Phage

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), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.

Genetics, 1993 Nov, 135(3), 619 - 29
Mutational analysis of a C-dependent late promoter of bacteriophage Mu; Chiang LW et al.; Late transcription of bacteriophage Mu initiates at four promoters, P(lys), PI, PP and Pmom, and requires the Mu C protein and the host RNA polymerase . Promoter-containing DNA fragments extending approximately 200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid . Promoter activity, assayed by beta-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage . beta-galactosidase activities were greatest for P(lys), intermediate for PI, and lowest for PP . Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity . Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR) . Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity.

Mol Biol (Mosk), 1993 Nov-Dec, 27(6), 1345 - 55
{Analysis of peptide fragment insertions in the basic proteins of the bacteriophage M13, f1, and fd envelopes . Interconnection of structural characteristics of the envelope protein and viability of mutant phages}; Eroshkina AM et al.; An analysis of 12 peptide fragment insertions into the major coat protein (protein pVIII) of bacteriophages M13, f1 and fd has been done . To elucidate the relations between protein structural characteristics and viability of mutant phages, we used the program Pro-Anal . Correlations were found between phage viability and different physicochemical and structural characteristics of protein N-termini . Thus peptide insertions in nonviable phages have high indexes of alpha-helicity, volumes, and polarity as well as high moments (alpha-helical and beta-structural) of isoelectric point . On the other hand, high beta-turn indexes are correlated with viability . The most important factor which determines phage viability is the lack of positively charged amino acid residues on the C-terminal ends of peptide insertions . The correlations found hold for the pVIII proteins of four related phages-M13, IKe, If1 and I2-2 . Based on these results, the rule of obtaining viable mutant phages with insertions in the major coat protein is suggested . A new site is described for peptide insertions--upstream of the first amino acid residue of the mature protein sequence.

Nippon Rinsho, 1993 Nov, 51(11), 3031 - 41
{Crystal structure and function of pyrimidine dimer specific excision repair enzyme: T4 endonuclease V}; Ariyoshi M et al.; Bacteriophage T4 endonuclease V is an enzyme which plays an important role in pyrimidine dimer specific excision repair of DNA . This enzyme possesses two distinct catalytic activities, pyrimidine dimer glycosylase and apyrimidinic endonuclease . The three dimensional structure (3D) of the wild type enzyme was determined at 1.45A resolution by X-ray crystallography . In combination with the results of site-directed mutagenesis, the refined structure revealed that Glu23 and the surrounding basic residues constitute the catalytic center of this enzyme . Furthermore, the 3D structure of active site mutants were determined and compared with that of the wild type . The results suggest that a precise configuration of Glu23 residue is required for glycosylation and that Arg3 plays an important role in the substrate binding.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9842 - 6
Use of a gene encoding a suppressor tRNA as a reporter of transcription: analyzing the action of the Nun protein of bacteriophage HK022; Sloan SB et al.; The Nun protein of phage HK022 blocks the expression of genes that lie downstream of the nut sites of phage lambda . Nun is believed to act by promoting premature termination of transcription at or near these sites . To test this hypothesis and to facilitate mapping the sites of termination, we inserted a gene encoding a suppressor tRNA immediately downstream of the lambda nutL site and determined the effect of Nun on tRNA level . We found that Nun severely reduced the accumulation of mature, biologically active tRNA and promoted the accumulation of short, promoter-proximal transcripts whose 3' ends were dispersed over a 100-nucleotide region downstream of nutL . These results are consistent with the hypothesis that Nun terminates transcription within the region immediately downstream of nutL and are inconsistent with the hypothesis that the only action of Nun is to prevent translation of genes located downstream of the nut site . The stability, small size, and easily assayable biological function of suppressor tRNA recommend it as a reporter of transcription in other systems.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10173 - 7
Bacteriophage T7 gene 2.5 protein: an essential protein for DNA replication; Kim YT et al.; The product of gene 2.5 of bacteriophage T7, a single-stranded DNA binding protein, physically interacts with the phage-encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities . Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth . T7 phages that contain null mutants of gene 2.5 were constructed by homologous recombination . These gene 2.5 null mutants contain either a deletion of gene 2.5 (T7 delta 2.5) or an insertion into gene 2.5 and cannot grow in Escherichia coli (efficiency of plating, < 10(-8)) . After infection of E . coli with T7 delta 2.5, host DNA synthesis is shut off, and phage DNA synthesis is reduced to < 1% of phage DNA synthesis in wild-type T7-infected E . coli cells as measured by incorporation of {3H}thymidine . In contrast, RNA synthesis is essentially normal in T7 delta 2.5-infected cells . The defects in growth and DNA replication are overcome by wild-type gene 2.5 protein expressed from a plasmid harboring the T7 gene 2.5.

Mol Immunol, 1993 Nov, 30(16), 1519 - 28
Bacterially expressed Fabs of monoclonal antibodies neutralizing tumour necrosis factor alpha in vitro retain full binding and biological activity; Orfanoudakis G et al.; Antibody fragments specific for the human tumour necrosis factor alpha (TNF alpha) have been cloned from lambda combinatorial expression libraries using total RNA obtained from three different hybridoma cell lines of therapeutic interest . The previously described bacteriophage lambda vectors, lambda HC2 and lambda LC1, were modified to create unique antibody cloning sites in the combinatorial construct and a novel tag peptide was inserted at the C-terminal end of the expressed Fd chain . Sequence analysis of the cloned Fabs indicated that two of them were derived from a single B cell . Expression in E . coli showed that the amount of recovered Fab in the bacterial culture medium was related to the sequences of the variable coding regions . Hybrid Fabs created by chain exchange of similar antibodies were as active as the originally paired Fabs in binding assays . The relative affinities and the capacities of the bacterially expressed Fabs to neutralize TNF alpha cytotoxicity in vitro were identical to those of the parental antibodies . The results demonstrate that, using an in vitro approach, it is possible to generate from existing hybridoma cell lines high affinity Fabs which retain antigen specificity . The cloning sites incorporated into the C-terminal parts of these Fabs will now permit their further modification to include additional functional characteristics not possible with the original hybridoma antibodies.

J Bacteriol, 1993 Nov, 175(21), 7081 - 5
Two overlapping genes encoding membrane proteins required for bacteriophage N4 adsorption; Kiino DR et al.; We present the nucleotide sequences of two genes whose products are required for bacteriophage N4 adsorption . The nfrA gene encodes a 122-kDa outer membrane protein which presumably serves as the phage receptor . The nfrB gene encodes an 85-kDa inner membrane protein and may be a component of the receptor.

J Bacteriol, 1993 Nov, 175(21), 7074 - 80
A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption; Kiino DR et al.; At least four genes are required for irreversible adsorption of bacteriophage N4 . nfrA and nfrB have been characterized previously and encode an outer membrane protein and inner membrane protein, respectively . The nfrC gene product is characterized in detail in this study . We have mapped the nfrD locus to min 52 on the Escherichia coli linkage map . Maxicell analysis of nfrC and a null allele (nfrC2) cloned into a high-copy-number plasmid shows its gene product to be 42 kDa in size . We determined the nfrC nucleotide sequence which predicts a gene product of 42 kDa . Western blots (immunoblots) of Escherichia coli proteins after cellular fractionation show NfrC to be a cytoplasmic protein which is required for irreversible bacteriophage N4 adsorption, an event occurring at the cell surface.

J Bacteriol, 1993 Nov, 175(21), 6996 - 7005
The active form of the KorB protein encoded by the Streptomyces plasmid pIJ101 is a processed product that binds differentially to the two promoters it regulates; Tai JT et al.; The korB gene of Streptomyces lividans plasmid pIJ101 is known to encode an autoregulated protein that also represses transcription of a gene, kilB, implicated in pIJ101 transfer and in spreading of the plasmid along mycelia of the recipient . Earlier work has indicated that the primary gene product of korB is a 10-kDa protein predicted from the gene sequence (D.S . Stein and S.N . Cohen, Mol . Gen . Genet . 222:337-344, 1990; S . Zamen H . Richards, and J . Ward, Nuleic Acids Res . 20:3693-3700, 1992) . We report here that the 10-kDa KorB protein product is processed in vivo into a 6-kDa peptide that has a 20-fold-greater binding affinity for its operator-promoter target; in addition, the 6-kDa peptide binds differentially to the regulatory regions of the two genes it controls, showing 50-fold-greater affinity for the kilB sequence . While both the processed and unprocessed forms of KorB were observed in Escherichia coli following korB gene expression under control of the bacteriophage T7 promoter, only the 6-kDa peptide was found in S . lividans containing pIJ101, implying that this peptide is normally the biologically active form of KorB . The footprint resulting from KorB binding to the korB operator sequence overlaps the sti locus, which affects pIJ101 copy number and incompatibility as well as the size of zones of inhibited recipient cell growth ("pocks") that form around donor cells during mating . The observed ability of the korB gene product to interact with both sti sequences and the kilB promoter region suggests that it may have a role in coordinating the replication and intramycelial spread of plasmids during and/or following bacterial mating.

EMBO J, 1993 Nov, 12(11), 4453 - 9
RNA-mediated specificity of DNA packaging into hybrid lambda/phi 29 proheads; Valpuesta JM et al.; A small RNA (pRNA, 174 nt) is known to be essential for DNA packaging in bacteriophage phi 29 . However, in an in vitro DNA packaging system based on hybrid lambda/phi 29 proheads (made up of head proteins from phage lambda and connectors from phage phi 29), the specificity of DNA packaging is lost, and different RNA molecules fulfil the requirements for DNA packaging, albeit with less efficiency than phi 29 pRNA . Competition assays with RNAs from different sources have shown that phi 29 connectors bind preferentially pRNA . An increase in the efficiency of phi 29 DNA packaging into hybrid proheads induced by phi 29 pRNA is observed because, when phi 29 pRNA is incubated with hybrid proheads, phi 29 DNA is packaged more efficiently than other DNAs of similar length . Furthermore, when hybrid proheads carrying phi 29 pRNA are incubated with a mixture of DNAs from different sources, phi 29 DNA is selectively packaged, thus indicating that phi 29 pRNA determines the specificity of DNA packaging.

Virology, 1993 Nov, 197(1), 320 - 7
Characterization of the foot-and-mouth disease virus 3C protease expressed in Escherichia coli; Bablanian GM et al.; We have constructed a clone encoding the foot-and-mouth disease virus (FMDV) 3C protease gene (p3C) using the polymerase chain reaction . The construct was engineered to contain initiation and termination codons and cloned into a plasmid under the control of the bacteriophage T7 promoter . The p3C gene was expressed both in an in vitro transcription-translation system and in vivo in an Escherichia coli system containing an inducible T7 RNA polymerase gene . In both systems the expressed products were of the appropriate molecular weight and immunologically reactive with bovine convalescent serum . E . coli-expressed 3C protein was mainly found in the insoluble fraction of cell lysates . The E . coli-expressed protease was assayed in an in vitro system with radiolabeled P1 capsid precursor protein and P2 precursor protein as substrates . E . coli-expressed 3C completely processed the P1 and P2 precursors into mature capsid and nonstructural proteins, respectively . The kinetics of processing of P1 by E . coli-expressed 3C revealed the following order of cleavage: VP3-VP1, VP0-VP3, VP1-2A.

Anal Biochem, 1993 Nov 1, 214(2), 517 - 20
A method for quantitative recovery of DNA from plate lysates of bacteriophage lambda-derived clones; Mitchell A; A method is described for the purification of DNA from recombinant clones in lambda vectors . Plate lysates are used as the starting material, with the phage suspension being either loaded directly onto mini-cesium chloride two-step gradients or first subjected to precipitation with polyethylene glycol-8000 before application to the gradients . A single centrifugation step is used and the DNA is isolated from the harvested bacteriophage by incubation at room temperature with formamide followed by ethanol precipitation . The small-volume gradients were effective in isolating phage particles in the range of 1.0 to 12.5 x 10(11) pfu with quantitative recovery of approximately 7 micrograms DNA/10(11) pfu . The method was tested with a cDNA clone in lambda gt10 (as an example of a lambda-derived vector which gives a plate lysate with a high titer) and rat genomic fragments cloned into EMBL3A (for which the plate lysates are of low titer) . For both vectors, the purified DNA appeared free of inhibitory contaminants as assessed by restriction endonuclease digestion and subcloning of fragments.

J Infect Dis, 1993 Nov, 168(5), 1300 - 3
A comparison of human and bovine Escherichia coli O157:H7 isolates by toxin genotype, plasmid profile, and bacteriophage lambda-restriction fragment length polymorphism profile; Paros M et al.; Foods of bovine origin have been linked to human disease outbreaks caused by Escherichia coli O157:H7 and may be linked to the more common sporadic cases as well . In this study, E . coli O157:H7 from the bovine reservoir (22 isolates: 12 from dairy and 10 from beef breed cows) and from human patients (50 isolates from sporadic human infections) were compared using Shiga-like toxin genotypes, plasmid profiles, and DNA restriction fragment length polymorphisms identified with a bacteriophage lambda probe (lambda-RFLP) . Twenty-three lambda-RFLP profiles, 4 Shiga-like toxin genotypes, and 8 plasmid profiles were identified among the isolates tested . Together the typing methods distinguished 43 strains, of which 3 were isolated from both humans (5 isolates) and cattle (6 isolates; 5 from dairy herds) . These data demonstrate the value of lambda-RFLP as a means of strain identification for E . coli O157:H7.

J Virol, 1993 Nov, 67(11), 6507 - 12
Novel mechanism for reverse transcription in hepatitis B viruses; Wang GH et al.; Reverse transcription of all retroviruses and most retroid elements requires tRNA as a primer for DNA synthesis . However, in hepatitis B viruses the viral polymerase itself acts as a primer for reverse transcription (G.-H . Wang and C . Seeger, Cell 71:663-670, 1992) . We have now demonstrated that in order to prime DNA synthesis, the polymerase binds to an RNA hairpin, which then serves as a template for the formation of a short DNA primer that is covalently linked to protein . Following its synthesis, the nascent DNA strand apparently dissociates from its template and reanneals with complementary sequences at the 3' end of the RNA genome, where DNA synthesis continues . Since this RNA hairpin also functions as a packaging signal for viral RNA, hepadnaviruses have adopted a replication strategy that relies on the same signal for two biochemically distinct events, RNA packaging and reverse transcription . This mechanism is without precedent among all known retroid elements and among other viruses and bacteriophages that use protein as a primer for RNA or DNA synthesis . It could provide an effective target for antiviral therapy, which is required for the treatment of more than 300 million carriers of hepatitis B virus.

Biochem Biophys Res Commun, 1993 Oct 29, 196(2), 943 - 9
A novel approach for isolation and mapping of intron mutations in a ribonucleotide reductase encoding gene (nrdB) of bacteriophage T4 using the white halo plaque phenotype; Lal SK et al.; The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 base pair self splicing intron which is closely related to other group I introns of T4 and eukaryotes . The screening, isolation and mapping of the nrbB intron mutations was conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dhydrofolate reductase or thymidylate synthase . We have isolated 159 hydroxylamine-induced nrdB mutants, determined which mutations are in nrdB by marker rescue with clones of the nrdB gene and have mapped these mutations by marker rescue using subclones of the nrdB intron . Thirty out of the 159 nrdB mutations are in or near the intron . These mutations cluster towards the ends, mainly the 3' end . We have performed deletion mapping to further map mutations in the 3' end of the intron . The mutations map in regions of conserved structural elements, thus supporting secondary structure predictions similar to those of the well studied td intron in the T4 gene coding for thymidylate synthase.

Gene, 1993 Oct 29, 133(1), 9 - 16
Synthesis of two bacteriophage lambda S proteins in an in vivo system; Chang CY et al.; Bacteriophage lambda has two genes which are essential for lysis: R, a gene encoding a 158-amino-acid (aa) transglycosylase that attacks the peptidoglycan, and S, a gene encoding two inner-membrane-associating proteins, designated S105 and S107 for their predicted lengths in aa residues . S105 and S107 are thought to have opposing roles in lysis, with the former acting as the lethal lysis effector and the latter as a lysis inhibitor . Here, we used a T7-polymerase-mediated expression system to show that S105 and S107 are synthesized at a constant ratio of about 2.5:1 throughout the period leading up to lysis, indicating that lysis scheduling does not require a translationally controlled switch from inhibitor (S107) to effector (S105) synthesis . However, evidence is presented that the mRNA sequences immediately 5' to the ribosome-binding site (RBS) of the S gene are required for the rather limited translation, but not the stability, of the S mRNA . No difference could be found in the pattern of ternary complex formation over the two S start codons in in vitro toe-printing assays with the wild-type mRNA and with mRNA deleted of the upstream sequences . Nevertheless, these results may suggest a role for translational control in S gene expression, if not in its temporal regulation or in the partition between S105 and S107 production.

J Biol Chem, 1993 Oct 25, 268(30), 22609 - 17
Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli; Wojtkowiak D et al.; We have used 14C-labeled bacteriophage lambda O-DNA replication protein as a probe to identify and purify Escherichia coli proteases capable of its degradation . In this manner, five different proteases (termed Lop) have been identified capable of degrading lambda O protein to acid-soluble fragments in an ATP-dependent fashion . One of these activities was purified to homogeneity and shown to be composed of two different polypeptides . The 23,000-Da component (LopP) was identified as the previously characterized ClpP protein, known to complex with ClpA to form the ClpAP, an ATP-dependent protease, capable of degrading casein . The second 46,000-Da component was identified as ClpX (LopC), coded by a gene located in the same operon, but promoter distal to that coding for ClpP (Gottesman, S., Clark, W . P., de Crecy-Lagard, V., and Maurizi, M . R . (1993) J . Biol . Chem . 268, 22618-22626) . This identification was based on the determination of the sequence of the first 24 amino acid residues of the purified ClpX protein and its identity with that predicted by the DNA sequence . The ClpXP protease is substrate specific, since it degrades casein (known to be degraded by ClpAP), lambda P, or DnaK proteins slowly or not at all . These results suggest that ClpX protein directs ClpP protease to specific substrates . It is estimated that 50% of all lambda O-specific protease activity present in crude E . coli extracts is due to the ClpXP protease . We propose that transient inhibition of lambda O degradation observed in vivo during the later stages of lambda-DNA replication in vivo is responsible for the switch from bidirectional to unidirectional replication . One round unidirectional replication will lead to strand separation resulting in a switch from early (theta) to late (sigma) mode of lambda-DNA replication.

J Mol Biol, 1993 Oct 20, 233(4), 682 - 94
Structural transitions during maturation of bacteriophage lambda capsids; Dokland T et al.; The three-dimensional structures of the procapsid and of the mature capsid of bacteriophage lambda were determined to a resolution of approximately 3.4 nm by cryo-electron microscopy and image processing . The mature lambda capsid contains two major proteins, gpE and gpD, arranged on a T = 7 lattice, with gpE arranged as hexamers and pentamers and gpD arranged as trimers . The hexamers and pentamers in the virion display a cartwheel-like structure, with skewed spokes (or arms) radiating out from a central hexameric hub . The thimble-shaped gpD trimers are superimposed on the trivalent interaction point of these arms . A reconstruction of a lambda D- mutant capsid to lower resolution shows no trace of these trimers, thus revealing the interactions of the underlying arms . The procapsid has elongated, irregularly shaped hexamers with gpE subunits set perpendicularly to the capsid surface.

Biochemistry, 1993 Oct 19, 32(41), 11235 - 46
Energetics of arginine-4 substitution mutants in the N-terminal cooperativity domain of T4 gene 32 protein; Villemain JL et al.; Gene 32 protein (gp32) from bacteriophage T4 is a sequence-nonspecific single-strand (ss) nucleic acid binding protein which binds highly cooperatively to ss nucleic acids . The N-terminal "B" or basic domain (residues 1-21) is known to be required for highly cooperative binding by gp32 (where K(app) = K(int) omega, omega > or = 500), since its removal results in a protein which binds ss nucleic acids noncooperatively (omega = 1) . In this paper, we probe the molecular details of cooperative binding by gp32 by physicochemical characterization of a set of four single amino acid substitution mutants of Arg4: Lys4 (R4K gp32), Gln4 (R4Q gp32), Thr4 (R4T gp32), and Gly4 (R4G gp32) . The qualitative ranking of binding affinities to poly(A) is wild-type > or = R4K > R4Q > R4T > R4G > gp32-B (gp32 lacking the first 21 amino acids) . The occluded site size is n(app) = 7.5 +/- 0.5 for all gp32s . Resolution of K(int) and omega for wild-type, R4K, R4Q, and R4T gp32s was estimated under conditions of low lattice saturation (v < or = 0.011) using multiple reverse fluorescence titrations collected at 10 mM Tris-HCl, pH 8.1, 20 degrees C, and a NaCl concentration where K(app) was (2-4) x 10(6) M-1 for each gp32 on the ribohomopolymer poly(A) . Binding parameters for all gp32s were obtained directly or compared by conservative extrapolation of the {NaCl} dependence of K(app) to 0.20 M NaCl, 20 degrees C, pH 8.1 . The magnitude of omega was then assumed not to vary with {NaCl} (shown for R4T gp32), allowing estimation of K(int) at 0.20 M NaCl . We find that R4K gp32 binds to poly(A) with an overall affinity (K(app)) which is 2-3-fold lower than wild-type gp32, while omega for each molecule seems indistinguishable (wild-type gp32, omega approximately 800-1300; R4K gp32, omega approximately 600-1200) . Surprisingly, R4Q gp32 is characterized by an omega also not readily distinguishable from the wild-type and R4K proteins (omega approximately 800-4400), while K(app) is reduced about 10-fold . This mutant also shows a significantly reduced {NaCl} dependence of the binding to poly(A) . R4T gp32 binds about 10-fold weaker than the Q mutant . It exhibits an omega ranging from 300 to 700 and a substantially reduced {NaCl} dependence (delta log K(int)/delta log {NaCl} = -1.4 from 0.10 to 0.20 M NaCl), indicative of significant perturbations in both K(int) and omega terms.(ABSTRACT TRUNCATED AT 400 WORDS)

Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9562 - 5
A single-base-pair mutation changes the specificities of both a transcription activation protein and its binding site; Retallack DM et al.; The C1 protein of bacteriophage P22 binds to a unique site in the -35 region of the PRE promoter and activates transcription of the phage c2 repressor gene . This -35 target has an approximate direct repeat that overlaps the 5' end of the c1 coding region . We have isolated a single-base-pair mutation in this region that changes the PRE -35 target as well as the amino-terminal region of the C1 protein . Although the mutant C1 protein activates the mutant PRE promoter, it fails to activate the wild-type PRE promoter . This suggests that a single-base-pair mutation changes the specificities of both a protein and its target site . These studies also indicate that C1 binding to DNA is influenced by contacts made through residues near the amino terminus.

