J Bacteriol, 1995 Dec, 177(23), 6844 - 53 Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4; Kreuzer KN et al.; We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome . Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4 . However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome . As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein . Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid . We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb) . For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene) . The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease) . The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome . As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins . These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p . 120-130, in C . K . Mathews, E . M . Kutter, G . Mosig, and P . B . Berget (ed.), Bacteriophage T4, 1983) . In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies. J Bacteriol, 1995 Dec, 177(23), 6810 - 9 Sequences of the Escherichia coli BtuB protein essential for its insertion and function in the outer membrane; Lathrop JT et al.; The Escherichia coli btuB gene encodes the outer membrane transporter for vitamin B12, the E colicins, colicin A, and bacteriophage BF23 . Several series of mutant forms of BtuB resulting from the insertion of dipeptide sequences and from overlapping in-frame deletions and duplications were constructed . Strains expressing the variant genes in single and multiple copy numbers were analyzed for BtuB function, for the level of BtuB polypeptide in the outer membrane, and for changes in the outer membrane permeability barrier . Most dipeptide insertions had normal transport function and assembly in the membrane . Only 2 of the 27 deletions spanning residues 5 and 514 possessed transport function, and most of the remainder were not stably inserted in the membrane . Most duplications (19 of 21) retained transport function and were inserted in the outer membrane, although some were subject to proteolysis . Even long duplications containing as many as 340 repeated amino-terminal residues retained function, suggesting considerable plasticity in the sequence requirements for membrane insertion of BtuB . Expression of many deletion and duplication proteins conferred increased susceptibility to structurally unrelated inhibitors that are normally excluded by the outer membrane . These results could be consistent with the mutational disruption of extracellular loops or transmembrane segments of BtuB that constitute a gated channel, but the finding that alterations throughout the length of BtuB affect membrane permeability properties suggests that the altered proteins might perturb the outer membrane structure itself. J Biol Chem, 1995 Dec 1, 270(48), 28541 - 50 Contribution of antibody heavy chain CDR1 to digoxin binding analyzed by random mutagenesis of phage-displayed Fab 26-10; Short MK et al.; We constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 of the anti-digoxin antibody 26-10 Fab to investigate sequence constraints necessary for high affinity binding in an antibody of known crystal structure . Phage were selected by panning against digoxin and three C-16-substituted analogues . All antigen-positive mutants selected using other analogues also bound digoxin . Among 73 antigen-positive clones, 26 different nucleotide sequences were found . The majority of Fabs had high affinity for digoxin (Ka 3.4 x 10(9) M-1) despite wide sequence diversity . Two mutants displayed affinities 2- and 4-fold higher than the parental antibody . Analysis of the statistical distribution of sequences showed that highest affinity binding occurred with a restricted set of amino acid substitutions at positions H33-35 . All clones save two retained the parental Asn-H35, which contacts hapten and hydrogen bonds to other binding site residues in the parental structure . Positions H30-32 display remarkable diversity, with 10-14 different substitutions for each residue, consistent with high affinity binding . Thus complementarity can be retained and even improved despite diversity in the conformation of the N-terminal portion of the H-CDR1 loop. Blood, 1995 Dec 1, 86(11), 4331 - 6 Virucidal short wavelength ultraviolet light treatment of plasma and factor VIII concentrate: protection of proteins by antioxidants; Chin S et al.; The use of solvent/detergent mixtures and various forms of heat treatment to inactivate viruses has become widespread in the preparation of blood derivatives . Because viruses that lack lipid envelopes and/or are heat resistant, eg, hepatitis A virus (HAV) or parvovirus B19 may be present, the use of two methods of virus elimination that operate by different mechanisms has been advocated . We now report on short wavelength ultraviolet light (UVC) irradiation for virus inactivation and enhancement of its compatibility with proteins by quenchers of reactive oxygen species (ROS) . Treatment of an antihemophilic factor (AHF) concentrate or whole plasma with 0.1 J/cm2 inactivated 10(5) to > or = 10(6) infectious doses (ID) of encephalomyocarditis virus (EMCV), HAV, bacteriophage M13, vesicular stomatitis virus (VSV), and porcine parvovirus . However, the recovery of factor VIII was 30% or lower on treatment of an AHF concentrate and 60% on treatment of plasma . Factor VIII recovery could be increased with little or no effect on virus kill by addition of rutin, a flavonoid known to quench both type I and type II ROS . On treatment of plasma in the presence of rutin, the recovery of several other coagulation factors was also enhanced by rutin addition and typically exceeded 75% . Electrophoretic analysis of treated AHF concentrate confirmed the advantage of rutin presence; UVC irradiation of plasma did not cause discernible changes in electrophoretic banding patterns, even in the absence of rutin . We conclude that addition of UVC treatment to existing processes used in the manufacture of blood derivatives will provide an added margin of safety, especially for nonenveloped or heat-stable viruses. Nucleic Acids Res, 1995 Nov 25, 23(22), 4707 - 11 Consistencies of individual DNA base-amino acid interactions in structures and sequences; Lustig B et al.; Amino acid-amino acid interaction energies have been derived from crystal structure data for a number of years . Here is reported the first derivation of normalized relative interaction from binding data for each of the four bases interacting with a specific amino acid, utilizing data from combinatorial multiplex DNA binding of zinc finger domains {Desjarlais, J . R . and Berg, J . M . (1994) Proc . Natl . Acad . Sci . USA, 91, 11099-11103} . The five strongest interactions are observed for lysine-guanine, lysine-thymine, arginine-guanine, aspartic acid-cytosine and asparagine-adenine . These rankings for interactions with the four bases appear to be related to base-amino acid partial charges . Also, similar normalized relative interaction energies are derived by using DNA binding data for Cro and lambda repressors and the R2R3 c-Myb protein domain {Takeda, Y., Sarai, A . and Rivera, V . M . (1989) Proc . Natl . Acad . Sci . USA, 86, 439-443; Sarai, A . and Takeda, Y . (1989) Proc . Natl . Acad . Sci . USA, 86, 6513-6517; Ogata, K . et al . (1995) submitted} . These energies correlate well with the combinatorial multiplex energies, and the strongest cases are similar between the two sets . They also correlate well with similar relative interaction energies derived directly from frequencies of bases in the bacteriophage lambda operator sequences . These results suggest that such potentials are general and that extensive combinatorial binding studies can be used to derive potential energies for DNA-protein interactions. Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 10909 - 13 Phage display selection of ligand residues important for Src homology 3 domain binding specificity; Rickles RJ et al.; The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction . It functions to direct protein-protein interactions through the recognition of proline-rich motifs on associated proteins . SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu . By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II . The class I sites have the consensus sequence ZP(L/P)PP psi P whereas the class II consensus is PP psi PPZ (where psi is a hydrophobic residue and Z is a SH3 domain-specific residue) . We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP psi P . These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells . In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues . These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains . By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides . The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct binding specificities . These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity. J Mol Biol, 1995 Nov 17, 254(1), 15 - 28 Dynamics of bacteriophage T4 DNA polymerase function: identification of amino acid residues that affect switching between polymerase and 3' --> 5' exonuclease activities; Stocki SA et al.; Many DNA polymerases are multifunctional with the ability to replicate DNA as well as to proofread misincorporated nucleotides . Since polymerase and 3'--> 5' exonuclease activities appear to reside in spatially distinct active centers, there must be some mechanism for coordinating replication with proofreading and for transferring DNA between the two active centers . We have designed a genetic selection scheme to isolate bacteriophage T4 mutant DNA polymerases that are defective in "switching" between polymerase and exonuclease activities . Amino acid residues that affected active-site-switching were identified in four regions of the T4 DNA polymerase: two regions in the proposed exonuclease domain . Representative mutant DNA polymerases from each region were purified for biochemical studies . We propose that amino acid substitutions identified by mutational analysis affect critical contacts between T4 DNA polymerase and DNA that are required for transfer of DNA between the polymerase and exonuclease active centers. EMBO J, 1995 Nov 15, 14(22), 5736 - 44 The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB; Parsons CA et al.; During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules . The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation . Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes . We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein . Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy . In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed . These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology. EMBO J, 1995 Nov 15, 14(22), 5724 - 35 Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site; Mueller JE et al.; I-TevI, the intron-encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence-tolerant fashion . We demonstrate here that the 28 kDa I-TevI binds the extensive 37 bp td homing site as a monomer and significantly distorts its substrate . In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the td homing site, I-TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site . Formation of the bent I-TevI-DNA complex is proposed to promote top-strand cleavage of the homing site . Furthermore, reductions in the degree of distortion and in the efficiency of binding base-substitution variants of the td homing site indicate that sequences flanking the cleavage site contribute to the I-TevI-induced conformational change . These results, combined with genetic, physical and computer-modeling studies, form the basis of a model, wherein I-TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top-strand cleavage site . The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine-containing DNA, as exists in the T-even phages. Genes Dev, 1995 Nov 15, 9(22), 2831 - 45 A protein-RNA interaction network facilitates the template-independent cooperative assembly on RNA polymerase of a stable antitermination complex containing the lambda N protein; Mogridge J et al.; The stable association of the N gene transcriptional antiterminator protein of bacteriophage lambda with transcribing RNA polymerase requires a nut site (boxA+boxB) in the nascent transcript and the Escherichia coli factors NusA, NusB, NusG, and ribosomal protein S10 . We have used electrophoretic mobility shift assays to analyze the assembly of N protein, the E . coli factors, and RNA polymerase onto the nut site RNA in the absence of a DNA template . We show that N binds boxB RNA and that subsequent association of NusA with the N-nut site complex is facilitated by both boxA and boxB . In the presence of N, NusA, and RNA polymerase the nut site assembles ribonucleoprotein complexes containing NusB, NusG, and S10 . The effects on assembly of mutations in boxA, boxB, NusA, and RNA polymerase define multiple weak protein-protein and protein-RNA interactions (e.g., NusB with NusG; NusA with boxB; NusA, NusB, and NusG with boxA) that contribute to the overall stability of the complex . Interaction of each component of the complex with two or more other components can explain the many observed cooperative binding associations in the DNA-independent assembly of a stable antitermination complex on RNA polymerase. Biochemistry, 1995 Nov 14, 34(45), 14918 - 31 Complexes between chaperonin GroEL and the capsid protein of bacteriophage HK97; Ding Y et al.; The 42 kDa capsid protein of bacteriophage HK97 requires the GroEL and GroES chaperonin proteins of its Escherichia coli host to facilitate correct folding, both in vivo and in vitro . In the absence of GroES and ATP, denatured gp5 forms a stable complex with the 14 subunit GroEL molecule . We characterized the electrophoretic and biochemical properties of this complex . In electrophoresis on a native (nondenaturing) gel, the band of the gp5-GroEL complex shifts to a slower migrating position relative to uncomplexed GroEL . The results show that there is only one subunit of gp5 bound to each GroEL 14-mer and that the shift in band position is due primarily to a change in the overall charge of the complex relative to uncomplexed GroEL, and not to a change in size or shape . GroEL forms similar complexes with proteolytic fragments of gp5, with a series of sequence duplication derivatives of gp5, and with other proteins . Electrophoretic examination of these complexes shows that a band shift occurs with proteins larger than 31-33 kDa but not with smaller proteins . For those proteins that cause a band shift upon complex formation, the magnitude of the shift is correlated with the predicted if the charge of the complex were simply the sum of the charge of GroEL and the charge of the substrate protein . We suggest that binding of a substrate protein to GroEL is accompanied by a net binding of solution cations to the complex, but only in the case of proteins above a minimum size of 31-33 kDa . The gp5-GroEL complex is in an association/dissociation equilibrium, with a binding constant measured in the range of 11-17 microM-1. J Mol Biol, 1995 Nov 3, 253(4), 517 - 29 Helix-destabilizing activity of phi 29 single-stranded DNA binding protein: effect on the elongation rate during strand displacement DNA replication; Soengas MS et al.; The single-stranded DNA (ssDNA)-binding protein (SSB) of bacteriophage phi 29 is one of the virus-encoded proteins required for viral DNA replication . We have found that phi 29 SSB has helix-destabilizing activity since it removes secondary structure of the ssDNA in phi 29 replicative intermediates, as revealed by electron microscopy, and displaces oligonucleotides annealed to M13 ssDNA . To investigate the mechanism of the SSB-dependent stimulation of phi 29 DNA replication we have characterized the helix-destabilizing activity of phi 29 SSB and measured its effect on the DNA elongation rate by phi 29 DNA polymerase, which does not require an accessory helicase . The use of replication reactions where strand displacement is either required (phi 29 DNA replication) or not (conversion of primed M13 ssDNA into double-stranded DNA (dsDNA)) has allowed us to find that (1) strand displacement DNA replication was affected by lowering the temperature or by increasing the salt concentration, since the DNA elongation rate on the phi 29 template was three to fourfold slower than on primed M13 ssDNA, (2) under those conditions, addition of phi 29 SSB stimulated to different extents the DNA elongation rate during phi 29 DNA replication, whereas it had a marginal effect on primed M13 ssDNA replication, and (3) phi 29 SSB increased four to sixfold the phi 29 DNA elongation rate by phi 29 DNA polymerase strand displacement mutants, reaching approximately 50% the rate of the wild-type enzyme . The implications of the helix-destabilizing properties of the phi 29 SSB under conditions in which DNA opening is impaired are discussed. Microb Pathog, 1995 Nov, 19(5), 351 - 64 Identification of Porphyromonas gingivalis prefimbrilin possessing a long leader peptide: possible involvement of trypsin-like protease in fimbrilin maturation; Onoe T et al.; Fimbriae of Porphyromonas gingivalis have been shown to be important as one of the virulence factors for colonization on mucosal surfaces . The gene (fimA) encoding the fimbrial subunit (fimbrilin) was overexpressed in Escherichia coli by using a bacteriophage T7 promoter-polymerase expression vector system . Analysis of the resulting fimA gene product revealed that the prefimbrilin had a 46 amino acid leader peptide . This extremely long leader peptide was cleaved from the prefimbrilin by treatment with trypsin or P . gingivalis extracts containing trypsin-like protease activity, resulting in production of a mature fimbrilin . We also found that some transposon-induced trypsin-like protease deficient mutants of P . gingivalis exhibited deficiency in fimbriation and that one of the mutants accumulated a fimbrilin precursor possessing a 25 amino acid leader peptide in the cell . The presence of an extremely long leader peptide and the requirement for a leader peptidase with a substrate specificity similar to that of P . gingivalis trypsin-like protease for fimbrilin maturation indicate that P . gingivalis fimbrilin is a novel type that is different from fimbrilins of type I and IV families. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1123 - 9 Upstream stimulators for recoding; Larsen B et al.; Recent progress in elucidation of 5' stimulatory elements for translational recoding is reviewed . A 5' Shine-Dalgarno sequence increases both +1 and -1 frameshift efficiency in several genes; examples cited include the E . coli prfB gene encoding release factor 2 and the dnaX gene encoding the gamma and tau subunits of DNA polymerase III holoenzyme . The spacing between the Shine-Dalgarno sequence and the shift site is critical in both the +1 and -1 frameshift cassettes; however, the optimal spacing is quite different in the two cases . A frameshift in a mammalian chromosomal gene, ornithine decarboxylase antizyme, has recently been reported; 5' sequences have been shown to be vital for this frameshift event . Escherichia coli bacteriophage T4 gene 60 encodes a subunit of its type II DNA topoisomerase . The mature gene 60 mRNA contains an internal 50 nucleotide region that appears to be bypassed during translation . A 16 amino acid domain of the nascent peptide is necessary for this bypass to occur. Vopr Virusol, 1995 Nov-Dec, 40(6), 279 - 82 {Detection of bovine infectious rhinotracheitis virus by hybridization using nonradioactive DNA-probes}; Oreshkova SF et al.; Biotin-labeled DNA probes for infectious bovine rhinotracheitis virus also known as bovine herpesvirus-1 (BHV-1) have been developed . The procedure is based on dot-blot hybridization using biotin-labeled bacteriophage M13 and plasmid probes containing cloned PstI and EcoRI-PstI restriction fragments of viral genome . The probes obtained were used to detect viral nucleic acids in specimens of bovine spermatic fluid or nasal swabs of calves . The method is simple and rapid, taking less than 24 h, and is highly specific and sensitive, this recommending it for practical veterinary. Biofizika, 1995 Nov-Dec, 40(6), 1265 - 80 {Base plate as a logical controller, matching receptor and other functions of bacteriophage T4}; Ivanitskii GR et al.; It has been shown that the complex functioning of the baseplate of the bacteriophage T4 is based on the high level hierarchy in the structure organization and on the interaction of protein components forming the hub and the "channels" of short and long fibers . The presence of structure proteins with stabilizing and destabilizing functions has been revealed . It has been shown that the process of binding of long tail fibers with cell receptors is a cooperative process . In general the baseplate can be considered as a logic module providing the transition from the adsorbtion to the nonreversible reorganization in the bacteriophage functioning. Genetics, 1995 Nov, 141(3), 825 - 32 The effects of trinucleotide repeats found in human inherited disorders on palindrome inviability in Escherichia coli suggest hairpin folding preferences in vivo; Darlow JM et al.; Unusual DNA secondary structures have been implicated in the expansion of trinucleotide repeat tracts that are associated with several human inherited disorders . We present evidence consistent with the folding of these trinucleotide repeats into hairpin loops at the center of a long DNA palindrome in vivo . Our assay utilizes a palindrome in bacteriophage lambda, the center of which determines its ability to inhibit plaque formation in a manner that is consistent with folding into a hairpin or cruciform structure . We show that central inserts of even numbers of d(CAG).d(CTG) repeats inhibit plaque formation more than do odd numbers . Both d(CAG)2.d(CTG)2 and d(CGG)2.d(CCG)2 central sequences behave like DNA sequences known to form two-base loops in vitro, suggesting that they may also form compact and stable loops . By contrast, repeats of d(GAC).d(GTC) do not show any evidence consistent with unusual loop stability . These results agree with in vitro evidence that the unstable repeats can form hairpin secondary structures and suggest a favored position of folding . We discuss the potential roles of secondary structures, DNA replication and recombination in models of repeat tract expansion. Endocr Res, 1995 Nov, 21(4), 793 - 802 A PCR-derived, non-isotopic labeled prolactin cRNA probe suitable for in situ hybridization; Wu H et al.; We have developed a novel vector-free method for the synthesis of nonisotopic (digoxigenin) labeled prolactin (PRL)-gene specific cRNA probe based on the direct in vitro transcription of DNA template amplified by polymerase chain reaction (PCR) . The T7 and T3 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the '5 end of the oligonucleotides used to prime the PCR . These promoters allowed the subsequent transcription of digoxigenin labeled antisense and sense cRNA probes from the amplified DNA . We successfully utilized these probes to detect specific PRL mRNA in human pituitary and decidua tissues by in situ hybridization (ISH) which can provide identification and localization of PRL-gene expression at a single cell level . This approach avoided time consuming steps which required subcloning of target DNA into the vectors that contains bacteriophage RNA polymerase promoter as well as the need for radioactive materials . This non-isotopic ISH procedure takes less than 72 h from specimen preparation to microscopic analysis and should prove to be useful for molecular biological studies of hormones and clinical diagnosis. Curr Biol, 1995 Nov 1, 5(11), 1312 - 21 Suicide substrates reveal properties of the homology-dependent steps during integrative recombination of bacteriophage lambda; Burgin AB Jr et al.; BACKGROUND: A fundamental feature of bacteriophage lambda site-specific recombination is the strict requirement for a region of sequence identity between recombining DNA duplexes . It has been difficult to understand how the recombination machinery identifies and responds to nonhomologies as subtle as a single base-pair substitution, because the reaction intermediates are transient and there are likely to be several different homology-dependent steps . In order to understand better how the recombination machinery compares parental sequences, we have used the recently developed 'suicide substances'--DNA containing 5'-bridging phosphorothioate linkages--to monitor the timing of homology-sensing relative to the strand cleavage reactions . RESULTS: The cleavage reactions for the two different strands of attB, the bacterial recombination locus for lambda integration, show very different degrees of dependence on homology with the partner locus, attP . Strand cleavage at the B binding site for Int recombinase is insensitive to homology . In contrast, cleavage at the B' binding site strongly depends on homology in the three base pairs adjacent to the B site . Strand cleavage at the B site is apparently required for the readout of this homology but, surprisingly, joining of the cleaved B site to a partner is not . CONCLUSIONS: Our finding that cleavage at the B site is insensitive to homology shows that effective