AIDS, 1992 Oct, 6(10), 1151 - 8 Progressive spastic myelopathy in a patient co-infected with HIV-1 and HTLV-II: autoantibodies to the human homologue of rig in blood and cerebrospinal fluid; Rosenblatt JD et al.; OBJECTIVE: Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) are closely related human retroviruses . HTLV-I has been implicated in a chronic progressive myelopathy, known as tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy (HAM) . We sought to determine whether autoantibodies to brain antigens were present in the cerebrospinal fluid (CSF) of a patient with chronic progressive spastic myelopathy with evidence of both HIV-1 infection and HTLV-I/II seropositivity . DESIGN: A 54-year-old bisexual man with clinical features of HAM/TSP of over 20 years' duration was followed . METHODS: We applied discriminatory DNA amplification (polymerase chain reaction) to distinguish HTLV-I from HTLV-II and to verify co-infection with HIV-1 . The patient's CSF was used to screen a human brain cDNA expression library to identify antibodies directed against brain antigens . Autoreactive bacteriophage clones were isolated and sequenced . RESULTS: The patient was found to be co-infected with both HIV-1 and HTLV-II, but not with HTLV-I . HTLV-II proviral levels in the peripheral blood remained relatively constant, despite therapy with zidovudine . Prominent oligoclonal banding of immunoglobulins was present in the patient's CSF . A single repeatedly reactive cDNA clone was identified, by screening with CSF antibody, sequenced, and found to be the human homologue of the rat insulinoma gene, rig . CONCLUSIONS: HTLV-II infection may predispose to development of a HAM/TSP-like illness . Autoimmune mechanisms, such as autoantibody formation, may play a role in pathogenesis. J Mol Biol, 1992 Oct 5, 227(3), 917 - 33 Structure of a hinge-bending bacteriophage T4 lysozyme mutant, Ile3-->Pro; Dixon MM et al.; The mutant T4 phage lysozyme in which isoleucine 3 is replaced by proline (I3P) crystallizes in an orthorhombic form with two independent molecules in the asymmetric unit . Relative to wild-type lysozyme, which crystallizes in a trigonal form, the two I3P molecules undergo large hinge-bending displacements with the alignments of the amino-terminal and carboxy-terminal domains changed by 28.9 degrees and 32.9 degrees, respectively . The introduction of the mutation, together with the hinge-bending displacement, is associated with repacking of the side-chains of Phe4, Phe67 and Phe104 . These aromatic residues are clustered close to the site of the mutation and are at the junction between the amino and carboxyl-terminal domains . As a result of this structural rearrangement the side-chain of Phe4 moves from a relatively solvent-exposed conformation to one that is largely buried . Mutant I3P also crystallizes in the same trigonal form as wild-type and, in this case, the observed structural changes are restricted to the immediate vicinity of the replacement . The main change is a shift of 0.3 to 0.5 A in the backbone of residues 1 to 5 . The ability to crystallize I3P under similar conditions but in substantially different conformations suggests that the molecule undergoes large-scale hinge-bending displacements in solution . It is also likely that these conformational excursions are associated with repacking at the junction of the N-terminal and C-terminal domains . On the other hand, the analysis is complicated by possible effects of crystal packing . The different I3P crystal structures show substantial differences in the binding of solvent, both at the site of the Ile3-->Pro replacement and at other internal sites. J Mol Biol, 1992 Oct 5, 227(3), 711 - 8 Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage; Christian RB et al.; An efficient strategy has been devised for the construction of diverse peptide libraries in bacteriophage vectors . This strategy was used to generate a library of 4 x 10(8) random decapeptide inserts in the pIII protein of bacteriophage fd . A novel method for evaluating the genetic diversity of bacteriophage libraries based on colony hybridization with partially degenerate oligonucleotides has been developed . The decapeptide library was affinity-selected with a previously characterized monoclonal antibody specific for the V3 loop of the gp120 protein of HIV-1 . Immunological screening, an efficient technique for the rapid identification of putative binding bacteriophage, is described . Hexapeptide sequences similar to those obtained from affinity selection of a hexapeptide bacteriophage library were obtained from the decapeptide library in all five frames . Immunological screening of 20,000 clones from the two libraries after two rounds of affinity selection rapidly identified antibody-binding sequences; 93% and 86% of the sequences obtained from the hexapeptide and decapeptide libraries, respectively, had IC50 values < or = 10 mM as free peptides. Biochim Biophys Acta, 1992 Oct 5, 1110(2), 218 - 24 Formation of non-bilayer structures induced by M13 coat protein depends on the conformation of the protein; Sanders JC et al.; A comparison is made of the interaction of the coat protein of bacteriophage M13 in a predominant alpha-helix conformation and in a predominant beta-sheet conformation . To perform a systematic study of the interaction between the protein in these two different forms of the surrounding lipid matrix, NMR spectra of 2H-nuclei of specific labelled phospholipid systems are measured . In addition 31P-NMR is employed to provide information about the morphological structure adopted by the reconstituted lipid/protein systems . From the 2H-NMR studies on specific headgroup and chain deuterium labelled phospholipids it is found that the protein in the predominant beta-sheet conformation causes a fraction of lipids to be trapped . By combining the results from the headgroup and acyl chains of the phospholipids, it is concluded that the trapped lipids are arranged in a non-bilayer structure, probably caused by a misfitting of the hydrophobic core of the protein and the membrane bilayer . The protein in the predominant alpha-helix conformation perfectly fits in the lipid bilayer and has only minor influences on the surrounding lipid matrix . A new model is proposed to explain the presence of the trapped lipids in the lipid/protein systems. J Biol Chem, 1992 Oct 5, 267(28), 20153 - 8 Inhibition of T7 RNA polymerase by T7 lysozyme in vitro; Ikeda RA et al.; The in vivo observation that the expression of bacteriophage T7 gene 3.5 (T7 lysozyme) inactivates T7 class II transcription and the in vitro observation that T7 lysozyme inhibits T7 RNA polymerase lead to the hypothesis that T7 lysozyme might preferentially inhibit transcription from T7 class II promoters . T7 lysozyme was cloned into a lambda pL expression vector, overproduced in Escherichia coli, and purified . The ability of purified T7 lysozyme to inhibit transcription from T7 DNA, the cloned T7 class II promoters, phi 2.5 and phi 4.7, and the cloned class III promoter, phi 10, was measured in vitro . It was observed that the effectiveness of T7 lysozyme as an inhibitor of T7 RNA polymerase is inversely related to the concentration of Mg2+; T7 lysozyme inhibits T7 RNA polymerase most effectively at low Mg2+ concentrations . In addition, no preferential inhibition of transcription from cloned T7 class II promoters was observed, nor was a strong T7 class III promoter preferred when transcriptional capacity was reduced by T7 lysozyme . These observations contradict the hypotheses that the temporal control of T7 gene expression is either due to direct and selective inhibition of the T7 class II promoters by T7 lysozyme or to preferential transcription of the strong T7 class III promoters when transcriptional capacity is reduced by T7 lysozyme . It appears that alternative mechanisms such as the involvement of additional proteins and/or cellular conditions to enhance transcription from T7 class III promoters or to inhibit transcription from T7 class II promoter are necessary to explain the temporal control of transcription of bacteriophage T7. Protein Eng, 1992 Oct, 5(7), 703 - 14 A molecular dynamics simulation of bacteriophage T4 lysozyme; Arnold GE et al.; An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented . The simulation was carried out with all hydrogen atoms modeled explicitly, the inclusion of all 152 crystallographic waters and at a temperature of 300 K . Temporal analysis of the trajectory versus energy, hydrogen bond stability, r.m.s . deviation from the starting crystal structure and radius of gyration, demonstrates that the simulation was both stable and representative of the average experimental structure . Average structural properties were calculated from the enzyme trajectory and compared with the crystal structure . The mean value of the C alpha displacements of the average simulated structure from the X-ray structure was 1.1 +/- 0.1 A; differences of the backbone phi and psi angles between the average simulated structure and the crystal structure were also examined . Thermal-B factors were calculated from the simulation for heavy and backbone atoms and both were in good agreement with experimental values . Relationships between protein secondary structure elements and internal motions were studied by examining the positional fluctuations of individual helix, sheet and turn structures . The structural integrity in the secondary structure units was preserved throughout the simulation; however, the A helix did show some unusually high atomic fluctuations . The largest backbone atom r.m.s . fluctuations were found in non-secondary structure regions; similar results were observed for r.m.s . fluctuations of non-secondary structure phi and psi angles . In general, the calculated values of r.m.s . fluctuations were quite small for the secondary structure elements . In contrast, surface loops and turns exhibited much larger values, being able to sample larger regions of conformational space . The C alpha difference distance matrix and super-positioning analyses comparing the X-ray structure with the average dynamics structure suggest that a 'hinge-bending' motion occurs between the N- and C-terminal domains. Biotechniques, 1992 Oct, 13(4), 554 - 6, 558-60, 562 High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones; Millar SJ et al.; Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome . We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family . Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify . Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates . In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors. Curr Opin Genet Dev, 1992 Oct, 2(5), 727 - 38 Interaction between bacteriophage lambda and its Escherichia coli host; Friedman DI; Bacteriophage lambda relies to a large extent on processes requiring interactions between viral- and host-encoded proteins for its lytic growth, establishment of lysogeny, and release from the prophage state . Both biochemical and genetic studies of these interactions have yielded new information about important host and lambda functions . In particular, mutations in Escherichia coli that compromise lambda DNA replication, genome packaging, transcription elongation, and site-specific recombination have led to the identification of bacterial genes whose products are chaperones, transcription factors, or DNA-binding proteins. J Protein Chem, 1992 Oct, 11(5), 467 - 73 Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain; Kan CC et al.; We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently in Escherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein . Human trifunctional enzyme is involved in de novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention . The GART domain was expressed in E . coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure . Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins . The active monofunctional GART protein produced in E . coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein. J Bacteriol, 1992 Oct, 174(20), 6539 - 47 Sequence and characterization of the bacteriophage T4 comC alpha gene product, a possible transcription antitermination factor; Sanson B et al.; We have sequenced a 1,340-bp region of the bacteriophage T4 DNA spanning the comC alpha gene, a gene which has been implicated in transcription antitermination . We show that comC alpha, identified unambiguously by sequencing several missense and nonsense mutations within the gene, codes for an acidic polypeptide of 141 residues, with a predicted molecular weight of 16,680 . We have identified its product on one- and two-dimensional gel systems and found that it migrates abnormally as a protein with a molecular weight of 22,000 . One of the missense mutations (comC alpha 803) is a glycine-to-arginine change, and the resulting protein exhibits a substantially faster electrophoretic mobility . The ComC alpha protein appears immediately after infection . Its rate of synthesis is maximum around 2 to 3 min postinfection (at 37 degrees C) and then starts to decrease slowly . Some residual biosynthesis is still detectable during the late period of phage development. J Bacteriol, 1992 Oct, 174(19), 6138 - 44 Bacteriophage P1 gene 10 is expressed from a promoter-operator sequence controlled by C1 and Bof proteins; Lehnherr H et al.; Gene 10 of bacteriophage P1 encodes a regulatory function required for the activation of P1 late promoter sequences . In this report cis and trans regulatory functions involved in the transcriptional control of gene 10 are identified . Plasmid-borne fusions of gene 10 to the indicator gene lacZ were constructed to monitor expression from the gene 10 promoter . Production of gp10-LacZ fusion protein became measurable at about 15 min after prophage induction, whereas no expression was observed during lysogenic growth . The activity of an Escherichia coli-like promoter, Pr94, upstream of gene 10, was confirmed by mapping the initiation site of transcription in primer extension reactions . Two phage-encoded proteins cooperate in the trans regulation of transcription from Pr94: C1 repressor and Bof modulator . Both proteins are necessary for complete repression of gene 10 expression during lysogeny . Under conditions that did not ensure repression by C1 and Bof, the expression of gp10-LacZ fusion proteins from Pr94 interfered with transformation efficiency and cell viability . Results of in vitro DNA-binding studies confirmed that C1 binds specifically to an operator sequence, Op94, which overlaps the -35 region of Pr94 . Although Bof alone does not bind to DNA, together with C1 it increases the efficiency of the repressor-operator interaction . These results are in line with the idea that gp10 plays the role of mediator between early and late gene transcription during lytic growth of bacteriophage P1. EMBO J, 1992 Oct, 11(10), 3797 - 806 Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding; Numrych TE et al.; We have performed a mutational analysis of the xis gene of bacteriophage lambda . The Xis protein is 72 amino acids in length and required for excisive recombination . Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision . Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA . In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision . However, these three mutants were unable to bind DNA cooperatively with Int . Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites . This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision. Curr Opin Biotechnol, 1992 Oct, 3(5), 474 - 80 Antibody expression in bacteriophage systems: the future of monoclonal antibodies? Garrard LJ, Zhukovsky EA. Bacteriophage systems have been utilized to express and isolate antibodies . This promising technology has been evolving rapidly and has the potential to revolutionize the way in which monoclonal antibodies are generated . This review focuses on the many recent advances that have been made in obtaining monoclonal antibodies from bacteriophage systems. Eur J Clin Microbiol Infect Dis, 1992 Oct, 11(10), 901 - 7 Emergence during unsuccessful chemotherapy of multiple drug resistance in a strain of Mycobacterium tuberculosis; Rastogi N et al.; Serial isolates of Mycobacterium tuberculosis were cultured from a patient who failed to respond to standard antituberculous chemotherapy . Isolates were cultured in March 1989, July 1989, December 1989 and May 1990 . Each successive isolate was found to be resistant to a wider range of antituberculous drugs than its predecessors . The initial isolate was resistant to isoniazid and rifampin, the second isolate was also resistant to ethambutol, the third was also resistant to pyrazinamide, ansamycin (= rifabutin) and ofloxacin and the last isolate was also resistant to ciprofloxacin and sparfloxacin . All four isolates' bacteriophage typing profiles and DNA restriction fragment patterns determined by Southern blot hybridization using the IS6110/IS986 probes and the new probe pTBN12 were concordant . It was concluded that this patient was persistently infected with a single strain of Mycobacterium tuberculosis which developed resistance to a number of families of drugs but did not show any significant change in typing patterns . The problem of acquired multiple drug resistance, particularly to fluoroquinolones and rifamycins, represents a new challenge in tuberculosis therapy. J Virol, 1992 Oct, 66(10), 6220 - 2 Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity; Gottlieb P et al.; Bacteriophage phi 6 contains three segments of double-stranded RNA . The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment . cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids . These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA . In this article, we report the presence of a nucleotide-binding site in protein P4 . The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates. Virology, 1992 Oct, 190(2), 824 - 33 Role of exonuclease in the specificity of bacteriophage T7 DNA packaging; Son M et al.; During morphogenesis in vivo, bacteriophage T7 packages and cuts to mature size an end-to-end concatemer of its nonpermuted, terminally repetitious, double-stranded, mature DNA . Efficient production (90-100%) and packaging (20-35%) of concatemers has also been demonstrated in extracts of T7-infected cells (in vitro) (Son, M., Hayes, S . J., and Serwer, P . {1988} Virology 162, 38-46) . By use of both this procedure of in vitro DNA packaging and in-gel hybridization to packaged DNA fractionated by agarose gel electrophoresis, the specificity of packaging in vitro is found to depend on the presence of T7 gene 6 exonuclease (p6) . In the absence of p6 in vitro, no concatemerization is detected and packaging of DNA nonhomologous to T7 DNA (bacteriophage P22 DNA) is as efficient (0.05-1.1%) as the packaging of monomeric T7 DNA . Addition of p6 in vitro both stimulates the concatemerization-packaging of T7 DNA and suppresses the packaging of P22 DNA . The packaging efficiency for concatemeric T7 DNA is 29-611 x higher than that for monomeric T7 DNA . Inhibition of the packaging of P22 DNA by p6 is correlated with the formation of single-stranded P22 DNA ends . These data are explained by the hypothesis that a DNA molecule with a single-stranded end is packaged less efficiently than the same DNA without the single-stranded end . Testing this hypothesis in vivo reveals that both p6 and gene 3 endonuclease contribute to suppressing the packaging of host DNA. J Biol Chem, 1992 Sep 25, 267(27), 19306 - 12 Transcription termination in vitro by bacteriophage T7 RNA polymerase . The role of sequence elements within and surrounding a rho-independent transcription terminator; Jeng ST et al.; rho-Independent transcription terminators in Escherichia coli contain a dG+dC-rich dyad-symmetrical structure that encodes an RNA hairpin structure and an adjacent, downstream dA+dT-rich region which encodes uridines at the 3'-end of the transcript . In the threonine (thr) attenuator, there are at least six sequence segments in the DNA that might affect termination: the sequence upstream of the attenuator, the deoxythymidine-rich stretch immediately preceding the G+C-rich region, the G+C-rich region itself and its hairpin loop-encoding region, the deoxyadenosine tract following the G+C-rich region, and the following downstream sequence . Our previous studies (Jeng, S.-T., Gardner, J.F., and Gumport, R.I . (1990) J . Biol . Chem . 265, 3823-3830) indicate that both the stability and sequence of the RNA hairpin formed by the G+C-rich region and the length of the uridine tract encoded by the deoxyadenosine stretch influence the termination of T7 RNA polymerase in vitro . In this report, we demonstrate that the template deoxythymidine run upstream of the G+C-rich region, the loop-encoding segment, and the sequences upstream and downstream of the thr attenuator also affect termination . These results indicate that: 1) a deoxythymidine tract is not absolutely required for termination, but increasing the number of deoxythymidines from one to nine base pairs causes T7 RNA polymerase to terminate more efficiently; 2) a template with the natural loop sequence reversed results in a higher termination efficiency than one encoded by the the wild-type attenuator; 3) the termination of T7 RNA polymerase is affected by sequences both proximal and distal to the thr attenuator. J Biol Chem, 1992 Sep 25, 267(27), 19418 - 26 Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda; Mason SW et al.; The N protein of phage lambda prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the lambda early operons . The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each lambda early operon, and involves the E . coli factors NusA, NusB, NusG, and ribosomal protein S10 . We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR . NusA is also sufficient for partial antitermination at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reaction . At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites . In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N-modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the lambda nut sites . This substantial processivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the lambda chromosome. J Biol Chem, 1992 Sep 25, 267(27), 19101 - 6 Pyruvic acid is attached through its central carbon atom to the amino terminus of the recombinant DNA-derived DNA-binding protein Ner of bacteriophage Mu; Rose K et al.; Ner protein of bacteriophage Mu, produced by recombinant DNA techniques in Escherichia coli, has been found to possess a molecule of pyruvic acid attached covalently through carbon-2 to the amino-terminal cysteine residue . The intact protein and the amino-terminal chymotryptic peptide were found by mass spectrometry to be 70 mass units heavier than expected . The modified peptide was unstable under mildly acid or mildly basic conditions . Two-dimensional nuclear magnetic resonance spectroscopy of the modified and unmodified forms of the amino-terminal chymotryptic peptide was consistent with the presence of pyruvate linked through carbon-2 to the amino-terminal Cys residue . Treatment of the modified form with 2,4-dinitrophenylhydrazine in acid medium led to the expected hydrazone of pyruvic acid, which was identified by high pressure liquid chromatography . Of the two proteins known to be modified by pyruvate through its central carbon (the other being human adult hemoglobin, in which the modified form represents only a very minor fraction), Ner is the first protein found to be modified quantitatively . Given the instability of the modification, it may be more prevalent than recognized hitherto . Incubation with 2,4-dinitrophenylhydrazine may offer a useful means of detecting the presence of pyruvate linked to proteins in this way. J Biol Chem, 1992 Sep 25, 267(27), 19521 - 8 Transmembrane signaling by the human insulin receptor kinase . Relationship between intramolecular beta subunit trans- and cis-autophosphorylation and substrate kinase activation; Frattali AL et al.; To examine the role of intramolecular beta subunit trans- and cis-autophosphorylation in signal transduction, the vaccinia virus/bacteriophage T7 expression system was used to generate insulin holoreceptors composed of a kinase-defective half-receptor precursor (alpha beta A/K or alpha beta A/K.delta CT) and a kinase-active half-receptor precursor (alpha beta delta CT or alpha beta WT) . In the alpha beta A/K-alpha beta delta CT hybrid insulin receptor, insulin stimulated a 20-fold increase in intramolecular beta subunit trans-phosphorylation, whereas cis-phosphorylation increased only 3-fold over the basal state . Similarly, in the alpha beta WT-alpha beta A/K.delta CT hybrid insulin receptor, insulin stimulated trans-phosphorylation approximately 30-fold and cis-phosphorylation only 3-fold over the basal state . Although cis-phosphorylation of the kinase-functional alpha beta half-receptor was observed within these hybrid receptor species, this was not sufficient to stimulate exogenous substrate kinase activity . These data demonstrate that insulin primarily activates an intramolecular beta subunit trans-phosphorylation reaction within the insulin holoreceptor and suggest that this reaction is necessary for activation of the holoreceptor . Furthermore, our results suggest a molecular basis for the dominant-negative phenotype observed in insulin-resistant patients possessing one kinase-defective insulin receptor allele. Biochemistry, 1992 Sep 22, 31(37), 9073 - 80 In vivo and in vitro activities of point mutants of the bacteriophage T7 RNA polymerase promoter; Ikeda RA et al.; Two compatible plasmids were recently reported {Ikeda et al . (1992) Nucleic Acids Res . 