Eur J Biochem, 1993 Oct 15, 217(2), 493 - 500
Proof-reading 3'-->5' exonucleases isolated from rat liver nuclei; Belyakova NV et al.; Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity . Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases . Two 3'-->5' exonucleases of 40 kDa and 50 kDa (exo-40 and exo-5) have been isolated from rat liver nuclei and purified to near homogeneity . They are shown to excise mismatched nucleotides from poly{d(A-T)} template, respectively, 10-fold and 2-fold faster than the matched ones . Upon addition of either of these exonucleases to the DNA polymerase alpha from rat liver or calf thymus, the fidelity of in-vitro reproduction of the primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, levels of exonuclease and DNA-polymerase activities being similar . Extrapolation of in-vitro DNA-replication fidelity to the cellular levels of activities of the exonucleases and the alpha-polymerase suggests that exonucleolytic proof-reading augments the accuracy of DNA synthesis by 2-3 orders of magnitude.

Biochem Biophys Res Commun, 1993 Oct 15, 196(1), 1 - 6
Mutagenesis of bacteriophage IKe major coat protein transmembrane domain: role of an interfacial proline residue; Williams KA et al.; The transmembrane (TM) domain of the 53-residue major coat protein of the M13-related bacteriophage IKe (residues 24-42: LISQTWPVVTTVVVAGVLI) has been subjected to randomized mutagenesis to probe the conformation and stability of the TM domain, as well as the effect of structurally-important residues such as proline . TM mutants were obtained by the Eckstein method of site-directed mutagenesis using the IKe genome as template so as to eliminate the need for subcloning . Over 40 single- and double-site viable mutants of bacteriophage IKe were isolated . Every residue in the TM segment, except the highly conserved Trp29, could be mutated to at least one other residue; polar and charged mutations occurred in the TM segment adjacent to the N-terminal domain (residues 24-28), while non-polar substitutions predominated in the C-terminal portion (residues 30-42) . The Pro30 locus tolerated four mutations-Ala, Gly, Cys, and Ser- which represent the four side chains of least volume . Mutant coat proteins obtained directly from the phage in milligram quantities were studied by circular dichroism spectroscopy and SDS-PAGE gels . Wild type IKe coat protein solubilized in sodium deoxycholate micelles was found to occur as an alpha-helical, monomeric species which is stable at 95 degrees C, whereas the mutant Pro30-->Gly undergoes an irreversible conformational transition at ca . 90 degrees C to an aggregated beta-sheet structure . The result that Pro30 stabilizes the TM helix in the micellar membrane suggests a sterically-restricted location for the wild type Pro pyrrolidine side chain in the bulky Trp-Pro-Val triad, where it may be positioned to direct the initiation of the subsequent TM core domain helix.

Biochim Biophys Acta, 1993 Oct 10, 1152(1), 171 - 6
The macroscopic organization of reconstituted M13 coat protein-phospholipid systems . An EPR spectroscopy and polarizing microscope study; Ottaviani MF et al.; The coat protein of the bacteriophage M13 in the alpha-helical state is reconstituted in macroscopically oriented systems of dioleoylphosphatidylcholine that are prepared by squeezing the reconstituted material between glass plates . The coat protein dramatically influences the macroscopic orientation of the multibilayers, as is investigated by polarizing microscopy and EPR spectroscopy of the cholestane spin label embedded in the bilayers . It is found that with increasing amounts of protein the spontaneous macroscopic orientation of the reconstituted system decreases . This effect is proposed to be due to an increase of the apparent viscosity of the lipid-protein systems with increasing amounts of protein . This is assumed to arise from a sticky effect of the C- and N-terminal protein parts that extend into the aqueous phase between the bilayers.

Cell, 1993 Oct 8, 75(1), 147 - 54
Bacteriophage T4 Alc protein: a transcription termination factor sensing local modification of DNA; Kashlev M et al.; Bacteriophage T4 Alc protein participates in shutting off host transcription after infection of E . coli . It is demonstrated that Alc acts as a site-specific termination factor . The Alc sites occur frequently in E . coli DNA, resulting in early cessation of elongation in several tested transcription units . Alc-dependent termination requires unimpeded propagation of the elongating complex as it approaches the Alc site . Temporary halting of RNA polymerase within 10-15 bp before the Alc site prevents termination . Bacteriophage T4 transcription is protected from the action of Alc by overall substitution of cytosine with 5-hydroxymethyl cytosine in T4 DNA . In vitro methylation of CpG sequences in the vicinity of an Alc site abolishes the effect of Alc . Thus, Alc-dependent termination involves local sensing of the state of cytosine modification and a short-term "memory" of recent pausing.

J Mol Biol, 1993 Oct 5, 233(3), 447 - 63
fd coat protein structure in membrane environments; McDonnell PA et al.; The membrane bound form of bacteriophage fd coat protein has a long hydrophobic membrane spanning helix and a shorter amphipathic helix in the plane of the bilayer . Residues near the N and C termini and in the turn connecting the two helices are mobile . The locations and orientations of the helical secondary structure elements and the protein backbone dynamics were characterized by combining results from multidimensional solution NMR experiments on protein samples in micelles and high resolution solid-state NMR experiments on protein samples in oriented and unoriented lipid bilayers . The coat protein is a monomer in micelles . The secondary structure of the membrane bound form of fd coat protein is very similar to that of the structural form found in the virus particles, since it is nearly all alpha helix . However, the membrane bound form of the protein differs from the structural form of the protein in virus particles in the arrangement of the secondary structure, since the membrane bound form of the protein has two distinct helical domains oriented perpendicular to each other and the structural form of the protein in the virus particles has a nearly continuous helix aligned approximately along the filament axis . In addition, there are substantial differences in the dynamics of residues in the bend between the two helices and near the C terminus, since they are mobile in the membrane bound form of the protein and not in the virus particles . Residues 1 to 5 at the N terminus are highly mobile and unstructured in both the membrane bound and structural forms of the coat protein.

J Mol Biol, 1993 Oct 5, 233(3), 429 - 46
Dual role of the sequence-specific bacteriophage T4 endoribonuclease RegB . mRNA inactivation and mRNA destabilization; Sanson B et al.; Gene regB of bacteriophage T4 encodes a sequence-specific endoribonuclease that introduces cuts in early phage messenger RNAs . Cutting takes place specifically in the middle of the tetranucleotide GGAG, as soon as the first minute of infection . Out of the 20 processing sites so far identified, seven are in Shine-Dalgarno sequences . The others are localized in intercistronic regions or within coding sequences . In the latter case, cutting efficiency is much lower . regB-dependent cleavages can occur within AU-rich sequences downstream of processed GGAG motifs that are not in effective translation initiation sites . We looked for possible consequences of regB-dependent cuts on gene expression in two early regions of the T4 chromosome . In the comC alpha region, none of the three major RegB cleavage sites is in a Shine-Dalgarno sequence, and in the motA region the unique regB-dependent processing site is found within the Shine-Dalgarno sequence of the gene . We find that in the region of gene comC alpha, RegB decreases two- to threefold the chemical half-life of early transcripts, but does not change the functional half-life of mRNAs coding for protein ComC alpha . The amount of MotA protein synthesized by the wild-type is half that obtained in a regB mutant infection . We show that this is a direct consequence of mRNA processing by RegB at the Shine-Dalgarno sequence of motA . This regB-mediated translation inhibition is not accompanied by an important modification in motA mRNA chemical half-life . We show that rapid shut-off of MotA protein synthesis that occurs soon after infection results both from RegB processing within the translation initiation region of motA and from early transcription inhibition followed by regB-independent breakdown of the motA mRNA.

J Mol Biol, 1993 Oct 5, 233(3), 414 - 28
Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained; Lehnherr H et al.; P1 lysogens of Escherichia coli carry the prophage as a stable low copy number plasmid . The frequency with which viable cells cured of prophage are produced is about 10(-5) per cell per generation . Here we show that a significant part of this remarkable stability can be attributed to a plasmid-encoded mechanism that causes death of cells that have lost P1 . In other words, the lysogenic cells appear to be addicted to the presence of the prophage . The plasmid withdrawal response depends on a gene named doc (death on curing), encoding a 126 amino acid protein . Expression of doc is not SOS-inducing and killing by Doc is recA-independent . In cells that retain P1 the killing is prevented by the product of a gene named phd (prevent host death), encoding a 73 amino acid protein . The genes phd and doc have been cloned and expressed from a 0.7 kb segment of P1 DNA . The two genes constitute an operon and the synthesis of Doc appears to be translationally coupled to that of Phd . Homologs of the P1 addiction genes are found elsewhere, but phd and doc are unrelated to previously described genes of other plasmids that also cause an apparent increase in plasmid stability by post-segregational killing.

J Mol Biol, 1993 Oct 5, 233(3), 349 - 58
A new start site for Escherichia coli RNA polymerase at an engineered short region of non-complementarity in double-stranded DNA; Tripatara A et al.; We have constructed versions of the bacteriophage PRM promoter containing short (9 or 12 base pairs) regions of DNA mismatches ("bubble") which include the authentic transcription start site of the unmodified promoter . These constructs direct transcription initiation at positions near the genuine PRM start site . In addition a new start site (designated Pbub) is observed in the region of non-complementarity, from which RNA synthesis proceeds in the opposite direction . The ability to initiate the divergent transcripts is specific to holo enzyme . Mapping of the Pbub start sites shows that they are but a few base pairs upstream of the edge of the bubble . Thus, with respect to the single-stranded region, the location of the start site is no different for Pbub than it is for open complexes at promoters . Compared with an unmodified PRM promoter, the region protected by RNA polymerase from digestion by DNase I is extended in the downstream direction (with respect to the PRM start) at the promoters bearing mismatches; this is consistent with the binding of the divergently transcribing RNA polymerase . Interestingly, cI protein represses rather than activates RNA synthesis originating in the PRM direction, indicating yet another aspect in which the complexes formed at these constructs differ from open complexes at the unmodified promoter.

Infect Immun, 1993 Oct, 61(10), 4510 - 3
Mitomycin-induced synthesis of a Shiga-like toxin from enteropathogenic Escherichia coli H.I.8; Yee AJ et al.; Escherichia coli H.I.8, an O128 infant diarrhea isolate, produces low titers of a unique Shiga-like toxin (SLT), called SLT-IIva, which is a variant of SLT-II . We investigated induction of toxin synthesis and the putative association of a bacteriophage with toxin synthesis . Induction of broth cultures of strain H.I.8 with mitomycin yielded a 3,000-fold increase in SLT-IIva, production of a colicin, and appearance of a bacteriophage . Southern hybridization demonstrated that the genes for SLT-IIva were not carried by the bacteriophage.

J Immunol, 1993 Oct 1, 151(7), 3894 - 901
Specific antibody production to a recall or a neoantigen by SCID mice reconstituted with human peripheral blood lymphocytes; Nonoyama S et al.; To explore the extent of immune reconstitution of SCID mice by human peripheral blood lymphocytes (hu-PBL-SCID mice), we studied the production of immunoglobulin isotypes and specific antibody (Ab) by the engrafted human cells . Human IgG was detectable in 94% of hu-PBL-SCID mice . IgE synthesis by hu-PBL-SCID mice correlated with the IgE levels observed in human donors . All SCID mice receiving PBL obtained from human donors previously immunized with the T-cell-dependent Ag, bacteriophage phi x 174 (phage), produced phage neutralizing antibody . Quantity and quality (Ig isotypes) of phage-specific Ab produced by hu-PBL-SCID mice correlated with that observed in the donor serum . Human B cells alone failed to engraft, and T cells were required for the production of Ig and anti-phage Ab . Phage-specific Ab production occurred without direct Ag exposure of the hu-PBL-SCID mice, suggesting that the specific Ab production was induced directly by polyclonal activation of the engrafted human cells . Intravenous phage injections given 4 wk after cell transfer failed to further increase the anti-phage Ab titer . Phage neutralizing Ab production could not be boosted if spleen cells obtained from hu-PBL-SCID mice were cultured in the presence of Ag . However, hu-PBL-SCID mice produced increased amounts of anti-phage Ab, providing they were injected with phage at the time of cell transfer . Injection of phage at the time of cell transfer, but not 4 wk later, to mice receiving PBL from nonimmunized donors induced production of minute amounts of anti-phage Ab . We conclude that human peripheral blood lymphocytes transferred into SCID mice become maximally stimulated presumably by xenogeneic murine Ag, resulting in polyclonal expansion of the graft and spontaneous production of Ab to Ag the human donor was previously exposed to, and in loss of responses to subsequent Ag exposure . Ab production to neoantigen, however, can be induced and that to recall Ag can be modified if PBL are exposed to Ag at the time of cell transfer.

Virology, 1993 Oct, 196(2), 910 - 3
Function of gene 49 of bacteriophage T4 III . Isolation of Holliday structures from very fast-sedimenting DNA; Flemming M et al.; Branched DNA molecules were identified in ClaI digests of cytosine containing very fast-sedimenting DNA (VFS-DNACYT) which was isolated from Escherichia coli infected with the multiple mutant 49-GT7 of phage T4 . In about 10% of randomly picked ClaI fragments branches with three arms (Y-structures) as well as four arms (Holliday structures) were seen in the electron microscope . Branched structures were absent from ClaI digests after treatment with purified endonuclease VII (gp49) in vitro.

Virology, 1993 Oct, 196(2), 896 - 9
Purification and characterization of giant empty proheads from packaging-defective 23ptg mutants of bacteriophage T4; Rao VB et al.; We constructed packaging-defective gene 23ptg mutants of bacteriophage T4 that produced giant proheads of varying lengths . The giant proheads constituted up to 20% of the total proheads . A purification protocol was developed to prepare large quantities of highly pure giant proheads . The purified giant proheads appeared to package both mature T4 DNA and concatemeric AD10 plasmid DNA in vitro.

Virology, 1993 Oct, 196(2), 758 - 68
Structural basis for bacteriophage phi X174 assembly and eclipse as defined by temperature-sensitive mutations; Ilag LL et al.; We present a screening procedure for classifying bacteriophage phi X174 mutants as defective in either "early" (genome delivery and RF DNA replication) or "late" (progeny ssDNA replication and morphogenesis) functions . When applied to 108 ts mutants, only five were classified as "early" mutants . Quantitative one-step growth curves confirmed the classification for 22 "late" mutants, while only one "early" mutant was correctly classified by the procedure . The other four "early" mutants gave burst sizes that required a third, or "intermediate," class of functional defects . The mutation in the "early" mutant mapped in gene A, which encodes an endonuclease required for RF replication . However, most of the mutations in the "late" and "intermediate" mutants mapped in genes for F, G, and H, three structural proteins found in mature virions . The amino acid substitutions for each mutation in F capsid and G "spike" genes were located in the atomic structure of wt virions . All but two replacements are on the outer surface of the virion or at interfaces between neighboring polypeptide chains, suggesting defects in protein-protein interactions involved in quaternary rearrangements during maturation . The kinetics of viral eclipse are also altered in most gene F "late" mutants, but only in one gene G "intermediate" mutant . Thus, the F capsid protein appears to have a central role in DNA ejection from the capsid.

J Virol, 1993 Oct, 67(10), 5740 - 8
Overexpression, purification, and late transcription factor activity of the 17-kilodalton protein encoded by the vaccinia virus A1L gene; Keck JG et al.; The A1L, A2L, and G8R open reading frames (ORFs) were previously shown by transfection assays to encode transactivators of late gene expression . We now present evidence that the 17-kDa protein product of the A1L gene can function in vitro as a transcription factor . Simultaneous overexpression of the transactivators was achieved by coinfecting HeLa cells with one recombinant vaccinia virus that encodes the bacteriophage T7 RNA polymerase and three recombinant vaccinia viruses that contain copies of A1L, A2L, and G8R ORFs regulated by T7 promoters . Extracts from the recombinant virus-infected cells exhibited greatly enhanced late in vitro transcription activity and served as a source of factors . The 17-kDa product of the A1L ORF represented approximately 8% of the ammonium sulfate-precipitated cell protein and copurified with a late transcription factor activity . The transcription factor activity could be specifically immunodepleted with immobilized antibody to the bacterially expressed A1L-encoded protein, providing additional evidence for its identity and role . A sequence encoding six consecutive histidines was added to the A1L ORF, which was then incorporated into the genome of a baculovirus expression vector . The 17-kDa protein, synthesized in insect cells and purified by binding to an Ni(2+)-chelating affinity column, could replace the vaccinia virus-overexpressed 17-kDa protein in transcription assays . In addition to the 17-kDa product of the A1L gene, which was named vaccinia virus late transcription factor 2, the proteins that stimulate specific transcription of late promoter-regulated templates included the viral multisubunit RNA polymerase, vaccinia virus late transcription factor 1 (the product of the G8R ORF), and at least one other partially purified protein.

Semin Hematol, 1993 Oct, 30(4 Suppl 4), 72 - 9; discussion 80-1
The role of adhesion molecules in the regulation of antibody responses; Ochs HD et al.; We used the T-cell-dependent antigen, bacteriophage (phage) phi X174, to study antibody synthesis in patients, guinea pigs, and dogs with complement component deficiencies (C2, C4, C3, C7); in patients with adhesion molecule deficiencies (CD11/CD18 or sialylated Lewisx); and in patients with the hyper IgM (HIM) syndrome (absence of functional gp39 expression by activated T cells) . Patients and guinea pigs deficient in early complement components, patients deficient in CD11/CD18, and patients lacking functional gp39 on activated T cells responded to repeated phage immunizations with depressed antibody titers, lack of or inadequate amplification, and failure to switch from IgM to IgG, suggesting that defective T-cell-B-cell interaction is the cause of the antibody deficiency observed in these patients.

Curr Opin Genet Dev, 1993 Oct, 3(5), 708 - 12
Protein-DNA assemblies controlling lytic development of bacteriophage Mu; Baker TA; Recent analysis of the mechanism and regulation of transposition by bacteriophage Mu has emphasized the importance of controlled assembly of specific protein-DNA complexes . Both the Mu transposase and the Mu repressor engage in multiple protein-protein and protein-DNA interactions that modulate the outcome of a phage infection.

Genome, 1993 Oct, 36(5), 944 - 53
Clusters of interspersed repeated DNA sequences in the rice genome (Oryza); Zhao X et al.; We have characterized a repeated DNA sequence (RTL122) from rice (Oryza sativa L.) with respect to its organization in the rice genome and its distribution among rice and other plants . The results indicate that the RTL122 sequence is interspersed in the rice genome and limited to the genus Oryza . It is highly polymorphic and can be used to fingerprint rice varieties . A structure was observed in which several repeated sequences were clustered in DNA regions of 15-20 kb . We characterized three bacteriophage lambda clones that contained the RTL122 sequence . Southern analysis using probes derived from restriction fragments of the three lambda clones indicated that all fragments except one are interspersed repeated sequences and belong to different repeated sequence families . Subsequent slot blot hybridization showed that most of them are only present within the genus Oryza . Some of the Oryza-specific, physically linked sequences show the same phylogenetic distribution, which suggests that these sequences might have evolved in a coordinate fashion . On the other hand, some of the repeated sequences have a different distribution even though they are physically adjacent in the genome . We speculate that such blocks of interspersed repeated sequences may serve as hotspots for rapid changes in the rice genome.

J Gen Microbiol, 1993 Oct, 139 ( Pt 10), 2517 - 24
The temperate phages RP2 and RP3 of Streptomyces rimosus; Rausch H et al.; The oxytetracycline-producing Streptomyces rimosus strains R6-65 and R7 (ATCC 10970) are lysogenic for the two narrow-host-range phages RP2 and RP3 . Both phages are released at low frequency from the lysogenic strains and form plaques on 'cured' S . rimosus strains . RP2 and RP3 are of similar shape with flexible tails and contain double-stranded DNA of about 70% G+C with cohesive ends (group B1 of bacteriophage classification) . The two phages also have identical, very slow, growth kinetics in S . rimosus, with a latent phase of about 6 h and a rise period of about 4 h . RP2 and RP3 are heteroimmune and they differ slightly in their size of phage particles and length of DNA (64.7 and 62.4 kb for RP2 and RP3, respectively) . The restriction maps of the two phages are completely different, and hybridization experiments showed only one short region of sequence similarity (less than 430 bp); the two phages are thus essentially unrelated . Both phages lysogenize their hosts by recombination via defined attachment (att) sites . The positions of the attP sites have been localized on the restriction maps of RP2 and RP3 to restriction fragments of 800 and 300 bp, respectively . The prophages did not affect the level of oxytetracycline production or the genetic instability of this trait.

Protein Expr Purif, 1993 Oct, 4(5), 412 - 6
A tightly regulated system for overproduction of bacteriophage T4 lysozyme in Escherichia coli; During K; Bacteriophage T4 lysozyme has been purified using the Ni-chelate affinity chromatography technique from overexpressing Escherichia coli cells by fusion to an N-terminal 6x His tail . Regulation of the lysozyme gene expression has been found to be critical during growth phase of the bacteria by comparing different plasmid constructions . Whereas a tac-promoter fusion construct alone did not lead to efficient production of T4 lysozyme because of early cell lysis, an improved repressor sequence and co-overproduction of the tac repressor resulted in high-level synthesis of the foreign protein after IPTG induction . Purification of the fusion protein from autolyzed crude cell extracts is possible in a simple one-step procedure.

Protein Expr Purif, 1993 Oct, 4(5), 358 - 66
High-level expression and purification of human leukotriene A4 hydrolase from insect cells infected with a baculovirus vector; Gierse JK et al.; Leukotrienes constitute a group of bioactive compounds derived from arachidonic acid which play important roles in immediate hypersensitivity and inflammation . Leukotriene A4 hydrolase (LTA4H) is an epoxide hydrolase, catalyzing the hydration of LTA4 to LTB4, and also acts an aminopeptidase, with the ability to cleave amides of p-nitroaniline . The cDNA for LTA4H was cloned using oligonucleotide-directed amplification of the cDNA sequence by polymerase chain reaction and by oligonucleotide-based screening of a bacteriophage lambda gt11 cDNA library derived from human placental tissue . High levels of biologically active LTA4H were expressed in cultured Spodoptera frugiperda insect cells infected with a baculovirus expression vector containing the LTA4H cDNA . Expression levels were approximately 100 mg per liter of cell-free culture media . LTA4H was recovered from the medium and purified to > 95% purity by ion-exchange and gel-filtration chromatography, with an overall yield of 76% . LTA4H produced by insect cells exhibits both hydrolase and aminopeptidase activities and has kinetic properties similar to those reported for enzyme isolated from human lung . Two major isoforms, with pI's of 5.3 and 5.1, were isolated by preparative chromatofocusing chromatography . NH2-terminal sequence analysis revealed that the two different by an NH2-terminal blocking group . Electrospray ionization mass spectrometry indicates that the two isoforms differ by a molecular mass of 42, indicating that the blocking group is an acetyl group.

