility of a temperate phage . Five of the nine acetogenic isolates showed a response to mitomycin C induction . Acetogenic isolate H3HH was chosen for further study because it produced the greatest lysogenic response to mitomycin C . The bacteriophage was induced with mitomycin C, examined by transmission electron microscopy, and shown to have a hexagonal head (diameter, 59 eta m), a long flexible tail (192 eta m), and a flat collar (diameter, 31 eta m) . The bacteriophage was classified within Bradley's group B . Bacteriophage DNA was determined to contain 36.2 kilobases of linear double-stranded DNA. Virology, 1995 Dec 1, 214(1), 235 - 8 Cucumber mosaic cucumovirus antibodies from a synthetic phage display library; Ziegler A et al.; Antibody fragments (scFv) that bind specifically to particles of cucumber mosaic cucumovirus (CMV) were obtained from a library which encodes a diverse array of synthetic antibody fragments, each displayed on the surface of filamentous bacteriophage . After four rounds of selection and enrichment, several clones were obtained which produced scFv that bound specifically to purified particles of CMV in ELISA . BstNI digestion of phagemid DNA resulted in the same restriction pattern for all clones . The nucleotide sequences of three of the clones showed that they belonged to the human VH1 family and that they had a complementarity determining region loop of 7 amino acids . Phage-displayed antibodies and soluble scFv secreted by these clones reacted with particles of CMV in sap from infected plants in ELISA . In immunoblotting tests, soluble scFv preparations reacted with SDS-denatured coat protein extracted from purified preparations of CMV isolates belonging to either subgroup I or II and also with protein extracted by SDS treatment of seeds harvested from naturally infected lupin plants . The results demonstrate the feasibility, and potential applicability, of recombinant antibody methods in plant pathology. Virology, 1995 Dec 1, 214(1), 150 - 8 Synthesis of a full-length infectious cDNA clone of cucurbit aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from other luteoviruses; Prufer D et al.; A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as an in vitro transcript, under control of a bacteriophage T7 RNA polymerase promoter, or in vivo, under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector . The biological activity of the cloned cDNA was demonstrated by the ability of its in vitro transcript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants . Virus in the agroinfected plants cold be transmitted by the aphid vectors Myzus persicae and Aphis gossypii . The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus . The resulting chimeric transcripts replicated in protoplasts and produced virions. Cell, 1995 Dec 1, 83(5), 773 - 82 Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition; Vassylyev DG et al.; T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway . The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution . The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60 degree inclination at the central thymine dimer . The adenine base complementary to the 5' side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface . These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
