Biochimie, 1994, 76(10-11), 1005 - 17 Multiple molecules of integration host factor (IHF) at a single DNA binding site, the bacteriophage lambda cos I1 site; Morse BK et al.; Integration host factor (IHF) is an E coli protein that binds DNA sequence-specifically and serves as a cofactor in many intracellular processes including lambda DNA packaging . In gel shift experiments, cos DNA, a DNA fragment containing the recognition signal for lambda DNA packaging, forms multiple protein-DNA complexes when combined with pure IHF . Copper(II)-1,10 orthophenanthroline footprinting of individual IHF-cos DNA complexes shows that multiple complex formation does not result from IHF binding to successive sites on the cos DNA fragment . Instead, the footprinting of DNA from two IHF-cos complexes shows protection at one site alone . DNA in the first complex is only partially protected from nucleolytic cleavage, while DNA in the second, slower-moving, complex is completely protected at the same binding site . Quantitative Western blotting experiments determined the relative stoichiometry of IHF to DNA in the two complexes . The results confirm that two molecules of IHF bind at a single site in the cos fragment . This site, cos I1, has two matches to the IHF consensus sequence, but the two matches overlap by eight of thirteen nucleotides . A search of the DNA sequence around cos, using an expanded IHF consensus sequence, has revealed additional, low-affinity consensus matches, contiguous to these . The extent of the copper(II)-1,10 orthophenanthroline footprint and the stoichiometry of the IHF-cos I1 complexes suggest that either two molecules of IHF bind to overlapping sites, or IHF binds to a site of low affinity contiguous to a strong site . Application of a thermodynamic model to the results of gel shift experiments with IHF and cos DNA suggests that multiple complex formation requires cooperative interaction between the two IHF binding sites. Vestn Ross Akad Med Nauk, 1994, (12), 24 - 9 {Genetic engineering in the bacterial synthesis of somatostatin}; Karpova SK et al.; Series of recombinant plasmids for expression of the synthetic gene somatostatin-14 (SST) as a fusion protein were obtained . The somatostatin gene was fused to chloramphenicol acetyltransferase (cat) or its deleted variant genes . Both parts of the resultant fusion protein were joined through a Met residue . The hybrid gene was expressed under the control of the cat gene promoter (Pcat), the tryptophan operon promoter (Ptrp) or the promoter of bacteriophage T5 (PT5) . These fusions gave insoluble polypeptide products amounting from 5-10% of the total cellular protein under constitutive biosynthetic conditions (Pcat) to 5-30% upon induction (Ptrp, PT5) . A correlation between the efficiency of expression and the length of cat, the power of the promoter used and the absence or presence of transcription terminators, was studied . The scheme for SST isolation from bacterial cells was developed . SST was liberated from the fused polypeptide by treatment with cyanogen bromide and purified to homogenity by a combination of chromatographic steps: gel filtration, ion-exchange and rpHPLC . The renaturated recombinant SST showed specific biological and immunological activities and had 98% purity . The yield was 1 mg of the purified cyclic SST/1 culture of E.coli. DNA Res, 1994, 1(6), 289 - 96 A novel method for generating nested deletions using the in vitro bacteriophage T3 DNA packaging system; Kawarabayasi Y et al.; To sequence a DNA segment inserted into a cosmid vector under the directed sequencing strategy, we established a simple and rapid method for generating nested deletions which uses the in vitro packaging system of bacteriophage T3 DNA . The principle is based on the previous finding that this system can translocate any linear double-stranded DNA up to 40 kb into the phage capsid in a time-dependent manner and the encapsulated DNA becomes DNase-resistant . For this purpose, we constructed a cosmid vector that carries two different antibiotic selection markers at both sides of the multiple cloning site, and after insertion of a DNA segment, the clone was linearized by lambda-terminase at the cos site . After the packaging reaction in vitro followed by DNase treatment, the encapsulated DNA was introduced into Escherichia coli cells to give clones with unidirectional deletions by differential antibiotic selection . Restriction and sequence analyses of deletion clones demonstrated that an ordered set of clones with nested deletions, ranging from less than 1 kb to 25 kb, was created from either the end of the DNA segment . Thus, nested deletion clones that cover the entire region of a approximately 40-kb cosmid insert can be obtained by a single packaging reaction, and its restriction map can be simultaneously obtained. Genet Anal Tech Appl, 1994, 11(5-6), 165 - 70 A single P1 clone bearing three genes from human chromosome 11p15.5: HRC1, HRAS1, and RNH; Weitzel JN et al.; Molecular genetic alterations of chromosome 11p15.5 are a common finding in human cancer . We previously reported the characterization of two cosmids representing a 55-kilobase (kb) region of DNA surrounding the protooncogene HRAS1 . A cluster of genes was identified adjacent to this locus, and one of these genes, HRC1, was divergently transcribed 30 kb upstream from HRAS1 . A recent report placed the gene for placental ribonuclease inhibitor (RNH, ribonuclease-angiogenin inhibitor) within 90 kb of HRAS1 by pulsed-field gel electrophoresis (PFGE) mapping . We used recombinant P1 bacteriophage clones for physical mapping to determine the position of RNH relative to the HRAS1 transcription unit and HRC1 on chromosome 11p15.5 . PFGE and Southern analysis of genomic DNA suggested the order of the genes (HRC1-HRAS1-RNH) . P1 clones confirmed this assignment, and placed RNH within 30-50 kb of the 3' end of HRAS1 . Furthermore, a single 80-kb P1 clone that bears all three genes was isolated and clarified the Not I restriction map for the HRAS1-RNH interval . Their close physical association was predicted by simple screening of an arrayed P1 library; the clone containing all three genes was selected from multiple positive signals obtained for each HRC1 and RNH because it mapped to the same library-well address. Genet Anal Tech Appl, 1994, 11(5-6), 158 - 64 Expression of P1 DNA in mammalian cells and transgenic mice; McCormick SP et al.; Because the P1 bacteriophage packages DNA inserts of 80-100 kb, which are much larger than inserts of bacteriophage lambda or cosmid vectors, P1 DNA can be used to express large genes in cultured cells and transgenic mice . We obtained a P1 bacteriophage clone with a 79.5-kb insert (p158) that spanned the entire human apolipoprotein (apo-) B gene . We used the insert from p158 to express the human apo-B gene in both cultured rat hepatoma cells and transgenic mice . In this article, we review our apo-B expression studies and discuss the techniques that we have used for these expression studies. Genet Anal Tech Appl, 1994, 11(5-6), 129 - 39 Isolation of P1 bacteriophage clones containing large contiguous segments of the human and mouse loci for the T-cell coreceptor molecule CD8; Zhang XL et al.; The T-lymphocyte coreceptor molecules CD8 (composed of CD8 alpha and CD8 beta chains) and CD4 undergo a complex pattern of regulated expression during T-cell maturation . In the thymus, the most immature cells progress from expressing neither molecule (the double-negative {DN} stage) to an intermediate stage at which both are coexpressed (the double-positive {DP} stage) . As a result of thymic selection and further differentiation, DP cells give rise to the most mature thymic cells and peripheral T cells that express either CD8 or CD4 (the single-positive {SP} stage) . Our previous studies of the transcriptional regulatory mechanisms controlling CD8 alpha expression during the DN-->DP and DP-->SP transitions suggest the existence of important cis-acting elements located a considerable distance from the CD8 alpha gene and that these elements might serve to regulate both CD8 alpha and CD8 beta . While both genes and intergenic DNA span approximately 60 kb in the mouse, the relevant cis elements could lie either within or beyond this region . As a result, we sought to isolate large contiguous segments of DNA in P1 bacteriophage that covered at least this region from the mouse and human CD8 locus . Our initial physical characterization of these clones demonstrates the value of the P1 system as all isolated clones were found to contain single contiguous 85- to 95-kb segments of DNA that are faithful replicas of the chromosomal locus . The presence of abundant native flanking DNA both upstream and downstream of the intact coding regions will make these clones extremely useful for identifying physiological CD8 cis-active regulatory elements by virtue of their ability to direct appropriate lineage- and stage-specific expression in transfected and transgenic T cells. Mol Biol Rep, 1994-95, 20(3), 109 - 27 Single-stranded DNA binding protein encoded by the filamentous bacteriophage M13: structural and functional characteristics; Stassen AP et al.; The single-stranded DNA binding protein, or gene V protein (gVp), encoded by gene V of the filamentous bacteriophage M13 is a multifunctional protein that not only regulates viral DNA replication but also gene expression at the level of mRNA translation . It furthermore is implicated as a scaffolding and/or chaperone protein during the phage assembly process at the hostcell membrane . The protein is 87 amino acids long and its biological functional entity is a homodimer . In this manuscript a short description of the life cycle of filamentous phages is presented and our current knowledge of the major functional and structural properties and characteristics of gene V protein are reviewed . In addition models of the superhelical complexes gVp forms with ssDNA are described and their (possible) biological meaning in the infection process are discussed . Finally it is described that the 'DNA binding loop' of gVp is a recurring motif in many ssDNA binding proteins and that the fold of gVp is shared by a large family of evolutionarily conserved gene regulatory proteins. Acta Microbiol Pol, 1994, 43(2), 145 - 53 Integration host factor, a histone-like Escherichia coli protein, binds to at least four sites of the DNA fragment containing the recA gene; Krawczyk B et al.; The integration host factor (IHF) 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 carried out mainly in bacteriophage lambda and other mobile genetic elements . Less is known concerning the role of 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 . In this paper we report data concerning the binding of IHF protein to the recA gene region . IHF binds to at least four sites of the DNA fragment containing the recA gene, as shown by gel mobility shift assays . On the basis of the ihf consensus sequences published, we have been able to identify two sequences of putative ihf sites (ihf 1 and ihf2) into the 1390 bp long sequence containing the recA gene, but only the ihf2 site was able to bind IHF, as measured by gel mobility shift experiments . The nonfunctional ihf1 sequence was found to overlap the -35 region of the recA promoter and the functional ihf2 sequence was found within the recA gene structure at nt +780 to +807 (both with three mismatches according to the consensus sequence of Kur et al., 1989) . This confirms our earlier results that the IHF-DNA interaction does not depend on any very rigid sequence, but also on the suitable sequences of the neighbouring regions, together with the proper DNA conformation.