Acta Virol, 1993 Oct, 37(5), 369 - 76
Cloning of toxic genes with mini-mu derivative of bacteriophage mu; Stuchlik S et al.; We describe an in vivo cloning method using mini-Mu phage for genes, which cannot be cloned on multicopy vectors, mainly for their toxicity . We have successfully cloned succinate dehydrogenase (sdh) gene E . coli which was inactivated with defined insertion of fragment Kmr by this method . The most of obtained Kmr clones of mini-Mu transductants have contained the sequence of whole sdh gene . The intact gene of sdh can be reconstructed by site-directed mutagenesis.

Mol Microbiol, 1993 Oct, 10(2), 293 - 8
Inhibition of bacteriophage Mu transposition by Mu repressor and Fis; van Drunen CM et al.; In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor . Fis can lower the transposition frequency of a mini-Mu 3-80-fold, but only if the Mu repressor is expressed simultaneously . In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site lead to the loss of the Fis effect . As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor . However, the role of Fis and repressor is not only to inactivate the IAS, since a 4 bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact . A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site . However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed . Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.

Genetics, 1993 Oct, 135(2), 255 - 64
Effects of uvsX, uvsY and DNA topoisomerase on the formation of tandem duplications of the rII gene in bacteriophage T4; Kumagai M et al.; We have characterized tandem duplications in the rII regions of phage T4 . The rII deletion r1589 blocks only the function of the rIIA cistron, although it extends into the B cistron . Another rII deletion, r1236, blocks the function of the rIIB cistron and overlaps r1589 . When a cross is made between r1589 and r1236, true rII+ progeny cannot form . Instead, anomalous phenotypically rII+ phages are detected carrying an rII region from each parent . Analyses of nucleotide sequences of the recombination junctions indicate that recombination takes place between short regions of homology (from 2 to 10 bp) . Open reading frames of the recombinants deduced from the nucleotide sequences reveal that they contain a normal rIIA cistron and one of a variety of fused, duplicated rIIB cistrons . The T4 uvsX and uvsY genes, which participate in homologous recombination, are involved in this duplication formation . T4 DNA topoisomerase is encoded by genes 39, 52 and 60 . Mutations in 52 and 60 reduced the frequency of such duplications, but mutations in gene 39 and some in gene 52 did not . Hence, the effects of topoisomerase mutations are allele-specific . Models are proposed in which these proteins are involved in tandem duplication.

Mutat Res, 1993 Oct, 294(3), 285 - 98
Mechanism of toxicity of 3-methyladenine for bacteriophage T7; Racine JF et al.; Treatment of bacteriophage T7 with methyl methanesulfonate perturbed phage-specific genetic expression in both repair-proficient and repair-deficient Escherichia coli cells . In wild-type cells (AB1157), the time course of protein synthesis was slowed down but an entire complement of phage proteins was synthesized . In cells (BK2114, tag-) unable to repair 3-methyladenine, the toxic lesion produced by methyl methanesulfonate, alkylated phage produced only early (class I) proteins . These results suggested that late transcription was inhibited in infected tag- cells . These cells were shown to contain a significant amount of active T7 RNA polymerase, a class I protein . Thus, the cause of inhibition appeared to be the inability of T7 RNA polymerase to use unrepaired DNA as template . In vitro transcription assays with alkylated T7 DNA as template supported this proposal . T7 RNA polymerase proved to be very sensitive to the presence of alkylation lesions . In addition, the phage enzyme was much more sensitive to these lesions than was its bacterial counterpart, E . coli RNA polymerase . These results suggest that 3-methyladenine exerts its toxic action, in the T7 system, at the level of transcription by T7 RNA polymerase . To further characterize the reduced activity of the T7 enzyme, an in vitro transcription assay using linearized plasmid DNA with one T7 promoter was devised . Gel electrophoresis revealed that only one transcript of well-defined length was synthesized by T7 RNA polymerase on this template . Alkylation of the template did not alter the size of the transcript produced . Simultaneous measurement of chain initiation and chain elongation confirmed this result by showing that both steps were reduced to the same extent by alkylation of template DNA . Thus T7 RNA polymerase does not appear to be blocked by 3-methyladenine . Rather the lesion must hinder translocation of T7 RNA polymerase along the DNA template during chain elongation.

Mutat Res, 1993 Oct, 294(3), 247 - 54
A forward mutation assay in phage T4: application to gene 42 mutator mutations; Ji J et al.; A forward mutation assay was developed to study mutagenic specificity induced by temperature-sensitive alleles of bacteriophage T4 gene 42, which encodes a thermolabile deoxycytidylate hydroxymethylase . Thymidine kinase (tk) mutations induced by T4 ts B3 at a semi-permissive temperature (34 degrees C) were selected under near-ultraviolet light on synthetic agar plates containing bromodeoxyuridine, and sequenced after PCR amplification of the tk gene . 21 of 23 tk- mutations identified were C-->T transitions, while the remainder were C-->A transversions . Analyses of the DNA sequence around each mutant site suggest that the mispairing of thymine with guanine in the template is suppressed when the next nucleotide is dGTP . The 5' neighbor nucleotide of the mismatch may influence mutation frequency as well; no mutations with dAMP residues on the upstream side were seen . Our observations with the forward mutation assay here are consistent with previous results from an rII reversion assay, supporting our model that the mutator phenotype displayed by tsLB3 is a consequence of perturbation of dNTP supplies to replication sites due to partial impairment of thermolabile deoxycytidylate hydroxymethylase at a semi-permissive temperature . The forward mutation assay described here is readily adapted for other studies of mutagenesis in T4 phage.

Mol Microbiol, 1993 Oct, 10(1), 1 - 6
The phage RNA polymerases are related to DNA polymerases and reverse transcriptases; McAllister WT et al.; The single subunit DNA-dependent RNA polymerase (RNAP) that is encoded by bacteriophage T7 is the prototype of a class of relatively simple RNAPs that includes the RNAPs of the related phages T3 and SP6, as well as the mitochondrial RNAPs . The T7 enzyme has been crystallized, and recent genetic and biochemical analyses have facilitated an interpretation of this structure . A growing body of evidence suggests that the phage-like RNAPs are related to other nucleotide polymerases such as DNA polymerases, RNA-dependent RNA polymerases, and reverse transcriptases . In this work, we review information concerning the structure and function of T7 RNAP, and evidence in support of its assignment to a broader class of nucleotide polymerases.

AIDS Res Hum Retroviruses, 1993 Oct, 9(10), 971 - 8
Use of the recombinant chimera proteins, LacZ-Env and Gag-Env, for immunological studies on HIV-1 infection; Morimoto M et al.; To use Env proteins as antigens for detection of the human immunodeficiency virus type-1 (HIV-1) antibodies, we attempted to overexpress the Env proteins in Escherichia coli . To study the epitopes in the Env proteins recognized by the sera of HIV carriers, various regions of the proviral DNA encoding the Env region were fused to the 3' end of the lacZ gene . The immunoblotting analysis of the LacZ-Env(512-611) and LacZ-Env(721-826) proteins with the 41 positive sera revealed that the former and the latter immunologically reacted with 100 and 78% of the sera, respectively . To avoid rare false-positive reactions due to the LacZ moiety of the fusion protein, we attempted to express the Env(512-611) alone or Gag-Env(512-611) under the control of bacteriophage T7 promoter . Although we could express only a low level of the Env(512-611) peptide in E . coli, we succeeded in producing large amounts of the Gag(121-406)-Env(512-611) and Gag(308-406)-Env(512-611) proteins as chimeric proteins . Both of these chimera proteins strongly reacted with the 41 positive sera . We purified these proteins and analyzed the immunological reactivity by dot blot with the 60 positive sera and the 84 normal sera . As little as 20 ng of the dotted proteins was enough for the reaction with the positive sera, whereas as much as 320 ng of them did not show false-positive reactions with the normal sera . We obtained highly purified Gag-Env proteins with highly specific seroreactivity, which should be useful for diagnosis and prognosis.

Gene, 1993 Sep 30, 132(1), 95 - 9
Isolation of enterohemolysin (Ehly2)-associated sequences encoded on temperate phages of Escherichia coli; Beutin L et al.; We have cloned and sequenced the enterohemolysin (ehl)-associated region from a temperate bacteriophage isolated from an Escherichia coli O26:H11 strain . Phage phi C3208 was isolated together with other temperate bacteriophages which transduce the enterohemolytic phenotype to non-hemolytic E . coli O26 strains . The nucleotide sequence of the 1245-bp phi C3208 DNA insert in plasmid pEO39, which mediates Ehly2 production in E . coli K-12, was determined and was found to be partially homologous to DNA of bacteriophage lambda but is completely unrelated to DNA sequences encoding the synthesis of Ehly1 {Stroeher et al . Gene 132 (1993), 89-94} . It was shown that part of this region can be used as an Hly2-associated specific DNA probe.

J Biol Chem, 1993 Sep 25, 268(27), 20046 - 54
DNA binding properties of the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli; Wurgler SM et al.; The dgt gene of Escherichia coli encodes a deoxyguanosine triphosphate triphosphohydrolase (dGTPase) that hydrolyzes dGTP to deoxyguanosine and tripolyphosphate . The enzyme is highly specific for dGTP which is hydrolyzed with a Km of 2-5 microM . Nitrocellulose filter binding assays demonstrate that, under physiological salt conditions, dGTPase binds with apparent cooperativity to single-stranded DNA with an association constant of 7.7 x 10(6) M-1 . In the presence of NaCl, dGTPase binds weakly to double-stranded DNA . In the absence of NaCl, dGTPase binds both single- and double-stranded DNA with an association constant of 1 x 10(7) M-1 . The dGTPase-double-stranded DNA complex, however, is readily dissociated with NaCl . Divalent cations such as Mg2+ or Mn2+ enhance, but are not required for DNA binding . The presence of dGTP or GTP does not effect the ability of dGTPase to bind DNA . dGTPase binds to oligonucleotides of length 17-35, but with lower affinities . The homopolymers poly(dT) and poly(rU) act as effective competitors of single-stranded DNA for binding to dGTPase . The bacteriophage T7 gene 1.2 protein, which specifically inhibits the enzymatic activity of dGTPase, also prevents dGTPase from binding to single-stranded DNA . dGTPase inhibits the activity of T7 DNA polymerase on a poly(dA)-oligo(dT) template . This inhibition is reversed by prior incubation of dGTPase with the T7 gene 1.2 protein.

Nucleic Acids Res, 1993 Sep 25, 21(19), 4621 - 6
Encapsidation of heterologous RNAs by bacteriophage MS2 coat protein; Pickett GG et al.; The RNA bacteriophages of E . coli specifically encapsidate a single copy of the viral genome in a protein shell composed mainly of 180 molecules of coat protein . Coat protein is also a translational repressor and shuts off viral replicase synthesis by interaction with a RNA stem-loop containing the replicase initiation codon . We wondered whether the translational operator also serves as the viral pac site, the signal which mediates the exclusive encapsidation of viral RNA by its interaction with coat protein . To test this idea we measured the ability of lacZ RNA fused to the translational operator to be incorporated into virus-like particles formed from coat protein expressed from a plasmid . The results indicate that the operator-lacZ RNA is indeed encapsidated and that nucleotide substitutions in the translational operator which reduce the tightness of the coat protein-operator interaction also reduce or abolish encapsidation of the hybrid RNA . When coat protein is expressed in excess compared to the operator-lacZ RNA, host RNAs are packaged as well . However, elevation of the level of operator-lacZ RNA relative to coat protein results in its selective encapsidation at the expense of cellular RNAs . Our results are consistent with the proposition that this single protein-RNA interaction accounts both for translational repression and viral genome encapsidation.

J Biol Chem, 1993 Sep 25, 268(27), 20198 - 204
Characterization of an RNA-binding domain in the bacteriophage phi 29 connector; Donate LE et al.; The connector of bacteriophage phi 29 is known to promote the viral prohead assembly, to bind DNA, and to drive DNA packaging into preformed viral shells in an RNA-dependent process . In this report, the phi 29 connector protein, p10, is shown to bind RNA in a sequence-independent fashion, and to possess an RNA recognition motif comprised approximately the region between residues 21 and 94 of the p10 sequence . Substitution mutants in specific amino acids of the RNA-binding domain obtained by site-directed mutagenesis showed that amino acids Phe23, His57, Phe59, and Tyr61 are critical for RNA binding and, subsequently, for DNA packaging into proheads . Proteolytic modified forms of the phi 29 connector have allowed us to conclude that the DNA- and RNA-binding domains are separated within the p10 sequence . It is also shown that RNA is stably associated to DNA-filled proheads during the DNA-packaging process.

Biochemistry, 1993 Sep 21, 32(37), 9735 - 44
Bacteriophage T4 gene 32 protein: modulation of protein-nucleic acid and protein-protein association by structural domains; Casas-Finet JR et al.; The cooperative binding of bacteriophage T4 gene 32 protein to single-stranded nucleic acids is dependent on homotypic protein-protein interactions between the N-terminus of a protein monomer with the core domain of an adjacent protein . In a previous report {Casas-Finet et al . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 1050-1054}, we demonstrated that synthetic peptides corresponding to various portions of the N-terminal B-domain (residues 1-21) formed a 1:1 complex with core domain and identified a sequence, residues 3-5, Lys-Arg-Lys-Ser-Thr (the LAST motif) strongly homologous to a sequence within the central portion of protein (core domain) that was likely to function in nucleic acid binding . On the basis of these observations, we proposed a model where cooperative binding involves an exchange of intramolecular protein-protein interactions involving the internal LAST sequence for intermolecular protein-protein interactions utilizing the N-terminal LAST sequence . In this paper, we have tested various predictions of the model, and utilizing several proteases, further have defined the domain structure of 32 protein . The interaction of peptides containing LAST sequences with 32 protein qualitatively reduces its binding cooperativity, indicating that the peptides bind at the same site within the core domain as the N-terminus of an adjacent intact protein bound to the polynucleotide lattice . As expected, these peptides bind to nucleic acids . The N-terminus of 32 protein is predicted to be largely alpha-helical, and the circular dichroism spectrum of a peptide corresponding to residues 1-17 is consistent with this prediction . On the basis of the magnitude of protein tryptophan fluorescence quenching, the conformational change in 32 protein brought about by LAST peptides may be similar to that effected by oligonucleotides . As predicted by our model, in the presence of interacting peptide, the binding of 32 protein to oligonucleotide becomes salt-dependent . Arg-C endoproteolysis of intact 32 protein indicates that the loss of as few as three or four amino acids from the N-terminus appears to eliminate binding cooperativity, although the remainder of the N-terminal B-domain appears to protect the core from proteolysis . In contrast, this enzyme will catalyze the breakdown of trypsin-generated core domain, which lacks the first 21 residues of the protein . Thus, the presence of residues 4/5-21 attached to core alters its conformation and/or accessibility to protease . Poly(dT) inhibits this digestion, whereas the presence of N-terminal peptide accelerates proteolysis, in agreement with our model.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem Biophys Res Commun, 1993 Sep 15, 195(2), 881 - 8
Cloning of a splice variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor; Svoboda M et al.; The rat pancreatic acinar cell line AR 4-2J is endowed with numerous PACAP type I receptors (PACAPR1) . The cDNA of this receptor was PCR amplified at low stringency using two degenerate primers based on conserved sequences in the TM2 and TM6 segments of secretin, parathormone and calcitonin receptors . One additional amplified band of 574 bp possessed an original 84 bp insert . This fragment, when used as probe for Northern blot analysis, revealed a high M(r) (about 7.5 kb) transcript in AR 4-2J cells and also in rat brain . Screening a lambda Uni-Zap bacteriophage library of AR 4-2J cell cDNA yielded one hybridizing clone with an ORF of 1254 bp . The translated 418 amino acid peptide showed 206 identities with rat VIP receptors and 176 identities with secretin receptors . This sequence homology, together with the mRNA distribution, and the expression study of a similar cDNA published very recently (8) indicated that we had cloned PACAPR1 except for its 77 N-terminal amino acids . Its putative I3 loop contained a unique additional 28 amino acid sequence (with four hemi-cystines and several serine, threonine and basic residues) . Using RT-PCR we then demonstrated the coexistence of a second form of mRNA, without this added insert, in DNAse-pretreated RNAs from both AR 4-2J cells and normal rat brain . This indicated that common alternative splicing provokes the diversification of PACAP type I receptors into PACAPR1A (unspliced) and PACAPR1B (spliced) variants.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8367 - 71
Engineering multiple properties of a protein by combinatorial mutagenesis; Sandberg WS et al.; A method for simultaneously engineering multiple properties of a protein, based on the observed additivity of effects of individual mutations, is presented . We show that, for the gene V protein of bacteriophage f1, effects of double mutations on both protein stability and DNA binding affinity are approximately equal to the sums of the effects of the constituent single mutations . This additivity of effects implies that it is possible to deliberately construct mutant proteins optimized for multiple properties by combination of appropriate single mutations chosen from a characterized library.

Biochem Biophys Res Commun, 1993 Sep 15, 195(2), 616 - 22
Interaction of ribonucleotides with T7 RNA polymerase: probable role of GTP in transcription initiation; Sen R et al.; Interaction of ribonucleotides (NTP where N = G, A, C or U) with bacteriophage T7 RNA polymerase (T7 RNAP) was studied by fluorescence emission spectroscopy of the enzyme . From the NTP-concentration-dependent quenching of fluorescence of the enzyme, apparent dissociation constants for NTP-T7 RNAP was found to be in following order: UTP>CTP>>ATP>GTP . Acrylamide quenching of tryptophan fluorescence of free and bound enzyme suggests a conformational change, particularly in the case of GTP (and ATP) . This is the first report of high affinity binding of the enzyme with purine ribonucleotides in the absence of promoter . These results also suggest that GTP may induce a promoter-specific conformation of the enzyme . The observation could account for specific requirement of GTP in transcription initiation reported earlier (1-4).

J Biol Chem, 1993 Sep 15, 268(26), 19299 - 304
Two base pairs at -9 and -8 distinguish between the bacteriophage T7 and SP6 promoters; Lee SS et al.; Bacteriophage T7 and SP6 RNA polymerases and their promoters share a high degree of their primary structure homology, but each polymerase exclusively recognizes its own promoter sequence . To reveal the molecular basis of this specificity, 4 base pairs at positions -12, -10, -9, and -8 of the T7 promoter were substituted individually and multiply by SP6 promoter-specific base pairs, and 3 base pairs at -10, -9, and -8 of the SP6 promoter were replaced by T7 promoter-specific base pairs . Promoter activities of 28 sequences were measured in vitro with T7 and SP6 polymerases separately under optimal conditions at 6 mM MgCl2 . Single and double substitutions at -12 and -10 do not significantly affect the T7 promoter activity, although they are almost exclusively conserved among T7 genomic promoters . Changes at -10 of SP6 promoter hardly affect the activity . However, any T7 variants that contain either or both changes at -9 and -8 show greatly reduced activity . Interestingly, the double substitution at -9 and -8 yields significant SP6 promoter activities and virtually no T7 promoter activity . Furthermore, the SP6 promoter variants with both T7-specific -9C and -8T show good T7 promoter activities, although they still show some SP6 promoter activities . However, under high salt conditions (either 20 mM MgCl2 or 100 mM NaCl plus 6 mM MgCl2), they show only slight SP6 promoter activity . No other SP6 variants show any T7 promoter activity . All these results indicate that the 2 base pairs at -9 and -8 of both the T7 and SP6 promoters are the primary (if not the only) determinants of specificity and that the hierarchy of importance of positions for promoter activity is -8, -9 > > -10 > -12 . Also, a phylogenic relationship among the T3, T7, K11, and SP6 promoters is suggested based on dissimilarities in their sequences from -12 to -8.

Biochemistry, 1993 Sep 14, 32(36), 9407 - 16
Exploring the DNA binding domain of gene V protein encoded by bacteriophage M13 with the aid of spin-labeled oligonucleotides in combination with 1H-NMR; Folkers PJ et al.; The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide . The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy . In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings . The DNA binding domain is principally situated on two beta-loops . The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label . The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73) . In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA . The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers . The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990) . These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA . In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found . Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.

FEBS Lett, 1993 Sep 13, 330(2), 111 - 3
Production of recombinant antibodies in lymphoid and non-lymphoid cells; Deyev SM et al.; A recombinant tandem of 'chimeric' mouse/human immunoglobulin (Ig) genes was constructed and inserted into plasmid pGEM1 under the control of the T7 bacteriophage RNA polymerase promoter . Lymphoid (Sp2/0) and non-lymphoid (CHO) cell lines used for transfection contained in their genomes a semisynthetic gene of T7 RNA polymerase and steadily expressed this enzyme . It was shown for the first time that a stable polycystronic transcription of the Ig gene tandem occurs under the control of a single T7 phage promoter, both in lymphoid and non-lymphoid cells . Synthesis of kappa-light and epsilon-heavy Ig chains and functionally active antibodies was observed in the above-mentioned transfected cell lines.

Gene, 1993 Sep 6, 131(1), 79 - 82
A bacteriophage T7-based expression vector, pBT7, with color selection for the recombinant; Maruyama IN et al.; A bacterial expression plasmid, pBT7, based on a bacteriophage T7 RNA polymerase/promoter system, has been devised for positive color selection of clones containing an insert . The T7 gene 10 promoter and its N-terminal portion, 89 nucleotides in length, have been amplified by the polymerase chain reaction and cloned into the multiple cloning site of plasmid pUC18 in the opposite direction to lacZ' . This insertion does not inactivate the lacZ alpha-peptide activity . Hence, bacteria carrying pBT7 without an insert form blue colonies on an indicator plate, while bacteria carrying recombinant clones with an insert appear as white colonies . Using pBT7, part of the Caenorhabditis elegans unc-13 gene product has been overproduced at a high level, approaching 50% of total Escherichia coli protein.

Biochim Biophys Acta, 1993 Sep 3, 1202(1), 161 - 8
Evaluation of transmembrane helix prediction methods using the recently defined NMR structures of the coat proteins from bacteriophages M13 and Pf1; Turner RJ et al.; Currently, there are a large number of hydropathy scales available to predict the presence of transmembrane segments within integral membrane proteins . These scales and their subsequent numerical manipulations provide an aid in the determination of topology in transmembrane proteins . In order to analyse the accuracy of these procedures to correctly identify the boundaries of a transmembrane segment, 13 methods were applied to the amino-acid sequence of the coat proteins from the bacteriophages Pf1 and M13 . These monotopic integral membrane proteins have been incorporated into detergent micelles and their structures have recently been solved using NMR . The predicted regions were then compared to their NMR-determined structures . All methods used were able to detect a transmembrane region within the protein sequence . However, there was considerable differences in their accuracy in determining the boundaries of the main transmembrane alpha-helix . Surprisingly, the methods which worked the best for Pf1 coat protein had poor accuracy in identifying the transmembrane region correctly in the M13 protein . It was concluded that a number of methods should be utilized in order to obtain a clear model of transmembrane protein topology, and that regardless of how closely related two proteins are, a different conclusion may be obtained from different prediction procedures.