20, 2517-2524} that together can be used to determine whether a mutant T7 RNA polymerase promoter is active or inactive in vivo . The first plasmid, pKGP1-1, carries T7 gene 1 (the gene encoding T7 RNA polymerase) ligated to a tac promoter, while the second plasmid, pCM-X#, carries the gene encoding chloramphenicol acetyltransferase (CAT) ligated to potential T7 promoters . If the pCM-X# plasmid carries a potential T7 promoter that can be utilized by T7 RNA polymerase, then CAT is produced from transcripts generated by T7 RNA polymerase from the potential promoter on the pCM-X# plasmid . To determine whether Escherichia coli growth characteristics and chloramphenicol (cam) resistance produced by the plasmids pKGP1-1 and pCM-X# reflect the T7 promoter activity of the possible promoters carried by the pCM-X# plasmids, the in vivo and in vitro strengths of the potential T7 promoters were compared and correlated . In vivo promoter strength was determined by measuring the relative amounts of CAT present in E . coli extracts, while relative in vitro promoter strength was measured in transcription assays . The in vivo and in vitro strengths of 22 point mutants of the consensus T7 promoter were shown to correlate with the growth characteristics and cam resistance conferred to E . coli harboring the plasmid pKGP1-1 and the respective pCM-X# plasmid.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1992 Sep 20, 227(2), 381 - 8 By-passing immunisation . Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro; Hoogenboom HR et al.; By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection . Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage . Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues . The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA) . Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range . The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3 . However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin . Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro. J Biol Chem, 1992 Sep 15, 267(26), 18623 - 30 Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites; Gabbara S et al.; EcoRII is unusual among type II restriction enzymes in that, while it cleaves substrates such as pBR322 and bacteriophage lambda that contain several recognition sites for the enzyme efficiently, substrates such as the genomes of bacteriophages T3 and T7 which contain a small number of recognition sites are cut poorly by it . Interestingly, pBR322, or a short DNA duplex containing a single site for the enzyme, can activate the enzyme to cleave resistant substrates . We show here that, at low concentrations, activator short duplexes are themselves cleaved poorly by the enzyme . Further, the reaction shows substrate cooperativity, and at high concentrations, the duplexes are both activators and good substrates for the enzyme . This supports the model that the activation of EcoRII involves binding of more than one DNA molecule and provides a simple system to study the mechanism of activation . Using a gel mobility shift assay, we show that the enzyme forms sequence-specific, methylation-sensitive complexes with the duplexes in the absence of activating DNA . Therefore, resistance of the short duplexes to the enzyme at low concentrations cannot be due to an inability of the enzyme to bind the duplexes . Interestingly, these complexes are stable in the presence of Mg2+, the cofactor for the enzyme, and the complexes obtained in the presence of Mg2+ do not contain DNA that is cleaved by the enzyme . The inefficient step in the action of EcoRII on resistant substrates must occur subsequent to initial substrate binding and it is this step that the activating DNA must regulate. Biochemistry, 1992 Sep 15, 31(36), 8619 - 28 Human hemoglobin expression in Escherichia coli: importance of optimal codon usage; Hernan RA et al.; The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time . Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter . In this system, beta-globin accumulated to approximately 10% of total E . coli proteins . alpha-Globin was not expressed in the T7 system using the native cDNA . For the expression of alpha-globin, synthetic genes containing optimal E . coli codons were constructed . Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter . Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter . The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E . coli protein . Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage . The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains . N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue . Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system . Functional and structural properties of the purified hemoglobins will be discussed in the following papers. Biochemistry, 1992 Sep 15, 31(36), 8397 - 405 A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis; Serwer P et al.; Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE) . For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA . When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin . Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band . The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE . For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA . During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases . During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8562 - 6 DNA helicase from mammalian mitochondria; Hehman GL et al.; In spite of the fact that a DNA helicase is clearly required for the predominantly leading-strand synthesis occurring during mammalian mtDNA replication, no such activity has heretofore been identified . We report the characterization of a mammalian mitochondrial DNA helicase isolated from bovine brain tissue . The sucrose gradient-purified mitochondria in which the activity was detected had less than 1 part in 2500 nuclear contamination according to Western blot analysis using nuclear- and mitochondrial-specific probes . Mitochondrial protein fractionation by DEAE-Sephacel chromatography yielded a DNA helicase activity dependent upon hydrolysis of ATP or dATP but not other NTPs or dNTPs . The mitochondrial helicase unwound 15- and 20-base oligonucleotides but was unable to unwind 32-base or longer oligonucleotides, and the polarity of the unwinding is 3'-to-5' with respect to the single-stranded portion of the partial duplex DNA substrate . This direction of unwinding would place the bovine mitochondrial helicase on the template strand ahead of DNA polymerase gamma during mtDNA replication, a situation analogous to that of the Rep helicase of Escherichia coli during leading-strand DNA synthesis of certain bacteriophages. Nucleic Acids Res, 1992 Sep 11, 20(17), 4451 - 5 Stoichiometry of the Cre recombinase bound to the lox recombining site; Mack A et al.; The site-specific recombinase Cre from bacteriophage P1 binds and carries out recombination at a 34 bp lox site . The lox site consists of two 13 bp inverted repeats, separated by an 8 bp spacer region . Both the palindromic nature of the site and the results of footprinting and band shift experiments suggest that a minimum of two Cre molecules bind to a lox site . We report here experiments that demonstrate the absolute stoichiometry of the Cre-lox complex to be one molecule of Cre bound per inverted repeat, or two molecules per lox site. J Mol Biol, 1992 Sep 5, 227(1), 29 - 37 The Escherichia coli rpoB60 mutation blocks antitermination by coliphage HK022 Q-function; Atkinson BL et al.; The lambdoid bacteriophage regulate gene expression by suppressing transcription terminators . Although similar in sequence to lambda, HK022 lacks an analogue to the lambda N antitermination gene and a distinct nutR sequence . To define the HK022 antitermination system, we plated the phage on Escherichia coli nus mutants that inhibit lambda N function . Only rpoB60 (also called nusC60) blocked HK022 lytic growth . Analyses of HK022-lambda hybrid phage suggested that a HK022 function analogous to lambda Q was inhibited by rpoB60 . This result was confirmed with pR'-tR'-galK fusions . HK022 Q-protein suppressed tR' in wild-type but not in rpoB60 mutants . The lambda Q-protein, although inhibited by rpoB60, was more active than the HK022 analogue . A single amino acid difference between the two Q-proteins accounts for the phenotype . Changing the penultimate residue of HK022 Q from alanine to the lambda threonine generated a phage that could propagate on rpoB60 hosts . Host and phage mutations that permitted HK022 growth in rpoB60 strains were characterized . The bacterial suppressors were located in the Escherichia coli nusB gene . The phage suppressors represented recessive mutations in a HK022 b-region sequence encoding an open reading frame of 73 codons. J Biol Chem, 1992 Sep 5, 267(25), 18080 - 4 Gene structure of semenogelin I and II . The predominant proteins in human semen are encoded by two homologous genes on chromosome 20; Ulvsback M et al.; The genes for semenogelin I and II, the major protein constituents of the human seminal fluid, have been characterized by three overlapping clones in bacteriophage lambda, encompassing 31.5 kilobases (kb) of genomic DNA . The two genes are located 11.5 kb apart in the region q12-q13.1 on chromosome 20 . Both genes are relatively compact, spanning only 2.7 and 3.1 kb, respectively . The transcription units are composed of three exons, of which the first encodes the signal peptide, the second encodes the secreted protein, while the third solely contains 3'-noncoding nucleotides . The nucleotide sequences exhibit a similarity of close to 90% in the exons and exceeding 80% in the introns and flanking nucleotides. J Biol Chem, 1992 Sep 5, 267(25), 17748 - 52 Critical functional role of the COOH-terminal ends of longitudinal hydrophobic strips in alpha-helices of T4 lysozyme; Rennell D et al.; The sensitivity of bacteriophage T4 lysozyme function to amino acid substitutions at defined positions in and around the longitudinal, hydrophobic strips of 9 alpha-helices was assessed after systematic replacement of each residue in the protein with a series of 13 amino acids . The hydrophobic strips were defined by identifying the longitudinal sectors in the helices with the highest mean residue hydrophobicities . Sensitivity to mutation (the percentage of replacements leading to loss of function) was calculated for each residue in the following positions: whole protein, helices, hydrophobic strips, other positions within the helices, and various positions within the hydrophobic strips as well as their extensions beyond the