(ABSTRACT TRUNCATED AT 250 WORDS) Virchows Arch, 1994, 424(1), 1 - 6 Bacteriophage epitope libraries . The generation of specific binding proteins and peptides in vitro; Fink LM et al.; New concepts and methodologies that can be used to generate proteins, such as specific variable regions of immunoglobulins and other binding peptides in an in vitro selection system are reviewed . These technologies can also be used to alter the kinetics, affinity and avidity of various binding interactions . The nature of epitopes recognized by specific antibodies or receptors can be delineated using selected epitopes displayed on bacteriophages . The basic principles of the technology is predicted upon the belief that if one has a large enough variety of keys, one can open any given lock . The range of utility of these systems to generate new reagents will impact upon the development of new diagnostic and therapeutic reagents . This technology should allow for a much wider range of probes which may have increased binding capacity and allow the development of more sensitive assays with higher signal to noise ratios . These reagents can be produced more efficiently without the use of animals and will be used in diagnostic and experimental pathology . This brief review presents a concise description of the concepts and uses of this new technology . Selected references and reviews are given as sources for further details. J Virol, 1994 Jan, 68(1), 6 - 13 Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase; Zoulim F et al.; All known DNA polymerases require primers for the initiation of DNA synthesis . While cellular polymerases and reverse transcriptases use free hydroxyl groups of RNA or DNA, the DNA polymerases of certain animal viruses and bacteriophages depend upon hydroxyl groups of amino acid residues within proteins as primers for DNA synthesis . Recently, the reverse transcriptase of a hepadnavirus has been shown to prime RNA-directed DNA synthesis from an internal site of the polypeptide (G.H . Wang and C . Seeger, Cell 71:663-670, 1992) . In this report we demonstrate that a tyrosine residue of the polymerase polypeptide is the site of a phosphodiester linkage with the first nucleotide of minus-strand DNA . This tyrosine residue is located within an amino-terminal domain of the polymerase polypeptide and is indispensable for the priming of reverse transcription . Our results demonstrate that the hepatitis B virus reverse transcriptase can initiate DNA synthesis without the requirement for tRNA as a primer. Gene, 1993 Dec 27, 137(1), 85 - 91 Cloning in a bacteriophage lambda vector for the display of binding proteins on filamentous phage; Hogrefe HH et al.; We have combined the efficiency and ease of use of bacteriophage lambda vectors with the power of phage display screening technology to create SurfZAP . The use of bacteriophage lambda allows the construction of large lambda expression libraries, which are rapidly and efficiently converted to stable plasmid libraries by mass excision . In SurfZAP, clones are expressed as fusions with amino acids 198-406 of the M13 minor coat protein (cpIII) and are displayed on the surface of filamentous phage . When produced with helper phage proteins, the fusion proteins are incorporated into the surface of phagemid particles . We demonstrate the utility of biopanning by isolating tetanus toxoid-binding mouse Fab clones from SurfZAP libraries . Approximately 10-100-fold enrichment of specific clones was observed after each panning round . The ability to create a large library of genotypes and screen the phenotypes by activity may be a potent methodology for basic research and drug discovery. Gene, 1993 Dec 27, 137(1), 77 - 83 Isolation and characterization of nucleic acid-binding antibody fragments from autoimmune mice-derived bacteriophage display libraries; Calcutt MJ et al.; The display of antibody fragments (Fab) on the surface of filamentous bacteriophage and selection of phage that bind to a particular antigen has enabled the isolation of Fab with numerous specificities, including haptens, proteins and viral particles . We have examined the possibility of isolating nucleic acid-binding Fab by constructing a combinatorial library of phage displaying Fab derived from autoimmune (MRL/lpr) mice . Autoimmune mice were chosen because they contain antibodies (Ab) reactive against nuclear components, including DNA, RNA and protein complexes . The library was panned against single-stranded (ss) calf thymus (CT) DNA and the selected Fabs were analyzed further . Characterization of the nucleic acid-binding phage led to the identification of two kinds of Fab with quite different properties . One Fab bound with high affinity a variety of ssDNA molecules, as well as several model RNA substrates . This Fab has been affinity purified to greater than 95% and competition studies revealed a marked preference for binding to poly(dT) . The second Fab showed a reduced binding to RNA ligands and a restricted number of ssDNA molecules . Analysis of the deduced amino acid (aa) sequences of the Fab variable (V) regions revealed that the heavy (H) chain V region from the strong nucleic acid-binding Fab was derived from a VH gene that is used recurrently in autoantibodies . This VH domain was most similar to an anti-ssDNA autoimmune monoclonal antibody (mAb) suggesting that antigen-binding specificities present in an autoimmune repertoire may be directly accessed by this approach.(ABSTRACT TRUNCATED AT 250 WORDS) Nucleic Acids Res, 1993 Dec 25, 21(25), 5934 - 9 Bacteriophage T4 and human type I DNA ligases relax DNA under joining conditions; Ciarrocchi G et al.; Both bacteriophage T4 and human type I DNA ligases in the presence of a mixture of ATP, AMP and PPi altered the topological properties of a supercoiled substrate by a step-wise reaction eventually leading to a population of fully relaxed, covalently closed products . In the presence of only AMP and PPi DNA products containing nicks with 3'OH/5'P termini accumulated in the presence of bacteriophage T4 DNA ligase, suggesting reversal of the entire joining reaction, but not in the presence of human DNA ligase I . Both DNA ligases became deoxyadenylylated in the presence of dATP, but the joining reaction did not proceed to completion . However, with both enzymes the full relaxing reaction took place in the presence of dAMP alone and in the presence of a mixture of dATP, dAMP and PPi . In no case could the joining reaction be reversed by dAMP and PPi . Related experiments with modified derivatives of deoxyribonucleoside 5'-triphosphates and PPi gave results in accord with these observations . The AMP dependent DNA relaxation catalysed by DNA ligases was insensitive to the presence of exonuclease III . These results indicate that controlled relaxation of the substrate by both DNA ligases occurs as a separate reaction rather than by simple reversal of the joining reaction . These findings support the hypothesis that in vivo the DNA topoisomerising ligases relax their substrate at the replication fork both during and separately from ligation of a pre-existing nick. J Biol Chem, 1993 Dec 25, 268(36), 27208 - 13 Evidence for distinct primase and helicase domains in the 63-kDa gene 4 protein of bacteriophage T7 . Characterization of nucleotide binding site mutant; Mendelman LV et al.; Gene 4 of bacteriophage T7 encodes two co-linear proteins, the 56- and 63-kDa gene 4 proteins . The 56-kDa protein translocates 5' to 3' on single-stranded DNA using nucleotide hydrolysis for energy and is a helicase . The 63-kDa gene 4 protein catalyzes all of the activities of the 56-kDa gene 4 protein and, in addition, catalyzes the synthesis of oligoribonucleotides on single-stranded DNA . Two conserved residues in a putative nucleotide binding site of the 63-kDa gene 4 protein were mutated by substituting Val and Met for wild-type residues Gly and Lys, at positions 317 and 318, respectively . The mutant 63-kDa gene 4 protein lacks the ability to catalyze the hydrolysis of a nucleoside 5'-triphosphate in a single-stranded DNA-dependent reaction and inhibits nucleotide hydrolysis by wild-type gene 4 proteins . The mutant primase contains 0.4% of the primase activity of the 63-kDa gene 4 protein on M13 single-stranded DNA and 12% of the wild-type primase activity on an oligonucleotide with a single primase recognition site . Addition of wild-type 56-kDa gene 4 protein stimulates the mutant primase activity over 50-fold on M13 single-stranded DNA and 8-fold on oligonucleotides . This increase in primase activity correlates with an increase in the affinity of the mutant primase-wild-type helicase complex for single-stranded DNA template. J Biol Chem, 1993 Dec 25, 268(36), 27198 - 207 The nucleotide binding site of the helicase/primase of bacteriophage T7 . Interaction of mutant and wild-type proteins; Notarnicola SM et al.; The helicase and primase activities of bacteriophage T7 are distributed between the 56- and 63-kDa gene 4 proteins . The 63-kDa protein catalyzes both helicase and primase activities . The 56-kDa gene 4 protein lacks the 63 amino acids at the N terminus of the colinear 63-kDa protein and catalyzes only helicase activity . Helicase activity is dependent on the hydrolysis of a nucleoside 5'-triphosphate . Sequence analysis reveals a single "A-type" nucleoside 5'-triphosphate binding site near the center of each gene 4 protein . We have examined the essential role of nucleoside triphosphate hydrolysis both in vivo and in vitro by using site-directed mutagenesis to alter the conserved, adjacent Gly and Lys residues within this nucleotide binding site . The mutant gene 4 proteins, expressed from plasmids carrying the cloned genes, do not complement a T7 phage lacking gene 4 . Moreover, the mutations are dominant-lethal: they block productive infection by wild-type T7 phage . A nucleotide binding site mutant 56-kDa gene 4 protein, purified to homogeneity from cells over-expressing the gene, binds but lacks the ability to hydrolyze nucleotides and cannot bind to single-stranded DNA . Consequently, this mutant gene 4 protein also lacks helicase activity . The mutant gene 4 proteins inhibit the nucleotide hydrolysis activity of wild-type gene 4 proteins in a stoichiometric manner . The apparent inhibition constant (Ki = 22 +/- 4.5 nM) of this interaction may reflect the gene 4 oligomer dissociation constant in the presence of nucleotide and single-stranded DNA . Analysis of the inhibition reaction indicates that this is a linear mixed-type inhibition, indicating that the mutant protein binds the wild-type protein to form an inactive complex on single-stranded DNA . Furthermore, the mutant 56-kDa gene 4 protein has the same affinity for both the wild-type 63- and 56-kDa gene 4 proteins, suggesting that there is no preference for the formation of homo-oligomeric complexes . The ability of the mutant proteins to inhibit the activity of the wild-type gene 4 proteins indicates that nucleotide hydrolysis is coordinated and cooperative among the members of the gene 4 protein complex as it binds and translocates on single-stranded DNA. J Biol Chem, 1993 Dec 25, 268(36), 27100 - 8 Genetic and biochemical studies of bacteriophage T4 DNA polymerase 3'-->5'-exonuclease activity; Reha-Krantz LJ et al.; DNA polymerase exonucleolytic proofreading is important in attaining high fidelity DNA replication . One of the most well characterized proofreading activities is the 3'-->5'-exonuclease activity of bacteriophage T4 DNA polymerase . We have used genetic analyses and protein sequence comparisons to Escherichia coli DNA polymerase I to identify amino acids in the N-terminal region of T4 DNA polymerase that are required for exonucleolytic proofreading . Mutant DNA polymerases with amino acid substitutions D112A/E114A, D219A, or D324A reduced 3'-->5'-exonuclease activity 10(2)-10(4)-fold in various in vitro assays and decreased DNA replication fidelity in vivo . DNA replication activity was also reduced for the exonuclease-deficient DNA polymerases in vitro and in vivo . Reduction in DNA replication appeared to be due primarily to the interdependence of T4 DNA polymerase replication and proofreading activities; T4 DNA polymerase requires 3'-->5'-exonuclease activity to repair primer termini that are not suitable substrates for extension . Observations reported here provide further evidence in support of the proposal that DNA polymerases have distinct 3'-->5'-exonuclease and polymerase active sites. J Biol Chem, 1993 Dec 25, 268(36), 26879 - 85 Kinetics of the uracil-DNA glycosylase/inhibitor protein association . Ung interaction with Ugi, nucleic acids, and uracil compounds; Bennett SE et al.; The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) inactivates Escherichia coli uracil-DNA glycosylase (Ung) by forming an Ung.Ugi protein complex with 1:1 stoichiometry . Stability of the Ung.Ugi complex was demonstrated by the inability of free Ugi to exchange with Ugi bound in preformed complex . Ung was reacted with fluorescein 5-isothiocyanate to produce fluorescent-Ung (F-Ung), which retained full uracil-DNA glycosylase activity and susceptibility to Ugi inactivation . Addition of Ugi to F-Ung under steady-state conditions resulted in saturable (15%) fluorescence quenching at a F-Ung.Ugi ratio of 1:1.4 . Dissociation constants determined for the F-Ung interaction with M13 DNA, uracil-containing DNA, and poly(U) equaled 600, 220, and 190 microM, respectively . While F-Ung associated with nucleic acid polymers was able to bind Ugi efficiently, F-Ung bound in the F-Ung.Ugi complex could no longer effectively bind nucleic acid . Stopped-flow kinetic analysis suggested the F-Ung/Ugi association was described by a two-step mechanism . The first step entailed a rapid pre-equilibrium distinguished by the dissociation constant Kd = 1.3 microM . The second step led irreversibly to the formation of the final complex and was characterized by the rate constant k = 195 s-1 . We infer Ugi inactivates Ung through the formation of an exceptionally stable protein-protein complex. Gene, 1993 Dec 22, 136(1-2), 211 - 3 Plasmid pBRINT: a vector for chromosomal insertion of cloned DNA; Balbas P et al.; Plasmid pBRINT is a pBR322 derivative {Bolivar et al., Gene 2 (1977) 95-113; Balbas et al., Gene 50 (1986) 3-40} that allows the insertion and replacement of DNA sequences into the Escherichia coli chromosome by homologous recombination . This method uses the inability of E . coli strain ATCC47002 (JC7623) to replicate covalently closed circular (ccc) pBR322-derived plasmids, and the convenience of XGal+IPTG screening for recombinants . The vector also contains suitable selection markers (Ap and Cm), as well as a multiple cloning site (MCS) derived from the pUC vectors {Yanisch-Perron et al., Gene 33 (1985) 103-119} to facilitate cloning . A simple PCR scheme was developed to scan for DNA insertions into the bacterial chromosome . Once introduced into the chromosome, the inserted DNA sequences can be transferred to other strains by bacteriophage P1-mediated transduction. Gene, 1993 Dec 22, 136(1-2), 193 - 8 An expression-packaging-processing vector which selects and maintains 7-kb DNA inserts in the blue T4 phage genome; Hong YR et al.; We have developed an efficient positive-selection vector to insert foreign DNA segments fused to the T4 ipIII gene (encoding internal protein IPIII) into the bacteriophage T4 genome . By using partial deletions of the T4 e gene, which encodes phage lysozyme, lysozyme activity required for plaque formation is used to select plasmid integrants which restore the e gene . In this work, we demonstrate that DNA inserts more than 7.0 kb in length can be incorporated into a T4 genome lacking the alt gene . In addition, the recombinant T4 not only contains a fusion gene driven by the T4 ipIII promoters, but also packages the fusion protein into the T4 capsid due to targeting by the IPIII portion . This expression-packaging-processing system shows that active IPIII::beta Gal fusion reporter protein is produced and packaged during phage infection. J Mol Biol, 1993 Dec 20, 234(4), 926 - 31 Suppression of bacteriophage T7 ssb mutation with host ssb; Shimizu K et al.; Three new mutants of bacteriophage T7 gene 2.5, which encodes a single-stranded DNA-binding protein (ssb), were isolated and characterized . One of them, ts2.5, which showed temperature-sensitive growth, was found to have two mutations in the gene: one a missense mutation generating a Gly143-->Ser change, and the other an amber mutation at Tyr215 . The other two mutants (am2.5-1 and am2.5-2) had amber mutations at Tyr15 and Ser201, respectively . None of these mutants produced a significant number of viable progeny under restrictive conditions, irrespective of whether the Escherichia coli ssb protein was functional . However, another gene 2.5 mutant, up2, which we had isolated previously, was found to be dependent on the function of host ssb for growth . Further analysis of the up2 mutation revealed that it had two additional mutations at genes 6 and 18 besides an opal mutation, op1, in gene 2.5 . Neither of the suppressor mutations for the op1 mutation suppressed other gene 2.5 mutations, ts2.5 and am2.5-2 . A mutant having the op1 mutation alone was unable to grow on nonsense suppressor-free hosts regardless of the presence of host ssb . These results indicate that the suppressors are specific for the op1 mutation and can make the host ssb usable during T7 phage development. J Mol Biol, 1993 Dec 20, 234(4), 1308 - 16 The DNA sequence of adenovirus type 40; Davison AJ et al.; The 34,214 bp DNA sequence of adenovirus type 40 strain Dugan was determined directly from random fragments of virion DNA cloned into a bacteriophage M13 cloning vector . The gene layout is similar to that of other human adenoviruses, and in addition contains two potential protein-coding regions that are conserved, but have not been recognized previously, in other adenovirus genomes . One is oriented rightward, contained within the intron in the protein-coding region for the L4 33K gene, and would encode a protein sharing N-terminal sequence with 33K . The other is a leftward oriented exon located between the E3 and L5 IV (fibre) regions which would specify the N terminus of a novel protein . The region encoding the C terminus of this protein is not apparent from sequence data. J Mol Biol, 1993 Dec 20, 234(4), 915 - 25 Sliding clamps of DNA polymerases; Kuriyan J et al.; The determination of the structure of the processivity factor (beta subunit) of Escherichia coli DNA polymerase III holoenzyme showed that this protein acts to clamp the polymerase onto DNA by forming a closed circular structure that can encircle duplex DNA (X.-P . Kong, R . Onrust, M . O'Donnell & J . Kuriyan . (1992) . Cell, 69, 425-437) . In this review we describe the features of the beta subunit that allow it to be linked tightly but non-specifically to DNA, and discuss the surprisingly symmetrical architecture of the molecule . The simple repeating pattern of the chain fold allows a connection to be made to the as yet unknown structures of eukaryotic proliferating cell nuclear antigen and the gene 45 protein of bacteriophage T4, which are the processivity factors of the corresponding DNA polymerases. Gene, 1993 Dec 15, 135(1-2), 161 - 6 Mechanisms of illegitimate recombination; Ehrlich SD et al.; Illegitimate recombination, which is one of the major causes of genome rearrangements, can occur in a number of ways . These might involve enzymes which cut and join DNA or enzymes which replicate DNA, as illustrated by two examples: (i) formation of deletions at the replication origin (ori) of an Escherichia coli bacteriophage, M13; and (ii) excision of E . coli transposon Tn10 . It is proposed that a common theme to various ways by which illegitimate recombination can occur might be the capacity to create ends in the DNA molecule and to make the ends meet. Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11648 - 52 Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein; Deber CM et al.; Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures . Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue . Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring . Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37) . The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs . those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins. J Biol Chem, 1993 Dec 15, 268(35), 26663 - 7 Efficient transcription of a DNA template associated with histone (H3.H4)2 tetramers; Puerta C et al.; A histone-DNA transcription template has been assembled, by dialysis against decreasing salt concentrations, from pGEMEX-1 (4 kilobases), a plasmid containing a promoter for bacteriophage T7 RNA polymerase, and from isolated histone (H3.H4)2 tetramers . Electron microscopy after psoralen cross-linking shows that each histone tetramer protects approximately 80 base pairs of DNA from psoralen action and that, under the employed conditions, an average of 15 tetramer particles are assembled per DNA molecule . This (H3.H4)2-DNA template is efficiently transcribed in vitro by T7 RNA polymerase as compared to naked DNA . The presence of (H3.H4)2 tetramers does not affect initiation, in contrast with the complete histone octamer, (H2A.H2B.H3.H4)2, assembled with the complementary addition of H2A.H2B dimers, which causes transcriptional inhibition mainly by blocking initiation. Biochim Biophys Acta, 1993 Dec 14, 1216(3), 455 - 9 Polyamines modulate streptomycin-induced mistranslation in Escherichia coli; Nastri HG et al.; The effects of intracellular levels of polyamines on both the in vivo inhibition of protein synthesis and the decrease of translation accuracy induced by streptomycin have been studied in polyamine-auxotrophic strains of Escherichia coli infected with the MS2 bacteriophage . The amount of viral coat protein formed was strongly reduced upon addition of increasing concentrations of streptomycin to polyamine-supplemented bacteria . In contrast, the antibiotic almost did not inhibit coat protein synthesis in polyamine-starved cells . The increase of mistranslation frequency elicited by streptomycin was only observed in bacteria grown with putrescine . In these cells several coat protein-satellites were detected after two-dimensional gel electrophoresis . These proteins, more basic than the normal MS2 coat protein, contain multiple substitutions of lysine for asparagine. Biochemistry, 1993 Dec 14, 32(49), 13399 - 404 Using circular permutation analysis to redefine the R17 coat protein binding site; Gott JM et al.; The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem . Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone . This method revealed that breakage of all but one phosphodiester bond within a well-defined binding site substantially reduced the binding affinity . This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions . One circularly permuted isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity . However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA . CPA also locates the 5' and 3' boundaries of protein binding sites on the RNA . The 5' boundary of the R17 coat protein site as defined by CPA was two nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2) . The smaller binding site was verified by terminal truncation experiments . A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein . The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens . Thus, the apparent site size of a protein binding site is dependent upon the method used. Nucleic Acids Res, 1993 Dec 11, 21(24), 5754 - 60 Direct genetic selection for a specific RNA-protein interaction; MacWilliams MP et al.; The decision between lytic and lysogenic development of temperate DNA bacteriophages is determined largely by transcriptional regulation through DNA-binding proteins . To determine whether a heterologous RNA-binding activity could control the developmental fate of a DNA bacteriophage, a derivative of P22 was constructed in which the chosen developmental pathway is regulated by an RNA-binding molecule interacting with its RNA target site located in a phage mRNA . In the example presented, lysogenic development of the phage relies upon R17 coat protein expression in the susceptible host cell and the availability of a suitable coat protein binding site encoded by the phage genome . Through the analysis of phage mutants that are able to grow lytically in susceptible cells that express the coat protein, additional insights were obtained regarding the specific interaction of the R17 coat protein with its RNA binding site . This study also suggests a novel and extremely sensitive strategy for selecting RNA-binding activities in vivo. Nucleic Acids Res, 1993 Dec 11, 21(24), 5705 - 11 An unstructured mRNA region and a 5' hairpin represent important elements of the E . coli translation initiation signal determined by using the bacteriophage T7 gene 1 translation start site; Helke A et al.; Gene 1 of bacteriophage T7 early region--the RNA polymerase gene--is very actively translated during the infectious cycle of this phage . A 29 base pair fragment of its ribosome binding site containing the initiation triplet, the Shine-Dalgarno sequence (S-D), 10 nucleotides (nt) upstream and 6 nt downstream of these central elements was cloned into a vector to control the expression of the mouse dihydrofolate reductase gene (dhfr) . Although all essential parts of this translation initiation region (TIR) should be present, this fragment showed only very low activity . Computer analysis revealed a potentially inhibitory hairpin binding the S-D sequence into its stem base paired to vector-derived upstream sequences . Mutational alterations demonstrated that this hairpin was not responsible for the low activity . However, addition of 21 nt of the T7 gene 1 upstream sequence to the 29 base pair fragment were capable of increasing the translational efficiency by one order of magnitude . Computer analysis of this sequence, including nucleotide shuffling, revealed that it contains a highly unstructured region lacking mRNA secondary structures but with a hairpin at its 5' end, here formed solely by T7 sequences . There was not much difference in activity whether the mRNA included or lacked vector-derived sequences upstream of the hairpin . Such highly unstructured mRNA regions were found in all very efficiently expressed T7 genes without any obvious sequence homologies . The delta G values of these regions were higher, i.e . potential secondary structural elements were fewer, than in TIR of genes from E . coli . This is likely due to the fact that T7 as a lytic phage is relying