Biopolymers, 1993 Sep, 33(9), 1327 - 36
Individual-site binding data and the energetics of protein-DNA interactions; Saroff HA; Individual-site isotherms for the binding of bacteriophage lambda repressor to the left and right lambda operators have been determined {D . F . Senear, M . Brenowitz, M . A . Shea, and G . K . Ackers (1986) Biochemistry, Vol . 25, pp . 7344-7354.} using the DNAse protection technique {footprinting; D . J . Galas and A . Schmitz (1978) Nucleic Acids Research, Vol . 5, pp . 3157-3170} . These extensive data have been interpreted with a quantitative model that emphasized cooperative interactions between adjacently bound ligands {occupied <==> occupied interactions; G . K . Ackers, A . D . Johnson, and M . A . Shea (1982) Proceedings of the National Academy of Science, USA, Vol . 79, pp . 1129-1133} . Overlooked in this model are the effects of cooperative interactions between a site containing a bound ligand and its neighboring unoccupied site (occupied <==> unoccupied interactions) . This paper reinterprets the existing data with a model that considers occupied <==> unoccupied as well as occupied <==> occupied interactions . The results yield parameters that differ substantially from those already reported . A discussion on the advisability of ignoring occupied <==> unoccupied interactions is included.

J Bacteriol, 1993 Sep, 175(18), 6059 - 61
Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity; Muller MM et al.; TolR is a 142-amino-acid protein required for the import of colicins and bacteriophage and for maintenance of cell envelope integrity . The topology of TolR in the inner membrane was analyzed by two methods . First, bacteria expressing a series of TolR-beta-galactosidase, TolR-alkaline phosphatase, and TolR-beta-lactamase fusions were assayed for the appropriate enzymatic activity . Second, the accessibility of TolR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited against the carboxyl-terminal 70% of TolR . The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exposed to the periplasm . Quantitative studies with the anti-TolR antibody indicated the presence of 2 x 10(3) to 3 x 10(3) TolR molecules per cell.

Eur J Biochem, 1993 Sep 1, 216(2), 507 - 17
Exploration of the single-stranded DNA-binding domains of the gene V proteins encoded by the filamentous bacteriophages IKe and M13 by means of spin-labeled oligonucleotide and lanthanide-chelate complexes; Van Duynhoven JP et al.; Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded . The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide . The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy . In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein . They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide . Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies . Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments . The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP . These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues . At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent . The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.

J Bacteriol, 1993 Sep, 175(17), 5648 - 54
A cryptic promoter in the O(R) region of bacteriophage lambda; Woody ST et al.; A cryptic promoter, designated P alpha, initiates transcription within the O(R) region of bacteriophage lambda . Transcription from P alpha proceeds in the direction of the cI repressor gene from sites 46 and 48 bp preceding the PRM transcription start site . P alpha is likely to compete with both PR and PRM for formation of open complexes, since it is only active when PR is mutated and can be suppressed by mutations that increase PRM activity . In addition, transcription initiation at P alpha is blocked by lambda repressor . Kinetic analysis of relative abundance of the products of in vitro transcription indicated that P alpha was approximately 1/3 as strong as PRM . However, a P alpha mutation had little effect on KBkf (the association rate constant) for PRM . These observations can be explained by the finding that open complexes formed at P alpha are relatively unstable (half-life = 20 to 25 min) . Dissociation of RNA polymerase from P alpha allows additional open complexes to form at PR or PRM, and thus the apparent strength of P alpha decreases with increasing preincubation times.

J Bacteriol, 1993 Sep, 175(17), 5314 - 23
Bacteriophage Mu Mor protein requires sigma 70 to activate the Mu middle promoter; Mathee K et al.; Transcription during the bacteriophage Mu lytic cycle occurs in three phases: early, middle, and late . Middle transcription requires the early gene product Mor for its activation . Mor protein overproduction was accomplished by fusing the mor gene to an efficient phage T7 promoter and translation initiation region . A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) from the Mor-overproducing strain was able to activate transcription from both the tac promoter (Ptac) and the Mu middle promoter (Pm) in vitro . Transcription initiation from Pm was Mor dependent, and the RNA 5' end was identical to that of in vivo RNA . Addition of anti-sigma 70 antibody to transcription reactions containing Ptac and Pm resulted in inhibition of transcription from both promoters; addition of purified sigma 70 restored transcription . These results indicate that Mor-dependent activation requires sigma 70 and therefore imply that Mor is not an alternate sigma factor . This conclusion was further substantiated by a reconstitution experiment with purified proteins in which all three components, Mor, sigma 70, and core RNA polymerase, were required for Pm-dependent transcription in vitro . The sigma 70 dependence of Mor-specific transcription and the amino acid sequence similarity between Mor and C (an activator for Mu late transcription) both support the hypothesis that Mor functions mechanistically as an activator protein.

J Immunol, 1993 Sep 1, 151(5), 2839 - 51
High affinity, thyroid-specific human autoantibodies displayed on the surface of filamentous phage use V genes similar to other autoantibodies; Portolano S et al.; Autoantibodies to thyroid peroxidase (TPO) are characteristic of thyroid inflammation in autoimmune thyroid disease . We have used the phage display, H and L chain combinatorial cDNA library approach to clone, from thyroid-infiltrating B cells, six new human Fab autoantibodies with high affinities (approximately 10(-10) M) for TPO . This library, in the pComb3 vector, was screened with viable, stably transfected Chinese hamster ovary cells expressing human TPO on their surface . The H and L chain genes in the six TPO-specific Fab were similar, but not identical, to those encoding Fab previously isolated from the same library by screening bacteriophage plaques in the Immunozap vector with purified TPO . The TPO-specific VK genes isolated with the phage display system are closer to germline than those obtained with Immunozap . Essentially all the V kappa isolated with pComb3 were 99 or 100% homologous with the germ-line genes KL012 and A3 that also code for low affinity systemic autoantibodies . There are two important implications of the study . First, the phage display system can be used with impure Ag to generate high affinity autoantibodies . This finding opens the way to cloning autoantibodies against other autoantigens, not previously possible with the bacteriophage lambda approach because of the lack of purified Ag . Second, germ-line L chain genes can code for very high affinity antibodies.

Virology, 1993 Sep, 196(1), 34 - 44
A novel terminase activity associated with the DNA packaging protein gp17 of bacteriophage T4; Bhattacharyya SP et al.; The mechanism of generation of circularly permuted ends in bacteriophage T4 by a strictly headful packaging process has remained unresolved since its proposal by Streisinger et al . (1967, Proc . Natl . Acad . Sci . USA 57, 292-295) . In this paper, we show that the phage T4 DNA packaging proteins gp16 and gp17 act as T4 terminase . Expression of gp16 and gp17 in Escherichia coli resulted in extensive cleavage of both plasmid as well as E . coli genomic DNAs . Analysis of a number of recombinant terminase constructs, and mutants in gene 17, demonstrated that the active site for cleavage of DNA is located in gp17, but not in gp16 . Consistent with the circularly permuted nature of phage T4 ends, cleavage by gp17 occurred in a sequence independent manner generating random ends . The terminase cutting occurred preferentially on a DNA substrate that is transcriptionally active . We propose that a structural feature in the transcriptionally active region provides a site for attachment of terminase to DNA following which the terminase moves along the DNA, and cleaves at a random sequence.

Virology, 1993 Sep, 196(1), 282 - 9
The direction and rate of bacteriophage T7 DNA packaging in vitro; Son M et al.; To determine the direction of the entry of DNA during in vitro bacteriophage T7 DNA packaging, incompletely packaged DNA (ipDNA) was fractionated by agarose gel electrophoresis after degradation of DNA outside of capsids and then release of packaged DNA from capsids . After fractionation, quantitative in-gel probing with a right end-specific oligonucleotide detects heterogeneous ipDNA (called right-end ipDNA) . Most of the right-end ipDNA appears with kinetics expected of a precursor to the mature T7 DNA . In-gel probing with a left-end-specific oligonucleotide detects ipDNA (left-end ipDNA); the molar amount of left end ipDNA is always at least 50x less than the molar amount of right-end ipDNA . Left-end ipDNA appears with the kinetics of an abortive end product of T7 DNA packaging . Thus, productive T7 DNA packaging occurs in a right-to-left direction . Quantitation of the conversion of right-end ipDNA to mature-length DNA yields an estimate of the mean rate of right-to-left in vitro T7 DNA packaging: 28 +/- 6 kbp/min for the last 20-50% of the DNA packaged.

Pharmacol Ther, 1993 Sep, 59(3), 343 - 89
Viral protein kinases and protein phosphatases; Leader DP; Certain large DNA viruses (e.g . herpesviruses and poxviruses) encode proteins related to cellular protein-serine/threonine kinases, and Hepatitis B virus and vesicular stomatitis virus may encode structurally different protein kinases . Other viruses activate cellular protein kinases, e.g . interferon-induced eukaryotic initiation factor-2 kinase, growth factor-induced kinases and protein kinases that regulate mitosis . Protein phosphatases are encoded by vaccinia virus and bacteriophage lambda and must also play a role in viral infection--as do cellular protein phosphatases . The functions of many of these viral enzymes remain to be determined, but they represent possible new targets for anti-viral therapy.

Virus Genes, 1993 Sep, 7(3), 297 - 303
Use of lambda gt11 and monoclonal antibodies to map the gene for the 60,000 dalton glycoprotein of infectious laryngotracheitis virus; Kongsuwan K et al.; To localize the gene encoding the 60 kD glycoprotein (gp60) of infectious laryngotracheitis virus (ILTV), a library of the ILTV genome was constructed in the lambda gt11 expression vector . Twelve recombinant bacteriophages expressing gp60 epitopes as fusion products with beta-galactosidase were detected by immunoscreening with monoclonal antibodies specific for gp60 . The ILTV DNA sequence contained in one of these recombinants lambda 24-4 was used as a hybridization probe for mapping the insert sequence on the viral genome . The gene for the gp60 was located at map unit 0.72-0.77 in the unique long region (UL) of the ILTV genome . The DNA sequence of the 1.2 kb insert of lambda 24-4 containing the gp60 epitope was determined . The majority of deduced gp60 amino acid sequence has no homology with any of the known alphaherpesvirus glycoproteins.

Biochem Mol Biol Int, 1993 Sep, 31(1), 153 - 9
Effects of multiple mutations at the conserved TATA sequence of bacteriophage SP6 promoter on transcription efficiency; Kim SS et al.; Mutations of A<==>T were introduced individually and multiple to TATA from -4 to -1 of the phage SP6 promoter and their effects on transcription initiation efficiency measured in vitro . All 15 mutants tested were less active than the wild type . Mutation at -4T nearly abolishes promoter activity independent of other changes, and alteration at -3A reduces promoter activity substantially . On the other hand, effects of mutations at -2T and -1A depend on other changes, suggesting their role should be associated with neighboring base pairs . These results suggest that -4T and -3A are involved in SP6 RNA polymerase binding and -2T and -1A are involved in DNA unwinding . This bipartite role of the SP6 promoter TATA contrasts with the single role of T7 promoter TATA on DNA unwinding . The polymerase binding region extends further downstream in the SP6 promoter than in the T7 promoter.

Biophys J, 1993 Sep, 65(3), 1180 - 7
Preparation of isolated biomolecules for SFM observations: T4 bacteriophage as a test sample; Droz E et al.; The T4 bacteriophage has been used to investigate protocols for the preparation of samples for scanning force microscopy in air, in order to obtaining reproducible images . The resolution of images and the distribution of bacteriophages on the substrate depends on the buffer type, its concentration, the surface treatment of substrate, and the method of deposition . The best imaging conditions for the phages require dilution in a volatile buffer at low ionic strength and adsorption onto hydrophilic surfaces . When imaging with the scanning force microscopy the quality of the images is influenced by the vertical and lateral forces applied on the sample and by the tip geometry.

Photochem Photobiol, 1993 Sep, 58(3), 450 - 4
Frequency of ultraviolet radiation-induced mutation at the hprt locus in repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4; Kusewitt DF et al.; The frequency of spontaneous and ultraviolet radiation (UVR)-induced mutation at the hprt locus was determined in control and denV-transfected, repair-proficient murine fibroblasts . Control cells removed an average of 25% of pyrimidine dimers induced by exposure to 150 J/m2 UVR from an FS40 sunlamp within 24 h; under the same conditions of induction and repair, denV-transfected cells removed an average of 71% of pyrimidine dimers . Control cells were somewhat more resistant than denV-transfected cells to killing by UVR . The average frequency of spontaneous mutation at the hprt locus for control and denV-transfected cells was 3 and 15 6-thioguanine (6-TG)-resistant colonies per 10(6) surviving cells, respectively; there was no statistically significant difference between control and denV-transfected cells . However, after exposure to 75 or 150 J/m2 UVR, denV-transfected cells had a significantly lower frequency of mutation to 6-TG resistance . After exposure to a fluence of 75 J/m2, the average frequency of UVR-induced mutation at the hprt locus was 166 mutant colonies per 10(6) surviving cells for control cells and 92 mutant colonies for denV-transfected cells; after 150 J/m2, control cells had 205 6-TG-resistant colonies per 10(6) cells, while denV-transfected cells had 61 mutant colonies . These results demonstrate that UVR-induced pyrimidine dimers are mutagenic photoproducts in mammalian cells.

Biotechniques, 1993 Sep, 15(3), 422 - 4, 426-8, 431
Rapid sequencing of viral DNA from filamentous bacteriophage; Haas SJ et al.; Several techniques have been combined to facilitate rapid sequencing of filamentous phage DNA . Up to 768 clones can be processed with excellent results in one week by a single worker.

J Mol Graph, 1993 Sep, 11(3), 193 - 9, 189-90
Global characterization of protein secondary structures . Analysis of computer-modeled protein unfolding; Arteca GA et al.; Analyses of structural and molecular shape changes undergone by a protein during an unfolding process are presented . The procedure, based on a spherical shape map method, provides a topological description of a three-dimensional macromolecular structure . Local properties of the backbone are used to derive a global characterization of its fold . A spherical shape map of backbone crossings is associated with a given macromolecular conformation . The map is built by classifying each point on the sphere according to the crossing pattern obtained when the backbone is observed along a direction defined by the center of the sphere and the chosen point . The surface of the sphere can be divided in equivalence classes . All the points within a given class correspond to directions from which the backbone has the same overcrossing pattern . Automatic computation and display of these equivalence classes is discussed, as is the implementation of the technique on a computer graphics workstation . The graphical manipulation simplifies the analysis of these maps when following a change in the conformation of the backbone . The procedure is illustrated with the results of a molecular dynamics computer simulation of the unfolding of the bacteriophage T4 glutaredoxin protein (in the form of its polyglycine model) . The method gives a novel description of the differential structural stability for the characteristic secondary structural elements (alpha-helices and beta-sheets) present in the protein . Recognition of the persistence of structural elements over the simulation time is performed in an unbiased manner.

Mol Microbiol, 1993 Sep, 9(5), 1079 - 88
Translational initiation at the coat-protein gene of phage MS2: native upstream RNA relieves inhibition by local secondary structure; de Smit MH et al.; Maximal translation of the coat-protein gene from RNA bacteriophage MS2 requires a contiguous stretch of native MS2 RNA that extends hundreds of nucleotides upstream from the translational start site . Deletion of these upstream sequences from MS2 cDNA plasmids results in a 30-fold reduction of translational efficiency . By site-directed mutagenesis, we show that this low level of expression is caused by a hairpin structure centred around the initiation codon . When this hairpin is destabilized by the introduction of mismatches, expression from the truncated messenger increases 20-fold to almost the level of the full-length construct . Thus, the translational effect of hundreds of upstream nucleotides can be mimicked by a single substitution that destabilizes the structure . The same hairpin is also present in full-length MS2 RNA, but there it does not impair ribosome binding . Apparently, the upstream RNA somehow reduces the inhibitory effect of the structure on translational initiation . The upstream MS2 sequence does not stimulate translation when cloned in front of another gene, nor can unrelated RNA segments activate the coat-protein gene . Several possible mechanisms for the activation are discussed and a function in gene regulation of the phage is suggested.

Mech Dev, 1993 Sep, 43(1), 57 - 70
A universal target sequence is bound in vitro by diverse homeodomains; Kalionis B et al.; To determine the number of DNA binding proteins capable of binding a consensus Engrailed binding site, this consensus sequence was used to screen a library of Drosophila cDNA clones in a bacteriophage expression vector . We retrieved clones encoding 20 distinct DNA binding domains, 17 of which are homeodomains . Binding to a variety of oligonucleotides confirms the related sequence specificity of the retrieved binding domains . Nonetheless, the homeodomains have remarkably diverse amino acid sequences . We conclude that during the evolutionary divergence of homeodomains, the specificity of DNA binding has been much more highly conserved than the amino acid sequence.

Enzyme Microb Technol, 1993 Sep, 15(9), 730 - 5
Stable expression plasmid for high-level production of GroE molecular chaperones in large-scale cultures; Kalbach CE et al.; A stable expression plasmid has been developed to overproduce the Escherichia coli GroES and GroEL molecular chaperones in large-scale cultures . This was achieved by cloning the groE operon under the transcriptional control of a bacteriophage T7 promoter to achieve regulated expression . Isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of a lacUV5 regulated chromosomal copy of T7 gene 1, encoding viral RNA polymerase, resulted in high-level expression of the groE operon from a multicopy plasmid . Induced cells harboring the pT7groE expression plasmid accumulated GroEL to levels of 30% total cell protein, and GroES to 4-5% . Both overproduced proteins were recovered primarily from the soluble fraction of lysed cells . The T7 expression plasmid was significantly more stable than other groE expression plasmids tested during scale-up experiments, and could be used successfully for large-volume cultures of up to 200 l . Strain stability was greatly improved, compared to rich media, when cells were grown in a supplemented minimal medium.

J Exp Med, 1993 Sep 1, 178(3), 1097 - 102
B cell activation via CD40 is required for specific antibody production by antigen-stimulated human B cells; Nonoyama S et al.; Costimulatory signals provided by T cells are required for B cells to produce specific antibody (Ab) to T-dependent antigen (Ag) bacteriophage phi x 174 . In this study, we demonstrate that if cultured in the presence of anti-CD40, interleukin 10 (IL-10), and Ag, purified B cells can produce antiphage Ab in quantities comparable to those synthesized by B cells cocultured with Ag and T cells . Isotypes produced by B cells in this culture system correspond to those observed in sera of B cell donors . Culture of immunoglobulin (Ig)D- and IgD+ B cells reveals that Ag-induced production of antiphage Ab is restricted to IgD- subset of B cells . In the absence of Ag, anti-CD40/IL-10-stimulated B cells produce only minute amounts of antiphage Ab, indicating that Ag stimulation is indispensable and provides a signal that is synergistic with anti-CD40 and IL-10 . Addition of a soluble form of the CD40 ligand (sgp39) to the culture system has a similar effect on specific Ab synthesis as anti-CD40; addition of the soluble construct, CD40 Ig, known to inhibit gp39/CD40 interaction, suppresses in vitro antiphage Ab production by Ag exposed peripheral blood mononuclear cells . Finally, in vivo requirement of gp39/CD40 interaction for specific Ab production was demonstrated by the finding that activated T cells from patients with x-linked hyper IgM syndrome express functionally defective gp39 and respond with depressed Ab titers and fail to switch from IgM to IgG after multiple phage immunizations . These observations illustrate that in vitro and possibly in vivo Ag-specific Ab synthesis requires the presence of Ag and IL-10, and activation signals via CD40.

J Biol Chem, 1993 Aug 25, 268(24), 17754 - 61
Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase; Zhuo S et al.; A protein phosphatase (PPase) from the bacteriophage lambda was overexpressed in Escherichia coli . The recombinant enzyme was purified to homogeneity yielding approximately 17 mg of enzyme from a single liter of bacterial culture . Biochemical characterization of the enzyme showed that it required Mn2+ or Ni2+ as an activator . The recombinant enzyme was active toward serine, threonine, and tyrosine phosphoproteins and phosphopeptides . Surprisingly, the bacterial histidyl phosphoprotein, NRII, was also dephosphorylated by the lambda-PPase . The lambda-PPase shares a number of kinetic and structural properties with the eukaryotic Ser/Thr phosphatases, suggesting that the lambda-PPase will serve as a good model for structure-function studies . Crystallization of the recombinant purified lambda-PPase yielded monoclinic crystals . The crystals diffract to 4.0 A when exposed to synchrotron x-ray radiation.

Nucleic Acids Res, 1993 Aug 25, 21(17), 4055 - 8
Relief of triple-helix-mediated promoter inhibition by elongating RNA polymerases; Skoog JU et al.; We have characterized triple-helix-mediated inhibition of an artificial bacteriophage promoter with respect to relief of inhibition by incoming RNA polymerases that initiate upstream or downstream from the operator sequence . Whereas oligonucleotide-directed triple-helix formation inhibits the test promoter, promoter activity is restored when the triple-helical complexes are disrupted by transcription of either strand of the homopurine operator sequence . The degree of relief from inhibition is related to the frequency of operator transcription . These observations demonstrate that this artificial repressor-operator complex is subject to antagonism by cis elements (other promoters) acting at a distance . Such antagonism might also arise between certain natural transcriptional control regions . Our results suggest that the efficiency of artificial repressors based on triple-helix formation may be limited by transcriptional activity in the gene control region.

J Mol Biol, 1993 Aug 20, 232(4), 1030 - 47
Termination and slippage by bacteriophage T7 RNA polymerase; Macdonald LE et al.; We have examined the termination efficiency of T7 and T3 RNA polymerases (RNAPs) at a variety of termination signals . In agreement with previous investigators we find that termination occurs after the synthesis of an RNA product with a stable secondary structure followed by a run of U residues . Stem-loop structures that lack a 3' U-tract fail to terminate the phage enzyme . The distance (or the sequence) between the start site for transcription and the termination signal may also be important, as placing the terminator at different locations downstream from the promoter, or changing the promoter sequence, results in alterations in termination efficiency . We have explored termination at extended runs of homopolymers in the absence of an apparent stem-loop structure, and have observed that the enzyme terminates (inefficiently) when synthesizing U-rich transcripts, but not A- or C-rich transcripts . This is especially true at low concentrations of UTP . Strikingly, when an elongation complex (EC) encounters a dA-tract in the template strand it is able to slide on the template, resulting in the synthesis of products that have more or fewer U residues than predicted by the sequence of the DNA . This observation suggests that the formation of an RNA: DNA hybrid may be important to the lateral stability of the EC (its ability to maintain proper register with the DNA template) . We have also explored the termination properties of a proteolytically nicked form of T7 RNAP . The nicked enzyme forms a less stable EC than the intact RNAP and dissociates more readily from the template in regions that encode inherently destabilizing RNAs (e.g . stem-loop structures, poly(U)-tracts) . However, the nicked enzyme terminates less efficiently at the late T7 terminator (T7-T phi) or at a termination signal in the human preproparathyroid hormone gene . These results suggest that termination is a highly specific event, and not merely a consequence of decreased stability of the EC . Our observations are not consistent with previous models of termination by the phage RNAP and indicate that revisions to these models may be required.