helices . Substitutions at positions in the hydrophobic strips led more frequently to loss of function than substitutions in the protein as a whole . One subset, the COOH-terminal hydrophobic strip residues, is apparently critical; substitutions of these residues (but not of their NH2-terminal counterparts) led at least as frequently to loss of function as substitutions of solvent-inaccessible residues, and nearly as frequently as substitutions of the most highly conserved residues. J Biol Chem, 1992 Sep 5, 267(25), 17827 - 35 Integration host factor activates the Ner-repressed early promoter of transposable Mu-like phage D108; Kukolj G et al.; The lytic-lysogenic switch in transposable, Mu-like bacteriophage D108 is governed by two divergent and slightly overlapping transcription units originating from the Pe and Pc promoters . DNase I footprinting and in vivo mutational analysis suggest that lysogeny is maintained by c-repressor occupancy of the O2 operator, which precludes RNA polymerase from binding to Pe . Lytic development is controlled by the Ner repressor, which binds to a site symmetrically situated between the converging promoters and, in the absence of other factors, prevents RNA polymerase from binding to either Pc or Pe . DNase I protection and potassium permanganate hypersensitivity in the presence of integration host factor (IHF), which binds and alters the DNA structure upstream of Pe, revealed that RNA polymerase was able to bind Pe irrespective of the Ner.DNA-bound complex, and partially unwind the Pe "-10 region." Ner repression of Pe transcription in vitro was significantly more effective in the absence of IHF . Using a cloned D108 early region-lacZ fusion in IHF-deficient and -proficient backgrounds, we also demonstrate this host factor's affect on ner-repressed Pe in vivo, and generate a system for isolating mutants in the regulatory genes and sites controlling this genetic switch . D108 lytic growth is proposed to occur through IHF-mediated activation of the phage Ner-repressed early operon. Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8145 - 9 The p50 subunit of NF-kappa B associates with the NF-IL6 transcription factor; LeClair KP et al.; The NF-kappa B-p50 polypeptide, a member of the Rel family of transcription factors, was produced as a fusion protein containing amino-terminal peptide additions that facilitate purification and detection with a monoclonal antibody and specific radiolabeling by phosphorylation in vitro . The 32P-labeled NK-kappa B-p50 fusion polypeptide was used as the probe in Western blotting experiments and in screenings of a bacteriophage expression library to isolate cDNAs encoding interacting protein domains . As expected, cDNAs encoding proteins of the Rel family were identified . Surprisingly, the 32P-labeled NF-kappa B protein also specifically bound to proteins encoded by cDNAs for the human NF-IL6 transcription factor . The NF-kappa B-p50 and NF-IL6 proteins directly interact, and the Rel homology domain and leucine-zipper motif, respectively, are important for this interaction . Since induction of the NF-kappa B and NF-IL6 factors are important events in immune and acute-phase responses, this interaction could permit coregulation of genes. J Bacteriol, 1992 Sep, 174(17), 5740 - 4 Tandem cloning of bacteriophage T4 nrdA and nrdB genes and overproduction of ribonucleoside diphosphate reductase (alpha 2 beta 2) and a mutationally altered form (alpha 2 beta 2(93)); Tseng MJ et al.; To investigate the role of ribonucleoside diphosphate reductase in the deoxyribonucleoside triphosphate synthetase multienzyme complex induced by bacteriophage T4 infection and to study the expression of the T4 nrdA and nrdB genes, we have constructed separate plasmid expression strains overproducing their respective alpha 2 and beta 2 protein products . Because complementation of the two proteins to form an active alpha 2 beta 2 enzyme presented complications, nrdA and nrdB, each with its own tac promoter, were also cloned in tandem into a single expression vector . The resulting plasmid (pnrdAB) overproduces ribonucleoside diphosphate reductase . Phage T4 nrdB93, described by Wirak et al . (D . O . Wirak, K . S . Cook, and G . R . Greenberg, J . Biol . Chem . 263:6193-6201, 1988) contains a lesion in exon II of the gene . The mutation causes not only a temperature-sensitive inactivation of the catalytic structure of the beta 2(93) protein and of its ability to interact with alpha 2 protein to form the alpha 2 beta 2(93) enzyme but also a profound non-temperature-sensitive decrease in the formation of the beta 2(93) protein . An expression vector overproducing active alpha 2 beta 2(93) was constructed by site-directed mutagenesis of the nrdB gene. Gene, 1992 Sep 1, 118(1), 5 - 11 Purification and characterization of the M.RsrI DNA methyltransferase from Escherichia coli; Kaszubska W et al.; The gene (rsrIM) encoding the RsrI DNA methyltransferase (M.RsrI) from Rhodobacter sphaeroides was cloned and expressed in Escherichia coli . Under the control of a bacteriophage T7 promoter, 2% of the total protein in a crude extract was M.RsrI . This level of expression represents an approximately 50-fold increase over that present in the natural host . Chromatography using DNA cellulose and the S-adenosylmethionine analogue, sinefungin, was useful in purifying the enzyme to homogeneity . The purification yielded 100 times more enzyme than was obtained from the same quantity of R . sphaeroides cell paste . M.RsrI deposits one methyl group per productive DNA-binding event, as does its functional but sequence-nonhomologous analogue, M.EcoRI . Unlike M.EcoRI, the R . sphaeroides enzyme is a dimer at micromolar concentrations. Gene, 1992 Sep 1, 118(1), 103 - 7 A vector for controlled, high-yield production of specifically mutated proteins in Escherichia coli: test of a putative cytidine-binding domain in Rho factor and its Thr16----Ala mutant; Richardson LV et al.; A derivative of the plasmid vector, pET-3a {Rosenberg et al., Gene 56 (1987) 125-135}, is described that contains the origin of replication from bacteriophage f1 . This plasmid is well-suited for oligodeoxyribonucleotide mutagenesis and controlled production of mutant proteins from a single vector . Its utility is demonstrated by the preparation of a mutational alteration of Thr16----Ala (T16A) of the Escherichia coli transcription termination factor, Rho . The altered protein (T16A Rho) binds oligo(C)7 with the same affinity as wild-type (wt) Rho, thus indicating that Thr16 is not critical for binding cytidine residues in RNA, in spite of its being part of a sequence that is similar to a sequence in the CTP-binding domain of aspartate transcarbamoylase . However, T16A Rho was less efficient in terminating transcription than was wt Rho and had a lowered kcat for ATP hydrolysis with cro RNA as co-factor . Thus, the change affects the coupling of ATP hydrolysis by Rho to actions on RNA that cause termination. Biochemistry, 1992 Sep 1, 31(34), 7948 - 56 Tryptophan contributions to the unusual circular dichroism of fd bacteriophage; Arnold GE et al.; The circular dichroism (CD) spectrum of fd bacteriophage has a deep minimum at 222 nm characteristic of highly alpha-helical protein, but there is a shoulder at 208 nm rather than a minimum, with a 222/208-nm amplitude ratio near 1.5 rather than near 1 . Oxidation of fd phage with the tryptophan reagent N-bromosuccinimide (NBS) changes the ratio . In this report, the NBS titration of fd is followed by CD and three other spectroscopies, the results of which yield an explanation of the unusual CD spectrum . Absorbance, fluorescence, and Raman data show the oxidation to have two phases, the first of which involves the destruction of tryptophan and the second, tryptophan and tyrosine . Raman spectra reveal the invariance of an environmentally-sensitive tyrosine Fermi resonance doublet during the first oxidative phase . Raman spectra also show that little or no change of alpha-helicity occurs in the first or second oxidation phase, although very slight changes in the helix parameters might be occurring . Concurrent with the destruction of tryptophan during the first phase is the appearance in CD difference spectra ({theta}NBS-treated fd - {theta}native fd) of positive maxima at 208-210 nm and negative maxima at 224 nm, with crossovers at 217 nm . Enormous difference ellipticities, per oxidized subunit of 50 amino acids, of +490,000 +/- 80,000 deg cm2 dmol-1 at 208 nm and -520,000 +/- 110,000 deg cm2 dmol-1 at 224 nm have been derived from the data.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Biol (Mosk), 1992 Sep-Oct, 26(5), 1022 - 35 {Site-specific mutagenesis of residue Lys-172 of phage T7 RNA polymerase: characterization of transcription properties of mutant proteins}; Liakhov DL et al.; Lys-172 residue of bacteriophage T7 RNA polymerase (T7RP) was substituted for Leu and Gly and Lys-172, Arg-173 were deleted by the site-directed mutagenesis using synthetic oligonucleotides . The specific activity of all mutant enzymes did not differ significantly from that of the wild-type (w.t.) T7RP while for Gly-172 mutant (G172) it was somewhat lower . Leu-172 (L172) and deletion (DEL172-3) mutants were able to direct RNA synthesis on the templates lacking the T7 promoter . DEL172-3 was not able to synthesize extraneous RNA sequences in addition to the expected run-off transcripts . L172 and DEL172-3 mutants revealed altered template specificity toward various DNA templates and showed the lower stability of enzyme-promoter complexes . The possible role of Lys-172 likely belonging to an interdomain "stretch" is discussed. Mol Microbiol, 1992 Sep, 6(18), 2557 - 63 The role of integration host factor in gene expression in Escherichia coli; Freundlich M et al.; Integration host factor is a sequence-specific, histone-like, multifunctional DNA-binding and -bending protein of Escherichia coli . The characterization and functional analysis of this protein has been done mainly in bacteriophage lambda and other mobile genetic elements . Less is known concerning the role of integration host factor (IHF) in E . coli, although it has been implicated in a number of processes in this organism including DNA replication, site-specific recombination, and gene expression . This review presents recent work which suggests that IHF alters the activity of an unusually large number of operons in E . coli . We discuss the possible physiological relevance of the involvement of IHF in gene expression and the hypothesis that IHF is a member of a class of functionally redundant proteins that participate in chromosome structure and multiple processes involving DNA. Appl Environ Microbiol, 1992 Sep, 58(9), 3180 - 2 A simple method to test condoms for penetration by viruses; Lytle CD et al.; A method by which virus penetration through condoms can be tested with simple, inexpensive equipment is described . The method uses chi X174 bacteriophage as the challenge virus and physiologically relevant pressure . Penetration by 0.1 microliters (or less) of challenge suspension can be readily detected . As examples, latex and natural-membrane condoms were examined. J Reprod Fertil, 1992 Sep, 96(1), 135 - 9 Rapid transport of seminal immunoglobulin to distal parts of the female reproductive tract in rabbits; Jappel D; Bucks immunized with the bacteriophage-cloning vector fd-tet display strong humoral seminal immunity of 1.5 x 10(6) fd-tet neutralizing units (nu) ml-1 . We used this model to study the distribution of seminal immunoglobulin in the female reproductive tract after copulation and artificial insemination . In contrast to artificial insemination, significant (P < 0.01) amounts (147 nu ml-1, i.e . about 1.25 x 10(-4) input ejaculate) of neutralizing activity reached the uterus within 20 min of a single copulation, which is evidence for rapid transport of seminal plasma to distal parts of the tract . Oviducts were also quickly impregnated with antibodies to fd-tet, which persist in the distal compartments for at least 24 h. Microbiol Rev, 1992 Sep, 56(3), 430 - 81 Bacteriophage lysis: mechanism and regulation; Young R; Bacteriophage lysis involves at least two fundamentally different strategies . Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review . The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer . This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm . The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains . The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis . The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself . In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized . Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope . These lysis proteins function by activation of cellular autolysins . A host locus is required for the lytic function of the phi X174 lysis gene E. J Gen Virol, 1992 Sep, 73 ( Pt 9), 2235 - 44 Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies; Jin H et al.; Bacterial fusion proteins containing portions of the Bunyamwera virus L protein were used as immunogens to prepare antisera in rabbits . Of five fusion proteins injected into rabbits, three yielded sera that reacted with the Bunyamwera virus L protein, detected by Western blotting or immunoprecipitation . Two of these antisera were specific for either the amino- or carboxy-terminal regions of the L protein . The specificity of these antisera was confirmed by their pattern of reactivity with full-length and truncated forms of the L protein . Plasmids containing the L gene cDNA under control of a bacteriophage T7 promoter were transfected into CV-1 cells which had previously been infected with a recombinant vaccinia virus, vTF7-3, that expresses T7 RNA polymerase . Antigenically authentic L protein was expressed . Using a nucleocapsid transfection assay developed previously, we showed that the transiently expressed L protein had RNA synthesis activity . Site-specific mutations were made in the L cDNA-containing plasmid to change certain amino acids in the putative polymerase domain of the L protein . The effects of these amino acid substitutions on the RNA synthesis activity of the L protein were monitored using the nucleocapsid transfection assay . These experiments showed that residues strictly conserved between the L proteins of different viruses in the family Bunyaviridae were obligatorily required for activity, whereas non-conserved residues could be substituted without abolishing RNA synthesis capability . Our results provide direct evidence for the functional significance of particular amino acids in the polymerase domain of a negative-strand virus RNA polymerase. Biotechniques, 1992 Sep, 13(3), 422 - 8 Multipurpose vectors for peptide expression on the M13 viral surface; Fowlkes DM et al.; We have developed a set of three cloning vectors for the expression of polypeptides on the surface of the M13 viral coat . The M13mp8 genome has been engineered for expression of foreign protein sequences near the NH2-terminus of the mature pIII protein, which is present in five copies on the outside of each M13 viral particle . All three of the vectors carry the same two useful restriction sites for directed cloning of inserts in the pIII coding region; in addition, one vector carries the bacterial gene conferring resistance to the antibiotic tetracycline, and another expresses the lacZ' polypeptide that allows functional complementation of beta-galactosidase activity within the host bacterial cell . All of these vectors propagate well in E . coli DH5 alpha F' cells and do not require helper phage . We demonstrate that a bacteriophage, expressing an eleven amino acid epitope (from human c-myc) at the NH2-terminus of pIII in one of our vectors, can be purified from a vast mixture of other M13 phage through panning techniques . In particular, we find that the c-myc-expressing viral particles can be easily recovered from phage mixtures with the biotinylated form of the monoclonal antibody, 9E10, and streptavidin-coated MagneSphere beads. Mutat Res, 1992 Sep, 275(3-6), 377 - 86 Interaction of singlet oxygen with DNA and biological consequences; Lutgerink JT et al.; To study the interaction of singlet oxygen (1O2) with DNA and the biological consequences of 1O2-induced DNA damage, we used the thermodissociable endoperoxide of 3,3'-(1,4 naphthalidene) dipropionate (NDPO2) as a generator of free 1O2 in reactions with (1) 2'-deoxynucleoside 3'-monophosphates (dNps), (2) an oligonucleotide (16-mer) having one deoxyguanine (dG), (3) native and denaturated rat kidney DNA and (4) single-stranded (ss) and double-stranded (ds) bacteriophage M13mp10 DNA . Using both anion exchange and reversed phase HPLC and 32P-postlabeling analyses, it was found that exposure of the various dNps to chemically generated 1O2 led to a detectable reaction with dGp and not with dAp, dCp, d5mCp or Tp . The reaction with dGp led to degradation of this nucleotide and the formation of a large number of reaction products, one of which could be identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp) . A second product could tentatively be identified as a formamido pyrimidine derivative of dGp (Fapy-dGp) . When ss DNA, ds DNA or the oligonucleotide were exposed to 1O2, the formation of 8-oxo-dG could also be demonstrated . With the oligonucleotide, we found a so far unidentified reaction product . Under the same reaction conditions the yield of 8-oxo-dG was about 8-fold higher in ss DNA than in ds DNA . In ss DNA 8-oxo-dG seemed to be a more prominent product than in the case of reaction of 1O2 with free dGp . Reaction of 1O2 with ss or ds M13mp10 DNA led to biological inactivation of these DNAs, ss DNA being at least 100-fold more sensitive than ds DNA . It could be concluded that inactivation of the ss DNA must be largely due to 1O2-induced DNA lesions other than 8-oxo-dG . In agreement with the observed preferential reaction of 1O2 with dG most of the so far sequenced mutations, induced by 1O2 in a 144 bp mutation target sequence inserted in the lacZ alpha gene of ss or ds M13mp10 DNA, occurred at a G or G/C base pair respectively . A preference for G(C) to T(A) transversions can be observed for which 8-oxo-dG might have been responsible . In ss DNA a significant number of the mutations are characterized by the fact that a G is deleted. Mutat Res, 1992 Sep, 275(3-6), 367 - 75 Singlet oxygen induced DNA damage; Sies H et al.; Singlet oxygen generated by photoexcitation and by chemiexcitation selectively reacts with the guanine moiety in nucleosides (kq + kr about 5 x 10(6) M-1s-1) and in DNA . The oxidation products include 8-oxo-7-hydro-deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) . Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA . The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity . Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells . Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols . Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G:C to T:A transversions. Mutat Res, 1992 Sep, 274(3), 163 - 76 Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation; Kusewitt DF et al.; Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp . Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers . After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells . DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure . Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU) . The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA . DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells . Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes. Mol Gen Mikrobiol Virusol, 1992 Sep-Oct, (9-10), 5 - 8 {Use of non-radioactive biotin-labelled probes for detecting hepatitis A virus}; Budiak EV et al.; The biotin-labeled DNA probes were constructed on the basis of the hybrid bacteriophage M13nip 9 single-stranded DNA containing the fragments of the hepatitis A viral cDNA . The probes were biotin treated by chemical modification of the DNA by the peraminating reagent or photochemically . The labeled DNA probes were used in molecular hybridization experiments with the nuclear acids fixed on the nitrocellulose filters . The biotin treated DNA was determined by the avidin-gold colloid conjugate with the subsequent physical silver amplification or by the streptavidin-alkaline phosphatase conjugate . The sensitivity of both probes was identical and permitted the determination of 5 x 10(-11)-5 x 10(-12) g of the control DNA and 10(-9) g of the hepatitis A virus . The developed test systems were used for detection of the viral RNA in blood from patients. Izv Akad Nauk SSSR Biol, 1992 Sep-Oct, (5), 744 - 52 {3'-->5'-exonucleases of the rat liver and the correction of replication errors}; Beliakova NV et al.; Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity . 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver . The exonucleases were shown to excise mismatched nucleotides from poly{d(A--T)} template 10 and 2 fold faster than matched ones . The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal . The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane . These data, taken together, are indicative of potent proofreading into hepatocytes. Microb Pathog, 1992 Sep, 13(3), 225 - 36 Cloning and nucleotide sequence of a variant Shiga-like toxin II gene from Escherichia coli OX3:H21 isolated from a case of sudden infant death syndrome; Paton AW et al.; Escherichia coli OX3:H21 expressing a toxin related to Shiga-like toxin (SLT) was isolated from the small bowel contents of a case of Sudden Infant Death Syndrome (SIDS) . This strain was lysogenic for a lambdoid bacteriophage, but this did not encode the toxin . Southern hybridization analysis of chromosomal DNA revealed that the SLT-related gene was located on a 4.6 kb PstI fragment, which was cloned into E . coli JM109 in both orientations, using the vector pUC19, to generate plasmids pJCP501 and pJCP502 . JM109 cells harbouring the recombinant plasmid produced SLT, as judged by cytotoxicity for Vero cells . Nucleotide sequence analysis revealed that the SLT gene was related to, but distinct from, previously reported variants of Shiga-like toxin type II, produced by E . coli from both human and animal sources . The A subunit of the SLT gene from OX3:H21 exhibited 95.9% homology (at both the DNA and derived amino acid sequence level) to the A subunit of the most closely related SLT-II variant . The B subunit was less similar, exhibiting 88.6 and 88.8% homology to the related gene at the DNA and amino acid level, respectively. Biochemistry, 1992 Aug 25, 31(33), 7587 - 94 Inhibition of T7 RNA polymerase initiation by triple-helical DNA complexes: a model for artificial gene repression; Maher LJ 3rd; An experimental approach is presented for the creation of an artificial and functional repressor/operator interaction that does not involve polypeptides . This in vitro approach confers oligonucleotide regulation upon a bacteriophage T7 RNA polymerase promoter by introducing an overlapping homopurine operator that can be recognized by oligonucleotide-directed DNA triple-helix formation . Recognition of optimized operator sequences in either of two triple-helix motifs is shown to efficiently inhibit T7 RNA polymerase transcription initiation in both a promoter- and oligonucleotide-specific manner . Inhibition due to triple helices of the pyrimidine motif is pH-dependent, as expected . Inhibition by purine motif triple helices is not pH-dependent and occurs efficiently under optimum T7 RNA polymerase transcription conditions . Repression by triple-helix formation can be observed rapidly after addition of purine motif repressor oligonucleotides, even when polymerase has been given prior access to the promoter . The mechanism of repression is shown to be occlusion of polymerase from the promoter rather than trapping of the polymerase in unproductive preinitiation or initiation complexes . In contrast to their inhibition of T7 RNA polymerase initiation, the triple-helical complexes studied here do not detectably inhibit transcription elongation. Biochim Biophys Acta, 1992 Aug 17, 1132(1), 17 - 25 Enriched sources of Escherichia coli replication proteins . The dnaG primase is a zinc metalloprotein; Stamford NP et al.; Primase, the product of the Escherichia coli dnaG gene, is the enzyme responsible for RNA primer synthesis on both template strands at replication forks during chromosomal DNA synthesis . The dnaG gene was modified by replacement of the natural ribosome-binding site with one complementary to the 3' end of 16S rRNA, and then inserted downstream of tandem bacteriophage lambda PR and PL promoters in the pUC9-derived vector pCE30 . Following thermal induction of transcription, the resulting plasmid pPL195 directed synthesis of primase activity to levels corresponding to approx . 