synapsis between partners does not depend on sequence matching . Cleavage at the B' site provides the earliest positive signal for a homology-dependent switch in the lambda recombination machinery . Because this switch can occur in the absence of strand joining, the results argue against models that invoke strand ligation as the critical element of homology-sensing . Alternative mechanisms are presented that involve varieties of non-covalent strand swapping . A synthesis of the present results and other recent experiments highlights the importance of the disannealing of complementary strands and their reannealing to new partners, a process traditionally described as branch migration . The reversibility of branch migration and its bias away from mismatched combinations are proposed to be the major mechanisms of homology-sensing during lambda integration. J Photochem Photobiol B, 1995 Nov, 31(1-2), 57 - 61 DNA UVB dosimeters; Regan JD et al.; DNA can be used to establish and monitor solar UVB dose . Since the principal molecular site of UVB damage in living organisms is DNA, it is logical to quantitate biologically effective solar UVB in DNA dosimeters . In addition to their particular sensitivity to UVB, DNA dosimeters have the advantage of a 2 pi geometry for collecting diffuse UVB radiation from all vectors, low cost, small size and portability, and no moving parts . Both molecular (cyclobutane pyrimidine dimers) and biological (bacteriophage plaques) dosimeters can be quantitated as endpoints to yield the total dose . DNA dosimeters integrate the absorbed energy of all UVB wavelengths (290-320 nm), are highly sensitive to the differential biological effectiveness of these wavelengths, and also integrate over time in hours, days or weeks of exposure . Our experiments have focused on the demonstration of DNA solar dosimeters in the ocean at various depths, the application of the dosimeters to the terrestrial monitoring of solar UVB under various conditions, and the development of a mini-dosimeter which uses nanograms of DNA and is assayed by polymerase chain reaction. Microbiol Res, 1995 Nov, 150(4), 337 - 46 Mutual adaptation of bacteriophage fd, pfd plasmids and their host strains; Geider K et al.; The synthetic plasmid pfdC1 with the replication origin of phage fd and fd gene 2 grows autonomously in E . coli cells . DNA sequencing revealed several mutations compared to the fd genome causing reduced expression of viral gene 2 protein, which can be toxic for the host cell . Another adaptation was noticed for E . coli strains with a copy of fd gene 2 on the F-episome and a pfdA-plasmid with a minimal fd replication origin, when maintained at 42 degrees C . The carrier cells adjusted their cellular metabolism to these stress conditions, whereas replication functions of the plasmid or expression of fd gene 2 on the F-episome were not changed . The filamentous bacteriophages tend to reduce their genome size into miniphages, which was also observed for phages with an antibiotic resistance gene . Bacteriophages with a transposon insertion in the viral gene 2 had a tendency to restore the mutated gene by exchange with the functional gene 2 carried in recA-host cells . Mobilization of pfd-plasmids with RP4 transfer functions was reduced due to interference of replication and transfer in the rolling circle mode . The vectors used in these studies can also be applied as cloning vectors, which are compatible with many other plasmid vectors. Protein Sci, 1995 Nov, 4(11), 2300 - 9 The use of side-chain packing methods in modeling bacteriophage repressor and cro proteins; Chung SY et al.; In recent years, it has been repeatedly demonstrated that the coordinates of the main-chain atoms alone are sufficient to determine the side-chain conformations of buried residues of compact proteins . Given a perfect backbone, the side-chain packing method can predict the side-chain conformations to an accuracy as high as 1.2 A RMS deviation (RMSD) with greater than 80% of the chi angles correct . However, similarly rigorous studies have not been conducted to determine how well these apply, if at all, to the more important problem of homology modeling per se . Specifically, if the available backbone is imperfect, as expected for practical application of homology modeling, can packing constraints alone achieve sufficiently accurate predictions to be useful? Here, by systematically applying such methods to the pairwise modeling of two repressor and two cro proteins from the closely related bacteriophages 434 and P22, we find that when the backbone RMSD is 0.8 A, the prediction on buried side chain is accurate with an RMS error of 1.8 A and approximately 70% of the chi angles correctly predicted . When the backbone RMSD is larger, in the range of 1.6-1.8 A, the prediction quality is still significantly better than random, with RMS error at 2.2 A on the buried side chains and 60% accuracy on chi angles . Together these results suggest the following rules-of-thumb for homology modeling of buried side chains . When the sequence identity between the modeled sequence and the template sequence is > 50% (or, equivalently, the expected backbone RMSD is < 1 A), side-chain packing methods work well . When sequence identity is between 30-50%, reflecting a backbone RMS error of 1-2 A, it is still valid to use side-chain packing methods to predict the buried residues, albeit with care . When sequence identity is below 30% (or backbone RMS error greater than 2 A), the backbone constraint alone is unlikely to produce useful models . Other methods, such as those involving the use of database fragments to reconstruct a template backbone, may be necessary as a complementary guide for modeling. Microbiology, 1995 Nov, 141 ( Pt 11), 2977 - 84 Can phage defence maintain colicin plasmids in Escherichia coli? Feldgarden M, Golden S, Wilson H, Riley MA. We examined the role of plasmid-based phage defence in maintaining plasmids, using colicin plasmids in Escherichia coli as a model system . Experimental data indicated that the possession of a colicin plasmid can confer limited protection against bacteriophages . A continuous culture model, using these experimental values, indicated that the observed limited protection alone could selectively maintain colicin plasmids, without requiring a competitive advantage due to colicinogeny . Phage defence might explain the current maintenance of colicin plasmids, given the naturally occurring high levels of resistance to colicins . This model also suggests that many plasmids might be maintained in natural populations, in part, by phage resistance, including 'cryptic' plasmids for which no phenotype is known. J Gen Virol, 1995 Nov, 76 ( Pt 11), 2859 - 62 Infectivity of algal viruses studied by chlorophyll fluorescence; Rohozinski J et al.; Algal virus infection proceeds via the specific recognition of the host cell wall, penetration of the cell wall and transfer of genetic material into the cytoplasm of the host cell . This process is similar to that which occurs when bacteriophage infect bacteria so that techniques and concepts developed to study bacteriophage are applicable to algal virus studies . By measuring virus-induced changes in chlorophyll fluorescence we have redefined classical studies on the distribution of infectivity . We show that infectivity does not follow a Poisson distribution with a fixed mean, n . By analysing the infectivity of algal viruses over a broad range of virus:cell ratios we have obtained a corrected Poisson distribution that reflects the probability of multiple virus particles attached per cell and is equally applicable to algal viruses and bacteriophage. J Virol, 1995 Nov, 69(11), 6729 - 34 Double-stranded RNA bacteriophage phi 6 protein P4 is an unspecific nucleoside triphosphatase activated by calcium ions; Paatero AO et al.; Double-stranded RNA bacteriophage phi 6 has an envelope surrounding the nucleocapsid (NC) . The NC is composed of a surface protein, P8, and proteins P1, P2, P4, and P7, which form a dodecahedral polymerase complex enclosing the segmented viral genome . Empty polymerase complex particles (procapsids) package positive-sense viral single-stranded RNAs provided that energy is available in the form of nucleoside triphosphates (NTPs) . Photoaffinity labelling of both the NC and the procapsid has earlier been used to show that ATP binds to protein P4 and that the NC hydrolyzes NTPs . Using the NC and the NC core particles (NCs lacking surface protein P8) and purified protein P4, we demonstrate here that multimeric P4 is the active NTPase . Isolation of multimeric P4 is successful only in the presence of NTPs . The activity of P4 is the same in association with the viral particles as it is in pure form . P4 is an unspecific NTPase hydrolyzing ribo-NTPs, deoxy NTPs, and dideoxy NTPs to the corresponding nucleoside diphosphates . The Km of the reaction for ATP, GTP, and UTP is around 0.2 to 0.3 mM . The NTP hydrolysis by P4 absolutely requires residual amounts of Mg2+ ions and is greatly activated when the Ca2+ concentration reaches 0.5 mM . Competition experiments indicate that Mg2+ and Ca2+ ions have approximately equal binding affinities for P4 . They might compete for a common binding site . The nucleotide specificity and enzymatic properties of the P4 NTPase are similar to the NTP hydrolysis reaction conditions needed to translocate and condense the viral positive-sense RNAs to the procapsid particle. Gene, 1995 Oct 27, 164(2), 323 - 7 The bovine DNA polymerase beta promoter: cloning, characterization and comparison with the human core promoter; Chen KH et al.; The core promoter of the human DNA polymerase beta (beta Pol)-encoding gene (POL beta) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins . The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells {Narayan et al., J . Biol . Chem . 269 (1994) 12755-12763} . To study the homologous promoter (ppol beta) in a bovine system, we cloned and characterized the 5'-flanking region of the bovine gene (pol beta) . A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5'-flanking region was isolated from a testis library in bacteriophage lambda EMBL3 . S1 nuclease mapping and primer extension analysis of the 5'-end of the pol beta mRNA identified the major transcription start point (tsp), which is located 142-bp 5' of the translational start codon . In transient expression assays using a bovine cell line, analysis of various 5'-deletion mutants demonstrated that a fragment of only 91-bp 5' of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx . 100-bp core promoter, as in the human promoter (pPOL beta) . Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Sp1-binding sites, and the spacing separating them. Nucleic Acids Res, 1995 Oct 25, 23(20), 4066 - 72 Mutagenic and genotoxic effects of DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II); Yarema KJ et al.; The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli . The adducts studied were cis-{Pt(NH3)2(d(GpG))} (G*G*), cis-{Pt(NH3)2(d(ApG))} (A*G*) and cis-{Pt(NH3)2(d(GpTpG))} (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA . Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA . Following transfection into E . coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes . Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively . Survival of the genome containing the G*TG* adduct did not increase upon SOS induction . In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base . In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts . The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct . No cis-{Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct. Virology, 1995 Oct 20, 213(1), 109 - 21 Bacteriophage P2: genes involved in baseplate assembly; Haggard-Ljungquist E et al.; The sequences of two previously defined tail genes, V and J, of the temperate bacteriophage P2, and those of two new essential tail genes, W and I, were determined . Their order is the late gene promoter, VWJI, followed by the tail fiber genes H and G, and a transcription terminator . The V gene product is the small spike at the tip of the tail, and the J gene product lies at the edge of the baseplate . The W gene product may be homologous to the product of gene 25 of T4 phage, which is part of the T4 baseplate . A temperature-sensitive mutation in gene V affects satellite phage P4 production more than it affects the production of P2 helper phage . P4 mutations that partially compensate for this defect of gene V lie in the P4 capsid size determination gene, sid. Biochim Biophys Acta, 1995 Oct 17, 1264(1), 115 - 20 Conserved sequence patterns in phages Mu and lambda DNA; Stern B et al.; The genetic maps of bacteriophages Mu and lambda can be aligned with respect to the functions of their genes . We were interested to ascertain whether the congruence of gene order is reflected at the nucleotide sequence level . A sliding window analysis of sequences from the early regions of both phages revealed a substantial degree of similarity . Equally high scores, however, were found when the early region of Mu was compared to the late region of lambda and in self-comparisons of either Mu or lambda . Hence, the similarity is due to a common pattern of nucleotides rather than to sequence similarities between functionally related genes . Employing degenerated scoring matrices we could show that primarily adenine and thymine residues contribute to the high scores and that a specific clustering of these residues is the basis for the conserved pattern . Since such a similarity was not observed with control sequences of other phages . Escherichia coli or eukaryotic viruses, the data support the notion that Mu and lambda have diverged from a common phage module . In general, our approach could offer a simple and sensitive way to trace distant relationships. Gene, 1995 Oct 16, 164(1), 149 - 52 Analysis of the DnaK molecular chaperone system of Francisella tularensis; Zuber M et al.; We have cloned the Francisella tularensis (Ft) grpE-dnaK-dnaJ heat-shock genes which are organized in that order . These genes allow heterologous genetic complementation of each respective mutant strain of Escherichia coli (Ec) for bacteriophage lambda growth . The nucleotide sequences of the Ft grpE-dnaK-dnaJ genes and the deduced amino-acid sequences share significant homologies with their respective Ec counterparts . The Ft DnaK and DnaJ proteins cross-react with polyclonal antibodies raised against the respective Ec proteins . The grpE-dnaK-dnaJ genes of Ft are organized in a fashion that is more characteristic of Gram+ bacteria. J Biol Chem, 1995 Oct 13, 270(41), 24509 - 17 Nucleotide and DNA-induced conformational changes in the bacteriophage T7 gene 4 protein; Yong Y et al.; The bacteriophage T7 gene 4 protein is a multifunctional enzyme that has DNA helicase, primase, and deoxyribonucleotide 5'-triphosphatase activities . Prior studies have shown that in the presence of dTTP or dTDP the gene 4 protein assembles into a functionally active hexamer prior to binding to single-stranded DNA . In this study, we have examined the effects of different nucleotide cofactors on the conformation of the gene 4 protein in the presence and absence of DNA . Gel retardation analysis, partial protease digestion, and DNA footprinting all suggest that the gene 4 protein undergoes a conformational change when dTTP is hydrolyzed to dTTP and that in the presence of dTDP the complex with DNA is more open or extended . We have also found that the dissociation constant of the gene 4 protein.DNA complex in the presence of dTDP was 10-fold lower than that determined in the presence of dTTP, further suggesting that these cofactors exerts different allosteric effects on the DNA-binding site of the gene 4 protein. J Mol Biol, 1995 Oct 13, 253(1), 86 - 99 Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system; Conway JF et al.; Bacteriophage capsid assembly pathways provide excellent model systems to study large-scale conformational changes and other mechanisms that regulate the formation of macromolecular complexes . These capsids are formed from proheads: relatively fragile precursor particles which mature by undergoing extensive remodeling . Phage HK97 employs novel features in its strategy for building capsids, including assembly without a scaffolding protein, and the formation of a network of covalent cross-links between neighboring subunits in the mature virion . In addition, proteolytic cleavage of the capsid protein from 42 kDa to 31 kDa is essential for maturation . To investigate the structural bases for proteolysis and cross-linking, we have used cryo-electron micrographs to reconstruct the three-dimensional structures of purified particles from four discrete stages in the assembly pathway: Prohead I, Prohead II, Head I and Head II . Prohead I has icosahedral T = 7 packing of blister-shaped pentamers and hexamers . The pentamers are 5-fold symmetric, but the hexamers exhibit an unusual departure from 6-fold symmetry, as if two trimers had undergone a shear dislocation of about 25 A . Proteolytic conversion to Prohead II leaves the outer surface largely unchanged, but a major loss of density from the inner surface is observed, which we infer to represent the excision of the amino-terminal domains of the capsid protein . Upon expansion to the Head I state, the capsid becomes markedly larger, thinner walled, and more polyhedral: moreover, the capsomer shapes change radically; especially notable is the disappearance of the large hexon dislocation . No differences between Head I and the covalently cross-linked Head II could be observed at the current resolution of about 25 A, from which we infer that it is the conformational rearrangements effected by expansion that create the micro-environments needed for the autocatalytic formation of the isodipeptide bonds found in the mature virions ("pseudo-active sites"). J Mol Biol, 1995 Oct 13, 253(1), 74 - 85 Assembly in vitro of bacteriophage HK97 proheads; Xie Z et al.; Bacteriophage HK97 is a lambdoid phage with a head assembled from 415 copies of a 42 kDa subunit arranged in an icosahedrally symmetrical lattice with a triangulation number of 7 . Prohead I, the first shell structure in the assembly pathway, is composed of 42 kDa coat protein subunits that have not yet undergone the proteolytic cleavage, conformational changes, and covalent cross-linking steps that occur later in the assembly of mature heads . Prohead I can be efficiently dissociated into capsomeres by treatment with 2 M KCl . The resulting capsomeres are a mixture of two species, identified as pentamers and hexamers of the 42 kDa subunit . These capsomeres were also detected as the products of chaperonin-assisted renaturation of 42 kDa polypeptide in vitro at room temperature or in the course of self folding and assembly in vitro at 0 degrees C . Pentamer and hexamer capsomeres can be interconverted in vitro by manipulating solvent conditions, and this makes it possible to carry out the in vitro shell assembly reaction at different input ratios of hexamer to pentamer . The Prohead I structures produced are always the normal (T = 7) size regardless of the input pentamer to hexamer ratio . Assembly is most efficient when the pentamer to hexamer ratio is 1:5 (a mass ratio of 1:6), or the same as the capsomere ratio in a T = 7 shell. J Immunol Methods, 1995 Oct 12, 186(1), 125 - 35 Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes; Davies EL et al.; In chickens, single functional immunoglobulin variable and joining gene segments at each of the heavy and light chain loci undergo V(D)J rearrangement . Diversity is subsequently introduced by conversions templated by upstream pseudo V region genes in such a way that practically all V regions in mature B cells have identical ends . This greatly simplifies the representative amplification of V region genes . Furthermore, the entire naive repertoire of the adult chicken is produced in the bursa of Fabricius of the young bird . These special properties of the generation of immunoglobulin diversity in chickens have been exploited in the development of procedures to produce large libraries of diverse antibody combining sites derived from chicken Ig genes and expressed on filamentous bacteriophage . The utility of this library was assessed by selection of specifically binding phage using three solid phase-bound protein antigens, hen egg white lysozyme, bovine thyroglobulin and bovine serum albumin . The sequences of the V region genes thus isolated demonstrated that selection was specific and that the library contained useful diversity of binding sites . This library provides access to a repertoire whose diversity is based on a mechanism different from that underlying previously available libraries . The demonstrated feasibility of generating chicken phage antibodies may lead to the production of monoclonal reagents from immunised chickens, and the derivation of reagents for studying immunoglobulin mediated selection in avian B cell development. Nucleic Acids Res, 1995 Oct 11, 23(19), 3816 - 21 Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase; Kanegae Y et al.; A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed . To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed . Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells . These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals . The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad. Biochemistry, 1995 Oct 10, 34(40), 13109 - 16 Domains of Mnt repressor: roles in tetramer formation, protein stability, and operator DNA binding; Waldburger CD et al.; The Mnt repressor of bacteriophage P22 is a member of the ribbon-helix-helix family of gene regulatory proteins . Proteolytic cleavage of Mnt with chymotrypsin reveals that it consists of two structural domains . Both domains are required for high-affinity operator binding . The N domain (residues 1-51) is dimeric and binds weakly but specifically to operator DNA . The C domain (residues 52-82) forms an independent alpha-helical, tetramerization domain and, by itself, has no DNA-binding activity . In intact Mnt, the N and C domains help to stabilize each other against denaturation but appear to be linked rather flexibly . Assays of the half-operator affinities of Mnt and the isolated N domain indicate that binding to adjacent half-sites in the whole operator is stabilized by protein-protein contacts between N domains in addition to protein-protein contacts between C domains. J Mol Biol, 1995 Oct 6, 252(5), 596 - 610 The modular character of a DNA junction-resolving enzyme: a zinc-binding motif in bacteriophage T4 endonuclease VII; Giraud-Panis MJ et al.; Bacteriophage T4 endonuclease VII is one of a class of structure-selective enzymes that resolve helical branchpoints in DNA molecules . The sequence of this protein suggests a modular organisation . We have expressed a synthetic gene encoding endonuclease VII, which has been used in a directed mutagenesis exercise, with the aim of understanding the role of different sections of the protein sequence . Towards the N-terminal end of the protein lies a section of polypeptide in which four cysteine residues distributed in a CxxC--CxxC pattern co-ordinate one atom of zinc . The N-terminal section composed of amino acid residues 1 to 65 isolated from the remaining C-terminal section also binds one mole of zinc, suggesting that this region folds autonomously . Mutation shows that the outer cysteine residues are essential for zinc binding, while the inner cysteine residues are partially degenerate in that either one of the two (but not both) can be replaced while retaining some zinc . The activity as a junction-resolving enzyme correlated qualitatively with the presence of the zinc . In the C-terminal part of the protein lies a section that is 48% identical with a sequence found in the DNA repair protein T4 endonuclease V . We can replace the section of T4 endonuclease VII with the corresponding sequence from T4 endonuclease V with no change in the pattern of cleavage on four-way junctions . The evidence supports a modular construction for T4 endonuclease VII. Immunol Rev, 1995 Oct, 147, 31 - 52 Antigen-presenting function of the TL antigen and mouse CD1 molecules; Cheroutre H et al.; The hallmark of all the nonclassical antigen-presenting molecules, including nonclassical class I and nonclassical class II (Karlsson et al . 1992) molecules, is their lack of polymorphism . It is presumed, therefore, that these nonclassical molecules must have a distinct antigen-presenting function in which polymorphism is not advantageous . In some cases this may involve presentation of a nonpeptide antigen, as has been demonstrated for human CD1b . It is possible that a molecule adapted to present bacterial lipids would remain relatively nonpolymorphic, because a lipid, which is the end product of a complex biosynthetic pathway, is likely to evolve less rapidly than a short stretch of amino acid sequence containing a T-cell epitope . Alternatively, the lack of polymorphism could reflect the presentation by these molecules of relatively invariant peptides, such as those derived from heat shock proteins . It also is possible that a nonpolymorphic molecule could be selected for the presentation of modified peptides . An example of this is the M3 molecule, which can bind even short peptides as long as they have a formylated N-terminus (Fischer Lindahl et al . 