Biochemistry, 1993 Aug 17, 32(32), 8322 - 8
Assignment of 1H, 15N, and backbone 13C resonances in detergent-solubilized M13 coat protein via multinuclear multidimensional NMR: a model for the coat protein monomer; van de Ven FJ et al.; The major coat protein (gVIIIp) of bacteriophage M13 complexed with SDS detergent micelles was used as a model system to study the lipid-bound conformation of the protein . Conditions were found that allowed the recording of good quality of NMR spectra . By making extensive use of three-dimensional heteronuclear (13C, 15N) NMR, we obtained a complete set of resonance assignments for 1HN, 1H alpha, 1H beta, 13C alpha, CO, and 15N and partially assigned the rest of the 1H spectrum . Analysis of NOE and chemical shift data reveals that gVIIIp is composed of two alpha-helical domains, one ranging from Pro-6 to Glu20 and the other ranging from Tyr-24 all the way to the C-terminus Ser-50 . In contrast to the results reported by Henry and Sykes {Henry, G.D., & Sykes, B.D . (1992) Biochemistry 31, 5285-5297}, at a high SDS to protein ratio the protein appears to be monomeric.

Gene, 1993 Aug 16, 130(1), 121 - 6
A plasmid system for high-level expression and in vitro processing of recombinant proteins; Pohlner J et al.; A novel plasmid expression system has been constructed that combines two useful functions: it facilitates single-step affinity purification of cytoplasmically overproduced fusion proteins and the in vitro processing of fusions with IgA protease (Igase) . The significant features directing the high expression rate of pEV41-based gene fusions in Escherichia coli are the lambda pL promoter for temperature-regulated transcription and the translation initiation region of the bacteriophage MS2 polymerase gene including a downstream box (db) within the first few codons of the open reading frame . Fusion proteins generated with this system contain a short N-terminal carrier peptide allowing convenient affinity purification by means of the His6 peptide . As exemplified by the production of the variable heavy (VH) and light (VL)-chain domains of a monoclonal antibody, the fusion proteins can be specifically processed with Igase either in purified form or simply by incubation with the culture medium of recombinant E . coli {pJP10} cells . Chemical cross-linking of processed VH and VL domains resulted in a recombinant antibody Fv fragment that can specifically bind to its antigen.

Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7744 - 8
Replication of UV-irradiated DNA in human cell extracts: evidence for mutagenic bypass of pyrimidine dimers; Thomas DC et al.; We have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells . Using as a mutational target the alpha-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2 DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate . Replication was inhibited by irradiation in a dose-dependent manner . Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication . These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication . When replicated, UV-irradiated DNA was used to transfect an E . coli alpha-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA . Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication . Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C-->T transition errors at dipyrimidine sites . A few mutants contained 1-nt frameshift errors or tandem double CC-->TT substitutions . The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer.

J Immunol, 1993 Aug 15, 151(4), 2050 - 61
Antigen presentation capacity of murine macrophages infected with Leishmania amazonensis amastigotes; Prina E et al.; Leishmania-infected M phi are potential candidates for the presentation of parasite Ag to Leishmania-specific CD4+ T lymphocytes . To assess whether infected cells could function as APC, we examined the ability of bone marrow-derived M phi infected with Leishmania amazonensis amastigotes to stimulate various CD4+, l-Ad- or l-Ed-restricted T-cell hybridomas specific for the bacteriophage lambda repressor cl protein, the human chorionic gonadotropin or OVA . A reduced capacity of infected M phi to present native Ag to most T-cell hybridomas tested was noted that was probably a result of a lower expression on their plasma membrane of stimulatory {la-peptide} complexes . Neither a reduced Ag uptake nor an altered Ag processing appeared to be at the origin of the partial inability of infected M phi to present Ag . As regards the level of plasma membrane la expression, no quantitative difference could be detected between uninfected and infected M phi . Moreover, after fixation with paraformaldehyde, the ability of plasma membrane la molecules to bind immunogenic peptides was apparently not reduced in infected M phi . So, these cells most likely expressed functional la molecules on their cell surface . Interestingly, infected M phi and M phi infected then cured by a treatment with a leishmanicidal compound were similarly impaired in their capacity to present native Ag or peptides to the hybridomas, and no recovery was noted even 24 h after the leishmanicidal treatment . Furthermore, infected M phi and M phi incubated with heat-killed amastigotes or with an amastigote homogenate exhibited similar inhibitions of Ag presentation . Taken together, these results suggest that the functional failure of infected M phi to present exogenous Ag could be because either of interferences with the events leading to the meeting of la molecules with peptides derived from these exogenous Ag or to a competition for binding to la molecules between these peptides and parasite molecules.

Nature, 1993 Aug 12, 364(6438), 593 - 9
Crystal structure of bacteriophage T7 RNA polymerase at 3.3 A resolution; Sousa R et al.; The crystal structure of T7 RNA polymerase reveals a molecule organized around a cleft that can accommodate a double-stranded DNA template . A portion (approximately 45%) of the molecule displays extensive structural homology to the polymerase domain of Klenow fragment and more limited homology to the human immunodeficiency virus HIV-1 reverse transcriptase . A comparison of the structures and sequences of these polymerases identifies structural elements that may be responsible for discriminating between ribonucleotide and deoxyribonucleotide substrates, and RNA and DNA templates . The relative locations of the catalytic site and a specific promoter recognition residue allow the orientation of the polymerase on the template to be defined.

Nucleic Acids Res, 1993 Aug 11, 21(16), 3725 - 30
In vitro replication of bacteriophage PRD1 DNA . Characterization of the protein-primed initiation site; Caldentey J et al.; Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism . Using single-stranded oligonucleotide templates carrying the sequence corresponding to the 25 first bases of the 3' end of PRD1 DNA, and Mg2+ as the activating metal ion of the phage DNA polymerase, we show that the fourth base from the 3' end of the template directs, by base complementarity, the dNMP to be linked to the phage terminal protein (TP) in the initiation reaction . This result suggests that phage PRD1 maintains its 3' end DNA sequences via a sliding-back mechanism . The single-stranded DNA templates could not be replicated by the PRD1 DNA polymerase, much in contrast to the natural TP-DNA . Nevertheless, the analysis of the transition products obtained with TP-DNA and origin-containing oligonucleotides suggests that sliding-back occurs stepwise, the fourth base being the directing position during the entire process.

J Mol Biol, 1993 Aug 5, 232(3), 826 - 38
The phage 434 OR2/R1-69 complex at 2.5 A resolution; Shimon LJ et al.; The crystal structure of the DNA-binding domain of bacteriophage 434 repressor (R1-69) in complex with a 20 base-pair DNA fragment has been determined to 2.5 A resolution . The DNA fragment contains the sequence of the OR2 operator site, which differs from the previously studied OR1 site at three of the variable six central base-pairs . Comparison of the two structures shows that the overall bent conformation of the DNA backbone as well as the pattern of DNA-protein interactions seen in the OR1/R1-69 complex are maintained in the OR2 complex . However, the conformations of the DNA base-pairs are different in the two structures . In particular, the central base-pairs of OR2/R1-69 structure are more co-planar than in OR1/R1-69, and there are no cross-strand "bifurcated" hydrogen bonds . These results show that binding of the protein causes operator DNA to adopt a particular, well-defined backbone conformation, and they reinforce the notion that the energetic cost of achieving this conformation, most likely different for different sequences, can determine, at least in part, the relative affinity of the repressor for different operator sites.

J Mol Biol, 1993 Aug 5, 232(3), 1005 - 6
Crystallization of bacteriophage fr and its recombinant capsids; Bundule M et al.; Single crystals of Escherichia coli bacteriophage fr and its recombinant capsids have been obtained by the vapour diffusion technique in the presence of ammonium sulphate . They diffract X-rays to at least 3.5 A . Electron microscopic observation of the crystals revealed a three-dimensional lattice of particles with RNA phage morphology and dimensions.

Biochemistry, 1993 Aug 3, 32(30), 7772 - 8
DNA-dependent adenosinetriphosphatase A: immunoaffinity purification and characterization of immunological reagents; Mesner LD et al.; We describe an immunoaffinity purification of DNA-dependent ATPase A from fetal calf thymus . The rapid purification increases the yield of enzymatically active enzyme approximately 4-fold, with up to a 7-fold increase in specific activity, and significantly improves the yield of a higher molecular weight species of ATPase A . In the presence of a denatured calf thymus DNA effector, the immunoaffinity-purified enzyme has a specific activity that is more than 10-fold higher than reported for any other eukaryotic DNA-dependent ATPase and 100-fold higher than most others . The improvement in yield has allowed several polypeptides to be identified using monoclonal antibodies, and these polypeptides are demonstrated to be structurally related by partial peptide mapping with N-chlorosuccinimide . The preferred DNA effector for ATP hydrolysis continues to be a DNA primer-template junction with an adjacent stretch of single-stranded DNA . We have used the immunoaffinity-purified enzyme to develop additional stable murine hybridoma monoclones, resulting in a bank of antibodies that recognize a number of different epitopes . All of the monoclonal antibodies react with both calf thymus DNA-dependent ATPase A and bacteriophage T4 gene 44 protein, a DNA-dependent ATPase essential for DNA replication in the bacteriophage T4 system . These monoclonal antibodies should facilitate the development of our understanding with respect to the role and regulation of DNA-dependent ATPases in eukaryotic DNA replication.

Genet Res, 1993 Aug, 62(1), 1 - 9
The genetics of the Luria-Latarjet effect in bacteriophage T4: evidence for the involvement of multiple DNA repair pathways; Hyman P; The Luria-Latarjet effect is an increase in resistance of a virus to DNA damage during infection of a host . It has often been assumed to involve recombinational repair, but this has never been demonstrated experimentally . Using nine bacteriophage (phage) T4 mutants, I present evidence indicating that, for phage T4, the Luria-Latarjet effect is due to three repair pathways-excision repair, post-replication-recombinational-repair (PRRR) and multiplicity reactivation (MR) (a second form of recombinational repair) . The results also show that the Luria-Latarjet effect develops in two stages . The first stage starts soon after infection . Damage which occurs during the first stage can be repaired by excision repair or PRRR . The second stage appears to start after the first round of DNA replication is complete . DNA damage which occurs during this stage can apparently be repaired by MR as well as the other two repair pathways . The results of this study support the hypothesis that recombinational repair has been selected to ensure that the progeny phage genomes which are packaged have minimum DNA damage . Since other viruses which infect bacterial, animal and plant cells show a Luria-Latarjet effect similar to that in phage T4, the conclusions from this study may have wide applicability.

Electrophoresis, 1993 Aug, 14(8), 720 - 4
Characterization of the electrophoretic properties of nucleosome core particles by transverse polyacrylamide pore gradient gel electrophoresis; Orban L et al.; Transverse pore gradient gel electrophoresis, previously applied to bent DNA, has extended the usefulness of the gel retardation assay in two ways: (i) by differentiating between different DNA conformations; (ii) by providing information regarding the physical properties of DNA . In the present study, similarly extended information is obtained with regard to a well-characterized DNA-protein complex, the chicken erythrocyte nucleosome core particle . (i) The winding of DNA around the protein core constrains the DNA which renders its Ferguson curve (migration distance vs . gel concentration) similar to that of kinetoplast DNA, i.e . it intersects sharply with the Ferguson curves of linear DNA standards . By contrast, the deproteinized nucleosome DNA exhibits a Ferguson curve similar to linear standards of the same length . (ii) Interpretation of the Ferguson curve based on a mathematical model shows that the nucleosome exhibits a linear Ferguson plot {log(mobility) vs . gel concentration} . This is similar to and characteristic of spherical proteins, contrasting with the concave plot typical for linear and bent DNA . (iii) The effective size of the nucleosome, evaluated in terms of an "equivalent sphere" (i.e . a hypothetical spherical particle with a radius, Res, having the same electrophoretic mobility as DNA for a particular set of experimental conditions), remains invariant across the gel concentration range of 3-9%T . This is similar to proteins and bacteriophages and contrasts with the progressive decline of Res with increasing gel concentration observed for linear DNA and the deproteinized nucleosomal DNA.

Mol Reprod Dev, 1993 Aug, 35(4), 339 - 44; discussion 344-5
Molecular dynamics of insulin/IGF-I receptor transmembrane signaling; Pessin JE et al.; To examine the molecular basis of ligand-stimulated intramolecular beta-subunit autophosphorylation, hybrid receptors composed of wild-type and mutant insulin and insulin-like growth factor-1 (IGF-I) half-receptor precursors were characterized . Previous studies have demonstrated that assembly of the IGF-I wild-type half-receptor (alpha beta WT) with a kinase-defective half-receptor (alpha beta A/K) produced a substrate kinase-inactive holoreceptor in vitro {Treadway et al . (1991): Proc Natl Acad Sci USA 88:214-218} . To extend these studies, the vaccinia virus/bacteriophage T7 expression system was used to generate various hybrid receptor complexes in cultured cells . As was observed for hybrid receptors assembled in vitro, the wild-type/mutant hybrid receptors formed in situ were also incapable of phosphorylating several peptide substrates . However, ligand-stimulated beta-subunit autophosphorylation was still observed . To determine the molecular basis for this discrepancy, hybrid receptors were assembled from a truncated beta-subunit insulin half-receptor (alpha beta delta 43) and a kinase-defective half-receptor (alpha beta A/K) . Under these conditions, insulin-stimulated autophosphorylation primarily occurred on the full-length kinase-inactive beta-subunit (alpha beta A/K) without significant labeling of the kinase-active truncated beta-subunit (alpha beta delta 43) . A similar IGF-I hybrid receptor species was characterized, and the same pattern of autophosphorylation was observed in response to IGF-I . These data demonstrate that both insulin and IGF-I stimulate an intramolecular trans-autophosphorylation reaction between two adjacent beta-subunits within the holoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetics, 1993 Aug, 134(4), 1023 - 30
Mutants of Escherichia coli with increased fidelity of DNA replication; Fijalkowska IJ et al.; To improve our understanding of the role of DNA replication fidelity in mutagenesis, we undertook a search for Escherichia coli antimutator strains with increased fidelity of DNA replication . The region between 4 and 5 min of the E . coli chromosome was mutagenized using localized mutagenesis mediated by bacteriophage P1 . This region contains the dnaE and dnaQ genes, which encode, respectively, the DNA polymerase (alpha subunit) and 3' exonucleolytic proofreading activity (epsilon subunit) of DNA polymerase III holoenzyme, the enzyme primarily responsible for replicating the bacterial chromosome . The mutated bacteria were screened for antimutator phenotype in a strain defective in DNA mismatch repair (mutL), using a papillation assay based on the reversion of the galK2 mutation . In a mutL strain, mutations result primarily from DNA replication errors . Among 10,000 colonies, seven mutants were obtained whose level of papillation was reduced 5-30-fold . These mutants also displayed decreased mutation frequencies for rifampicin or nalidixic acid resistance as well as for other markers . Mapping by P1 transduction and complementation showed each to reside in dnaE . These observations support the idea that the mutants represent antimutators which replicate their DNA with increased fidelity . Mutation rates were reduced in both mutL and mutT backgrounds, but mutagenesis by ultraviolet light was not significantly affected, suggesting that the antimutator effect may be largely restricted to normal DNA replication.

Genetics, 1993 Aug, 134(4), 1003 - 11
Second-site suppressors of a cold-sensitive external scaffolding protein of bacteriophage phi X174; Fane BA et al.; This report describes the isolation and characterization of second-site suppressors of a cold-sensitive (cs) external scaffolding protein, gpD, of bacteriophage phi X174 . Seven genetically distinct suppressors were isolated . Six of them are located in gene F which encodes the major coat protein of the virus . The seventh is located in gene J which encodes the DNA-binding protein . A subset of the suppressors are trans-acting . These second-site suppressors do not exhibit allele specificity; they are able to suppress defects associated with a csD protein for which they were not selected . The initial characterization of the second-site suppressors and their locations within the major coat protein suggest that the mechanism of suppression may involve both structural and stoichiometric phenomena.

J Bacteriol, 1993 Aug, 175(16), 5145 - 52
Expression analysis of cloned chromosomal segments of Escherichia coli; Sankar P et al.; The novel transcription system of bacteriophage T7 was used to express Escherichia coli genes preferentially with a new low-copy-number plasmid vector, pFN476, to minimize toxic gene effects . Selected E . coli chromosomal fragments from an ordered genomic library (Y . Kohara, K . Ikiyama, and K . Isono, Cell 50:495-508, 1987) were recloned into this vector, and their genes were preferentially expressed in vivo utilizing its T7 promoter . The protein products were analyzed by two-dimensional gel electrophoresis . By using DNA sequence information, the gel migration was predicted for the protein products of open reading frames from these segments, and this information was used to identify gene products visualized as spots on two-dimensional gels . Even in the absence of DNA sequence information, this approach offers the opportunity to identify all gene products of E . coli and map their genes to within 10 kb on the E . coli genome; with sequence information, this approach can produce a definitive expression map of the E . coli genome.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7010 - 4
Genetic fusion of subunits of a dimeric protein substantially enhances its stability and rate of folding; Liang H et al.; The gene V protein of bacteriophage f1 is a single-stranded DNA and RNA-binding protein composed of two identical subunits . We have constructed single-chain variants of the protein using short peptide linkers of five or six amino acids to connect the carboxyl terminus of one monomer to the amino terminus of the second monomer . The resulting subunit-fusion gene V proteins were found to bind single-stranded DNA nearly as tightly as the wild-type protein . Denaturation measurements show that the subunit-fusion gene V proteins are 5 kcal/mol (1 kcal = 4.18 kJ) more stable than the wild-type protein at a protein concentration of 10 microM . The rate of unfolding of the protein is essentially unaffected by the fusion of monomeric subunits, whereas the rate of folding is greatly enhanced . Our results suggest a simple way of obtaining a substantial thermodynamic stabilization for some oligomeric proteins.

Virology, 1993 Aug, 195(2), 511 - 20
Expression of the Bunyamwera virus M genome segment and intracellular localization of NSm; Nakitare GW et al.; Bunyamwera (BUN) virus is the prototype of the family Bunyaviridae and contains a trisegmented, single-stranded RNA genome of negative polarity . The medium (M) RNA segment encodes the two virion glycoproteins, G1 and G2, and a nonstructural protein, NSm, in the form of a polyprotein precursor which is cotranslationally cleaved . The gene order of the M segment is 5' G2-NSm-G1 3' . We have raised a monospecific antiserum in rabbits to a branched chain synthetic peptide to a region of the NSm protein which specifically immunoprecipitates NSm from BUN-infected cells . Indirect immunofluorescence experiments on BUN-infected cells using this antiserum gave a perinuclear staining pattern, suggesting that like the viral structural proteins, NSm localizes to the Golgi complex . An essentially full-length M segment cDNA was cloned into a recombinant vaccinia virus under control of bacteriophage T7 promoter and terminator sequences and expressed in cells co-infected with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase . G1, G2, and NSm were detected in cells dually infected with the recombinant vaccina viruses, indicating that processing of the M segment-encoded precursor does not require other BUN proteins . Immunofluorescence experiments showed that the BUN glycoproteins expressed from this recombinant vaccinia virus system localized to the Golgi complex like authentic BUN proteins.

J Bacteriol, 1993 Aug, 175(15), 4738 - 43
In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein; Marsic N et al.; The interaction between the RecBCD enzyme of Escherichia coli and the lambda Gam protein was investigated . Two types of experiments were done . In one type, Gam protein was produced by transient induction of the cells lysogenic for lambda cI857gam+ . The presence of Gam protein, which inhibits RecBCD nuclease, enabled these cells to support the growth of a gene 2 mutant of bacteriophage T4 (T4 2) . The lysogens overproducing the RecB subunit of RecBCD enzyme could titrate Gam protein and thus prevent the growth of T4 2 . In contrast, the lysogens overproducing either RecC or RecD retained their capacity for growth of T4 2 . It is therefore concluded that the RecB subunit is capable of binding Gam protein . In the second type of experiments, Gam protein was provided by derepressing the gamS gene on the plasmid pSF117 (S . A . Friedman and J . B . Hays, Gene 43:255-263, 1986) . The presence of this protein did not interfere with the growth of wild-type cells (which were F-) . Gam protein had a certain effect on recF mutants, whose doubling time became significantly longer . This suggests that the recF gene product plays an important role in maintenance of viability of the Gam-expressing cells . Gam protein exerted the most striking effect on growth of Hfr bacteria . In its presence, Hfr bacteria grew extremely slowly, but their ability to transfer DNA to recipient cells was not affected . We showed that the effect on growth of Hfr resulted from the interaction between the RecBCD-Gam complex and the integrated F plasmid.

J Virol, 1993 Aug, 67(8), 4914 - 22
RNA structure and heterologous recombination in the double-stranded RNA bacteriophage phi 6; Onodera S et al.; Bacteriophage phi 6 has a genome of three segments of double-stranded RNA, designated L, M, and S . A 1.2-kbp kanamycin resistance gene was inserted into segment M but was shown to be genetically unstable because of a high recombination rate between segment M and the 3' ends of segments S and L . The high rate of recombination is due to complementary homopolymer tracts bounding the kan gene . Removal of one arm of this potential hairpin stabilizes the insertion . The insertion of a 241- or 427-bp lacZ' gene into segment M leads to a stable Lac+ phage . The insertion of the same genes bounded by complementary homopolymer arms leads to recombinational instability . A stable derivative of this phage was shown to have lost one of the homopolymer arms . Several other conditions foster recombination . The truncation of a genomic segment at the 3' end prevents replication, but such a damaged molecule can be rescued by recombination . Similarly, insertion of the entire 3-kb lacZ gene prevents normal formation of virus, but the viral genes can be rescued by recombination . It appears that conditions leading to the retardation or absence of replication of a particular genomic segment facilitate recombinational rescue.

J Virol, 1993 Aug, 67(8), 4696 - 703
Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle; Savilahti H et al.; Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism . Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats {ITRs}) . The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses . This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences . These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments . The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis . Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybrid phages made in vitro verified this observation . However, genomes in which the dissimilar DNA termini had regained identical sequences were also detected . These observations are explained by a DNA replication model involving two not mutually exclusive pathways . The generality of this model in protein-primed DNA replication is discussed.