120,000 molecules per cell . The overproduced protein was soluble and was readily purified in high yield (31 mg per 1 of culture) . Purified primase was monomeric, was fully active in priming replication at the bacteriophage G4 complementary strand origin, and was shown to contain 0.92 +/- 0.08 g atom of tightly-bound zinc per mol of protein . Potential zinc-binding amino-acid residues near the N-terminus of the protein were identified . Although a mutant primase lacking 27 amino acid residues from the N-terminus was partly soluble, it was completely inactive. Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1663 - 8 Delimitation of cohesive ends site (cos) of Streptomyces temperate bacteriophage R4; Mitsui H et al.; The cohesive ends site (cos) of actinophage R4 was delimitated by assaying the in vivo packaging activity of cosmid derivatives in Streptomyces lividans . A region of 66 bp from -30 to +36 from the center of cohesive ends was required for the basal level of packaging activity . Two additional regions outside the basal sequences from -39 to -31 and from +37 to +97 were necessary for the high level of activity, defined as the accessory sequences . Direct- or inverted-repeat sequences were found within the delimitated region, which might be involved in the recognition by the terminase of actinophage R4. Nucleic Acids Res, 1992 Aug 11, 20(15), 3971 - 6 In vitro replication of bacteriophage PRD1 DNA . Metal activation of protein-primed initiation and DNA elongation; Caldentey J et al.; Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism . Compared to Mg2+, the use of Mn2+ as the metal activator of the phage DNA polymerase results in a great stimulation of the initiation reaction . The molecular basis of the observed stimulatory effect is an increase in the velocity of the reaction . The phage DNA polymerase is also able to catalyze the formation of the initiation complex in the absence of DNA template . Although the presence of Mn2+ does not affect either the polymerization activity or the processivity of the DNA polymerase, this metal is unable to activate the overall replication of the phage genome . This can be explained by a deleterious effect of Mn2+ on the 3'-5'-exonucleolytic and/or the strand-displacement activity, the latter being an intrinsic function of the viral DNA polymerase required for protein-primed DNA replication. J Mol Biol, 1992 Aug 5, 226(3), 889 - 96 Selection of phage antibodies by binding affinity . Mimicking affinity maturation; Hawkins RE et al.; We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen . For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (less than 1 microgram) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kd) of the antibody . Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads . The process can distinguish between antibodies with closely related affinities . For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidin-coated paramagnetic beads . To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genes in vitro using an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity . Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VH gene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)-caproic acid (mutant Kd = 9.4(+/- 0.3) nM compared with B1.8 Kd = 41.9(+/- 1.6) nm) . The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3-nitrophenylacetyl/NIP-caproic acid murine secondary immune response. J Mol Biol, 1992 Aug 5, 226(3), 661 - 73 Cre-lox recombination in Escherichia coli cells . Mechanistic differences from the in vitro reaction; Adams DE et al.; The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction . Lambda infection was used to introduce the cre gene into cells containing plasmid substrates . The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin . Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently . These results are strikingly different from those found with purified enzyme in vitro . Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo . We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo. Gene, 1992 Aug 1, 117(1), 113 - 7 A T7 expression vector optimized for site-directed mutagenesis using oligodeoxyribonucleotide cassettes; Tanhauser SM et al.; Site-directed mutagenesis is widely used to examine structure/function relationships in proteins . We have designed a bacterial expression vector series which is optimized for efficient site-directed mutagenesis and subsequent protein synthesis without intervening subcloning steps . The vectors, derived from the T7 expression vectors of Studier and his collaborators {Studier et al., Methods Enzymol . 185 (1990) 60-89}, are small and have a bacteriophage f1 origin of replication for production of single-stranded (ss) DNA . Both single-site mutants {using ssDNA and mutating oligodeoxyribonucleotides (oligos)} and cassette mutants (mutagenesis of a short region by inserting double-stranded oligos into unique restriction sites) are rapidly synthesized and expressed with these vectors . Vector construction and use are detailed with examples showing the expression of the sequences encoding human carbonic anhydrases II and III . Production levels of greater than 60 mg of protein per liter of culture have been obtained. Genes Dev, 1992 Aug, 6(8), 1373 - 85 Activation of the catalytic core of a group I intron by a remote 3' splice junction; Michel F et al.; Over 1000 nucleotides may separate the ribozyme core of some group I introns from their 3' splice junctions . Using the sunY intron of bacteriophage T4 as a model system, we have investigated the mechanisms by which proximal splicing events are suppressed in vitro, as well as in vivo . Exon ligation as well as cleavage at the 5' splice site are shown to require long-range pairing between one of the peripheral components of the ribozyme core and some of the nucleotides preceding the authentic 3' splice junction . Consistent with our three-dimensional modeling of the entire sunY ribozyme, we propose that this novel interaction is necessary to drive 5' exon-core transcripts into an active conformation . A requirement for additional stabilizing interactions, either RNA-based or mediated by proteins, appears to be a general feature of group I self-splicing . A role for these interactions in mediating putative alternative splicing events is discussed. Virology, 1992 Aug, 189(2), 474 - 82 Bacteriophage T4 gene 21 encodes two proteins essential for phage maturation; Hintermann E et al.; The T4 prohead protease (T4 PPase) is the key enzyme in the morphopoietic pathway of the T4 phage head . It is responsible for the proteolytic processing of all head proteins allowing protein rearrangement and head expansion . To study its biochemistry and gene regulation, T4 gene 21 was cloned into an expression vector under the control of the inducible tac promoter . Two proteins of apparent molecular weights of 21.5 and 27.5 kDa were detected after induction . These proteins are synthesized using two different start codons in the same reading frame . Destruction of either start codon resulted in the loss of the respective protein . Complementation experiments with bacteriophage T4 21(-)-infected cells showed that both proteins are functional in vivo and essential for T4 phage assembly. J Virol, 1992 Aug, 66(8), 5013 - 7 Dependence of minus-strand synthesis on complete genomic packaging in the double-stranded RNA bacteriophage phi 6; Frilander M et al.; Bacteriophage phi 6 has a segmented genome consisting of three pieces of double-stranded RNA (dsRNA) . The viral procapsid is the structure that packages plus strands, synthesizes the complementary negative strands to form dsRNA, and then transcribes dsRNA to form plus-strand message . The minus-strand synthesis of a particular genomic segment is dependent on prior packaging of the other segments . The 5' end of the plus strand is necessary and sufficient for packaging, while the normal 3' end is necessary for synthesis of the negative strand . We have now investigated the ability of truncated RNA segments which lack the normal 3' end of the molecules to stimulate the synthesis of minus strands of the other segments . Fragments missing the normal 3' ends were able to stimulate the minus-strand synthesis of intact heterologous segments . Minus-strand synthesis of one intact segment could be stimulated by the presence of two truncated nonreplicating segments . The 5' fragments of each single-stranded genomic segment can compete with homologous full-length single-stranded genomic segments in minus-strand synthesis reactions, suggesting that there is a specific binding site in the procapsid for each segment. Genetics, 1992 Aug, 131(4), 769 - 81 Genetic recombination in bacteriophage T4: single-burst analysis of cosegregants and evidence in favor of a splice/patch coupling model; Shcherbakov VP et al.; To reveal the structure of penultimate DNA intermediates in T4 bacteriophage recombination, resolution of which produces free recombinant molecules, a single-burst analysis of the recombinant progeny was made in multifactor crosses, enabling one to determine quantitatively the different recombinants generated by one or two exchanges within the same chromosome segment . It was found that double and single exchanges are highly correlated in T4 recombination . These results were interpreted as evidence for simultaneous formation of a splice/patch pair as the primary recombination products . A recombination model called here the "splice/patch coupling model" is presented according to which resolution of a single DNA intermediate results in two linear heterozygous molecules containing a patch and a splice, respectively, in homologous positions. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6866 - 70 Mutations induced by methylene blue plus light in single-stranded M13mp2; McBride TJ et al.; Reactive oxygen species are generated by a variety of cellular processes . These endogenously generated, reactive intermediates produce a multiplicity of DNA alterations and mutations and have been implicated in the pathogenesis of several human diseases . We report here that treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light generates increased levels of 8-hydroxydeoxyguanosine and that mutagenesis is both highly specific and dependent on the SOS response . Lesions produced block the progression of DNA synthesis one base preceding template guanines . In SOS-induced Escherichia coli, 97% of all methylene blue-induced mutations in the lacZ alpha gene of M13mp2 DNA are single-base substitutions opposite template guanines . The most frequent mutations are G----C transversions . The G----T transversions expected from the presence of 8-hydroxydeoxyguanosine in the template strand occur, but at a lower frequency . Sequence data together with SOS dependency and the presence of replication blockage demonstrate that while 8-hydroxydeoxyguanosine may serve as an important marker to monitor oxygen-induced DNA damage in humans, it does not account for either the observed blockage to replication or the mutagenesis by methylene blue plus light in SOS-induced E . coli . Instead, an as yet unidentified lesion generated by active oxygen species is a more potent mutagenic event. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6861 - 5 Tissue- and site-specific DNA recombination in transgenic mice; Orban PC et al.; We have developed a method of specifically modifying the mammalian genome in vivo . This procedure comprises heritable tissue-specific and site-specific DNA recombination as a function of recombinase expression in transgenic mice . Transgenes encoding the bacteriophage P1 Cre recombinase and the loxP-flanked beta-galactosidase gene were used to generate transgenic mice . Genomic DNA from doubly transgenic mice exhibited tissue-specific DNA recombination as a result of Cre expression . Further characterization revealed that this process was highly efficient at distinct chromosomal integration sites . These studies also imply that Cre-mediated recombination provides a heritable marker for mitoses following the loss of Cre expression . This transgene-recombination system permits unique approaches to in vivo studies of gene function within experimentally defined spatial and temporal boundaries. Acta Crystallogr B, 1992 Aug 1, 48 ( Pt 4), 499 - 511 Structure determination of the bacteriophage phiX174; McKenna R et al.; The structure of the single-stranded DNA phage phiX174 has been determined to 3.4 A resolution . The crystal space group was P2(1) with one icosahedral particle per asymmetric unit, giving 60-fold noncrystallographic redundancy . Oscillation diffraction photographs were collected using synchrotron radiation at various wavelengths . The particle orientations in the unit cell were determined with a rotation function . Because cowpea mosaic virus has a similar external envelope to phiX174, it was used as a search model to find the approximately positions of the phiX174 particles in the unit cell relative to the crystallographic symmetry axes . An initial phase set to 12 A resolution was then based on the cowpea mosaic virus atomic structure . These phases were improved by 20 cycles of real-space molecular replacement averaging . The phase information was gradually extended to 3.4 A resolution by molecular replacement electron density averaging . One reciprocal lattice point was used for each extension followed by four cycles of averaging . The unusual particle capsid, with its 12 pentameric spikes, required the careful determination of a precise molecular envelope . This was redetermined at regular intervals, as was the particle center . The resultant electron density map was readily interpreted in terms of the F, G and J polypeptides in the capsid . A difference electron density map between full and partially empty particles showed some ordered DNA structure. Virus Genes, 1992 Aug, 6(3), 229 - 46 Expression of biologically active HIV glycoproteins using a T7 RNA polymerase-based eucaryotic vector system; Wilk T et al.; Bacteriophage T7 RNA polymerase and a derivative containing a nuclear localization signal were transiently expressed in CV-1 cells and were shown to localize to the cytoplasm and nucleus, respectively . A vector was constructed containing T7 promoter and transcription terminator sequences flanking a picornaviral 5' untranslated sequence for cap-independent translation and a polyA signal . Expression of the HIV-1 envelope glycoproteins in this vector system gave high levels of specific transcripts and translation products, independent of the subcellular localization of T7 RNA polymerase . The synthesis of HIV glycoproteins was also completely independent of the coexpression of the HIV rev protein, which is normally required for the expression of HIV structural proteins . In addition, a polyA signal was not required, whereas the presence of the picornaviral 5' untranslated region was necessary for efficient expression . Different possibilities to account for these findings are discussed . The HIV glycoproteins synthesized in this system were normally processed and assembled; they could induce syncytium formation and complement an env-deletion mutant of HIV-1. Comput Appl Biosci, 1992 Aug, 8(4), 401 - 4 DNABIND: an interactive microcomputer program searching for nucleotide sequences that may code for conserved DNA-binding protein motifs; Mrazek J et al.; This paper presents a simple program for interactive searching for nucleotide sequences that may code for the helix-turn-helix, zinc finger or leucine zipper motifs in proteins . The helix-turn-helix motifs are predicted using the recently published method of Dodd and Egan, while zinc fingers and leucine zippers are searched for by our original methods . DNABIND is shown to detect all four known helix-turn-helix motifs in bacteriophage lambda genes and both zinc fingers of the adr1 gene of yeast. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7139 - 43 Partial loss of function mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, affect highly conserved amino acids implicated in ATP binding and hydrolysis; Wild J et al.; A set of 37 mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, was isolated using a selection for high constitutive expression of heat shock proteins . Of these, 11 mutants were able to carry out some but not all functions of DnaK . These partial function mutants were divided into two classes . Class I mutants are recessive and permit replication of bacteriophage lambda and growth of cells up to 40 degrees C . Class II mutants are dominant, do not permit growth of lambda, and are temperature-sensitive for growth above 34 degrees C . Mutations in both classes alter amino acids that are highly conserved in the 70-kDa heat shock protein family . The dominant negative mutations provide strong genetic evidence that at least one form of DnaK is multimeric . Moreover, every dominant negative mutation occurs at an amino acid that has been hypothesized to be intimately involved in the process of ATP binding and hydrolysis . Our findings provide strong support for the hypothesis that such mutations are excellent tools for identifying amino acids that play critical roles in protein function. Mutat Res, 1992 Aug, 282(4), 247 - 52 UV resistance of E . coli K-12 deficient in cAMP/CRP regulation; Puyo MF et al.; Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E . coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm) . Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium . This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria . Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon. PCR Methods Appl, 1992 Aug, 2(1), 21 - 7 Ligation-mediated PCR of restriction fragments from large DNA molecules; Smith DR; A general method is described for PCR amplification of single restriction fragments from large DNA molecules . The method involves sequence-specific ligation of synthetic oligonucleotides to ambiguous 4-base 5' overhangs produced by type IIS restriction endonucleases . Such "adapter-tags" provide one target for primer annealing in subsequent PCR reactions . The second target for primer annealing is provided by a universal "bubble-tag" ligated to blunt ends produced with another endonuclease . The key advantage of this approach is that specific fragments can be isolated without any prior knowledge of the nucleotide sequence of the target . Using bacteriophage lambda DNA as a test system, unique PCR products could be generated consistently . Conditions of temperature, ionic strength, and substrate concentration in the adapter-tag ligations--which affect sequence specificity--were found to have a major influence on the purity of PCR-generated fragments . In principle, the method permits the amplification of virtually any sequence from purified cosmid or YAC DNA using a library of only 240 adapter-tags. Genomics, 1992 Aug, 13(4), 1133 - 42 Isolation of 1001 new markers from human chromosome 11, excluding the region of 11p13-p15.5, and their sublocalization by a new series of radiation-reduced somatic cell hybrids; Gerhard DS et al.; The determination of the physical map of human chromosome 11 will require more clones than are currently available . We have isolated an additional 1001 new markers in a bacteriophage vector from a somatic cell hybrid cell line that contains most of chromosome 11, except the middle of the short arm . These markers were localized to five different regions, 11p15-pter, 11p12-cen, 11q11-q14, 11q14-q23, and 11q23-qter, by a panel of previously characterized somatic cell hybrids . The region 11q11-14 harbors genes that have been shown to be important in breast cancer, B-cell lymphomas, centrocytic lymphomas, asthma, and multiple endocrine neoplasia, type 1 (MEN1) . To determine the positions of the recombinant clones located there, we developed a new series of radiation-reduced somatic cell hybrids . These hybrids, together with those previously characterized, allowed us to map the 11q11-q14 markers into 11 separate segregation groups. Infect Control Hosp Epidemiol, 1992 Aug, 13(8), 463 - 71 Rapid inactivation of infectious pathogens by chlorhexidine-coated gloves; Modak S et al.; OBJECTIVE: Gloves containing chlorhexidine gluconate in an instant-release matrix on their inner surface (CHG gloves) were tested to determine their ability to rapidly inactivate infectious pathogens that may permeate or leak through the latex surface . DESIGN: CHG gloves were exposed for 1 to 10 minutes to blood or media containing infectious pathogens (e.g., bacteria, fungi, parasites, and viruses) as well as to lymphocytes and macrophages that are known to be the primary carriers of human immunodeficiency virus (HIV) . Inactivation of pathogens was determined either by in vitro assay or in vivo infectivity . Stressed control and CHG glove fingers were submerged in a viral pool (retrovirus or bacteriophage) and after a set time, the glove interiors were checked for presence of permeated virions . RESULTS: CHG gloves rapidly inactivate all the pathogens tested including retrovirus and hepatitis B virus (90% to 100%) . In the stressed glove fingers, live virus was detected in 26% of the control group but not in any of the CHG group . CONCLUSIONS: The use of CHG gloves may reduce the risk of exposure to infectious fluid-borne pathogens should the integrity of the latex barrier be compromised by overt failure or by permeation of viruses . Rapid destruction of lymphocytes and macrophages may facilitate inactivation of HIV associated with these cells . Tests have shown that CHG coating does not alter physical properties of the glove, and, furthermore, CHG gloves do not show potential for dermal irritation or sensitization. FEBS Lett, 1992 Jul 28, 307(2), 181 - 4 Raman spectroscopic study on the conformation of a peptide fragment representing the DNA-binding domain of filamentous virus Pf3 coat protein; Miura T et al.; Raman spectra have been measured of a nonapeptide which has an amino acid sequence identical to that of the C-terminal region of the major coat protein subunit of filamentous bacteriophage Pf3 . The peptide shows a strong tendency to form a beta-sheet structure in aqueous solution . The beta-sheet formation is significantly promoted by complexation with single-stranded DNA but not with double-stranded DNA . It is suggested that the C-terminal region of the Pf3 coat protein binds to the single-stranded DNA genome in the virion with a beta-sheet conformation, in sharp contrast with the alpha-helical binding in other filamentous bacteriophages. FEBS Lett, 1992 Jul 27, 307(1), 66 - 70 Peptide display on filamentous phage capsids . A new powerful tool to study protein-ligand interaction; Cesareni G; Peptides can be displayed on the surface of filamentous bacteriophages by fusion to phage coat proteins . It was recently shown that vast (10(8)) collections of phages, each exposing a variant of the original peptide, can be constructed and utilized as a general source of peptide ligands . By panning these libraries on a target molecule linked to a solid support it is possible to select, out of the hundreds of millions of clones, those few phages that display a peptide that binds the target molecule . Searching these libraries is a powerful tool to be applied in many areas of fundamental and applied biology. Nucleic Acids Res, 1992 Jul 25, 20(14), 3591 - 8 Transcription by an immobilized RNA polymerase from bacteriophage T7 and the topology of transcription; Cook PR et al.; It is often assumed that a polymerase moves along the template as it synthesizes RNA . However, a polymerase that tracks along a helical strand will generate a transcript that is entwined about the template . No such interlocking results if the polymerase is immobile and the template moves past it . Therefore we investigated whether immobilization inhibits the RNA polymerase of T7 bacteriophage using a hybrid protein, in which the polymerase is connected through a peptide linker to an immobilizing domain, which in turn was attached through an antibody to protein A covalently linked to plastic beads . Polymerase could be released by cleaving the linker with a protease, factor Xa . Comparison of the activity of the bound and free enzymes showed that immobilization reduced the rate of initiation about fivefold . However, when re-initiation was eliminated by removing excess template, immobilization was found to have little effect on the rate of elongation . Perhaps the untwining problem is sidestepped in vivo by immobilizing the polymerase. J Biol Chem, 1992 Jul 25, 267(21), 15032 - 40 Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7; Kim YT et al.; Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy . T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl . In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl . Thioredoxin does not bind to gene 2.5 protein . Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1 . Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin . The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E . coli single-stranded DNA-binding protein, T4 gene 32 protein, and E . coli recA protein . All except E . coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E . coli recA protein inhibits these activities. J Biol Chem, 1992 Jul 25, 267(21), 15022 - 31 Purification and characterization of the bacteriophage T7 gene 2.5 protein . A single-stranded DNA-binding protein; Kim YT et al.; Bacteriophage T7 gene 2.5 protein has been purified to homogeneity from cells overexpressing its gene . Native gene 2.5 protein consists of a dimer of two identical subunits of molecular weight 25,562 . Gene 2.5 protein binds specifically to single-stranded DNA with a stoichiometry of approximately 7 nucleotides bound per monomer of gene 2.5 protein; binding appears to be noncooperative . Electron microscopic analysis shows that gene 2.5 protein is able to disrupt the secondary structure of single-stranded DNA . The single-stranded DNA is extended into a chain of gene 2.5 protein dimers bound along the DNA . In fluorescence quenching and nitrocellulose filter binding assays, the binding constants of gene 2.5 protein to single-stranded DNA are 1.2 x 10(6) M-1 and 3.8 x 10(6) M-1, respectively . Escherichia coli single-stranded DNA-binding protein and phage T4 gene 32 protein bind to single-stranded DNA more tightly by a factor of 25 . Fluorescence spectroscopy suggests that tyrosine residue(s), but not tryptophan residues, on gene 2.5 protein interacts with single-stranded DNA. J Biol Chem, 1992 Jul 25, 267(21), 15005 - 12 Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase; Rosenberg AH et al.; T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein . To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon . These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells . They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein . Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth . Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity . The mutation at amino acid 64 of 4A' appears to have little effect on 4A function . The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth. J Mol Biol, 1992 Jul 20, 226(2), 455 - 70 Three-dimensional structure of a cloning vector . X-ray diffraction studies of filamentous bacteriophage M13 at 7 A resolution; Glucksman MJ et al.; Filamentous bacteriophage M13 is a single-stranded DNA phage about 65 A in diameter and 9300 A long . X-ray diffraction studies of magnetically oriented fibers of native, mercury and iodine-labeled phage particles have been used to determine the arrangement of the major coat protein, the gene 8 product, in the virion . The coat protein is made up of a single gently curving alpha-helix extending from approximately Pro6 to near the carboxyl terminus . The axis of the alpha-helix is tilted about 20 degrees from the viral axis and wraps around the axis in a right-handed helical sense . The surface of the virus is made up largely of polar residues in the amino-terminal half of the protein including the segment of alpha-helix extending from Pro6 to Tyr24 . The interior surface of the protein coat faces the DNA and consists of an amphipathic helical segment extending from Thr36 to Ser50 . The alpha-helices form a tightly packed 15 to 20 A thick cylindrical coat around the DNA . This structural model provides insight into the potential sites for incorporating foreign protein domains that may act as functional binding sites on the surface of M13. J Mol Biol, 1992 Jul 20, 226(2), 311 - 7 Formation of the right before the left mature DNA end during packaging-cleavage of bacteriophage T7 DNA concatemers; Serwer P et al.; During bacteriophage T7 morphogenesis in a T7-infected cell, mature length T7 DNA molecules join end-to-end to form concatemers that are subsequently both packaged in the T7 capsid and cut to mature size . In the present study, the kinetics of the appearance in vivo of the mature right and left T7 DNA ends have been analyzed . To perform this analysis, the intercalating dye proflavine is used to interrupt DNA packaging . When used at 0.5 to 8.0 micrograms/ml, proflavine progressively inhibits events in the T7 DNA packaging pathway, without either altering protein synthesis or degrading intracellular T7 DNA . Restriction endonuclease kinetic analysis reveals that proflavine (8 micrograms/ml) completely blocks formation of the mature T7 DNA left end, but only partially blocks formation of the mature T7 DNA right end . Both these and other observations are explained by the hypothesis that, in the T7 DNA packaging pathway, events occur in the following sequence: (1) formation of a mature right end; (2) packaging of at least some of the genome; (3) formation of the mature left end. FEBS Lett, 1992 Jul 20, 306(2-3), 129 - 32 On the functional role of the Tyr-639 residue of bacteriophage T7 RNA polymerase; Rechinsky VO et al.; Substitution of Asp for a Tyr residue normally present at position 639 of the bacteriophage T7 RNA polymerase leads to a drastic drop in the enzymatic activity . This mutation does not affect the enzyme-promoter interaction but decreases the ability of the RNA polymerase to discriminate between GTP and ATP molecules, resulting in a decrease in the rate of the incorporation of the nucleotide into the RNA chain. Proc Natl Acad Sci U S A, 1992 Jul 15, 89(14), 6658 - 62 Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage; Sharma M et al.; The bacteriophage T4 segA gene lies in a genetically unmapped region between the gene beta gt (beta-glucosyltransferase) and uvsX (recombination protein) and encodes a protein of 221 amino acids . We have found that the first 100 amino acids of the SegA protein are highly similar to the N termini of four other predicted T4 proteins, also of unknown function . Together these five proteins, SegA-E (similar to endonucleases of group I introns), contain regions of similarity to the endonuclease I-Tev I, which is encoded by the mobile group I intron of the T4 td gene, and to putative endonucleases of group I introns present in the mitochondria of Neurospora crassa, Podospora anserina, and Saccharomyces douglasii . Intron-encoded endonucleases are required for the movement (homing) of the intron DNA into an intronless gene, cutting at or near the site of intron insertion . Our in vitro assays indicate that SegA, like I-Tev I, is a Mg(2+)-dependent DNA endonuclease that has preferred sites for cutting . Unlike the I-Tev I gene, however, there is no evidence that segA (or the other seg genes) resides within introns . Thus, it is possible that segA encodes an endonuclease that is involved in the movement of the endonuclease-encoding DNA rather than in the homing of an intron. J Biol Chem, 1992 Jul 15, 267(20), 14157 - 66 Processive proofreading is intrinsic to T4 DNA polymerase; Reddy MK et al.; DNA replication occurs in vivo with very high processivity, meaning that the replication complex assembles at the origin(s) of replication and then performs template-directed synthesis of DNA over virtually the entire genome without dissociation . Such processivity also characterizes reconstituted replication holoenzyme complexes in vitro . However, the isolated DNA polymerases are much less processive, especially under physiological conditions . In this paper we monitor the degree of processivity displayed by the bacteriophage T4-coded DNA polymerase while in its proofreading mode by asking whether an isolated polymerase can "edit-out" the 3'-terminal nucleotide from the primer (using the 3'----5'-exonuclease activity of the polymerase) and then switch into the synthesis mode without dissociating from the DNA template . This "switch experiment" is accomplished by using mismatched primer/template substrates as an experimental tool to mimic the situation that T4 DNA polymerase encounters after a misincorporation event has occurred . By performing experiments under single-turnover conditions (obtained using a heparin trap), we demonstrate that T4 DNA polymerase, upon encountering a misincorporated base, neither synthesizes the next base nor dissociates into solution . Instead, with a greater than 80% probability, it removes the misincorporated base and then continues synthesis in a fully processive manner . We also show that the removal of a doubly mispaired sequence from the 3'-terminus of the primer, followed by synthesis, is comparably processive . In contrast, the apparent processivity of removing a triply mispaired terminus is much reduced . Taken together, these observations are consistent with the notion that the "editing active site" of the T4 enzyme optimally accommodates only two unpaired nucleotide residues . Our results do not support the idea that the exonuclease activity of T4 DNA polymerase is highly selective for mismatched termini; they suggest instead that the dwell time at a misincorporated base determines overall editing efficiency . The integrated results of this study provide additional insight into the structure of the T4 DNA polymerase, as well as into the interactions between the polymerase and the polymerase accessory proteins that are required to provide the holoenzyme complex with full processivity. Gene, 1992 Jul 15, 116(2), 195 - 203 DNA-binding activity of the murine homeodomain protein Hox-2.3 produced by a hybrid phage T7/vaccinia virus system; de Jong R et al.; Homeobox-containing genes encode transcription factors that, via the homeodomain, bind specifically to DNA . To study the DNA-binding properties of the murine homeodomain-containing protein, Hox-2.3, a hybrid expression system was used, combining gene expression by recombinant vaccinia virus (reVV) with bacteriophage T7 transcription . Expression was achieved by co-infecting HeLa cells with two reVVs, one expressing the T7-RNA polymerase-encoding gene directed by the VV promoter, P7.5, and another containing the Hox-2.3 coding sequence under control of a T7 promoter {Fuerst et al., Mol . Cell . Biol . 7 (1987) 2538-2544} . Co-infected HeLa cells produced large amounts of full-length Hox-2.3 protein . Cytoplasmic and nuclear extracts from these cells were used to examine DNA-binding specificity in vitro . reVV-produced Hox-2.3 protein bound to oligos that contained one or several copies of the common homeodomain-binding site, 5'-TCA-ATTAAAT, and to a lesser extent to multiple (TAA) repeats . Using Southwestern blot analysis, no Hox-2.3-binding sites were detected in a region of the Hox-2 cluster containing the Hox-2.3, Hox-2.4 and Hox-2.5 genes. J Mol Biol, 1992 Jul 5, 226(1), 37 - 45 Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity; Osumi-Davis PA et al.; To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity . The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases . Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants .