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 l