for successful infection on much stronger signals which a cell cannot afford because of the indispensable balanced equilibria of its interdependent biochemical processes . When the 5' ends of efficient T7 gene mRNA are formed by the action of RNase III they generally start with an unstructured region . Efficiently expressed T7 genes within a polycistronic mRNA, however, always contain a hairpin preceding the structure free sequence . We suggest that the formation of this 5' hairpin is releasing enough energy to keep the unstructured regions free of secondary RNA structures for sufficient time to give ribosomes and factors a good chance for binding to the TIR . In addition, sequences further downstream of the start codon give rise to an additional increase in efficiency of the TIR by almost two orders of magnitude. J Mol Biol, 1993 Dec 5, 234(3), 620 - 39 The refined structure of bacteriophage MS2 at 2.8 A resolution; Golmohammadi R et al.; Bacteriophage MS2 is an icosahedral virus with 180 copies of a coat protein forming a shell around a single-stranded RNA molecule . The coat protein subunits form a lattice with the triangulation number T = 3 . The coat protein has a fold which is different from the fold of all other viral coat proteins so far known . It consists of a five-stranded beta sheet facing the inside of the particle, and a hairpin and two helices on the outside . The crystal structure has been refined at 2.8 A resolution . The final R-factor was 0.189 for reflections with F > 2 sigma, and the root-mean-square deviation from idealized bond lengths and bond angles was 0.015 A and 2.9 degrees, respectively . The three chemically identical conformers A, B and C are largely similar . The B conformer has a unique conformation in one loop, which is involved in 5-fold interactions, while the A and C conformers, which are involved in the quasi-6-fold contacts, are similar throughout the structure . One cis-proline has been identified in the B conformer but the corresponding prolines in A and C are of the trans isomer . This residue is conserved within small RNA coliphages and it is proposed that this isomerization enables a less elongated loop (FG) around the 5-fold axis, thus creating a channel . The extensive dimer contact supports the idea of dimers as initial building blocks . An assembly pathway is proposed where five dimers converge into a pentamer and 12 pentamers are linked together with free dimers creating a complete particle. J Mol Biol, 1993 Dec 5, 234(3), 594 - 609 The role of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, in the nicking reaction; Cue D et al.; cosB is the binding site for terminase, the DNA packaging enzyme of ai-12581mbda, and cosN is the adjacent site at which terminase gm-07228es staggered nicks to generate mature lambda DNA molecules . There are three binding sites (R3, R2 and R1) within cosB for gpNu1, the small subunit of terminase . A particular transition mutation of R1, known to weaken binding of gpNu1 to R1, has been introduced into the other R sites, and in the present work the effects of R site mutations on nicking of cosN have been examined . Nicking experiments performed in the presence of ATP suggest that the most profound cosB mutation tested (the R3-R2-R1- mutation) would, at most, reduce cos nicking to congruent to 30% of the level observed for the wild-type substrate . In the presence of ATP, the R3-R2-R1- mutation had no significant effect on terminase nicking of the 1 strand and reduced r-strand nicking to 35% of the wild-type level . The other cosB mutations had no effect on the nicking of either DNA strand when nucleotide was added, but in the absence of ATP, most of the cos mutations resulted in some form of cosN nicking defect; the nicking defects, however, are milder than the in vivo packaging defects that result from the mutations . Quantitatively, only the effect of the R3-R2-R1- mutation on in vitro cosN nicking is reflective of the growth defect exhibited by a R3-R2-R1- phage but the nicking defect is only observed when ATP is omitted from the reaction . The proposal that the cosB mutations primarily affect DNA packaging rather than cosN nicking is discussed . All of the cosB mutations affect r-strand nicking to a greater extent than 1-strand nicking, implying that the interaction of terminase with the left half of cosN occurs via the direct recognition of cosNL by terminase . The level of DNA substrate required for half-maximal cos nicking is approximately equivalent for reactions performed in the presence or absence of ATP, indicating that ATP does not increase the affinity of terminase for cosB . ATP does accelerate the rate of cos nicking, suggesting that the role of ATP in promoting nicking of the cosB- DNAs is primarily to increase the rate of conversion of a cosN-terminase complex into product . A possible fourth R site, R4, is located on the other side of cosN from cosB.(ABSTRACT TRUNCATED AT 400 WORDS) Science, 1993 Dec 3, 262(5139), 1561 - 3 Visualization of single molecules of RNA polymerase sliding along DNA; Kabata H et al.; Transcription requires that RNA polymerase binds to promoters buried in nonspecific sites on DNA . The search for promoters may be facilitated if the polymerase slides along the molecule of DNA . Single molecules of Escherichia coli RNA polymerase were visualized, and their movements on immobilized bacteriophage lambda and T7 DNAs were examined . Deviating from drifts by bulk flow, about 40 percent of the enzyme molecules moved along the extended DNA . The results provide direct evidence for sliding as a mechanism for relocation of the enzyme on DNA. Protein Sci, 1993 Dec, 2(12), 2226 - 32 Structures of randomly generated mutants of T4 lysozyme show that protein stability can be enhanced by relaxation of strain and by improved hydrogen bonding via bound solvent; Pjura P et al.; The structures of three mutants of bacteriophage T4 lysozyme selected using a screen designed to identify thermostable variants are described . Each of the mutants has a substitution involving threonine . Two of the variants, Thr 26-->Ser (T26S) and Thr 151-->Ser (T151S), have increased reversible melting temperatures with respect to the wild-type protein . The third, Ala 93-->Thr (A93T), has essentially the same stability as wild type . Thr 26 is in the wall of the active-site cleft . Its replacement with serine results in the rearrangement of nearby residues, most notably Tyr 18, suggesting that the increase in stability may result from the removal of strain . Thr 151 in the wild-type structure is far from the active site and appears to sterically prevent the access of solvent to a preformed binding site . In the mutant, the removal of the methyl group allows access to the solvent binding site and, in addition, the Ser 151 hydroxyl rotates to a new position so that it also contributes to solvent binding . Residue 93 is in a highly exposed site on the surface of the molecule, and presumably is equally solvent exposed in the unfolded protein . It is, therefore, not surprising that the substitution Ala 93-->Thr does not change stability . The mutant structures show how chemically similar mutations can have different effects on both the structure and stability of the protein, depending on the structural context . The results also illustrate the power of random mutagenesis in obtaining variants with a desired phenotype.(ABSTRACT TRUNCATED AT 250 WORDS) Invest Ophthalmol Vis Sci, 1993 Dec, 34(13), 3669 - 78 An opsin homologue in the retina and pigment epithelium; Jiang M et al.; PURPOSE: The aim of this project was to investigate the retinal pigment epithelium (RPE) at the molecular level by identification of novel RPE-specific cDNAs that may encode proteins of signal transduction pathways or other proteins that are expressed preferentially in the RPE . METHODS: A bovine RPE cDNA library was constructed in bacteriophage lambda g10 using RPE-enriched poly(A)+ RNA . The library was screened by differential hybridization to bovine RPE and kidney cDNA probes . RESULTS: A member of the hepatahelical receptor family was identified in bovine RPE by molecular cloning . Its deduced amino acid sequence predicts a protein that has 291 amino acid residues and resembles most closely the family of visual pigments . A lysine residue, analogous to the retinaldehyde attachment site in rhodopsin, is conserved in the seventh hydrophobic segment of the novel sequence . Messenger RNA encoding the putative G protein-coupled receptor was detected by in situ hybridization in the RPE, inner nuclear layer, and specific cells of the ganglion cell layer . Immunohistochemical staining of bovine retina showed that the receptor protein is localized in Muller cells, as well as in the RPE . CONCLUSIONS: A novel heptahelical receptor defines a distant evolutionary branch of the visual pigment tree . The selective localization of this putative receptor, its abundance in RPE and retina, and its homology to the visual pigments suggest that the function of this receptor is important in a visual process involving the RPE and Muller cells. J Clin Invest, 1993 Dec, 92(6), 3029 - 37 Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a); Linton MF et al.; The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic {e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)} . Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100 . A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs . 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl) . The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation . When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a) . Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis. J Bacteriol, 1993 Dec, 175(24), 7848 - 55 The Cox protein is a modulator of directionality in bacteriophage P2 site-specific recombination; Yu A et al.; The P2 Cox protein is known to repress the Pc promoter, which controls the expression of the P2 immunity repressor C . It has also been shown that Cox can activate the late promoter PLL of the unrelated phage P4 . By this process, a P2 phage infecting a P4 lysogen is capable of inducing replication of the P4 genome, an example of viral transactivation . In this report, we present evidence that Cox is also directly involved in both prophage excision and phage integration . While purified Cox, in addition to P2 Int and Escherichia coli integration host factor, was required for attR x attL (excisive) recombination in vitro, it was inhibitory to attP x attB (integrative) recombination . The same amounts of Int and integration host factor which mediated optimal excisive recombination in vitro also mediated optimal integrative recombination . We quantified and compared the relative efficiencies of attB, attR, and attL in recombination with attP and discuss the functional implications of the results . DNase I protection experiments revealed an extended 70-bp Cox-protected region on the right arm of attP, centered at about +60 bp from the center of the core sequence . Gel shift assays suggest that there are two Cox binding sites within this region . Together, these data support the theory that in vivo, P2 can exert control over the direction of recombination by either expressing Int alone or Int and Cox together. Proc Natl Acad Sci U S A, 1993 Dec 1, 90(23), 11019 - 23 Autoregulation of the Escherichia coli heat shock response by the DnaK and DnaJ heat shock proteins; Liberek K et al.; All organisms respond to various forms of stress, including heat shock . The heat shock response has been universally conserved from bacteria to humans . In Escherichia coli the heat shock response is under the positive transcriptional control of the sigma 32 polypeptide and involves transient acceleration in the rate of synthesis of a few dozen genes . Three of the heat shock genes--dnaK, dnaJ, and grpE--are special because mutations in any one of these lead to constitutive levels of heat shock gene expression, implying that their products negatively autoregulate their own synthesis . The DnaK, DnaJ, and GrpE proteins have been known to function in various biological situations, including bacteriophage lambda replication . Here, we report the formation of an ATP hydrolysis-dependent complex of DnaJ, sigma 32, and DnaK proteins in vitro . This DnaJ-sigma 32-DnaK complex has been seen under different conditions, including glycerol gradient sedimentation and co-immunoprecipitation . The DnaK and DnaJ proteins in the presence of ATP can interfere with the efficient binding of sigma 32 to the RNA polymerase core, and are capable of disrupting a preexisting sigma 32-RNA polymerase complex . Our results suggest a possible mechanism for the autoregulation of the heat shock response. Oncogene, 1993 Dec, 8(12), 3333 - 42 Timing of SV40 oncogene activation by site-specific