1991) . Based upon their structural differences, we believe it is likely that the TL antigen and mCD1 are likely to present different types of ligands . The presence in the TL antigen of the conserved amino acids, which in class I normally from hydrogen bonds with peptides, suggests that the TL antigen also can present nanomeric peptides . A peptide antigen-presenting function also is suggested by the expression of the TL antigen by at least one antigen-presenting cell type, the epithelial cell of the intestine, and by the ability of alloreactive T cells to recognize the TL molecule . While we favor the hypothesis that the TL antigen presents peptides, the data cited above do not constitute formal proof of any kind of antigen-presenting function, and it remains possible that the TL antigen does something else . As noted above, no attempts to elucidate the structure of the ligands bound to the TL antigen have so far succeeded, including the screening of bacteriophage display libraries (Castano, A.R., Miller, J.E., Holcombe, H.R., unpublished data) . In contrast, our recent work has demonstrated that mCD1 presents relatively long peptides with a structured motif distinct from classical class I molecules . This mCD1-binding motif, which is present in a wide range of proteins, does not by itself provide a simple explanation for the lack of mCD1 polymorphism and, as noted above, it remains possible that the natural ligand for mCD1 is a nonpeptide structure . Besides their lack of polymorphism, the TL antigen and mCD1 molecules share two additional features in common which might give insight into their their biological role . First, their surface expression does not depend upon the presence of a functional TAP transporter, and they probably can reach the cell surface as empty molecules . Second, both molecules are expressed by epithelial cells in the intestine . This leads to the speculation that these two nonclassical class I molecules could be involved in sampling or uptake of lumenal peptides for their ultimate presentation to cells of the systematic immune system . For example, longer lumenal peptides could be taken up by mCD1, and perhaps by the TL antigen, and then further processed to nonamers for presentation by classical class I molecules . They also could be transported across the epithelial cell by the TL antigen or mCD1 and subsequently presented by either class I or class II molecules expressed by cells in the lamina propria . This sampling or uptake mediated by either the TL antigen or mCD1 could play a role in the induction of immune responses, or more likely perhaps, in the induction of systemic oral tolerance to peptide antigens.(ABSTRACT TRUNCATED) Farmaco, 1995 Oct, 50(10), 669 - 78 Synthesis and photobiological properties of 3-acylangelicins, 3-alkoxycarbonylangelicins and related derivatives; Iester M et al.; Convenient synthesis of 3-acyl-2H-furo{2,3-h}-1-benzopyran-2-ones, esters of 2-oxo-2H-furo{2,3-h}-1-benzopyran-3-carboxylic acid and 2H-furo{2,3-h}-1-benzopyran-3-carboxamides was accomplished via aromatization of the adducts obtained by a reaction between (E)-5-dimethyl-aminomethylene-6,7-dihydrobenzofuran-4(5H)-one and the appropriate acylacetate or dialkyl malonate . These compounds are angelicin derivatives which were prepared with the aim of obtaining intrinsically monofunctional drugs for photochemotherapy, with only one photoreactive site in their molecule . The new angelicins appear to be free of the known phototoxicity of furocoumarins on the skin and at a genetic level . The 3-carboxylic esters showed significant antiproliferative activity in Ehrlich ascites cells and T2 bacteriophage; the other derivatives were only slightly effective . The features of these compounds are such that they represent a new model for non-toxic agents for photochemotherapy. Res Microbiol, 1995 Oct, 146(8), 601 - 16 Mu transposase-stimulated illegitimate recombination of Tn3kan- and IS101-containing plasmids; Cameron RK et al.; The transposable bacteriophage Mu and the mobile genetic elements Tn3 and IS101 replicatively transpose to random target sites, produce 5 bp target site duplications, and contain the sequence 5'-PuCGAAAPu-3' starting at bp 21 from their ends . The presence of these shared characteristics, plus the fact that Mu transposase can specifically bind to the termini of Tn3 and IS101 in vitro, suggests that the elements may be evolutionarily conserved and retain some functional capacity to transpose each other's DNA . To examine this proposition, in vivo transposition-mating assays were performed and demonstrated that Mu transposase stimulated the formation of recA-independent recombination products between Tn3kan- or IS101-containing plasmids and a target plasmid (pOX38cam) up to 200-fold . However, when transferred to recA+ hosts, these recA-independent products yielded resolution products suggestive of illegitimate recombination, as similar recombination and resolution products were generated, at reduced frequencies, in the absence of Mu transposase . Thus, Mu transposase may stimulate a host-mediated, recA-independent illegitimate recombination reaction . As adjacent pSC101 sequences, including a formerly unknown but functional IHF site (bp 2238-2251), were required for Mu transposase-stimulated IS101 illegitimate recombination, IHF may be one of the putative host factors involved in these recombination reactions. Mamm Genome, 1995 Oct, 6(10), 714 - 24 A small-insert bovine genomic library highly enriched for microsatellite repeat sequences; Stone RT et al.; A bovine genomic phagemid library was constructed with randomly sheared DNA . Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones . The 14% of positive clones with (CA.GT) > 12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries . Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers . The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library . Only GCT, GGT, and GGAT were estimated to have a frequency of > 100 per genome . Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine . Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA . In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers. Protein Sci, 1995 Oct, 4(10), 1998 - 2005 Crystal structure of thioltransferase at 2.2 A resolution; Katti SK et al.; We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution . The protein is known for its thiol-redox properties and dehydroascorbate reductase activity . Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique . The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives . The protein folds into an alpha/beta structure with a four-stranded mixed beta-sheet in the core, flanked on either side by helices . The fold is similar to that found in other thiol-redox proteins, viz . E . coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins . The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface . Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule . Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration . Mutational data available on the protein are in agreement with the three-dimensional structure. Protein Expr Purif, 1995 Oct, 6(5), 625 - 31 Expression and functional characterization of Escherichia coli NusA and lambda Q as glutathione S-transferase fusion proteins; Zhang Y et al.; The Escherichia coli transcription factor NusA and the bacteriophage lambda antiterminator Q proteins were expressed as inducible glutathione S-transferase (GST) fusion proteins . The fusion proteins were purified under nondenaturing conditions by affinity chromatography on glutathione agarose . Thrombin cleavage of the glutathione agarose-bound fusion proteins yielded homogeneously pure NusAN+15 (5 mg/g cells) and almost homogeneously pure QN+13 protein (0.7 mg/g cells), where N+x indicates the presence of x additional amino acids at the N-terminus of the protein . The purified NusAN+15 exhibited the same activities as wildtype NusA in enhancement of transcriptional pausing, enhancement of termination at Rho-independent terminators, and enhancement of Q-mediated antitermination in vitro . The QN+13 protein exhibited both anti-pausing and antitermination activities in Q-mediated transcription antitermination . However, the antitermination activity of QN+13 was lost gradually during storage if the thrombin used for cleavage of the GST fusion protein was not removed . This was due to cleavage by thrombin after Arg22 within the Q protein itself, at a noncanonical thrombin cleavage site, so the truncated protein (QN+22) lacked the first 22 amino acids at the N-terminus of Q . The expression vectors described here can be used to rapidly produce large quantities of these proteins, and the truncated Q protein can be used to evaluate the requirement for the N-terminus of Q in antitermination, anti-pausing, interactions with the DNA template (qut site), and interaction with RNA polymerase itself. Protein Expr Purif, 1995 Oct, 6(5), 609 - 18 Purification of recombinant human rhinovirus 14 3C protease expressed in Escherichia coli; Birch GM et al.; A gene encoding the human rhinovirus 14 (HRV14) sequence for expression of the viral polypeptide protein delta 3ABC was inserted into a plasmid driven by the heat-inducible bacteriophage lambda PL promoter . The coding sequence was also inserted into a pET vector for expression in the T7 system to produce 13C, 15N-labeled protein . The expressed HRV14 3C protease (3Cpro) autocatalytically cleaved itself from the polyprotein delta 3ABC, and the mature HRV14 3Cpro partitioned predominantly, in the case of the T7 system, in the insoluble fraction and exclusively, in the case of the PL system, in the insoluble fraction . The insoluble HRV14 3Cpro was solubilized in urea and purified using anion- and cation-exchange chromatography . The protease was refolded/activated and further purified using a size-exclusion column . HRV14 3Cpro was purified to > 90% homogeneity as shown by SDS-PAGE and to 95% by HPLC . A continuous fluorescence assay was developed which utilized an intramolecularly quenched 9-amino-acid substrate . The substrate anthranilic acid (Anc)-Thr-Leu-Phe-Gln-Gly-Pro-Val-(p-NO2)-Phe-Lys mimicked the natural 2C/3A cleavage site (Thr-Leu-Phe-Gln-Gly-Pro-Val-Tyr-Phe) using an N-terminal anthranilic acid donor group on one side of the scissile bond (Gln/Gly) and a p-NO2-Phe acceptor group at the P4 position . Measured by the fluorescence assay, HRV14 3Cpro had a Km of 300 microM for the substrate. Protein Expr Purif, 1995 Oct, 6(5), 559 - 69 Requirements for optimal expression of secreted and nonsecreted recombinant proteins in vaccinia virus systems; Pfleiderer M et al.; Selection of an optimal promoter is necessary for efficient expression of foreign genes with vaccinia virus . Since a variety of powerful (homologous) vaccinia virus promoters and foreign (heterologous) promoter systems have been described for use in vaccinia, we have addressed the question of whether a general rule exists that allows the prediction of the optimal promoter/gene combination . We have compared the expression properties of four secreted proteins, the human blood clotting factor IX (FIX), the human blood glycoprotein Protein S (ProtS), the human von Willebrand factor (vWF), and the Hepatitis B virus (HBV) middle surface glycoprotein preS2, with proteins that were reported not to be secreted, the HBV large surface glycoprotein preS1 and the murine leukemia virus (MuLV) BM-5 Eco gag protein . In addition, we have included in our study an internal control protein, the vaccinia virus p11 protein, to monitor possible side effects of the promoter system used . Genes encoding the foreign proteins were placed either under control of a synthetic vaccinia virus early/late promoter (selP) or under control of the bacteriophage T7 promoter (T7/emc system) . The secreted proteins were more efficiently expressed when fused to the homologous promoter . Direct comparison of the two promoters indicated that the expression level ranged between 1.4 (ProtS) and 3.9 (FIX)-fold higher with the selP than with the T7 promoter . In contrast, the cell-associated HBV preS1 was more efficiently expressed under the T7 promoter and the MuLV BM-5 Eco gag polypeptide was expressed equally well from both promoters . These data indicate that a careful prediction of optimal promoter/foreign gene combinations for the vaccinia virus expression system is possible . The choice of the optimal promoter/expression system is based on a simple classification scheme, discriminating secreted and nonsecreted proteins. Biophys J, 1995 Oct, 69(4), 1382 - 6 A Monte Carlo model of fd and Pf1 coat proteins in lipid membranes; Milik M et al.; A Monte Carlo Dynamics simulation was used to investigate the behavior of filamentous bacteriophage coat proteins in a model membrane environment . Our simulation agrees with the previous experimental observations that despite the low sequence similarity between the major coat proteins of Pf1 and fd bacteriophages, their structure in the membrane environment is very similar . These results support the hypothesis that the hydrophobic effect exerts an important influence on membrane protein structure . The model may also be used for modeling the insertion and transport processes in protein-membrane systems . The example of fd protein was also used as a test of sensitivity of our model to temperature, thickness of the hydrocarbon phase, and simulation time . In all cases, the results were independent (over the tested range) of the particular values of the parameters. Biophys J, 1995 Oct, 69(4), 1355 - 62 Double-stranded DNA organization in bacteriophage heads: an alternative toroid-based model; Hud NV; Studies of the organization of double-stranded DNA within bacteriophage heads during the past four decades have produced a wealth of data . However, despite the presentation of numerous models, the true organization of DNA within phage heads remains unresolved . The observations of toroidal DNA structures in electron micrographs of phage lysates have long been cited as support for the organization of DNA in a spool-like fashion . This particular model, like all other models, has not been found to be consistent will all available data . Recently we proposed that DNA within toroidal condensates produced in vitro is organized in a manner significantly different from that suggested by the spool model . This new toroid model has allowed the development of an alternative model for DNA organization within bacteriophage heads that is consistent with a wide range of biophysical data . Here we propose that bacteriophage DNA is packaged in a toroid that is folded into a highly compact structure. J Bacteriol, 1995 Oct, 177(20), 5937 - 42 Characterization of Mu prophage lacking the central strong gyrase binding site: localization of the block in replication; Pato ML et al.; Bacteriophage Mu contains an unusually strong DNA gyrase binding site (SGS), located near the center of its genome, that is required for efficient Mu DNA replication (M . L . Pato, Proc . Natl . Acad . Sci . USA 91:7056-7060, 1994; M . L . Pato, M . M . Howe, and N . P . Higgins, Proc . Natl . Acad . Sci . USA 87:8716-8720, 1990) . Replication of wild-type Mu initiates about 10 min after induction of a lysogen, while replication in the absence of the SGS is delayed about an hour . To determine which step in the replication pathway is blocked in the absence of the SGS, we inactivated the SGS by deletion and by insertion and studied the effects of these alterations on various stages of Mu DNA replication . Following induction in the absence of a functional SGS, early transcription and synthesis of the Mu-encoded replication proteins occurred normally . However, neither strand transfer nor cleavage at the Mu genome termini could be detected 40 min after induction . The data are most consistent with a requirement for the SGS in the efficient synapsis of the Mu prophage termini to form a separate chromosomal domain. Mutat Res, 1995 Oct, 338(1-6), 203 - 13 Use of transgenic mouse models for studying somatic mutations in aging; Martus HJ et al.; Theories on the causes of aging, based on the accumulation of somatic mutations in tissues of an organism, were formulated decades ago, but remain insufficiently tested . Transgenic animals, equipped with integrated bacterial reporter genes that can be efficiently rescued from total genomic DNA of all tissues and organs, represent ideal tools for investigating the types and frequencies of spontaneous mutants accumulating during aging . The first of such systems, based on the transgenic integration of bacteriophage lambda shuttle vectors that contain the bacterial lacZ gene as mutational target, was constructed in our laboratory and is now routinely used . Results obtained with this and the related LacI system that are relevant for the somatic mutation theory of aging will be discussed . One conclusion is that, due to the nature of the transgene, lambda-based systems have the disadvantage that deletion type mutations are underrepresented in comparison to point mutations . To overcome those limitations, we constructed a new transgenic mouse model carrying a pUR288 plasmid shuttle vector with the lacZ reporter gene . Some preliminary data obtained with this model serve to illustrate its potential use to extensively test the somatic mutation theory of aging. Infect Immun, 1995 Oct, 63(10), 4016 - 23 Association of lysogenic bacteriophage MAV1 with virulence of Mycoplasma arthritidis; Voelker LL et al.; Mycoplasma arthritidis causes a severe polyarthritis under natural conditions in rats and under experimental conditions in both rats and mice . Although the disease itself has been extensively studied, M . arthritidis virulence factors remain uncharacterized . Comparison of relative arthritogenicity of 20 strains of M . arthritidis revealed that the strains tended to fall into two groups, a highly arthritogenic group, inducing maximum arthritis scores of > or = 11 in rats, and a low-virulence group, inducing maximum scores of < 6 . Chromosomal DNA from the more highly arthritogenic strains possessed sequences that hybridized by Southern analysis with a probe prepared from lysogenic M . arthritidis bacteriophage MAV1, while DNA from low-virulence strains did not . One of the low-virulence strains, 158, was experimentally lysogenized with MAV1 . Lysogenized 158 showed a significant increase in arthritogenicity over nonlysogenized 158 . These data suggest that MAV1 carries a factor that is important in pathogenesis of M . arthritidis-induced arthritis of rats. J Virol, 1995 Oct, 69(10), 6131 - 9 Vaccinia virus gene A18R encodes an essential DNA helicase; Simpson DA et al.; The vaccinia virus A18R protein is a DNA-dependent ATPase that contains the canonical sequence motifs associated with the DEXH group of DNA and RNA helicases . Investigation of A18R protein function during infection indicated it functions in the early and late phases of vaccinia virus transcription . The A18R protein shares sequence similarity with the mammalian DNA helicase ERCC3 . The ERCC3 protein has a dual function: it is a component of the transcription factor TFIIH and is an essential participant in the cellular nucleotide excision repair pathway . Here we present evidence that the A18R protein is a DNA helicase that unwinds duplex DNA in a 3'-to-5' direction . The A18R helicase was inactive on RNA-DNA and RNA-RNA hybrids . The A18R unwinding activity was most efficient on DNA substrates with lengths of 20 nucleotides or less, and its unwinding activity was not stimulated by the addition of Escherichia coli single-strand-binding protein (SSB), the bacteriophage T4 gene 32 SSB, or the vaccinia virus I3L protein, a putative SSB . We have used an electrophoretic gel mobility shift assay to show that the A18R protein forms a stable complex with single-stranded DNA, and to a lesser extent RNA, in a reaction that does not require ATP. Biochemistry, 1995 Sep 26, 34(38), 12388 - 97 Accessibility and dynamics of Cys residues in Bacteriophage IKe and M13 major coat protein mutants; Khan AR et al.; The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion . To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein Cys mutants was exploited to probe the mobility and environment of this residue at several loci within the hydrophobic domain of these proteins . IKe mutants P30C, G39C, and G39C-V36A and M13 mutant Y24C-V31A, each previously obtained from randomized mutagenesis, were characterized in the intact virion, the intermediate spheroidal S-form, and in membrane-mimetic sodium dodecyl sulfate (SDS) micelles . The accessibility of the Cys sulfhydryl in the virion was examined by reaction with {14C}iodoacetamide (14C-IAN) and other alkylating agents . The IKe mutants G39C and G39C-V36A were found to be the most reactive with 14C-IAN and thus the most accessible, although this accessibility was subject to strict steric constraints since only the smallest sulfhydryl-specific alkylating agents were able to modify the Cys39 locus . The spin probe proxyliodoacetamide (PIAN) was used to characterize Cys side chain mobility by electron paramagnetic resonance (EPR) spectroscopy . The M13 mutant Y24C-V31A Cys side chain in the phage was observed to be the most mobile, with slightly less mobility for IKe mutant P30C and considerably less for G39C mutants . The SDS micelle-bound forms of the Cys mutants all exhibited enhanced side chain mobility compared to the virion form, with the extent of mobility dependent upon the specific location of the Cys residue . EPR and fluorescence quenching results show that the Cys side chain in the Y24C-V31A S-form is largely immobilized and inaccessible in comparison to the virion and micelle-solubilized forms . The overall results are interpreted in terms of the structural changes accompanying disassembly and insertion of the coat protein into the Escherichia coli inner membrane. Nucleic Acids Res, 1995 Sep 25, 23(18), 3764 - 70 Replacing tryptophan-128 of T4 endonuclease V with a serine residue results in decreased enzymatic activity in vitro and in vivo; Valerie K; Endonuclease V of bacteriophage T4 possesses two enzymatic activities, a DNA N-glycosylase specific for cyclobutane pyrimidine dimers (CBPD) and an associated apurinic/apyrimidinic (AP) lyase . Extensive structural and functional studies of endonuclease V have revealed that specific amino acids are associated with these two activities . Controversy still exists regarding the role of the aromatic amino acid stretch close to the carboxyl terminus, in particular the tryptophan at position 128 . We have expressed wild-type and mutant W128S endonuclease V in Escherichia coli from an inducible tac promoter . Purified W128S endonuclease V demonstrated substantially decreased N-glycosylase (approximately 5-fold) and AP lyase (10- to 20-fold) activities in vitro compared to the wild-type enzyme when a UV-irradiated poly(dA)-poly(dT) substrate was used . However, a much smaller difference in AP lyase activity between the two forms was observed with a site-specific abasic oligonucleotide . The difference in enzymatic activity was qualitatively, but not quantitatively, reflected in the survival of UV-irradiated bacteria, that is the W128S cells were slightly less UV resistant than wild-type cells . No difference was observed in the complementation of UV repair using UV-damaged denV- T4 phage . A more pronounced difference between the wild-type and W128S proteins was observed in human xeroderma pigmentosum group A cells by host-cell reactivation of a UV-irradiated reporter gene . The relatively large discrepancy between the in vitro and in vivo results observed with bacteria may be because saturated levels of DNA repair are obtained in vivo with relatively low levels of endonuclease V . However, under limiting in vitro conditions and in human cells in vivo a considerable difference between the W128S mutant and wild-type endonuclease V activities can be detected . Our results demonstrate that tryptophan-128 is important for endonuclease V activity. J Biol Chem, 1995 Sep 22, 270(38), 22541 - 7 Identification, cloning, and characterization of the bacteriophage N4 gene encoding the single-stranded DNA-binding protein . A protein required for phage replication, recombination, and late transcription; Choi M et al.; The coliphage N4-coded single-stranded DNA-binding protein (N4SSB) is essential for phage replication and for expression of the phage late genes, which are transcribed by the Escherichia coli sigma 70 RNA polymerase . As a first step in investigating the role of N4SSB in replication and transcriptional activation, we have identified and sequenced the N4SSB gene . The gene encodes a 265-amino acid protein with no apparent sequence homology to other single-stranded DNA-binding proteins . We present data indicating that N4SSB is also essential for phage recombination . Mutational analysis of the carboxyl terminus of the protein indicates that this region is required for protein-protein interactions with the N4 replication, N4 recombination, and E . coli transcriptional machineries, while the rest of the protein contains the determinants for single-stranded DNA binding. J Biol Chem, 1995 Sep 22, 270(38), 22236 - 42 Bacteriophage T4 Dda helicase translocates in a unidirectional fashion on single-stranded DNA; Raney KD et al.; The T4 bacteriophage Dda helicase is believed to be involved in early events in T4 DNA replication and has been shown to stimulate genetic recombination processes in vitro . Dda unwinds double-stranded DNA with 5' to 3' polarity but its ability to translocate on DNA has not been established . The DNA stimulated ATPase activity of Dda helicase has been used to probe translocation on single-strand DNA (ssDNA) . Dda exhibits higher ATPase activity in the presence of poly(dT) than oligo(dT)16, indicating that Dda translocates on ssDNA . Oligonucleotides containing biotin/streptavidin blocks on the 5' or 3' end were used to probe directionality of translocation . The Kact (Km for DNA) for Dda ATPase activity was reduced in the presence of a streptavidin block on the 3' end, whereas a streptavidin block on the 5' end had only a small effect on the steady-state ATPase parameters . These results suggest that Dda translocates unidirectionally in a 5' to 3' manner and upon encountering the block remains bound to the oligonucleotide rather than sliding off the 3' end . The direction of translocation on ssDNA is consistent with the direction in which Dda unwinds duplex DNA and is not dependent on duplex structure. Biochemistry, 1995 Sep 19, 34(37), 11970 - 8 Relationship between in vivo activity and in vitro measures of function and stability of a protein; Sandberg WS et al.; The in vivo activities of mutant proteins are readily measured and can potentially be used to estimate changes in in vitro properties such as stability or function, but this connection has not been rigorously established . Gene V protein is a small protein produced by bacteriophage f1 that binds to single-stranded DNA and to RNA and for which fitness can be assayed both in vivo and in vitro . We have assembled a large number of temperature-sensitive mutants of the gene V protein of bacteriophage f1 and measured their ability to support phage growth and replication in vivo . We have also purified many of these mutant gene V proteins and measured their stabilities and ssDNA binding affinities in vitro . Mutations at surface residues frequently yielded temperature-sensitive mutants, but remarkably, no overall correlation between in vivo activity and in vitro measures of either stability or function was found for this group . Mutations at buried residues often lead to the temperature-sensitive phenotype . At buried sites temperature sensitivity was strongly correlated with in vitro stability changes, but not with in vitro ssDNA binding affinity . The implication of these observations for protein engineering efforts is that phenotypes conferred by amino acid substitutions at buried sites can be used to identify mutants whose stabilities fall into ranges of interest, while phenotypes of mutants with surface substitutions may be much less readily interpreted, even in the case of a single-stranded-DNA-binding protein. J Mol Biol, 1995 Sep 15, 252(2), 178 - 88 Identifying determinants of recombination specificity: construction and characterization of mutant bacteriophage integrases; Dorgai L et al.; The Integrases of bacteriophages lambda and HK022 promote recombination between DNA molecules that carry attachment sites . The two integrases are about 70% identical in sequence and catalyze nearly identical reactions, but recognize different sets of sites . To identify the amino acids that determine this difference in specificity, we selected mutants of lambda integrase with increased ability to recombine HK022 sites . This selection yielded eleven different amino acid substitutions at eight different positions . Three of the positions belong to a larger set that were identified as important for the lambda/HK022 specificity difference by analysis of chimeric integrases . Substitution of the HK022 for the corresponding lambda residue at each of these three positions increased recombination of HK022 sites, and one double substitution, N99D-E319R, increased recombination to nearly wild-type HK022 levels . Mutations at the other five positions changed residues that are identical in the wild-type proteins or are at positions identified by chimera analysis as unimportant for the lambda/HK022 specificity difference . All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine lambda sites . However, we found that substitution of HK022 for lambda residues at three additional positions, S282P, G283K, and R287K, specifically reduced recombination of lambda sites . These three substitutions when combined with N99D and E319R were sufficient to change the specificity of lambda to that of HK022 integrase . The first three substitutions act principally to prevent recombination of lambda sites, and the second two to remove a barrier to recombination of HK022 sites . We suggest that many natural alterations in the specificity of protein-DNA interactions occur by multi-step changes that first relax and then restrict specificity. J Mol Biol, 1995 Sep 15, 252(2), 163 - 77 Identifying determinants of recombination specificity: construction and characterization of chimeric bacteriophage integrases; Yagil E et al.; Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites . Phages lambda and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment sites . To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of lambda and HK022 sites in vivo . A chimera that is of lambda origin except for one HK022 residue at position 99 and 12 HK022 residues located between positions 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination . Chimeras containing certain subsets of these 13 residues had incomplete specificity . The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis . Many chimeras were inactive in recombining either HK022 or lambda sites . Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other. Cancer Res, 1995 Sep 15, 55(18), 4023 - 8 Genetic suppressor elements: new tools for molecular oncology--thirteenth Cornelius P . Rhoads Memorial Award Lecture; Roninson IB et al.; Genetic suppressor elements (GSEs) are short biologically active gene fragments that encode dominantly acting peptides or inhibitory antisense RNAs . GSEs can be isolated from a single gene or from a multigene complex by constructing a library of short random fragments of the target gene(s) in an expression vector, followed by expression selection for the desired phenotype in a suitable cellular system . GSE selection from a single gene allows one to develop efficient and specific inhibitors of the gene function and to identify functional protein domains . GSE selection from a multigene complex, such as a normalized (uniform abundance) cDNA population from mammalian cells, makes it possible to identify genes that are involved in selectable cellular phenotypes . The potential of GSE selection for uncovering novel gene functions was first demonstrated using bacteriophage lambda as a model system . GSE selection in retroviral expression vectors has been applied in mammalian cells to identify genes responsible for sensitivity to etoposide and other chemotherapeutic drugs . GSE selection is also useful for cloning and analysis of tumor suppressor genes and can be applied to identifying tumor-specific targets for future anticancer drugs . Investigators should find this experimental strategy applicable to many different areas of medical and biological research. Virology, 1995 Sep 10, 212(1), 213 - 7 Packaging of multiple copies of reduced-size genomic segments by bacteriophage phi 6; Mindich L et al.; Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid . The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction . Packaging of individual segments is dependent upon unique packaging sequences of about 300 nucleotides near the 5' ends of the segments . We have prepared segments L, M, and S with internal deletions that decrease their size by as much as sixfold without affecting either their packaging sequences or their 3' ends . Although packaging of genomic segments is normally very precise, with only one of each in a procapsid, these smaller segments are packaged in multiples such that the total number of nucleotides for each segment class approaches that of the normal genomic segment. Virology, 1995 Sep 10, 212(1), 204 - 12 Acquisition of a fourth genomic segment in bacteriophage phi 6, a bacteriophage with a genome of three segments of dsRNA; Onodera S et al.; Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid . Packaging of individual segments is dependent upon unique packaging sequences near the 5' ends of the segments . We have prepared deletions in segments L and M that decrease their size by half . Phages with these deletions can be propagated on host strains carrying plasmids with complementing genes . The deletion segments are present in two copies per virion . Phage carrying a deletion segment can acquire the transcript of the complementing plasmid if the latter has a packaging sequence . If the packaging sequence is homologous to that of the deletion segment, acquisition occurs at high frequency . If it is heterologous, then recombination exchanges the heterologous packaging sequence for a homologous one or it attaches the transcript to one of the other genomic segments. Virology, 1995 Sep 10, 212(1), 128 - 33 DNA sequence of tail fiber genes of coliphage 186 and evidence for a common ancestor shared by dsDNA phage fiber genes; Xue Q et al.; We present here the nucleotide sequence of the tail fiber genes of phage 186 . Marker rescue was used to associate an open reading frame (ORF) of 462 codons with the previously known tail gene K . A downstream ORF, encoding a 166-amino-acid product, was designated orf 45 . Comparative studies suggested that K encodes the tail fiber protein and that orf 45 encodes an assembly protein . K protein contains a succession of short amino acid sequences (motifs) that are homologous with sequences from the tail fiber proteins of unrelated bacteriophages . The fact that these sequence motifs are variously present in the tail fiber proteins of unrelated bacteriophages has been advanced as evidence for horizontal transfer in the evolution of the associated tail fiber genes . However, the fact that the order of the various motifs in the proteins is invariant emphasizes the probability that independent divergence from a common ancestor also played a major role in the evolution of the tail fiber genes. Cell, 1995 Sep 8, 82(5), 701 - 8 Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA; Mol CD et al.; Uracil-DNA glycosylase inhibitor (Ugi) is a B . subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG) . The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket . The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules . Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop . The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications. J Mol Biol, 1995 Sep 8, 252(1), 70 - 85 DNA palindromes adopt a methylation-resistant conformation that is consistent with DNA cruciform or hairpin formation in vivo; Allers T et al.; Long DNA palindromes present a threat to genomic stability and are not tolerated in Escherichia coli . It has been suggested that this is a consequence of cruciform or hairpin formation by palindromic sequences . This work describes a methylation inhibition assay for unusual DNA secondary structure in vivo that is both internally controlled and non-invasive . If a palindrome with a central GATC target site for Dam methylase assumes a cruciform or hairpin conformation in vivo, then the GATC sequence will be located in a single-stranded loop and will consequently not be modified . The centre of a long perfect palindrome located in bacteriophage lambda is shown to be methylation-resistant in vivo . Changes to the central sequence and insertions of 10 base-pairs of asymmetric sequence do not alter the degree of under-methylation, but insertions of 20 base-pairs or more of asymmetric sequence reduce the under-methylation of the palindrome centre . We also show that the centres of long palindromes are more under-methylated than equivalent sequences in a non-palindromic context . These results are consistent with an unusual secondary structure, such as DNA cruciform or hairpin, and indicate that the formation pathway of the structure detected is independent of the composition and symmetry of the central 10 base-pairs of the palindrome. J Mol Biol, 1995 Sep 8, 252(1), 6 - 14 Packing of coat protein amphipathic and transmembrane helices in filamentous bacteriophage M13: role of small residues in protein oligomerization; Williams KA et al.; Filamentous bacteriophage M13, an important cloning and phage display vector, is encapsulated by ca 2700 copies of its 50-residue major coat protein (gene 8) . This protein occurs as a membrane protein while stably inserted into its E . coli host inner membrane, and as a coat protein upon assembly and packing onto phage DNA in the lipid-free virion . To examine the specific protein-protein interactions underlying these processes, we used a combination of randomized and saturation mutagenesis of the entire gene 8 to assess the susceptibility of each position to mutation . In the resulting library of ca 100 viable M13 mutants, "small" residues (Ala,Gly,Ser), which constitute the non-polar face of the N-terminal amphipathic helical segment, and a face of the hydrophobic (effective transmembrane) helical segment, were found to be highly conserved . These results support a model in which coat protein packing is stabilized by the presence within each protein subunit of two "oligomerization segments", i.e . specific helical regions with faces rich in small residues which function to promote the close approach of alpha-helices. J Mol Biol, 1995 Sep 8, 252(1), 47 - 58 Characterization of the interaction between the lambda intasome and attB; Patsey RL et al.; Bacteriophage lambda DNA integrates into the chromosome of Escherichia coli by first forming an intasome at the phage attachment site on the phage DNA with the integrase Int and integration host factor . This intasome searches the host chromosome for the bacterial attachment site (attB) and then orchestrates two sequential strand exchange reactions to achieve integration . This study characterizes the weak interaction of the intasome and attB . The hypothesis that all of the proteins necessary for integration are brought to the reaction site by the intasome is given additional support by showing that the concentration of phage attachment site and not attB determines the optimal concentration of proteins for integration . The value of the dissociation constant of the complex formed between the intasome and attB is determined in two different ways . First, the rate of the integration reaction is measured as a function of the attB DNA concentration . The saturation constant reflects the dissociation constant of the complex . Second, a recombination reaction is inhibited by the introduction of varying amounts of a second attB with a sequence change that blocks recombination with this site . The inhibition constant reflects the dissociation constant of the intasome and altered attB in this experiment . The two methods agree and give a dissociation constant of approximately 300 nM . attB contains two core binding sites for the intasome; it is shown that both are necessary for efficient capture . The value of the dissociation constants are considerably lower when a mutant integrase, IntE174K, is used . This increased affinity for core sites can explain how IntE174K can function in the absence of integration host factor . The inhibition constants also show dependence on the exact sequence of the inhibiting attB . Possible implications of this dependence are discussed. Oncogene, 1995 Sep 7, 11(5), 825 - 31 Lack of evidence for the activation of the Ras/Raf mitogenic pathway by 14-3-3 proteins in mammalian cells; Suen KL et al.; We have isolated cDNA clones encoding the rodent 14-3-3 zeta and epsilon isoforms by screening bacteriophage expression libraries with a probe derived from the carboxy-terminus of the Vav oncoprotein . These isoforms, however, did not recognize the full length Vav protein under physiological conditions . In agreement with previous studies (see D Morrison, Science 266, 56-57, 1994), these 14-3-3 proteins bound very efficiently to Raf . The interaction between 14-3-3 zeta and Raf involves the central region of 14-3-3 zeta which includes a motif related to annexins . 14-3-3 zeta binds to Raf independently of Ras and forms stable ternary complexes with these two molecules . In contrast to published reports, we have observed that the catalytic activity of Raf was not activated in Raf/14-3-3 zeta immunocomplexes . Likewise, purified preparations of 14-3-3 zeta had no effect on the kinase activity of Raf immunoprecipitates . In addition, Ras activated Raf regardless of whether it was bound or not to 14-3-3 zeta . Finally, overexpression of 14-3-3 zeta cDNA clones in NIH3T3 cells did not result in detectable morphologic transformation even when co-transfected with plasmids encoding Raf and/or Ras proteins . These observations argue against a critical regulatory role of the 14-3-3 proteins in the Raf mitogenic pathway. Bioconjug Chem, 1995 Sep-Oct, 6(5), 587 - 95 Cell-specific delivery of bacteriophage-encapsidated ricin A chain; Wu M et al.; We have used covalent coupling of deglycosylated ricin A chain (RAC) to the assembly initiation/translational repression RNA stem-loop (TR) of the bacteriophage MS2 to direct encapsulation of the toxin in bacteriophage capsids . Multiple copies of the TR-RAC conjugate can be incorporated into single capsid shells . The resultant particles can then be directed to specific cells by receptor-mediated endocytosis (RME) of complexes formed with anti-MS2 coat protein antibodies or by further covalent modification of the capsids by addition of human transferrin molecules . The results suggest that bacteriophage encapsulation and targeting is an efficient way to deliver toxins in a cell-specific fashion . The system may have widespread application in the field of targeted drug delivery, including antisense reagents. Eur Respir J Suppl, 1995 Sep, 20, 633s - 648s Mobile genetic elements in mycobacteria; Dale JW; Mobile genetic elements, ranging from plasmids and bacteriophages to insertion sequences and transposons, have come to play key roles in many aspects of basic and applied research in mycobacteriology . Plasmids and bacteriophages have been widely used as cloning vectors, especially for constructing recombinant vaccines based on bacille Calmette-Guerin (BCG); composite transposons have also been used for this purpose . At the same time, insertion sequences have proved invaluable for diagnostic and epidemiological studies, and transposon mutagenesis provides a useful method for inactivating and marking selected mycobacterial genes . Plasmids are commonly found in many mycobacterial species, notably M . avium, although not in M . tuberculosis; the biological significance of these plasmids (if any) is mostly unknown . Insertion sequences, and other repetitive elements, have also been characterized from many mycobacterial species . Special attention is paid to IS6110/IS986, from M . tuberculosis, and the IS900 family from M . avium and related organisms; the latter includes the recently described highly mobile element IS1110 . The emphasis of the paper is on the molecular biology and significance of plasmids and insertion sequences/transposons, in mycobacteria and in bacteria of plasmids and insertion sequences/transposons, in mycobacteria and in bacteria in general, and their applications as cloning vectors and in transposon mutagenesis. Int J Pept Protein Res, 1995 Sep-Oct, 46(3-4), 333 - 40 Design, synthesis, and characterization of HIV-1 enhancer-binding polypeptides derived from bacteriophage 434 repressor; Stadler K et al.; We have designed and synthesized HIV-1 enhancer-binding polypeptides that were derived from bacteriophage 434 repressor . These peptides were 39-54 residues long and contained either the recognition helix or the entire helix-turn-helix motif of the DNA-binding domain of 434 repressor . The dissociation constant of the complex formed between the standard peptide (R42) and a synthetic 70-bp HIV enhancer DNA was ca . 10(-8) M . The specificity of the interaction of R42 with the two HIV enhancers was demonstrated by competitive band shift assays, stepwise displacement of the p50 subunit of transcription factor NF-kappa B from its two HIV enhancer binding sites, and DNase I footprinting; R42 seemed to protect best the two TTTCC sequences of the HIV enhancers against digestion by DNase I . R42 analogues with mutated recognition helix had lower DNA binding specificity . It remains to be investigated whether our artificial HIV enhancer-binding polypeptides are active in vivo. Med Phys, 1995 Sep, 22(9), 1369 - 75 The relaxation of supercoiled DNA molecules as a biophysical dosimeter for ionizing radiations: a feasibility study; Chen W et al.; In this paper we explore the feasibility of using DNA molecules as a biophysical radiation dosimeter . Supercoiled phi X174 bacteriophage DNA molecules were irradiated with different gamma radiation doses . The strand breakage produced by ionizing radiation within supercoiled double-stranded DNA molecules (RFI) yields relaxed circular DNA molecules (RFII) and linear DNA molecules (RFIII) as a result of single-strand breaks and double-strand breaks, respectively . The irradiated samples were subjected to electrophoresis on agarose gels to separate the three forms . A proprietary fluorescent dye was used to detect DNA bands within the gel, which was photographed under UV transillumination . The negative was scanned with a computerized imaging densitometric system for DNA band quantitation . The relative fractions of the three molecular forms are dose dependent, and can be modeled mathematically with five parameters . The values of the parameters were determined by optimizing the fit of the model to the data, using a nonlinear regression procedure of a commercial statistical analysis package . Once the parameters of DNA breakage have been determined, absorbed dose can be measured by this technique, which we have termed supercoil relaxation dosimetry . The average accuracy of dose determination for our system over the range of 1-40 Gy was about 5% . Supercoil relaxation dosimetry may be well suited to certain difficult dosimetric problems. Genomics, 1995 Sep 1, 29(1), 195 - 9 Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11 beta-hydroxysteroid dehydrogenase; Agarwal AK et al.; 11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney . Recent studies indicate the presence of at least two isozymes of 11 beta HSD . In vitro, the NAD(+)-dependent kidney (type 2) isozyme catalyzes 11 beta-dehydrogenase but not reductase reactions, whereas the NADP(+)-dependent liver (type 1) isozyme catalyzes both reactions . We have now characterized the human gene encoding kidney 11 beta HSD (HSD11K) . A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA . The gene consists of 5 exons spread over 6 kb . The nucleotide binding domain lies in the first and the second exon, and the catalytic domain in the fourth exon . The 5' flanking sequences and first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences . Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22 . In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical . HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes . Different transcriptional start sites are utilized in kidney and placenta . These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11 beta HSD. Mutat Res, 1995 Sep, 331(1), 89 - 97 Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model; Gossen JA et al.; Transgenic mouse mutation models carrying bacterial marker genes in bacteriophage lambda shuttle vectors have been applied to study spontaneous or induced mutations in vivo . However, due to the nature of the shuttle vector these models are insensitive to large deletions . Clastogenic agents, which predominantly induce large deletions, were therefore found to yield very low responses in these assays . Here we report the use of LacZ plasmid-based transgenic mice, allowing the detection of a broad spectrum of mutations . Treatment of mice with X-rays (5 x 50 rads) resulted in induction of up to about 5-fold higher mutation frequencies in lung, spleen and liver . Analysis of spontaneous and induced mutant LacZ genes indicated that at least 40-50% of all mutations were caused by deletions . The possibility of detecting a broad spectrum of mutations with this system suggests that the LacZ plasmid-based transgenic mouse may be the mammalian model of choice for studying spontaneous and induced mutations in vivo. J Bacteriol, 1995 Sep, 177(17), 5166 - 8 The interaction of T4 endonuclease V E23Q mutant with thymine dimer- and tetrahydrofuran-containing DNA; Latham KA et al.; The interaction between endonuclease V, the cyclobutane pyrimidine d