recombination determines subsequent tumor progression during murine lens development; Pichel JG et al.; We generated mice that carry copies of a dormant transgene encoding the SV40 tumor antigens . The transgenes are specifically targeted to the lens and contain features that render their expression dependent on the action of Cre, a site-specific bacteriophage DNA recombinase . Timing of oncogene activation was controlled by making Cre available either prior to, or coincident with, the onset of primary fiber differentiation in the embryonic lens vesicle . Early expression of Cre resulted in oncogene activation in undifferentiated lens epithelial cells that rapidly proliferated inside the lens capsule . By contrast, when Cre accumulation was delayed to coincide with the onset of primary lens fiber differentiation, SV40 oncogenes were activated in cells that had begun to elongate and to accumulate lens-specific crystallins . During subsequent proliferation inside the lens capsule, transformed progeny cells maintained the profile of fiber differentiation that their parent cells had acquired at the time of oncogenic conversion . Developing lens tumors were confined within the capsule of the embryonic lens . However, if the capsule was perforated in an embryonic eye in organ culture, cells rapidly grew out while still maintaining features of differentiation . Our findings show that the differentiated state of the primary target cells is an important parameter of subsequent lens oncogenesis, and that an intact lens capsule can restrict invasive neoplastic growth. J Immunol, 1993 Dec 1, 151(11), 6318 - 28 Antigen-dependent stimulation by bone marrow-derived mast cells of MHC class II-restricted T cell hybridoma; Frandji P et al.; This paper describes a new role for mast cells as being able to present Ag to immune T cells . A mouse bone marrow-derived mast cell population obtained after 3 wk of culture in a conditioned medium has been shown to express a variety of membrane-associated Ag, including MHC class II and class I Ag, CD23, CD32, high affinity receptor for IgE, and CD4 . Expression of MHC class II molecules was up-regulated upon stimulation with LPS but not with IFN-gamma and was down-regulated after exposure of mast cells to IL-3 treatment . We have demonstrated that mast cells were able to present native Ag as well as immunogenic peptides to MHC class II-restricted T cell hybridoma . The inhibition of Ag presentation after mast cells have been treated with ammonia suggests that Ag catabolism in intracytoplasmic compartment as a key step in Ag handling takes place in these cells . The MHC class II molecule is the restricting element for the presentation of OVA and the lambda repressor from bacteriophage lambda to a panel of specific T cell hybridomas, as demonstrated by the blocking effect of anti-MHC class II mAb on the Ag-presenting function . A characteristic feature of mast cells is the generation of a narrower immunogenic peptide repertoire as compared with A20 and LBB 3.4.16, a B lymphoma cell line, and a B cell hybridoma, respectively . This novel function of mast cells brings to a much closer connection inflammatory and immunologic processes and sheds new light on the biology of mast cells and particularly on the specific allergic responses. J Bacteriol, 1993 Dec, 175(23), 7741 - 2 The FhuA protein is involved in microcin 25 uptake; Salomon RA et al.; A chromosomal Tn5 insertion resulting in complete resistance to the peptide antibiotic microcin 25 was mapped to the min 4 region of the Escherichia coli genetic map . Additional experiments showed that the insertion disrupted the fhuA gene, which encodes the multifunctional outer membrane receptor for ferrichrome, the antibiotic albomycin, colicin M, and bacteriophages T5, T1, and phi 80 . Thus, microcin 25 and all of these agents share the same receptor. J Bacteriol, 1993 Dec, 175(23), 7724 - 6 Mutational analysis of the bacteriophage P2 Ogr protein: truncation of the carboxy terminus; Gebhardt K et al.; The Ogr protein is a 72-residue, zinc-binding transcription factor essential for activation of late gene expression in bacteriophage P2 . Analysis of C-terminal truncated proteins generated by stop codon mutagenesis shows that deletion of residues distal to position 51 had negligible effects on Ogr function . More-extensive deletion resulted in unstable products with severely reduced activity . These results, as well as the effects of other mutations in this region, support the idea that the 21 C-terminal residues are not required for transactivation. J Bacteriol, 1993 Dec, 175(23), 7720 - 3 Energy-dependent degradation of lambda O protein in Escherichia coli; Bejarano I et al.; Protein O of bacteriophage lambda is a short-lived protein which has a key role in the replication of the phage DNA in Escherichia coli . Here we present evidence that lambda O degradation is energy dependent: it is impaired by cyanide and alpha-methylglucoside, both of which inhibit cellular energy metabolism . Removal of these inhibitors restored the degradation of lambda O . Our experiments suggest that limited amounts of cellular energy are sufficient to support lambda O degradation . In addition, degradation of lambda O protein is prevented by a mutation in the E . coli clpP gene, but not by a mutation in the clpA gene . These results suggest that the ClpP protease is involved in the energy-dependent degradation of the lambda O protein. J Bacteriol, 1993 Dec, 175(23), 7673 - 82 Genetic and molecular analyses of the C-terminal region of the recE gene from the Rac prophage of Escherichia coli K-12 reveal the recT gene; Clark AJ et al.; The nucleotide sequence of the C-terminal region of the recE gene of the Rac prophage of Escherichia coli K-12 reveals the presence of a partially overlapping reading frame we call recT . Deletion mutations show that recT is required for the RecE pathway of conjugational recombination . By cloning recT with a plasmid vector compatible with pBR322, we showed by cis-trans tests that the portion of the recE gene encoding ExoVIII DNA nuclease activity is also required for RecE pathway conjugational recombination . The recT gene can replace the redB gene of lambda for recA-independent plasmid recombination . A Tn10 insertion mutation previously thought to be in recE is located in recT and is renamed recT101::Tn10 . Discrepancies between the molecular mass estimates of wild-type ExoVIII protein determined from mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and calculated from the predicted amino acid sequence are discussed . The hypothesis that wild-type ExoVIII protein results from fusion of RecE and RecT proteins is disproved genetically, thus supporting a previous hypothesis that the discrepancies are due to abnormal protein mobility in SDS-PAGE . A computer-performed scan of the bacteriophage nucleotide sequence data base of GenBank revealed substantial similarity between most of recE and a 2.5-kb portion of the b2 region of lambda . This suggests interesting speculations concerning the evolutionary relationship of lambda and Rac prophages. J Bacteriol, 1993 Dec, 175(23), 7541 - 9 A novel antivirulence element in the temperate bacteriophage HK022; Carlson NG et al.; Lysogens of the temperate lambdoid phage HK022 are immune to superinfection by HK022 . Superinfection immunity is conferred in part by the action of the HK022 CI repressor at the O.R operators . In this work, we have identified an additional regulatory element involved in immunity . This site, termed OFR (operator far right), is located just downstream of the cro gene, more than 250 nucleotides distant from OR . The behavior of phage containing a mutation in OFR suggests that the wild-type site functions as an antivirulence element . HK022 OFR- mutants were able to form turbid plaques indistinguishable from those of the wild type . However, they gave rise to virulent derivatives at a far higher frequency than the wild type (approximately 10(-5) for OFR- versus about 10(-9) for the wild type) . This frequency was so high that cultures of HK022 OFR- lysogens were rapidly overgrown by virulent derivatives . Whereas virulent mutants arising from a wild-type OFR+ background contained mutations in both OR1 and OR2, virulent derivatives of the OFR- mutant phage contained a single mutation in either OR1 or OR2 . We conclude that the wild-type OFR site functions to prevent single mutations in OR from conferring virulence . The mechanism by which OFR acts is not yet clear . Both CI and Cro bound to OFR and repressed a very weak rightward promoter (PFR) . It is unlikely that repression of PFR by CI or Cro binding to OFR can account in full for the antivirulence phenotype conferred by this element, since PFR is such a weak promoter . Other models for the possible action of OFR are discussed. EMBO J, 1993 Dec, 12(12), 4577 - 84 Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site; Han YW et al.; Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products . One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA . Int is the catalytic recombinase and has two different DNA binding domains . When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place . The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond . In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases . We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site . It is likely that the active site is formed by two Int monomers. EMBO J, 1993 Dec, 12(12), 4567 - 76 Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites; Segall AM et al.; Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome . The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites . A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes' . Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex . These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein . The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites . These complexes should enable a detailed analysis of synapsis for this pathway. Biochem Genet, 1993 Dec, 31(11-12), 507 - 20 Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4; Brown MD et al.; The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here . Mutations were mapped in 10 regions of the td gene by recombinational marker rescue using plasmid or M13 subclones of the td gene . Phage crosses using deletion mutants with known breakpoints in the 3' end of the td intron subdivided HA and NA mutations which mapped in this region . At least 31 of the mutations map within the 1-kb group I self-splicing intron . Intron mutations mapped only in the 5' and 3' ends of the intron sequence, in accordance with the hypothesis that the 5' and 3' domains of the T4 td intron are essential for correct RNA splicing . RNA sequence analysis of a number of mapped td mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur . These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue . Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within the td gene. Am J Infect Control, 1993 Dec, 21(6), 289 - 96 Permeability of latex and thermoplastic elastomer gloves to the bacteriophage phi X174; Hamann CP et al.; BACKGROUND: Most studies challenging the integrity of the glove barrier have compared the permeability of vinyl and latex gloves . However, no studies of a new nonlatex, nonvinyl thermoplastic elastomer have been reported . This pilot study therefore compared the protective barriers provided by latex and thermoplastic elastomer surgical gloves against penetration of the bacteriophage phi X174 . METHODS: Twenty thermoplastic elastomer gloves and 25 commercially available latex gloves (20 brand 1, 5 brand 2) were filled with a sterile serum surrogate and exposed to the phi X174 virus in a flask, with shaking for 180 minutes at 37 degrees C . Aliquots of 5 ml were withdrawn at baseline, 30, 60, 120, and 180 minutes and assayed by a standard plaque assay . The remaining contents of the gloves were then tested by an extremely sensitive qualitative assay (plaque assay without dilution of the sample) . RESULTS: With the standard plaque assay, virus was detected in 30% of the brand 1 latex gloves, in 80% of the brand 2 latex gloves, but in none of the thermoplastic elastomer gloves . The qualitative assay, which can detect even a single virus in the entire glove contents, had positive results for 30% of the thermoplastic elastomer gloves, 70% of the brand 1 gloves, and 100% of the brand 2 gloves . CONCLUSIONS: Despite the small sample, the results of this stringent assay suggest that the mechanical barrier offered by thermoplastic elastomer gloves is equal to or better than that provided by the latex gloves tested . Clinical studies are needed to evaluate thermoplastic elastomer gloves, which may withstand mechanical stress better than latex or vinyl . Thermoplastic elastomer gloves may therefore be a desirable alternative for health care workers in high-risk settings or for individuals with latex allergies. Trends Genet, 1993 Dec, 9(12), 413 - 21 Site-specific recombinases: tools for genome engineering; Kilby NJ et al.; Site-specific recombinases from bacteriophage and yeasts have been developed as novel tools for manipulating DNA both in the test-tube and in living organisms . We discuss the characteristics of these enzyme systems, review their application in genetic and developmental studies and speculate on their future potential for large-scale directed modifications of eukaryotic genomes. Am J Vet Res, 1993 Dec, 54(12), 2031 - 9 Assembly pathway of avian infectious laryngotracheitis virus; Guo P et al.; Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly contagious upper respiratory tract infection in chickens . At present, ILTV vaccines are not satisfactory because of development of a latent carrier status in vaccinated birds . Development of recombinant virus vaccines has been hampered by the limited information available on the molecular level and organization of this virus . We isolated 3 assembly intermediates, designated A, B, and C from ILTV-infected cells . Analysis of {3H}thymidine-and {35S}methionine-labeled particles, and electron microscopic studies indicated that particle A was the empty capsid, particle B was the procapsid containing scaffolding protein, and particle C was the DNA-filled capsid . The ILTV procapsids could only be found in the nucleus, which indicated that procapsids could not translocate through the nuclear membrane until they packaged the DNA . The DNA-filled capsids migrated through the nuclear membrane and obtained an envelope from the inner membrane of the nucleus . The enveloped particles then migrated through the lumen of the endoplasmic reticulum into vacuoles in the cytoplasm . Infective virions were isolated from within the infected cells, indicating that budding through the cytoplasmic membrane is not a necessary step in ILTV maturation . Abundant arrays composed of tubules about 45 to 50 nm wide were found in the cytoplasm of chicken embryonic liver cells about 30 to 38 hours after infection . Comparison of the assembly intermediates and the DNA packaging pathway of ILTV with that of bacteriophage pi 29 indicates that similarity exists.(ABSTRACT TRUNCATED AT 250 WORDS) J Clin Microbiol, 1993 Dec, 31(12), 3179 - 83 Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster; Samadpour M et al.; Genomic DNAs prepared from 168 isolates of Escherichia coli O157:H7 were analyzed for restriction fragment length polymorphisms on Southern blots probed with bacteriophage lambda DNA . The isolates analyzed included strains from a recent large multistate outbreak of E . coli O157:H7 infection associated with consumption of poorly cooked beef in restaurants, a day-care center cluster, and temporally and geographically unrelated isolates . E . coli O157:H7 isolates recovered from the incriminated meat and from 61 (96.8%) of 63 patients from Washington and Nevada possessed identical lambda restriction fragment length patterns . The lambda restriction fragment length polymorphisms observed in 11 (91.7%) of 12 day-care center patients were identical, but they differed from that of the strain associated with the multistate outbreak . E . coli O157:H7 from 42 patients temporally or geographically unrelated to either cluster of infection possessed unique and different lambda restriction fragment length patterns, except for paired isolates from three separate clusters of infection . These data demonstrate that the hybridization of DNA digests of E . coli O157:H7 with radiolabelled bacteriophage lambda DNA can be a useful, stable, and discriminatory epidemiologic tool for analyzing the linkage between strains of E . coli O157:H7. Protein Sci, 1993 Dec, 2(12), 2217 - 25 Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability; Pjura P et al.; An M13 bacteriophage-based in vivo screening system has been developed to identify T4 lysozyme mutants of enhanced thermal stability . This system takes advantage of easy mutagenesis in an M13 host, the production of functional T4 lysozyme during M13 growth, and the ability to detect lysozyme activity on agar plates . Of several mutagenesis procedures that were tested, the most efficient was based on misincorporation by avian myeloma virus reverse transcriptase . This one-step mutagenesis and screening system has been used to find 18 random single-site mutant lysozymes, of which 11 were heat resistant . Each of these had a melting temperature within 0.8-1.4 degrees C of wild type, suggesting that the screening system is quite sensitive. Protein Sci, 1993 Dec, 2(12), 2103 - 11 Identification of the amino acids comprising a surface-exposed epitope within the nucleotide-binding domain of the Na+,K(+)-ATPase using a random peptide library; Malik B et al.; Monoclonal antibodies that bind native protein can generate considerable information about structure/function relationships, but identification of their epitopes can be problematic . Previously, monoclonal antibody M8-P1-A3 has been shown to bind to the catalytic (alpha) subunit of the Na+,K(+)-ATPase holoenzyme and the synthetic peptide sequence 496-HLLVMK*GAPER-506, which includes Lys 501 (K*), the major site for fluorescein-5'-isothiocyanate labeling of the Na+,K(+)-ATPase . This sequence region of alpha is proposed to comprise a portion of the enzyme's ATP binding domain (Taylor, W . R . & Green, N . W., 1989, Eur . J . Biochem . 179, 241-248) . In this study we have determined M8-P1-A3's ability to recognize the alpha-subunit or homologous E1E2-ATPase proteins from different species and tissues in order to deduce the antibody's epitope . In addition the bacteriophage random peptide or "epitope" library, recently developed by Scott and Smith (1990, Science 249, 386-390) and Devlin et al . (Devlin, J . J., Panganiban, L . C., & Devlin, P . E., 1990, Science 249, 404-406), has served as a convenient technique to confirm the species-specificity mapping data and to determine the exact amino acid requirements for antibody binding . The M8-P1-A3 epitope was found to consist of the five amino acid 494-PRHLL-498 sequence stretch of alpha, with residues PRxLx being critical for antibody recognition. Virology, 1993 Dec, 197(2), 825 - 8 A chimeric human immunodeficiency virus type 1 TAR region which mediates high level trans-activation in both rodent and human cells; Newstein M et al.; Trans-activation of the HIV-1 LTR by the Tat protein functions by a novel mechanism which involves the direct interaction of the Tat protein and cellular factors with nascently transcribed viral RNA encoding the Tat responsive element (TAR) . Rodent cells do not efficiently support HIV-1 Tat activity because of a deficiency of human-specific factor(s) . Human chromosome 12 appears to encode one of these Tat cofactors . We have designed chimeric TAR sequences which contain the heterologous RNA sequence derived from the bacteriophage R17 genome which binds to the bacteriophage MS2 coat protein . These chimeric TAR constructs were co-transfected into rodent and human cells with a plasmid encoding a chimeric Tat protein which contains the RNA binding domain of the MS2 coat protein . TAR constructs which contain the MS2 coat protein binding region inserted into the three nucleotide "bulge" region support a high level of trans-activation by Tat-MS2 coat protein chimeras in both human and rodent cells . This result suggests that the human-specific Tat cofactor(s) may act to allow Tat to interact effectively in a ribonucleoprotein complex which includes Tat, cellular factors, and TAR RNA. Virology, 1993 Dec, 197(2), 652 - 8 Topology of the major capsid protein P3 of bacteriophage PRD1: analysis using monoclonal antibodies and C-terminally truncated proteins; Bamford JK et al.; Trimeric capsomeres of protein P3 (395 aa) are the main component of the phage PRD1 capsid, which encloses a lipid-protein vesicle containing the viral dsDNA genome . In this study we characterize a panel of monoclonal antibodies (MAb) against P3 . The epitopes recognized by the MAbs are analyzed by immunoprecipitation of intact virions or of released P3 trimers, and by Western blotting using a series of C-terminally truncated P3 molecules . Nine of the MAbs recognize epitopes on the virion surface, whereas five require unmasking of epitopes by disruption of the virions . Several of the MAbs are capable of neutralizing the virus; this is most probably due to virus aggregation by the antibodies . Analysis of the C-terminal truncations (the 6 Western blot-positive MAbs were used) delineates three major antigenic regions of the protein . The epitope of MAb 3T74 is included in the 66 N-terminal amino acids, and is not accessible on the virion surface, suggesting that the N-terminus is internally located in the capsid . MAbs 3N81 and 3R2 recognize epitopes in the region of amino acids 159-168, which is part of the first predicted beta-barrel structure of P3 . The third antigenic region is in the second predicted beta-barrel, between amino acids 217-242, where the epitopes of 3N180, 3P4, and 3T5 map . The trimerization of P3 was found to be independent of the non-structural assembly factor proteins P10 and P17 . Functional studies of the truncated proteins reveal that molecules comprising of 294 or more residues from the P3 N-terminus are capable of trimer formation. Biochemistry, 1993 Nov 30, 32(47), 12793 - 801 Mutagenic and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-ethenoadenine, 3,N4-ethenocytosine, and 4-amino-5-(imidazol-2-yl)imidazole; Basu AK et al.; The mutagenic and genotoxic properties of 1,N6-ethenoadenine (epsilon Ade), 3,N4-ethenocytosine (epsilon Cyt), and 4-amino-5-(imidazol-2-yl)imidazole (beta) were investigated in vivo . The former two modified bases are known DNA adducts formed by the human carcinogen vinyl chloride; beta is formed by pyrimidine ring-opening of epsilon Ade . Chemically synthesized deoxyhexanucleotides containing epsilon Ade and beta, d{GCT-(epsilon A)GC}, and d{GCT(beta)GC}, respectively, were described previously {Biochemistry (1987) 26, 5626-5635} . epsilon Cyt was inserted into an oligonucleotide, d{GCTAG(epsilon C)}, by a mild enzymatic synthetic procedure, which avoided exposure of the base to alkaline conditions . 3,N4-Etheno-2'-deoxycytidine 3',5'-bisphosphate coupled with reasonable efficiency (30-40%) to the 3'-nucleoside of an acceptor pentamer, d(GCTAG), in a reaction catalyzed by T4 RNA ligase in the presence of ATP . Each of the three modified hexanucleotides and an unmodified control were inserted into a six-base gap positioned at a known site in the genome of bacteriophage M13-NheI . A nick was placed in the DNA strand opposite that containing the single DNA lesions, enabling the formation of singly adducted single-stranded genomes by denaturation . After transfection of the adducted phage DNAs into Escherichia coli, each of the adducts was found to be genotoxic . The most toxic lesion was beta, which reduced survival of the genome by 97% . epsilon Cyt and epsilon Ade reduced survival by 90% and 65%, respectively . An examination of the surviving phage populations revealed that each of the three adducts was mutagenic . The least mutagenic lesion was epsilon Ade (0.1% of the survivors were mutant), which showed primarily A-->G transitions.(ABSTRACT TRUNCATED AT 250 WORDS) Gene, 1993 Nov 30, 134(1), 135 - 6 The sequences of gene rIII of bacteriophage T4 and its mutants; Raudonikiene A et al.; The nucleotide sequences of bacteriophage T4 gene rIII, from six different rIII mutants, have been determined . We show that the mutations r67, rES35 and rES40 cause basic amino acid changes in the rIII protein, while the mutations rBB9, rCR28 and rCP24 cause chain termination. Biochemistry, 1993 Nov 23, 32(46), 12538 - 47 Characterization of the Pf3 single-strand DNA binding protein by circular dichroism spectroscopy; Powell MD et al.; We have used circular dichroism (CD) spectroscopy and gel electrophoresis to characterize the single-strand DNA binding protein (ssDBP) of the bacteriophage Pf3 and its complexes with Pf3 DNA and various DNA and RNA homopolymers . The secondary structure of Pf3 ssDBP had < 1% alpha-helix and therefore was probably a beta-sheet structure like the fd gene 5 protein (g5p) . From CD titrations, the binding stoichiometry of Pf3 ssDBP was two nucleotides per protein monomer (n = 2) for complexes formed with all of the nucleic acids except poly{r(U)}, for which n = 3 (in a buffer of 10 mM Tris-HCl and 70 mM NaCl, pH 8.2) . Evidence of an additional binding mode of n = 4 for complexes formed with Pf3 DNA was found by gel electrophoresis experiments . Pf3 ssDBP showed a marked sequence dependence in binding affinities similar to that known for the fd g5p. Biochemistry, 1993 Nov 23, 32(46), 12478 - 87 Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs; Hingorani MM et al.; Protein-DNA interactions of bacteriophage T7 DNA primase/helicase protein 4A' with small synthetic oligodeoxynucleotides were investigated using a 20-base-paired hairpin duplex, and 10-, 30-, and 60-base-long single-stranded DNA . The effect of nucleotide cofactors on DNA binding was examined using membrane binding assays which showed that 4A' binds DNA optimally only in the presence of MgdTMP-PCP, the nonhydrolyzable analog of dTTP . About 20% of single-stranded DNA binding was observed in the presence of MgdTDP, but none was detectable in the absence of nucleotides . Native polyacrylamide gel electrophoresis showed that the DNAs bind predominantly to the hexameric form of 4A' . Larger oligomers of 4A' can bind DNA, but no DNA binding was observed to species smaller than the hexamer . Quantitative equilibrium binding studies at increasing 4A' concentrations and at increasing DNA concentrations showed tight binding of one 10-mer or 30-mer per hexamer . The 4A' hexamer can bind a second strand of DNA, but with a 50-fold weaker affinity than the first strand . The 60-mer showed tight binding to two 4A' hexamers, suggesting that a hexamer may interact with only 30-40 bases of single-stranded DNA . This was corroborated by nuclease protection experiments where the smallest length of DNA protected by 4A' or 4B protein was found to be about 30 bases . Equilibrium binding studies and competitive DNA binding data are consistent with a weaker affinity of 4A' for the duplex DNA . Only 20-25% of duplex DNA binding was observed at increasing 4A' protein in the presence of MgdTMP-PCP . About four duplex DNAs can bind each 4A' hexamer at increasing DNA concentrations, but their weaker binding was evident from their facile dissociation from 4A' in the presence of competing single-stranded DNA. FEBS Lett, 1993 Nov 22, 334(3), 355 - 9 Effects of amino acid substitution on the thermal stability of MS2 capsids lacking genomic RNA; Stonehouse NJ et al.; The thermal stability of capsids of the bacteriophage MS2, lacking genomic RNA, has been investigated using electron microscopy . Coat protein mutants with amino acid substitutions at residues involved in making contracts at both inter-molecular interfaces and within the coat protein submit are also capable of forming 'empty' capsids of the same size and symmetry as the wild-type protein . Mutations have been characterised which are neutral, deleterious or advantageous in terms of thermal stability . In some cases, the results can be rationalised by reference to the recently refined X-ray crystal structure of the wild-type particle. J Mol Biol, 1993 Nov 20, 234(2), 493 - 5 Crystallization and preliminary crystallographic characterization of bacteriophage T4 baseplate protein encoded by gene 9; Strelkov SV et al.; The structural protein, gene product 9 (gp9), of bacteriophage T4 controls baseplate expansion at the first steps of virus attachment onto its host bacterial cell with subsequent tail contraction . Gp9, which has an M(r) of 30.8 kDa and contains 287 amino acids, has been purified from a recombinant Escherichia coli strain and crystallized at 25 degrees C using the hanging drop vapor diffusion method at pH 4.0 with ammonium sulfate as precipitant . The crystals of gp9 belong to the space group R32 with hexagonal cell dimensions a = b = 86.5 A and c = 156.2 A and diffract X-rays to at least 2.7 A . There is one molecule per asymmetric unit. J Mol Biol, 1993 Nov 20, 234(2), 331 - 46 Monitoring of the cooperative unfolding of the sunY group I intron of bacteriophage T4 . The active form of the sunY ribozyme is stabilized by multiple interactions with 3' terminal intron components; Jaeger L et al.; We have studied the mechanism by which the 3' terminal domain of the sunY intron of bacteriophage T4 activates the group I ribozyme core of this intron, from which it is separated by some 800 nucleotides . As shown by monitoring either UV absorbance or self-splicing reaction kinetics as a function of temperature, intron transcripts undergo highly cooperative unfolding/inactivation upon heating: the two methods yield similar estimates of the thermodynamic parameters associated with this process . Such cooperativity makes it possible in turn to assess the energetic contribution of specific interactions to the overall structure, by comparing the sensitivity to heat inactivation of molecules carrying various nucleotide substitutions . By combining this approach with chemical modification, we have probed several proven or putative interactions between the core and 3' terminal domain of the intron and conclude that the role of the 3' terminal domain is to stabilize the active form of the ribozyme . Interestingly, the P9.0 interaction, which brings 3' terminal nucleotides next to the core site that binds the guanosine cofactor of the self-splicing reaction, is now shown to be composed in fact of two distinct pairings . An isolated base-pair (P9.0a), involving a residue located only six nucleotides upstream of the 3' splice site, participates in the stabilization of the ribozyme and appears to persist during the second stage of self-splicing (exon ligation) . In contrast, formation of the previously demonstrated P9.0b pairing, which involves the two penultimate intron nucleotides, contributes no additional stability and results in no detectable rearrangement of the core structure . Implications for the concept of a static ribozyme are discussed in the light of a slightly revised three-dimensional model of the sunY intron. Cell, 1993 Nov 19, 75(4), 717 - 28 Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP; Blond-Elguindi S et al.; We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides . BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule . Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM. Biochemistry, 1993 Nov 16, 32(45), 11992 - 7 Kinetic characterization of the ATPase activity of the DNA packaging enzyme from bacteriophage lambda; Tomka MA et al.; Terminases are enzymes common to all of the complex double-stranded DNA viruses and are required for viral assembly . These enzymes function to excise a single viral genome from a concatemeric DNA precursor and package it into a preformed protective protein shell or capsid . ATP hydrolysis by these enzymes has been described and appears to be critical to the packaging process . We have previously characterized the endonuclease activity of purified terminase from bacteriophage lambda {Tomka, M . A., & Catalano, C . E . (1993) J . Biol . Chem . 268, 3056-3065}, and we describe here a kinetic characterization of the ATPase activity of the enzyme . lambda Terminase possesses a DNA-stimulated ATPase activity and hydrolyzes ATP to ADP and Pi . This activity requires divalent metal and is supported by all of the group IIa metals examined, as well as Mn2+ . The reaction is also stimulated by NaCl, GTP, and dGTP . Of note is that neither of the guanosine nucleotides is hydrolyzed by the enzyme, while dATP is hydrolyzed at a rate comparable to that of ATP . Kinetic analysis of the ATPase activity revealed two apparent binding sites for ATP hydrolysis . The high-affinity site (Km = 5 microM) and low-affinity site (Km approximately 1.3 mM) hydrolyze ATP with kcat = 3 and 16 min-1, respectively . While the high-affinity site is unaffected by the presence of DNA, ATP hydrolysis at the low-affinity site is stimulated by DNA, which results from both a decrease in the Km and a concomitant increase in the kcat of the reaction.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1042 - 8 Efficient gene transfer with less cytotoxicity by means of cationic multilamellar liposomes; Yagi K et al.; A simple procedure for the preparation of cationic multilamellar vesicles (MLV) consisting of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride, dilauroyl phosphatidylcholine, and dioleoyl phosphatidylethanolamine in a molar ratio of 1:2:2 was devised . When bacteriophage lambda DNA was encapsulated into these liposomes, entrapment efficiency was found to be nearly 100%, and digestibility of the DNA was less than 10% . Upon encapsulation of the plasmid pCH110 into cationic MLV, efficient expression was comparable to that obtained with cationic vesicles prepared by reverse-phase evaporation method (REV) . Cytotoxicity of the present liposomes was less than that of cationic REV and far less than that of Lipofectin. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10881 - 5 Rapid assembly of the bacteriophage T4 core replication complex on a linear primer/template construct; Kaboord BF et al.; DNA synthesis on a primed DNA substrate by bacteriophage T4 requires the assembly of a core replication complex consisting of the T4 DNA polymerase, a single-stranded binding protein (32 protein), and the accessory proteins 44/62 and 45 . In this paper, we demonstrate the successful assembly of this core complex on a short linear primer/template system at levels of accessory proteins equivalent to the concentration of primer 3' ends . The key to this assembly is the presence of streptavidin molecules bound at each end of the DNA substrate via biotin moieties incorporated into the template strand . Streptavidin serves to block the ends of the primer/template, thus preventing translocation of the accessory proteins away from the site of assembly and their subsequent dissociation from the ends of the primer/template . Complex assembly on this substrate requires ATP and the presence of both the 44/62 and 45 proteins . The time required for assembly of a full enzyme equivalent of complex in our system is approximately 2 s. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10866 - 70 The Escherichia coli hflA locus encodes a putative GTP-binding protein and two membrane proteins, one of which contains a protease-like domain; Noble JA et al.; The hflA (high frequency of lysogenization) locus of Escherichia coli governs the lysis-lysogeny decision of bacteriophage lambda by controlling stability of the phage cII protein . hflA contains three genes, hflX, hflK, and hflC, encoding polypeptides of 50, 46, and 37 kDa, respectively . We have determined the nucleotide sequence of 3843 base pairs containing hflA and have found three large open reading frames corresponding to hflX, hflK, and hflC . HflX contains the three sequence motifs typical of GTP-binding proteins and appears to be a member of a distinct family of putative GTPases . HflC and HflK appear to be integral membrane proteins which show some similarity to each other and to a human membrane protein . The C-terminal region of HflC contains a domain resembling the catalytic domain of ClpP, a bacterial ATP-dependent protease . We hypothesize that HflK and HflC constitute a distinct membrane-bound protease whose activity may be modulated by HflX GTPase. J Biol Chem, 1993 Nov 15, 268(32), 24481 - 90 Escherichia coli topoisomerase IV . Purification, characterization, subunit structure, and subunit interactions; Peng H et al.; DNA sequence analysis of Escherichia coli parC and parE, encoding the subunits of topoisomerase IV (Topo IV) (Kato, J.-I., Suzuki, H., and Ikeda, H . (1992) J . Biol . Chem . 267, 25676-25684), showed that ParC was 22 amino acids longer on the N terminus and ParE was 29 amino acids longer on the C terminus than reported previously . E . coli strains bearing bacteriophage T7 RNA polymerase-based expression plasmids carrying both intact and truncated parC and parE were used to overproduce the ParC and ParE proteins . Full-length ParC and ParE were required to reconstitute Topo IV activity, whereas the truncated ParC and ParE were inactive . Topo IV activity was supported only by ATP or dATP . The {ATP}1/2 for DNA relaxation was 0.45 mM, almost 25-fold higher than the {ATP}1/2 for decatenation of kinetoplast DNA . Topo IV activity was inhibited by the quinolone and coumarin antibiotics, although the concentrations required for 50% inhibition of activity were 3-30-fold higher than those required to inhibit DNA gyrase . The norfloxacin-induced DNA cleavage patterns of Topo IV and DNA gyrase were distinct but overlapping . The native forms of ParC and ParE were a dimer and a monomer, respectively; whereas the active form of Topo IV was a heterotetramer, ParC2ParE2 . The inactivity of the truncated forms of ParC and ParE could be attributed to their failure to form the heterotetramer. J Biol Chem, 1993 Nov 15, 268(32), 23997 - 4004 Amplification of bacteriophage Mu DNA by rolling circle DNA replication in vitro; Nakai H; When mini-Mu DNA was allowed to transpose and replicate in vitro over a prolonged period, the products consisted not only of simple inserts and cointegrates but also high molecular weight DNA many times the unit length of mini-Mu . This high molecular weight product contained predominantly full-length mini-Mu DNA and relatively little non-Mu DNA (the vector harboring the mini-Mu element and the target for transposition in the reaction system) . It arose from rolling circle DNA replication of templates created by intramolecular strand transfer, which is catalyzed by Mu transposition proteins . A donor substrate, which is a supercoiled plasmid bearing a mini-Mu element, gave rise to large amounts of the high molecular weight product provided that the vector segment outside the mini-Mu element was 2 kilobase pairs or more . When a donor substrate had a vector segment of only 600 base pairs, the mini-Mu element first had to transpose to a larger circular target before giving rise to the high molecular weight product . These results suggest a mechanism by which Mu DNA can be amplified for lytic development without transposing multiple times . By establishing a circular template, multiple copies of Mu can be processively generated from a single initiation event. J Biol Chem, 1993 Nov 15, 268(32), 23830 - 6 The mechanism of sequence-specific DNA cleavage and strand transfer by phi X174 gene A* protein; Hanai R et al.; We have examined the biological role and catalytic function of two juxtaposed tyrosyl residues in the bacteriophage phi X174 gene A protein, Tyr-343 and Tyr-347, which have been implicated in the catalysis of sequence-specific DNA strand transfer . Site-directed mutagenesis changing either tyrosine to phenylalanine abolishes phage viability . The biochemical basis of this inviability was studied using purified A* protein containing the carboxyl-terminal 341 amino acids of the A protein, as well as purified A* protein with a Y343F or Y347F mutation . All three proteins can cleave the phi X174 replication origin and perform strand transfer between oligodeoxynucleotides bearing the recognition sequence of the A protein; however, both Tyr-343 and Tyr-347 appear to be required for coordinated DNA strand transfer by a single A* protein molecule . The chirality of a phosphorothioate group at the site of strand transfer in the DNA was found to be retained following the strand-transfer reaction, which argues against transfer of Tyr-343-linked DNA to Tyr-347 on the same protein molecule or vice versa . These results support the current model of gene A protein function in which the two tyrosines of a single protein molecule alternate in catalyzing DNA strand transfer at the viral replication origin. J Biol Chem, 1993 Nov 15, 268(32), 23812 - 7 Differential recognition of OR1 and OR3 by bacteriophage 434 repressor and Cro; Koudelka GB et al.; The developmental decisions of bacteriophage 434 depend on the ability of 434 repressor and Cro to bind OR1 and OR3 with different relative affinities; repressor binds OR1 tighter than OR3, whereas Cro slightly prefers OR3 over OR1 . Studies with operator mutants show that repressor's lower relative affinity for OR3 results from a deviation in the sequence of OR3 from consensus; an A-->G change at position 4 in one half-site (OR1: A-C-A-A-A-C-T-T-T-C-T-T-G-T; OR3: A-C-A-G-T-T-T-T-T-C-T-T-G-T) . Similar experiments show that Cro binds operators containing either A.T or G.C bases pairs at position 4 equally well, but cannot bind operators containing C.G or T.A base pairs at this position . A Gln33-->Ala mutation in 434 repressor diminishes, but does not eliminate, its ability to distinguish between purines at position 4 . This shows that a glutamine at amino acid 33 is not the sole determinant of repressor's position 4 specificity . Changing Gln33-->Leu, the amino acid at the homologous position in Cro, does not confer "Cro-like" position 4 base specificity on repressor . Similarly, a Cro protein bearing Gln at this position does not exhibit repressor's position 4 base preferences . The residual specificities of these mutant proteins indicates that in each protein, more than 1 amino acid is responsible for recognizing bases at position 4 . These were identified by analyzing the binding specificities of multiply mutated repressors, in vitro . The types of substitutions made were guided by sequence homologies between 434 repressor and Cro . At least three mutations are needed to eliminate repressor's position 4 base specificity; Gln33-->Ala, Glu32-->Gln, and Thr27-->Lys, although no set of amino acid substitutions in repressor was able to confer Cro-like position 4 specificity to repressor . These results indicate that at least the amino acids at these positions are involved in recognition of the position 4 base . Other evidence suggests that Cro and repressor use identical amino acids present at homologous positions in the DNA recognition helix in different ways. Nucleic Acids Res, 1993 Nov 11, 21(22), 5212 - 20 Fidelity of DNA synthesis catalyzed by human DNA polymerase alpha and HIV-1 reverse transcriptase: effect of reaction pH; Eckert KA et al.; The accuracy of DNA synthesis catalyzed by the Thermus aquaticus DNA polymerase and the 3'-->5' exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I varies as a function of reaction pH (Eckert, K.A . and Kunkel, T.A . (1990) Nucleic Acids Res . 18, 3739-3744; Eckert, K.A . and Kunkel, T.A . (1993) J . Biol . Chem . 268, 13462-13471) . In the current study, we demonstrate that the fidelity of human DNA polymerase alpha increases 10-fold when the pH of the in vitro synthesis reaction is lowered from pH 8.6 to pH 6.1 (37 degrees C), as determined using a base substitution reversion assay to score polymerase errors within the lacZ alpha gene of bacteriophage M13mp2 . Similarly, the base substitution fidelity of DNA-dependent DNA synthesis by the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was improved nine-fold at pH 6.5 relative to pH 8.0 (37 degrees C) . A detailed comparison of HIV-1 RT error specificity at neutral and low pH in a lacZ alpha forward mutation assay revealed that low pH suppresses both mispairing-mediated and misalignment-mediated mutations; however, the characteristic HIV-1 RT pattern of mutational hotspots at homopolymeric sequences is retained at the lower pH . Consistent with the presumption that these mutations result, in part, from increased termination of DNA synthesis within the hotspot sequences relative to other homopolymeric sequences, the HIV-1 RT termination pattern during processive DNA synthesis is not altered by low pH . The HIV-1 RT results are in agreement with our previous hypothesis that the observed increase in polymerase fidelity at low pH results from a decreased efficiency of continuing DNA synthesis from premutational DNA intermediates. J Mol Biol, 1993 Nov 5, 234(1), 124 - 39 A programmed translational frameshift is required for the synthesis of a bacteriophage lambda tail assembly protein; Levin ME et al.; Two proteins, one of 31 kDa and one of 16 kDa, are encoded by a segment of the phage lambda tail gene region that contains two overlapping reading frames, neither of which is long enough to encode the larger protein . We show that the abundant 16-kDa protein (gpG) is encoded by the upstream open reading frame, gene G . The 31-kDa protein, gpG-T, is encoded jointly by gene G and the overlapping downstream T open reading frame . gpG-T is synthesized as the result of a translational frameshift that occurs when a ribosome translating the G gene slips back by one nucleotide at a position six codons from the C terminus of the gene and thereby bypasses the G termination codon to continue on in the T open reading frame . The resulting protein shares 135 residues of N-terminal amino acid sequence with gpG, followed by 144 amino acid residues of unique sequence . The frameshift event occurs with a frequency of approximately 4% at the sequence G GGA AAG, which encodes the dipeptide -Gly-Lys- in both the zero and -1 reading frames . The frameshift frequencies of point mutants in this "slippery sequence" argue that codon-anticodon interactions with both the glycyl and the lysyl-tRNA are important for frameshifting to occur . We find no clear evidence for a pausing mechanism to enhance frameshifting, as is seen in other well-characterized frameshifts . No simple secondary structure has been predicted for the region downstream from the slippery sequence, but this downstream sequence does contribute to the frameshifting rate . Our results together with those of Katsura and Kuhl show that the frameshift product, gpG-T, has an essential role in lambda tail assembly, acting prior to tail shaft assembly . The role of gpG in tail assembly is not known . We find that both gpG and the gpG-T are absent from mature virions. J Biol Chem, 1993 Nov 5, 268(31), 23025 - 30 Selection of targeted biological modifiers from a bacteriophage library of random peptides . The identification of novel calmodulin regulatory peptides; Dedman JR et al.; The interaction of short amino acid sequences is the basis of molecular recognition and biological regulation in many cellular systems . Libraries of random peptides provide an approach to identify peptides that can be used to modulate, in a targeted fashion, the function of specific gene products . We have used a library of random peptides designed and constructed in the M13 bacteriophage to select calcium-dependent calmodulin binding-peptides . Twenty-eight independent sequences were obtained; all contained a tryptophan within the fifteen-amino acid insert . In 11 sequences, the tryptophan was located in the first possible variable position of the inserted sequence and was followed by a proline . The tryptophan-proline combination was also present in six additional isolates but at various other positions within the peptide insert . Synthetic peptides, representative of the calmodulin binding sequences, bound to calmodulin in a calcium-dependent fashion, competed with known calmodulin inhibitors and, when introduced via a patch pipette, inhibited calcium-activated chloride conductance of the colonic epithelial cell line, T84 . This report demonstrates the utility of identifying modifiers of biological function and should prove to be a valuable approach in understanding the cellular role of proteins of unknown function. Nature, 1993 Nov 4, 366(6450), 33 - 9 The DNA replication fork can pass RNA polymerase without displacing the nascent transcript; Liu B et al.; Replication proteins encoded by bacteriophage T4 generate DNA replication forks that can pass a molecule of Escherichia coli RNA polymerase moving in the same direction as the fork in vitro . The RNA polymerase ternary transcription complex remains bound to the DNA and retains a transcription bubble after the fork passes . The by-passed ternary complex can resume faithful RNA synthesis, suggesting that the multisubunit RNA polymerase of E . coli has evolved to retain its transcript after DNA replication, allowing partially completed transcripts to be elongated into full-length RNA molecules. Protein Eng, 1993 Nov, 6(8), 883 - 91 Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis; Pushko P et al.; A structure-function analysis of the icosahedral RNA bacteriophage fr coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis . Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid . This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles indistinguishable morphologically and immunologically from native phage particles . A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their assembly properties investigated . The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69) . The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of the fr CP . A third type of assembled structure was formed by a variant with a single amino acid substitution I104T . The alpha A-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E . coli . The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed. Genetics, 1993 Nov, 135(3), 719 - 30 Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family; Paulovich AG et al.; The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine . A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene . The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe . The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene . The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46 . Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered . Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype . Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype . (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits . (3) CRY2 mRNA is present at lower levels than CRY1 mRNA . (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion . Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine . Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1),