Biol Chem Hoppe Seyler, 1993 Aug, 374(8), 657 - 64
Rifampicin resistance of Mu lysogenic strains by rpoB mutations; Kirschbaum TM et al.; The resistance of Escherichia coli against the antibiotic rifampicin is caused by mutations in the rpoB gene which alter the structure of the beta subunit of the DNA-dependent RNA polymerase . By insertion-mutagenesis with bacteriophage Mu rifampicin-resistant mutants that were believed to have a sensitive RNA polymerase had been generated . For closer analysis of this putative rpoB-independent mechanism of resistance we cloned and sequenced the insertion sites from two of the Mu lysogens . One of them showed > 95% sequence similarity with the phn locus of E . coli B, and was mapped between nt 3824 and 3825 within the phnB gene . This positions the phn locus between 4397 and 4410 kb of the Kohara map of E . coli K-12 . Since both analysed insertion sites differ in respect to each other and to the findings of previous work we determined the rifampicin sensitivity of the RNA polymerase of the Mu lysogens with partially purified enzyme . For all mutants the IC50 as a measure for sensitivity was significantly higher than for the parent strain . Sequence analysis of part of the rpoB gene (nt 1519 to 1716) revealed single point mutations in the investigated Mu lysogens . The rpoB mutation is necessary and sufficient for the observed resistance, while the prophage alone does not evoke resistance and has no synergistic effect together with the rpoB mutation . Our work supports the assumption that rifampicin like other hydrophobic molecules enters the cell via simple diffusion through the outer membrane with LPS being the main barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Probes, 1993 Aug, 7(4), 269 - 75
Single-stranded RNA probes generated from PCR-derived DNA templates; Bales KR et al.; The following report outlines the use of the polymerase chain reaction (PCR) in combination with in vitro transcription to generate single-stranded radiolabelled RNA probes useful for nuclease protection and in situ hybridization experiments . Specific DNA fragments with bacteriophage promoter (T3 and/or T7) sequences at the 5' or 3' end are generated by repeated rounds of amplification . Following purification, these PCR-generated DNA products are used as templates for in vitro transcription with the correct DNA-dependent RNA polymerase . The resultant radiolabelled, single-stranded RNA (ssRNA) can be used for in situ hybridization, Southern or Northern blot analysis, and ribonuclease protection experiments . Sub-cloning or hydrolysis of large fragments is not required . Probes can be made from virtually any sequence using a variety of template sources.

J Cell Biochem, 1993 Aug, 52(4), 375 - 83
Association of a human H1 histone gene with an H2A pseudogene and genes encoding H2B.1 and H3.1 histones; Kardalinou E et al.; A cluster of human histone genes was found on three overlapping clones isolated from cosmid and bacteriophage libraries . These three overlapping segments of the human genome comprise genes coding for H3.1, an H2A pseudogene, and an H2B.1 gene downstream of the previously characterized H1.2 gene . The cosmid clone covers 30 kb upstream of the H1.2 gene and overlaps with two phage clones covering the core histone genes and the pseudogene . The same arrangement of an H3 gene, an H2A pseudogene and an H2B gene downstream of an H1 gene has been described within a mouse histone gene cluster {Yang et al.:J Biol Chem 262:17118-17125, 1987; Gruber et al.:Gene 95:303-304, 1990}.

Zentralbl Bakteriol, 1993 Aug, 279(3), 307 - 15
Bacteriophages induced by ciprofloxacin in a Borrelia burgdorferi skin isolate; Neubert U et al.; Two types of tailed bacteriophages were detected by electron microscopy in a Borrelia burgdorferi strain which had been isolated from infected human skin and exposed to the gyrase inhibitor ciprofloxacin . One of these phages displayed an isometric 30 nm head, a neck and a contractile tail 50-64 nm in length with a baseplate, according to an A-1 morphology . The other phage showing a B-1 morphology had a 30 nm isometric head as well and a long non-contractile straight tail 115-130 nm in length without neck and baseplate . Both types of phages could be found together within one plasmolysed spirochetal cell . We conclude that we are dealing with a lysogenic strain of Borrelia burgdorferi carrying at least two different prophages inducible by ciprofloxacin.

J Egypt Soc Parasitol, 1993 Aug, 23(2), 431 - 5
Isolation, purification and ultrastructure of bacteriophages from natural mosquito breeding places in Egypt; Ali SM et al.; Two bacteriophages were isolated from field collected samples representing two different mosquito breeding places . The phage AB-1 (isolated from Abheit village, Faiyoum Governorate "seepage water") and the phage GA-2 (isolated from El-Gabal El-Asfer Qualyobia Governorate "sewage drain water") were purified . Both bacteriophages were ultrastructurally described with respect to their morphology, dimensions, phases of bacterial attack and lysogeny . No major differences were observed between both isolated phages in relation to specificity, however; they were isolated from two different types of breeding places and two different geographic areas as well . This study may assume a wide host range of the isolated phages and reflect how bacterial insecticides used for mosquito larval control could be inhibited by such bacteriophage.

Genetics, 1993 Aug, 134(4), 1013 - 21
Efficient double-strand break-stimulated recombination promoted by the general recombination systems of phages lambda and P22; Poteete AR et al.; To examine bacteriophage recombination in vivo, independent of such other processes as replication and packaging, substituted lambda phages bearing restriction site polymorphisms were employed in a direct physical assay . Bacteria were infected with two phage variants; DNA was extracted from the infected cells and cut with a restriction endonuclease . The production of a unique recombinant fragment was measured by Southern blotting and hybridization with a substitution sequence-specific probe . High frequency recombination was observed under the following conditions: the substituted lambda phages infected a wild-type host cell bearing a lambda repressor-expressing plasmid designed to shut down phage transcription and inhibit phage DNA replication as well . The same plasmid expressed the lambda red and gam genes . In addition, the host cell bore a second plasmid which expressed the EcoRI restriction-modification system . Both phage chromosomes possessed a single EcoRI site in the middle of the marked substitution sequence; however, as the site was modified in one of the parent phages, only the other partner was cut . Recombination was found to be dependent upon (1) red, (2) recA, (3) inactivation of the host recBCD function, either by Gam protein or by mutation and (4) double-strand breaks . The homologous recombination system of phage P22 could substitute for that of lambda.

Int J Radiat Biol, 1993 Aug, 64(2), 157 - 64
Uracil-DNA glycosylase produces excess lethal damage induced by an Auger cascade in BrdU-labelled bacteriophage T1; Furusawa Y et al.; T1 phages with and without 5-bromo-2'-deoxyuridine (BrdU) labelling were irradiated in solids with monochromatic X-rays at 12.40 and 13.51 keV, below and just above the K-absorption edge of bromine (13.47 keV) in vacuum and wet states . Irradiated phages were assayed on uracil-DNA glycosylase (Udg) deficient (ung-1) and sufficient (ung+) host strains of Escherichia coli, in order to investigate the nature of the lethal damage induced by Auger cascade following X-ray absorption at the K-shell of bromine as a key atom . The results were: (1) An Auger-specific enhancement (1.15) was observed only when BrdU-labelled phages were irradiated in the wet state and assayed on ung+ host cells . (2) A Udg-specific enhancement was observed only for BrdU-labelled phages, not for unlabelled phages . (3) The sensitivities of BrdU-unlabelled phages were almost the same, despite the irradiation states and strains of the host cell, indicating that this sensitivity was a common fraction of the sensitivity under all conditions . (4) The lethal damage for the T1 phage was categorized into four fractions according to the sensitivities under different conditions: the general fraction was defined as being the sensitivity of unlabelled phages (G-fraction); BrdU-specific, but unrecognizable by Udg (B-fraction); Udg specific, but not Auger-specific (U-fraction); and Auger-specific (A-fraction) . (5) Although the so-called indirect action of water radicals increased only the G-fraction by about three-fold, the B- and U-fractions were not affected by any change in the irradiation states, thus indicating that these two fractions were caused by the so-called direct action.

Mutat Res, 1993 Aug, 292(1), 69 - 81
ENU-induced mutagenesis at a single A: T base pair in transgenic mice containing phi X174; Burkhart JG et al.; Transgenic mice containing the bacteriophage phi X174 am3 as a chromosomally integrated and recoverable marker for in vivo mutation have been produced to measure spontaneous and induced substitutions at an A:T base pair among single copies . phi X174 was chosen for its small size (5 kb), unique sequence, and the opportunity to take advantage of previously reported in vitro data on mutation and repair; the am3 site provides sequence specificity in a reversion assay for mutation of an A:T base pair . Inbred C57Bl/6 mice have been made homozygous for approximately 100 copies of the the phage sequence without any apparent detrimental effects on the homozygous individuals . Recoveries of phage from mouse tissues are in the range of 1-5 x 10(7) PFU per micrograms mouse DNA; both recovery and mutation are independent of endogenous CpG methylation . Background mutation frequencies are 2-4 x 10(-7) among phage recovered from liver, brain, spleen, and kidney . Adult mice were treated with 200 mg/kg N-ethyl-N-nitrosourea, and phage were recovered at 2 and 14 days after treatment . At 2 days after treatment we observed a slight increase only among phage isolated from the brain of one mouse out of four . At 14 days after ENU treatment, there were significant increases in mutation frequencies among phage recovered from the liver (6 x) and spleen (10 x) . These results demonstrate (1) response of a single A:T base pair to alkylation-induced mutation in a nonexpressed gene, (2) the role of cell proliferation in somatic mutagenesis, and (3) provide a model for a transgenic approach for study of site-specific mutagenesis in vivo in higher eukaryotes.

Mutat Res, 1993 Aug, 288(2), 301 - 10
Deletion between direct repeats in bacteriophage T7 gene 1.2; Scearce LM et al.; Deletion mutagenesis in bacteriophage T7 was studied with an insertion-reversion assay involving phage containing inserts of foreign DNA that form 10-bp direct repeats . The precise deletion of the insert restores the function of the non-essential gene and is easily assayed by growth on selection strains of E . coli . Similar inserts with unique direct repeats were placed in either gene 1.2 (dGTPase Inhibitor) or gene 1.3 (DNA ligase) . The deletion rates of the inserts were quantified with Luria and Delbruck fluctuation tests . Deletion rates were similar for inserts in both genes indicating that the rates of deletion were not unique to either specific site, or the sequence of the direct repeats . Deletion was independent of functional T7 ligase at 37 degrees C, while an increase in the rate of deletion was noted in some ligase-deficient phage at 43 degrees C . The effect of E . coli DNA Polymerase I on deletion rate was tested and found to decrease deletion rate 60% with the polA1 mutation and 90% with the polA546ex mutation.

Mutat Res, 1993 Aug, 288(2), 207 - 14
Chemically altered apurinic sites in phi X174 DNA give increased mutagenesis in SOS-induced E . coli; Bockrath R et al.; Single-strand DNA from bacteriophage phi X174 am3 is treated with mild acid and heat to produce increasing numbers of apurinic sites per molecule . Samples are assayed, either directly or after additional chemical reactions, by electroporation into the recipient E . coli strain HF4714(su-1+) . Modified apurinic sites are produced by reactions with O-methyl- or O-benzyl-hydroxylamine, and reduced apurinic sites by reactions with sodium borohydride . Reversion mutation frequencies are significant only if the recipient strain is SOS-induced (by growth after UV irradiation) . A simple apurinic site at the target gives rise to mutation (a transversion) with a probability of 0.07, while the modified or reduced apurinic site has a mutagenic efficiency of 0.22-0.27 or 0.29, respectively . The open ring form of deoxyribose may account for the 3-4-fold increased mutagenicity with altered apurinic lesions . Also considered are effects by temperature and cyclobutane pyrimidine dimers on mutagenicity and the relatively invariant survival curves that obtain regardless of chemical alterations at the apurinic sites and/or SOS induction.

Nature, 1993 Jul 29, 364(6436), 401 - 6
Transcriptional antitermination; Greenblatt J et al.; Antiterminator proteins control gene expression by recognizing control signals near the promoter and preventing transcriptional termination which would otherwise occur at sites that may be a long way downstream . The N protein of bacteriophage lambda recognizes a sequence in the nascent RNA, and modifies RNA polymerase by catalysing the formation of a stable ribonucleoprotein complex on its surface, whereas the lambda Q protein recognizes a sequence in the DNA . These mechanisms of antitermination in lambda provide models for analysing antitermination in viruses such as HIV-1 and in eukaryotic genes.

Biochemistry, 1993 Jul 27, 32(29), 7549 - 58
Mutational analysis of a ribonuclease III processing signal; Chelladurai B et al.; A mutational approach was employed to identify sequence and structural elements in a ribonuclease III processing signal that are important for in vitro enzymatic cleavage reactivity and selectivity . The substrate analyzed was the bacteriophage T7 R1.1 processing signal, a 60 nucleotide irregular RNA hairpin exhibiting an upper and lower dsRNA stem, separated by an asymmetric internal loop which contains the scissile phosphodiester bond . Altering the length of either the upper or lower dsRNA segment in R1.1 RNA dose not change the site of RNase III cleavage . However, decreasing the size of either the upper or lower dsRNA segment causes a progressive inhibition of processing reactivity . Omitting monovalent salt from the reaction buffer promotes cleavage of otherwise unreactive R1.1 deletion mutants . Accurate processing is maintained with R1.1 variants containing specific point mutations, designed to disrupt Watson-Crick (WC) base-pairing in a conserved sequence element within the upper dsRNA stem . The internal loop is not required for processing reactivity, as RNase III can accurately and efficiently cleave R1.1 variants in which this structure is WC base-paired . Moreover, an additional cleavage site is utilized in these variants, which occurs opposite the canonical site, and is offset by two nucleotides . The fully base-paired R1.1 variants form a stable complex with RNase III in Mg(2+)-free buffer, which can be detected by a gel electrophoretic mobility shift assay . In contrast, the complex of wild-type R1.1 RNA with RNase III is unstable during nondenaturing gel electrophoresis . Thus, a functional role of the T7 R1.1 internal loop is to enforce single enzymatic cleavage, which occurs at the expense of RNase III binding affinity.

Nucleic Acids Res, 1993 Jul 25, 21(15), 3391 - 8
Analysis of the Escherichia coli genome . III . DNA sequence of the region from 87.2 to 89.2 minutes; Plunkett G 3rd et al.; The DNA sequence of 96.5 kilobases of the Escherichia coli K-12 genome has been determined, spanning the region between rrnA at 87.2 minutes and katG at 89.2 minutes on the genetic map . The sequence includes 84 open reading frames, of which 46 code for unidentified proteins . Six previously mapped but unsequenced genes have been identified in this span: mob, fdhD, rhaD, rhaA, rhaB, and kdgT . In addition, five new genes have been assigned: the heat shock genes hsIU and hsIV, and the genes fdoG, fdoH, and fdoI, which encode the three subunits of formate dehydrogenase-O . The arrangement of the genes relative to possible promoters and terminators suggests 57 potential transcription units . Other features include the precise location of the bacteriophage P2 attachment site attP2II, and eleven REP elements, including one containing 9 REP sequences--one of the largest such elements known . This segment brings the total length of contiguous finished sequence to 325 kilobases.

J Biol Chem, 1993 Jul 25, 268(21), 15721 - 30
Laser cross-linking of proteins to nucleic acids . II . Interactions of the bacteriophage T4 DNA replication polymerase accessory proteins complex with DNA; Hockensmith JW et al.; In this paper we examine the interactions of the polymerase accessory proteins subassembly of the bacteriophage T4 DNA replication complex, using single-pulse ultraviolet laser excitation to induce protein-nucleic acid cross-links . The laser-induced cross-linking permits effective "freezing" of the instantaneous equilibrium state of the complex and thus provides a mechanism to dissect the individual protein-nucleic acid interactions involved in complex assembly . We find that the binding of the gene 44, 62, and 45 proteins is dependent not only on the presence of each of the other proteins, but also on the presence of adenine nucleotide cofactors . We find that the nonhydrolyzable analogs of ATP often behave more like ADP than ATP in these experiments . Gene 45 protein is able to induce an increase in cross-linking of the gp44/62 complex to nucleic acids, and this increased cross-linking correlates with changes in the apparent Km of the gp44/62 complex for polynucleotides and with changes in Vmax during ATP hydrolysis . Our results suggest that the enhanced DNA binding is predominately through the gene 62 protein and not the ATPase catalytic subunit (gene 44 protein) . Thus the gene 62 protein seems to play an integral role in gp45-mediated enhancement of the ATP hydrolytic activity of gp44 . These results are summarized and integrated in the form of a model for the multiple interactions of the accessory proteins with DNA and one another in the presence of mononucleotide cofactors and substrates.

J Mol Biol, 1993 Jul 20, 232(2), 512 - 21
Different mechanisms of recognition of bacteriophage Q beta plus and minus strand RNAs by Q beta replicase; Barrera I et al.; Our earlier work on the recognition of Q beta plus strand RNA by replicase had shown by RNase degradation and by electron microscopic techniques that specific binding interactions occurred at two internal sites, the S-site and the M-site, but not at the 3'-end, i.e . the site of initiation of synthesis . Using essentially similar methods, we have found now for binding complexes of replicase with the minus strand a completely different pattern, namely considerable terminal binding, whereas binding to internal sites was without detectable specificity . In the case of plus strand complexes, simultaneous binding at the two internal sites and at a terminal site could be demonstrated by electron microscopy after initiation of RNA synthesis in the presence of host factor, GTP and ATP . A variant plus strand RNA containing a 490 nucleotide duplication near the 5'-end resulted in similar double-looped complexes, however with an elongated free arm, showing that the protein-bound terminal site was the 3'-end of the RNA . Interestingly, the same two-looped structures were also found for complexes consisting of plus strand RNA and host factor without replicase . This suggests that the role of the host factor on the plus strand template is to bring the 3'-end into the proximity of the S-site/M-site domain, where replicase can initiate on it . In contrast, the 3'-end of the minus strand appears to be directly available to the enzyme.

Gene, 1993 Jul 15, 129(1), 1 - 8
Expression of the Rz gene and the overlapping Rz1 reading frame present at the right end of the bacteriophage lambda genome; Hanych B et al.; The Rz lysis gene of bacteriophage lambda was cloned into the expression vectors, pT7-3 and pT7-7 . The recombinant plasmids expressed either a protein of an unexpected 6.5-kDa size (pT7-3H and pSB54) or two proteins of 6.5 and 17.2 kDa (pBH21) . The 6.5-kDa protein alone did not complement the lysis defect of the lambda Rz mutant; hence, this protein was not the Rz gene product . Complementation observed as a result of pBH21 expression thus can be ascribed to the 17.2-kDa protein, which agrees with the size based on the nucleotide sequence of Rz . The 6.5 kDa is a product of an open reading frame entirely encompassed within the Rz sequence and denoted by us Rz1 . Both proteins were detectable only by autoradiography, which may mean that the genes are expressed at low rates . Polyclonal anti-Rz antibodies (Ab) were obtained by rabbit immunization with a synthetic polypeptide corresponding to an antigenic determinant of Rz defined by a computer program . The Ab reacted with the 17.2-kDa protein resulting from pBH21 expression, as well as with the 17.2-kDa protein present in the induced Escherichia coli W3350(lambda cI857Sam7) lysate.

FEBS Lett, 1993 Jul 12, 326(1-3), 39 - 41
Atomic force microscopy of bacteriophage T4 and its tube-baseplate complex; Ikai A et al.; Bacteriophage T4 was imaged by atomic force microscopy with the finest resolution to date with a clear image of tail fibers of an estimated diameter of 2-3 nm . T4 phages were spread on a clean surface of silicon wafer and dried under air before observation with an atomic force microscope . The head, tail and tail fibers were routinely imaged with somewhat distorted dimensions . The ease of imaging isolated phage particles with a good resolution raised our expectation for the further use of AFM in biomedical applications.

J Mol Biol, 1993 Jul 5, 232(1), 89 - 104
Translational repression by the bacteriophage T4 gene 32 protein involves specific recognition of an RNA pseudoknot structure; Shamoo Y et al.; An RNA pseudoknot has been shown to form the 5'-end of bacteriophage T4 gene 32 mRNA that is essential to autoregulation of gene 32 (g32) mRNA by gene product 32 (gp32), a single-stranded nucleic acid binding protein . Structure-mapping of RNA oligonucleotides with structure-specific RNases indicate that two stem regions consisting of nucleotides -67 to -64 base-paired to -52 to -55 (stem-1) and nucleotides -62 to -56 base-paired to -40 to -46 (stem-2) can fold into a "pseudoknotted" structure that may be analogous to the semi-continuous a-helical pseudoknot . Our results suggest that the g32 mRNA pseudoknot can form under conditions where specific autoregulation by gp32 is observed . Although the g32 mRNA pseudoknot is stabilized by Mg2+, it exists in equilibrium with a 3'-hairpin structure . Gel mobility studies carried out with defined length oligonucleotides indicate the gp32 does, in fact, bind tightly to the pseudoknot . These studies agree with the proposal of McPheeters et al., that the pseudoknot represents a nucleation site essential for autogenous gp32 translation regulation . Although disruption of tertiary structure interactions in this pseudoknot (with EDTA) significantly reduces the ability of gp32 to specifically recognize its own mRNA, in vitro mutagenesis studies suggest the sequence of stem-2 and of the loop region (nucleotides -47 to -50) also represent important determinants for specific gp32 autoregulation . Based on a competition assay relying on gel mobility shifts, the order of importance of the major elements of the pseudoknot are stem-1 > sequence of stem-2 or loop-2 > stem-2 . In this assay, disruption of stem-1 decreased the ability of the resulting structure to compete for gp32 binding by approximately fourfold . Both stem-1 and stem-2 appear to be essential to maintain high-level expression from gp32 mRNA in an in vitro transcription/translation system . Taken together, these results support the translation control model in which the pseudoknot region is a nucleation point for cooperative gp32 binding, which then proceeds in a 3' direction through a long stretch of single-stranded RNA that includes the initiation codon for gene 32.

J Mol Biol, 1993 Jul 5, 232(1), 301 - 4
The MotA protein from bacteriophage T4 contains two domains . Preliminary structural analysis by X-ray diffraction and nuclear magnetic resonance; Finnin MS et al.; Controlled protease cleavage experiments and N-terminal sequence analyses were used to show that the transcriptional activator MotA from bacteriophage T4 has a two-domain structure . The N and C-terminal domains have M(r) values of 10,300 and 11,800, respectively, and were separately cloned and overexpressed in Escherichia coli . One and two-dimensional NMR spectroscopy indicate that both domains have stably folded structures and contain extensive secondary structure . The N-terminal domain is substantially alpha-helical, whereas the C-terminal domain has a high content of beta-strand . The N-terminal domain has been crystallized under three different conditions, all with the space group P3(1(2))21 and similar unit cell dimensions . The best crystals are grown from ammonium sulfate, have cell dimensions a = b = 46.7 A, c = 139.6 A, and diffract to beyond 2.4 A . The high quality of the NMR and diffraction data will allow a complete structural analysis of MotA by a combination of these techniques.

J Mol Biol, 1993 Jul 5, 232(1), 105 - 22
Bacteriophage T7 RNA polymerase . 19F-nuclear magnetic resonance observations at 5-fluorouracil-substituted promoter DNA and RNA transcript; Rastinejad F et al.; We have substituted 5-fluorodeoxyuridine (5-FdU) in place of thymidine in defined positions along synthetic bacteriophage T7 promoter DNA sequences . None of the fluoro-substitutions in the promoter DNA sequence reduced transcription yields with T7 RNA polymerase significantly . Substitutions on the coding template strand reduced transcription yields when placed at +3, but not at +4 . 19F-n.m.r . spectra from transcription reactions and gel analysis of transcription products show that T7 RNA polymerase correctly and efficiently utilizes 5-FUTP as a RNA substrate analog . The fluorine atom provides a sensitive probe for monitoring the local environment, base sequence and solvent exposure at the DNA major groove through its 19F-n.m.r . resonance . Buffer dependencies of the fluorine chemical shift and digestion patterns with DNase I suggest that the T7 promoter base-pairs near the transcription start site are distorted with a more open minor groove and less solvent accessible major groove . Previous chemical footprinting data of promoter-polymerase complexes yield a picture that T7 RNA polymerase recognizes major groove features in the region from positions -7 to -11 and minor groove features on the same side of DNA flanking both sides of this region . Consistent with this, 19F-n.m.r . observations identify two additional positions, -8 and -17, involved in promoter recognition on this side of the DNA helix . On the other hand, our observations also implicate the opposite side of the DNA helix, primarily at positions -14 and -15, as major groove recognition sites for T7 RNA polymerase . In addition, n.m.r . spectra from 5-FdU-substituted base-pairs -2 and -3, suggest either additional interactions on the same side of the DNA helix as -14 and -15, or distortions in the DNA structure.

J Mol Biol, 1993 Jul 5, 232(1), 1 - 4
DNA conformation induced by the bacteriophage T4 UvsX protein appears identical to the conformation induced by the Escherichia coli RecA protein; Yu X et al.; The study of homologous genetic recombination has been dominated by the RecA protein of Escherichia coli, which is involved in DNA recombination and repair, as well as phage induction, in vivo . The active form of the RecA protein is a helical filament formed on DNA in the presence of ATP, and within this filament, the DNA is extensively stretched to about 5.1 A rise per base-pair and untwisted to about 19 base-pairs per turn . The bacteriophage T4 UvsX protein is only weakly homologous to RecA, but it has very similar ATP-dependent DNA binding and strand-exchange activities . We can now show that the UvsX protein forms helical filaments that are very similar to those made by RecA, and induces the same extended DNA conformation within these filaments that is induced by RecA . This implies that the unusual conformation of DNA in the RecA filament may be a universal structure in homologous recombination.

J Mol Biol, 1993 Jul 5, 232(1), 35 - 49
Nucleation of RNA chain formation by Escherichia coli DNA-dependent RNA polymerase; Metzger W et al.; We have studied the early steps in RNA synthesis . The kinetic behaviour of the nascent RNA, having chain lengths between 3 and 11 bases, and the transcription fidelity were analysed using the bacteriophage T7 A1 promoter . By moving the stop-inducing base at position +12 in the wild-type template in single base steps upstream, a set of closely related templates was constructed which allowed stalling of the complexes in the registers 11, 10, 9 and 8 . Using this set of templates sigma-factor release was determined . It occurs when RNA synthesis has proceeded to base position +9 . Analysis of the RNA synthesis both with and without heparin yielded the following results: there are three kinds of complexes, (a) the well-known abortively transcribing complex, which is present until the RNA has reached a length of 5 bases, (b) an intermediate complex having RNA chain lengths between 6 and 8 bases, which is stably bound but has high forward as well as back reaction rates, (c) complexes with RNA chains consisting of more than 8 bases, which are stably bound and do not contain the sigma-factor . In general, the likelihood of chain elongation and the stability of the complexes increases with increasing RNA chain length in the early stages of RNA synthesis . Also the transcription fidelity increases correspondingly . Lack of fidelity leads to additional RNA products during the abortive state of transcription . "Read through" of RNA polymerase at stall positions of +8 to +11 also result from misincorporation.

J Biol Chem, 1993 Jul 5, 268(19), 14208 - 14
Molecular cloning and expression of a cDNA encoding a novel isoenzyme of protein kinase C (nPKC) . A new member of the nPKC family expressed in skeletal muscle, megakaryoblastic cells, and platelets; Chang JD et al.; At least seven bacteriophage lambda clones encoding structurally related but unique polypeptides with PKC activity have been isolated from mammalian brain, epidermis, and lung cDNA libraries . The possibility that additional isoenzymes are expressed in human blood platelets or megakaryoblastoid human erythroleukemia cells was examined by polymerase chain reaction amplification of reverse transcribed RNA employing oligonucleotide primers corresponding to conserved peptide sequences . cDNAs encoding a novel PKC-related sequence, designated PKC-theta, and four (alpha, beta, delta, and eta) previously identified isoenzymes were isolated from reverse transcribed total RNA of human erythroleukemia cells and platelets . PKC-theta lacks a conserved region (C2) that is present in the calcium-dependent isoenzymes and therefore belongs to the group of novel, or nPKC, isoenzymes . Significantly increased {3H} phorbol 12,13-dibutyrate binding and cytoskeleton-associated calcium-independent PKC activity were found in COS cells expressing the transfected cDNA . Northern transfer analysis of mRNA from various human tissues revealed high level expression of PKC-theta in skeletal muscle, lung, and brain, and minimal expression in cardiac muscle, placenta, and liver . These findings extend the PKC family and suggest a novel approach to the study of diversity within this pathway of intracellular signal transduction.

Mol Microbiol, 1993 Jul, 9(2), 357 - 64
Generation of isogenic K54 capsule-deficient Escherichia coli strains through TnphoA-mediated gene disruption; Russo TA et al.; Transposon mutagenesis, using IS50L::phoA(Tn-phoA), was performed in a K54/O4/H5 blood isolate of Escherichia coli (CP9), to generate a library of random mutants . Five hundred and twenty-six independent CP9 TnphoA mutants were isolated with active gene fusions to alkaline phosphatase . From this mutant library, eight capsule-deficient strains were detected and were found to have a single copy of TnphoA . Sixteen additional capsule deficient mutants with TnphoA inserts were subsequently obtained that did not possess active PhoA fusions . In conjunction with the initial eight capsule-deficient isolates we have defined genes on three different XbaI fragments as being involved in capsule production . Generalized transduction with the bacteriophage T4 established that these insertions were responsible for the loss of capsule and that they are linked . These capsule-deficient strains can be used to assess the pathogenic role of the K54 capsular polysaccharide.

Mol Microbiol, 1993 Jul, 9(2), 261 - 71
The int genes of bacteriophages P22 and lambda are regulated by different mechanisms; Wulff DL et al.; Bacteriophage P22 and lambda are related bacteriophages with similar gene organizations . In lambda the cll-dependent Pl promoter is responsible for lambda int gene expression . The only apparent counterpart to pl in P22 is oriented in the opposite direction, and cannot transcribe the P22 int gene . We show that this promoter, called P(al), is active both in vivo and in vitro, and is dependent upon the P22 cll-like gene, called c1 . We have also determined the DNA sequence of a 3.3 kb segment that closes the gap between previously reported sequences to give a continuous sequence between the P22 pL promoter and the int gene . The newly determined sequence is densely packed with genes from the pL direction, and the proteins predicted by the sequence show excellent correlation with the proteins mapped by Youderian and Susskind in 1980 . However, the sequence contains no apparent genes in the opposite (p(al)) direction, and no additional binding motifs for the P22 c1 protein . We conclude that int gene expression in P22 is regulated by a different mechanism than in lambda.

Photochem Photobiol, 1993 Jul, 58(1), 1 - 10
Laser cross-linking of proteins to nucleic acids: photodegradation and alternative photoproducts of the bacteriophage T4 gene 32 protein; Kubasek WL et al.; Pulsed laser cross-linking provides a means of introducing a covalent bond between proteins and the nucleic acids to which they are bound . This rapid cross-linking effectively traps the equilibrium that exists at the moment of irradiation and thus allows examination of the protein-nucleic acid interactions that existed . Laser irradiation may also induce photodestruction of protein and we have used the bacteriophage T4 gene 32 protein to investigate this phenomenon . Our results show that both nonspecific and specific photoproducts can occur, specifically at wavelengths where the peptide backbone of proteins is known to absorb . These results demonstrate that nonspecific photodegradation can be correlated with the formation of a specific photodegradation product . The formation of this product was monitored to show that product yield is nonlinearly dependent on laser power and wavelength . We have also investigated an unexpected photoproduct whose formation is dependent on the length of the polynucleotide to which the gene 32 protein binds and that further demonstrates the complexities of analyzing protein-nucleic acid interactions through the use of UV laser cross-linking . These data provide essential information for the establishment of appropriate conditions for future studies that use UV cross-linking of protein-nucleic acid complexes.

Mol Biol (Mosk), 1993 Jul-Aug, 27(4), 798 - 804
{Calorimetric studies of the effect of amino acid replacements 16Gln-Leu and 26Tyr-Asp on the structural organization and stability of the Cro-repressor from phage lambda}; Rogov VV et al.; The scanning calorimetry technique has been used to study the influence of amino acid substitutions on thermodynamic parameters of formation and stabilization of a cooperative structure of Cro repressor of bacteriophage lambda molecule . It is shown that substitutions 16Gln-Leu and 26Tyr-Asp enhances the protein molecule stability by 32 degrees C as compared with the wild-type protein . It is also demonstrated that the denaturation enthalpy of the mutated Cro differs slightly from that of the wild-type protein at the same temperature, while the effective enthalpy value is significantly lower . The analysis of excess heat capacity of the mutated protein shows that this complex function determined experimentally is approximated by two functions, which is indicative of the presence of two quasi-independent transitions . Thus, the stabilization and redistribution of intermolecular interactions evoked by the above substitutions leads to disintegration of the single cooperative repressor molecule in two interacting cooperative domains . The plausible mechanism of formation of such a domain structure is discussed on the basis of the available calorimetric data.

Appl Environ Microbiol, 1993 Jul, 59(7), 2299 - 303
Estimation of ruminal bacteriophage numbers by pulsed-field gel electrophoresis and laser densitometry; Klieve AV et al.; To investigate phage activity in the rumen, a method for quantifying phage has been developed . By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid . Linear double-stranded DNA from this fraction containing predominantly tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE) . Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated . Phage numbers were estimated to be between 3 x 10(9) and 1.6 x 10(10) particles ml of ruminal fluid-1 . The phage population, as gauged by the appearance of DNA on PFGE gels, had two major components . A broad region of DNA between 30 and 200 kb was always present on PFGE gels . It appears this region comprises DNA from a great many different phages and would include most of the temperate phages . In addition, discrete DNA bands ranging in size from 10 to 850 kb were frequently observed . DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originated from a specific phage . It is postulated that the discrete bands are due to epidemics or blooms of phage activity from specific, probably lytic, phages . The method that has been developed will greatly enhance future investigations into the interactions between the ruminal phage population, the ruminal bacterial population, and animal nutrition and growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Jikken Dobutsu, 1993 Jul, 42(3), 337 - 42
{Application of DNA fingerprinting to investigation of genetic relationships between laboratory rabbit strains}; Sudo T et al.; It is well known that laboratory rabbits are not controlled genetically like laboratory mice and rats . In order to test the usefulness of DNA fingerprinting in investigation of genetic uniformity of the laboratory rabbits strains and their relationships, we applied DNA fingerprinting using bacteriophage M13 probe to five strains (2 inbreds (JWY-NIBS and DuY-NIBS) and 3 outbreds (JW-NIBS, Icl:JW and WHHL)) . DNA fingerprints of 2 inbred strains showed the same banding patterns within each strain but the strain-specific patterns . Although there were no rabbits showing the same banding patterns in 3 outbred strains, average percent differences (APD) were 13.7 to 18.6 . A dendrogram based on APD of DNA fingerprints was constructed by 2 large clusters, JW group and DuY . The dendrogram was essentially similar to that based on rabbit mandible measurements . These results suggest that DNA fingerprinting is available not only for the genetic monitoring of the laboratory rabbit strains but also for the investigation of their genetic relationships.

J Bacteriol, 1993 Jul, 175(14), 4354 - 63
Complete nucleotide sequence of the bacteriophage K1F tail gene encoding endo-N-acylneuraminidase (endo-N) and comparison to an endo-N homolog in bacteriophage PK1E; Petter JG et al.; Endo-N-acylneuraminidase (endo-N) is a phage-encoded depolymerase that degrades the alpha (2-8)-linked polysialic acid chains of K1 serotypes of Escherichia coli and vertebrate neural cell adhesion molecules . We have determined the DNA sequence of the bacteriophage K1F tail protein structural gene, which codes for a polypeptide of 920 residues . Purification of the tail protein yields a 102-kDa species upon denaturing gel electrophoresis and detection by Western immunoblot analysis . An identical polypeptide was detected by Western blot analysis of K1F virions . Peptide sequencing confirmed that the open reading frame determined by nucleotide sequencing encodes endo-N . Immunoelectron microscopy with neutralizing antibodies raised against the depolymerase confirmed that endo-N is a component of the K1F tail apparatus . Antibodies in the serum cross-reacted with endo-N from another K1-specific phage, PK1E, demonstrating the presence of shared epitopes . Homology between K1F and PK1E endo-N was confirmed by Southern, Northern (RNA), and Western blot analyses . The endo-N amino-terminal domain is homologous to the amino termini of phage T7 and T3 tail proteins, indicating by analogy that this domain functions in attachment of endo-N to the K1F virion's head . A central domain of 495 residues has weak similarity to sea urchin aryl sulfatase, suggesting that this region may contain the endo-N catalytic site . Failure to detect homology between the PK1E homolog and the carboxy-terminal domain of K1F endo-N is consistent with the central domain's involvement in binding and catalysis of polysialic acid . These results provide the initial molecular and genetic description of polysialic acid depolymerase, which has so far been detected only in K1-specific phage.

Carcinogenesis, 1993 Jul, 14(7), 1297 - 302
The role of prostaglandin H synthase-mediated metabolism in the induction of oxidative DNA damage by BHA metabolites; Schilderman PA et al.; The carcinogenicity of the phenolic food antioxidant butylated hydroxyanisole may be related to its oxidative biotransformation in vivo . In order to determine the ability of BHA, 2-tert-butyl(1,4)hydroquinone (TBHQ) and 2-tert-butyl(1,4)paraquinone (TBQ) to induce oxidative DNA damage, biological inactivation of single-stranded bacteriophage phi X-174 DNA, as well as induction of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in dG by these compounds was studied in vitro, in the presence and absence of peroxidases . Both test systems showed that BHA and TBQ (probably due to lack of reductase activity in vitro) were not capable of inducting oxidative DNA damage . TBHQ, however, appeared to be a strong inactivator of phage DNA as well as a potent inducer of 8-oxodG formation . Addition of radical scavengers showed that this damage was due to formation of superoxide anion, hydrogen peroxide and hydroxyl radicals . Addition of iron chelators and metal ions showed that the one-electron oxidations of TBHQ via the semiquinone radical into TBQ are toxic via the formation of oxygen radicals and are not directly due to the hydroquinone itself or the formation of semiquinone radicals . Although peroxidation of TBHQ by prostaglandin H synthase (PHS) is indicated to result in a superoxide anion burst, this is not accompanied by an increase in oxidative DNA damage in vitro . This might be due to the use of hydrogen peroxide as a substrate by PHS itself, consequently resulting in less formation of hydroxyl radicals . Oxidation of TBHQ by lipoxygenases showed that no semiquinone radicals or oxygen radicals were formed, probably due to a two-electron oxidation of TBHQ directly into TBQ . The present results indicate that metabolic activation of BHA yielding reactive oxygen species may induce a carcinogenic potential, since the BHA metabolite TBHQ, appeared to be a strong inducer of oxidative DNA damage.

J Bacteriol, 1993 Jul, 175(13), 3998 - 4007
Analysis of the structure and subcellular location of filamentous phage pIV; Russel M et al.; The gene IV protein of filamentous bacteriophages is an integral membrane protein required for phage assembly and export . A series of gene IV::phoA fusion, gene IV deletion, and gene IV missense mutations have been isolated and characterized . The alkaline phosphatase activity of the fusion proteins suggests that pIV lacks a cytoplasmic domain . Cell fractionation studies indicate that the carboxy-terminal half of pIV mediates its assembly into the membrane, although there is no single, discrete membrane localization domain . The properties of gene IV missense and deletion mutants, combined with an analysis of the similarities between pIVs from various filamentous phage and related bacterial export-mediating proteins, suggest that the amino-terminal half of pIV consists of a periplasmic substrate-binding domain that confers specificity to the assembly-export system.

Int J Biol Markers, 1993 Jul-Sep, 8(3), 147 - 50
Monoclonal antibodies by genetic engineering: novel reagents for in vivo diagnosis and therapy; Canevari S et al.; In only a few years, the technology of antibody engineering has demonstrated its power and a variety of recombinant monoclonal antibodies are now being developed . Recent developments in gene manipulation have allowed the isolation of antibodies, including human antibodies, with or without immunization, by displaying functional antibody fragments on the surface of bacteriophage particles and directly selecting with antigen . In the present review some recent achievements in these areas are highlighted.

J Struct Biol, 1993 Jul-Aug, 111(1), 9 - 16
4-A projection map of bacteriophage T4 DNA helix-destabilizing protein (gp32*I) crystal by 400-kV electron cryomicroscopy; Soejima T et al.; Ice-embedded crystals of bacteriophage T4 DNA helix-destabilizing protein gp32*I were imaged by computer-controlled spot-scanning on a 400-kV electron cryomicroscope . gp32*I crystals generally have different steps of thickness within a crystal; each step can have different symmetry . Multivariate statistical analysis enabled us to unambiguously select spot-scan images that consist entirely of one motif which were processed subsequently by crystallographic Fourier-averaging techniques . The computed phases of the resulting reflections were evaluated for symmetry in projection, and some of those images were correlated with independent thickness measurements of freeze-dried samples of the same crystals . The structure factors with pgg symmetry from nine spot-scan images were merged, and the mean figure of merit of merged phases was better than 0.9 for data at resolution up to 4 A . A projection map was generated and showed multiple density peaks that corresponded to the high-resolution features of gp32*I.

Turk J Pediatr, 1993 Jul-Sep, 35(3), 221 - 6
Studies of immune response in a patient with selective complete C1q deficiency; Berkel AI; Immune response in a six-year-old girl with a rare complement defect, namely selective complete C1q deficiency, was studied . Her cell-mediated immune response (delayed hypersensitivity skin tests, E, EAC rosettes, in vitro lymphocyte transformation with phytohemagglutinin), isohemagglutinin titers, serum immunoglobulin levels, antibody titers to tetanus, Epstein-Barr virus, keyhole limpet hemocyanin, pneumococcus and bacteriophage 0 x 174 were all normal . However, she had abnormal kinetics of the antibody response to bacteriophage following primary and secondary immunizations . Her antibody titers reached a peak four weeks after primary immunization and three weeks after secondary immunization, while the titers of controls peaked at two weeks and one week, respectively . In this study we could not prove the hypothesis that defective antibody response caused recurrent infections in this patient . An alternative hypothesis is the possible role of defective clearance of immune complexes in the development of severe infections . Patients with selective complete C1q deficiency form immune complexes with bacterial or viral antigens, thus activating the classical complement pathway . As a result, very low C1q levels decrease even further, leading to a severe immunodeficiency and recurrent infections . Future studies in this area may help to explain the mechanism(s) responsible for infections in this rare type of complement defect.

J Clin Microbiol, 1993 Jul, 31(7), 1700 - 3
Use of digoxigenin-labelled oligonucleotide DNA probes for VT2 and VT2 human variant genes to differentiate Vero cytotoxin-producing Escherichia coli strains of serogroup O157; Thomas A et al.; Digoxigenin-labelled oligonucleotide DNA probes specific for B-subunit genes of Vero cytotoxin 2 (VT2) and a variant of VT2 (VT2vha) were used to differentiate 116 strains of Escherichia coli serogroup O157 belonging to phage types 1, 2, 4, 8, 14, and 49 . Of these strains, 38% had sequences for both VT2 and VT2vha, 38% had sequences for VT2 only, and 24% had sequences for VT2vha only . Oligonucleotide probe hybridization subdivided strains of all of the phage types except phage type 1 . The greatest variation in toxin gene pattern was observed with strains of phage type 14, for which there were six distinct patterns when the presence or absence of VT1 genes was also considered . Two strains from each phage type group were examined for bacteriophages encoding VT production . Two of the six VT2vha-producing strains carried phage from which DNA hybridized with the VT2vha-specific probe . Phages were not detected in the remaining four VT2vha strains, suggesting that genes may be chromosomally located or associated with a defective prophage . In contrast, seven of the eight VT2 strains carried phages from which DNA hybridized with the VT2-specific probe . Two strains of E32511 (O157:H-) were also investigated . One strain (E32511A) possessed gene sequences for both VT2 and VTvha and was shown to carry phage possessing gene sequences for VT2 . With strain E32511B, however, phages were not detected and DNA hybridized only with the VT2vha probe . Analysis of total genomic DNA digested with restriction endonuclease EcoRI showed that polymorphisms were seen with VT2 strains and not with VT2vha strains.

J Biol Chem, 1993 Jun 25, 268(18), 13462 - 71
Effect of reaction pH on the fidelity and processivity of exonuclease-deficient Klenow polymerase; Eckert KA et al.; We have examined as a function of pH the fidelity of DNA synthesis catalyzed by the 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I . Increasing the pH of in vitro gap-filling reactions from pH 6.2 through 9.8 (37 degrees C increased the frequency of base substitution and minus-one-base frameshift mutations 50- and 40-fold, respectively, as measured by reversion of a nonsense or frameshift mutation within the lacZ alpha gene of bacteriophage M13mp2 . To understand the mechanisms of high fidelity at low pH, we have examined the biochemical events associated with DNA synthesis at pH 6.2 that might be responsible for the observed accuracy in vitro . We show that while the steady-state frequency of T.dGTP misinsertion at the lacZ alpha opal codon is 20-fold lower at pH 6.2 than at pH 7.6, pH-dependent changes in the frequencies of G-dATP and A-dCTP base misinsertions at the lacZ alpha nonsense codon are insufficient to explain the fidelity changes observed in the gap-filling assay . However, the efficiency of steady-state extension synthesis from template-primers containing 3'-terminal T.G, G.A, and A.C (template-primer) mispairs was reduced up to 160-fold at pH 6.2 relative to pH 7.6 . Analyses of the processivity of DNA polymerization versus pH demonstrated that at low pH the termination probability was decreased at specific template positions . Concomitantly, at sites where the termination probability was lower at pH 6.2, a decreased error rate was observed for base substitution mutations at three template positions and for minus-one-base frameshift mutations at two homopolymeric sequences relative to pH 7.6 . We suggest that the observed increase in error discrimination by the exonuclease-deficient Klenow polymerase results from altered template binding properties of the enzyme at pH 6.2.

Nucleic Acids Res, 1993 Jun 25, 21(12), 2867 - 72
Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes; Gao X et al.; Expression of bacteriophage T7 RNA polymerase in mammalian cells can efficiently drive the transcription of a foreign gene controlled by the T7 promoter (Elroy-Stein et al., Proc . Natl . Acad . Sci . USA . 86, 6126-6130, 1989) . We have tested the hypothesis that purified T7 RNA polymerase can be co-delivered into mammalian cells together with a reporter gene (chloramphenicol acetyltransferase, CAT) controlled by the T7 promoter (pT7-EMC-CAT) using DC-chol cationic liposomes . Indeed, significant level of CAT activity was observed in human lung adenocarcinoma (A549-1) cells which had been incubated with a complex of T7 RNA polymerase, pT7-EMC-CAT DNA and DC-chol cationic liposomes . The expression was specific in that T3 RNA polymerase could not replace the T7 RNA polymerase, and that co-delivered T7 RNA polymerase did not enhance the expression of a CAT gene controlled by the SV40 early promoter . The system was optimized in terms of enzyme, DNA and liposome concentrations . Time course experiment indicated that the expression of the T7 system was about 8-10 hours sooner than the SV40 system, consistent with the notion that T7 RNA polymerase does not enter into the nucleus and the transcription takes place in the cytoplasm of the transfected cells . The expression of the T7 system was transient; it declined after 30 hours post transfection, probably due to turnover of the phage enzyme in the mammalian cells . The expression system described here should be useful for gene transfer experiments which require a fast but transient expression of a foreign gene . We have also compared our delivery system with a commercial reagent, Lipofectin, which has been used to deliver T3 or T7 RNA polymerase with a reporter plasmid encoding the T3 or T7 promoter.

Biochemistry, 1993 Jun 22, 32(24), 6179 - 89
The primary self-assembly reaction of bacteriophage lambda cI repressor dimers is to octamer; Senear DF et al.; Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage . It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites . As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution . Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan {Ross et al . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 12023-12027} and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied . We report here that the primary assembly mode of all four proteins is dimer to octamer . It is not dimer to tetramer as previously assumed . While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution . Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape . This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL . Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions . Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer {Laue et al . (1993) Biochemistry 32, 2469-2472} . Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.

J Mol Biol, 1993 Jun 20, 231(4), 999 - 1008
Reversible pressure dissociation of R17 bacteriophage . The physical individuality of virus particles; Da Poian AT et al.; In the absence of urea, pressures up to 2.5 kbar promote only 10% dissociation of the whole particles of R17 bacteriophage . In the presence of concentrations of urea between 1.0 and 5.0 M, pressure promotes complete, reversible dissociation of the virus particles . At the lower urea concentrations reversible dissociation of R17 virus particles shows no dependence on protein concentration indicating a high degree of heterogeneity of the particles, but higher urea concentrations, 2.5 to 5.0 M, result in progressive restoration of the protein concentration dependence of the pressure dissociation . At still higher urea concentrations, 5.0 to 8.0 M, irreversible dissociation of virus takes place at atmospheric pressure . In contrast, the dissociation of the isolated dimers of the capsid protein was dependent on protein concentration to the extent predicted for a stochastic equilibrium, and dimers were much less stable than the whole virus both to dissociation by pressure or urea . In contradistinction, the reversible whole-virus dissociation observed at urea concentrations below 2.5 M appears to be a typical deterministic equilibrium, without appreciable dynamic exchange between whole particle and subunits during the lengthy experiments . The experiments demonstrate that the "thermodynamic individuality" of the virus particles arises in conformational differences in the assembled viruses, and that there is a direct relation between the stability of the particles and their heterogeneity.

Cell, 1993 Jun 18, 73(6), 1155 - 64
Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting; Gu H et al.; We have employed a method based on the Cre-loxP recombination system of bacteriophage P1 to generate a mouse strain in which the JH segments and the intron enhancer in the IgH locus are deleted . By analysis of immunoglobulin isotype switch recombination in heterozygous mutant B cells activated by lipopolysaccharide plus interleukin-4, we show that, on the mutant chromosome, switch recombination at the mu gene switch region is strongly suppressed, whereas the switch region of the gamma 1 gene is efficiently rearranged . These data demonstrate an independent control of switch recombination at individual switch regions and suggest that, in the process of switch recombination, the alignment of the recombining strands occurs independently of and probably after the introduction of double-strand breaks into the switch regions involved.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5509 - 13
The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes?
Lerbs-Mache S.
Highly purified RNA polymerase preparations from spinach chloroplasts contain seven major polypeptides of 150, 145, 110, 102, 80, 75, and 38 kDa . I find that RNA polymerase activity can be separated under defined conditions into three different fractions by heparin-Sepharose chromatography . Immunological analysis has shown that the first fraction contains RNA polymerase activity associated with all seven major polypeptides, and other studies have shown that some of these polypeptides (150, 145, 80, and 38 kDa) are associated with an RNA polymerase similar to the Escherichia coli enzyme . However, similar analyses of the remaining fractions show activity associated only with the 110-kDa polypeptide, suggesting the existence of a second kind of chloroplast RNA polymerase . Samples of this 110-kDa polypeptide purified by SDS/PAGE actively synthesize RNA in a reaction dependent on a supercoiled DNA template and the four ribonucleoside triphosphates . Hence, this polypeptide has all of the properties expected of a single-subunit RNA polymerase of the T7 bacteriophage type.

J Biol Chem, 1993 Jun 15, 268(17), 12970 - 6
Primase from Escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins . Template sequence requirements based on the bacteriophage G4 complementary strand origin and Okazaki fragment initiation sites; Swart JR et al.; We find that neither the hairpins nor bound single-stranded DNA-binding protein (SSB) are required for in vitro primase activity at the G4 complementary strand origin . Primase has the ability to prime synthetic DNA templates in the absence of any auxiliary proteins, including SSB, and in the absence of any DNA secondary structure . primase does, however, initiate primer synthesis starting at a specific nucleotide sequence present in both the G4 origin system and at sites of Okazaki fragment initiation . We tested a series of single-stranded DNA templates that consisted of various portions of the G4 complementary strand origin for the ability to support primer synthesis . Primer synthesis activity was determined using an assay in which primer length and quantity are monitored by the incorporation of {gamma-32P}ATP once per primer and the products analyzed on a denaturing polyacrylamide gel . We find that primase requires only a short DNA template, 5'-(AC)7CTG CAA AGC-3', to synthesize a 17-nucleotide primer starting at the thymidine residue . We also report primase's ability to incorporate dideoxyribonucleotide triphosphates into primers, which has allowed us to determine directly both length and sequence of primers synthesized.

J Biol Chem, 1993 Jun 15, 268(17), 12736 - 43
Phage and Escherichia coli expression of the human high affinity immunoglobulin E receptor alpha-subunit ectodomain . Domain localization of the IgE-binding site; Robertson MW; The high affinity IgE receptor is a multisubunit complex that participates in IgE-dependent activation of mast cells and basophils . The IgE-binding portion of the receptor resides exclusively in the alpha-subunit and specifically within the 180 residues of the mature extracytoplasmic portion . In this study the contiguous two-domain human alpha-subunit has been displayed on the surface of a filamentous bacteriophage in a form that specifically binds both human and murine IgE but not other isotypes . Phage display of the individual immunoglobulin-like domains reveals that the IgE-binding portion resides exclusively in the COOH-terminal domain and that this domain appears to bind IgE with lower affinity than the comparably displayed two-domain fragment . Phage display results using a chimeric rat-human alpha-subunit fragment suggest that structural elements within the NH2-terminal domain are necessary to impart high affinity IgE binding activity to the alpha-chain ectodomain . The two-domain human receptor fragment was also expressed in a soluble form from Escherichia coli and purified in one step by affinity chromatography . The solution binding of the purified receptor fragment to IgE was measured by fluorescence quench which afforded an approximate equilibrium affinity constant of 2 x 10(9) M-1 together with a stoichiometry of 1 receptor molecule/molecule of IgE . The complementary approach of phage display and E . coli expression used in this study represents a useful strategy for further analysis of discrete receptor epitope(s) that contribute to IgE binding activity.

Gene, 1993 Jun 15, 128(1), 37 - 42
A ribonuclease S-peptide antagonist discovered with a bacteriophage display library; Smith GP et al.; From a filamentous phage library displaying random hexapeptides, we selected clones displaying peptides that bind S-protein, a 104-amino-acid (aa) fragment of bovine pancreatic ribonuclease (RNase) . The selected peptides show a sequence motif, (F/Y)NF(E/V)(I/V)(L/V), that bears little resemblance to S-peptide, a 20-aa fragment of RNase that is S-protein's natural ligand . One of the displayed peptides, YNFEVL, was synthesized chemically and shown by isothermal titration calorimetry to bind S-protein with a dissociation equilibrium constant of 5.5 microM at 25 degrees C, an affinity comparable to that of previously studied S-peptide variants . The YNFEVL peptide is an antagonist of S-peptide, in that it blocks the ability of S-peptide to restore enzyme activity to S-protein . The S-protein/S-peptide system preserves the essential features of a pharmacologically significant receptor/hormone couple, and the S-peptide antagonist can therefore be regarded as a new RNase-specific 'drug' . This work illustrates the potential value of phage display libraries for discovering novel classes of pharmaceuticals.

Gene, 1993 Jun 15, 128(1), 29 - 36
M13 bacteriophage displaying disulfide-constrained microproteins; McLafferty MA et al.; A display-phage library (TN2), displaying an 18-residue peptide fused to coat protein III, represents a collection of up to 8.55 x 10(6) peptides encoded by only 1.68 x 10(7) DNA sequences . Each displayed peptide has two fixed cysteine residues (allowing disulfide formation) and six variegated residues, four between the cysteines and one either side of the cysteines . Screening this library against streptavidin (Sv) and the anti-beta-endorphin monoclonal antibody, 3-E7, yielded phage displaying disulfide-constrained microproteins with sequences similar to those published for the linear-peptide display phage . Analysis of selected clones indicated that a disulfide bond is required for high-affinity binding to each of the target proteins . The microproteins selected for binding to Sv and 3-E7 show more stringent sequence specificity than do linear peptides selected for binding to the same targets.

Gene, 1993 Jun 15, 128(1), 129 - 34
Trypsin display on the surface of bacteriophage; Corey DR et al.; The gene III and VIII-encoded coat proteins (pIII and pVIII) from bacteriophage M13 have been fused to the C terminus of the serine protease, trypsin (Tsn) . The genes encoding the fusions were then inserted directly into M13mp18 to create vectors which expressed both the Tsn-coat protein hybrids and the wild-type (wt) coat proteins . Immunoblot analysis confirmed that the bacteriophage express Tsn on their surface . Isolated fusion phage possess kinetic parameters which approximate those of the wt enzyme . An endogenous Escherichia coli protease inhibitor, ecotin, copurifies with the Tsn phage . Immobilized ecotin can be used to selectively bind bacteriophage which express Tsn::pIII fusion proteins.

Gene, 1993 Jun 15, 128(1), 119 - 26
A bacteriophage lambda vector for the cloning and expression of immunoglobulin Fab fragments on the surface of filamentous phage; Hogrefe HH et al.; We have combined the high cloning efficiency of the lambda bacteriophage vectors with the surface expression screening method for the display of combinatorial antibody fragment (Fab) libraries on the surface of filamentous phage particles . The utility of the herein described ImmunoZAP 13 system for the isolation of Fabs that specifically bind antigen is demonstrated using two phagemid display libraries prepared from a previously characterized human combinatorial library . The percentage of clones that specifically bind antigen is maintained throughout the process of subcloning the LC and VH genes into ImmunoZAP 13, in vivo mass excision to convert the lambda library to a phagemid library, and preparation of phagemid particles displaying Fabs . Specific phagemid were isolated from libraries containing 0.6% and 0.03% tetanus toxoid (TT)-binding clones after two and three rounds of biopanning, respectively . Relative binding curves determined on a small sample of isolated clones indicate that several unique immunoglobulin Fab fragments have been isolated.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5414 - 7
A site-specific endonuclease encoded by a typical archaeal intron; Dalgaard JZ et al.; The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene . This enzyme, I-Dmo I, is unusual among the intron endonucleases in that it is thermostable and is expressed only from linear and cyclized intron species and not from the precursor RNA . However, in analogy to its eukaryotic counterparts, but unlike the bacteriophage enzymes, I-Dmo I makes a staggered double-strand cut that generates 4-nt 3' extensions . Additionally, although the archaeal and group I introns have entirely different structural properties and splicing pathways, I-Dmo I shares sequence similarity, in the form of the LAGLI-DADG motif, with group I intron endonucleases of eukaryotes . These observations support the independent evolutionary origin of endonucleases and intron core elements and are consistent with the invasive potential of endonuclease genes.

Gene, 1993 Jun 15, 128(1), 103 - 9
Selection of an anti-IGF-1 Fab from a Fab phage library created by mutagenesis of multiple CDR loops; Garrard LJ et al.; Diverse Fab libraries containing 2-3 x 10(8) members were generated by randomizing amino acid residues within four of the six complementarity determining regions of a humanized version of an anti-HER-2 Ab (hu4D5) . These libraries were subsequently displayed on the surface of the filamentous bacteriophage M13 and selected for binding to three proteins: CD4, insulin-like growth factor 1 (IGF-1), and tissue plasminogen activator . An Fab-bacteriophage was isolated that showed specific binding to IGF-1 . The affinity of this Fab was determined to be 3.5 microM.

Gene, 1993 Jun 15, 128(1), 85 - 8
Design of specific immunogens using filamentous phage as the carrier; Minenkova OO et al.; Earlier, we developed an expression vector allowing exposure of short peptides on the surface of bacteriophage M13 . It was used to obtain a recombinant phage carrying an antigenic determinant of HIV1 p17 Gag protein . Immunoglobulin elicited by immunizing rabbits with the phage reacted with the 17-kDa core protein of the virus and with its polyprotein precursor, p55, on Western blots of HIV1 viral proteins . The results of present experiments may be useful in vaccine development.

Gene, 1993 Jun 15, 128(1), 79 - 83
Immunological properties of foreign peptides in multiple display on a filamentous bacteriophage; Willis AE et al.; The genome of bacteriophage fd has been engineered to permit construction of hybrid virus particles in which the wild-type major coat protein (gpVIII) subunits were interspersed with coat proteins displaying one or other of two foreign peptides (fdMAL1, sequence NANPNANPNANP or fdMAL2, sequence NDDSYIPSAEKI) in the exposed N-terminal segments {Greenwood et al., J . Mol . Biol . 220 (1991) 821-827} . These sequences represent major antigenic determinants of the circumsporozoite protein of the malaria parasite, Plasmodium falciparum . The peptide epitopes in the hybrid bacteriophages were found to be strongly immunogenic in four different strains of mice without the use of external adjuvants, and the antibodies (Ab) were highly specific to the individual epitopes in ELISA assays . When tested in nude (nu/nu) and heterozygote (nu +/-) BALB/c mice, the immune response was found to be T-cell dependent and to undergo class-switching from IgM to IgG . Proliferation assays of T-cells taken from lymph nodes of BALB/c mice injected with bacteriophage particles in the presence or absence of Freund's complete adjuvant indicated no difference in the immune response . This way of generating Ab against peptide epitopes is simpler and much less expensive than the conventional method of peptide synthesis and coupling to a carrier protein for injection . The specificity of the immune response, the ability to recruit helper T-cells and the lack of need for external adjuvants suggest that it will also be an inexpensive and simple route to the production of effective vaccines.

Gene, 1993 Jun 15, 128(1), 51 - 7
Mimicking of discontinuous epitopes by phage-displayed peptides, I . Epitope mapping of human H ferritin using a phage library of constrained peptides; Luzzago A et al.; We have constructed a random nonapeptide library in the N-terminal region of the major coat protein VIII of bacteriophage f1, with two cysteines flanking the insert, and preliminary data suggest that many of the clones display at least some of their peptides in cyclized form . This library was used to select oligopeptides binding to the monoclonal antibody (mAb) H107, recognising the assembled native conformation of recombinant human H-subunit ferritin (H Fer), whose three-dimensional structure is known . Comparison of the selected oligopeptides with one another allowed us to derive two consensus sequences characterized by conserved amino acid (aa) residues . Analysis of the distribution of the aa side chains exposed on the surface of H Fer reveals that most of the aa defining both consensus sequences are present either at the end of the big loop or at the end of the A helix . These two regions of the H Fer, though separated in the linear sequence, are very close in the folded molecule . Interestingly, each consensus sequence derived from the selected phage-displayed peptides is characterized by aa present both at the end of the big loop and at the end of the A helix . These two H Fer regions are good candidates for mimicry by the selected peptides and therefore for constituting part of the H107 epitope . To provide support to this hypothesis, we constructed several H Fer mutants carrying point mutations in different positions of these two regions.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1993 Jun 5, 231(3), 581 - 93
Coupling of rRNA transcription and ribosomal assembly in vivo . Formation of active ribosomal subunits in Escherichia coli requires transcription of rRNA genes by host RNA polymerase which cannot be replaced by bacteriophage T7 RNA polymerase; Lewicki BT et al.; Transcription of a plasmid-located rrnB operon and the corresponding formation of ribosomes in vivo were studied using either T7 RNA polymerase or host RNA polymerase as transcriptase . The 23 S rRNA gene on the plasmid carried an A1067-->T mutation, which confers resistance against the drug thiostrepton . The proportion of particles containing plasmid-borne 23 S rRNA versus chromosome-borne rRNA was quantified with a precision of better than 10% by scanning sequence autoradiograms around nucleotide 1067 . The activity of these particles was determined in the presence of thiostrepton which exclusively abolishes the activity of chromosomal wild-type ribosomes . When the plasmid rrnB operon was transcribed with phage T7 RNA polymerase, up to 80% of the rRNA synthesis was plasmid-directed (pulse labelling) in the late induction phase, most of which (about 85%) became degraded . The cells accumulated 50 S particles with plasmid-borne intact rRNA that was hardly found in 70 S ribosomes, i.e . particles harbouring plasmid-borne rRNA did not enter the pool of active ribosomes . The particles with plasmid-derived rRNAs were also practically inactive in protein synthesis in vitro . However, the rRNA was functional as shown by reconstitution analysis . The same patterns were found at various expression levels of the plasmid rrnB operon, indicating that not the overproduction of rRNA but rather the T7 transcriptase was responsible for the observed effects . However, when the plasmid rrnB operon was transcribed with host RNA polymerase, growth was not affected upon induction, the 30 S to 50 S to 70 S ratios in the cell were not altered, both 50 S subunits and 70 S ribosomes contained large amounts of plasmid-borne rRNA, and the particles with plasmid-derived rRNA were active in vitro . When the induction of rRNA transcription by T7 RNA polymerase was performed at 25 degrees C instead of 37 degrees C, an almost normal pattern was observed . Inactive 50 S particles did not accumulate, and large amounts of plasmid-borne rRNA were found in the pool of 70 S ribosomes . Lowering the induction temperature reduces the transcription rate by T7 RNA polymerase, which is five times faster at 37 degrees C than the host polymerase . The results suggest that the formation of active ribosomal subunits in vivo requires a fine adaptation of the transcription rate of rRNAs and the assembly process, underlining the importance of a coupling between rRNA transcription and ribosome assembly in vivo . T7 RNA polymerase cannot replace the host RNA polymerase in this process at 37 degrees C.

J Bacteriol, 1993 Jun, 175(12), 3909 - 12
Phi X174 E complements lambda S and R dysfunction for host cell lysis; Roof WD et al.; Hybrid lambda phages which have the E lysis gene of the bacteriophage phi X174 in cis to defective nonsense and deletion alleles of the normal lambda lysis genes S and R have been constructed and shown to be fully competent for plaque-forming ability, which demonstrates that the single-gene, lysozyme-independent lysis system of phi X174 and related phages can serve the lytic function for large complex phages . These hybrid phages are unable to form plaques on a slyD host . Moreover, plaque morphology indicates that in E-mediated lysis the soluble lambda R endolysin can participate in lysis, indicating that the protein E-mediated lesions are not completely sealed off from the periplasm.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5262 - 6
Identification of the gene associated with the recurring chromosomal translocations t(3;14)(q27;q32) and t(3;22)(q27;q11) in B-cell lymphomas; Baron BW et al.; Chromosomal translocations involving chromosome 3, band q27, are among the most common rearrangements in B-cell non-Hodgkin lymphoma . From a bacteriophage lambda library prepared from a lymphoma characterized by a t(3;14)(q27;q32), genomic clones were isolated using a probe from the immunoglobulin heavy chain locus (IGH) joining region . In addition to clones containing an apparently normal IGH rearrangement, others were found to contain one of the translocation breakpoint junctions . Normal chromosome 3 sequences and the reciprocal breakpoint junction were subsequently isolated . DNA probes on each side of the chromosome 3 breakpoint hybridized at high stringency to the DNA of various mammalian species, demonstrating evolutionary conservation . One such probe from the presumptive der(3) chromosome detected an 11-kilobase transcript when hybridized to RNA of B- and T-cell lines . A probe made from partial cDNA clones isolated from a T-cell line hybridized with genomic DNA from both sides of the chromosome 3 breakpoint, indicating that the t(3;14) is associated with a break within the gene on chromosome 3 . In situ chromosomal hybridization revealed that the same gene is involved in the t(3;22)(q27;q11) . Preliminary nucleotide sequencing shows no identity of the cDNA to gene sequences in available data banks . We propose the name BCL6 (B-cell lymphoma 6) for this gene, since it is likely to play a role in the pathogenesis of certain B-cell lymphomas.

Virology, 1993 Jun, 194(2), 682 - 7
Capsid localization of the bacteriophage P4 Psu protein; Dokland T et al.; In addition to its polarity-suppressing activity, the Psu protein of bacteriophage P4 also serves to stabilize the capsid against heat treatment and binds externally to the phage capsid . However, the heat stability is lost upon purification of the virus, indicating a loss of Psu protein from the capsid . By using three-dimensional reconstruction from cryo-electron micrographs of P4 psu1 amber mutants lacking Psu, and of P4 virions, which have been saturated with Psu protein to regain heat stability, we have determined the position of this protein on the virus surface . Our results are consistent with the hypothesis that the function of Psu is to stabilize the hexameric capsomer assembly.

Virology, 1993 Jun, 194(2), 674 - 81
Characterization of the capsid associating activity of bacteriophage P4's Psu protein; Isaksen ML et al.; The Psu (Polarity suppression) protein of satellite bacteriophage P4 was first characterized as an anti-terminator of transcription termination in Escherichia coli . Psu is also a structural component of mature P4 capsids, where it is present as a decoration protein . Psu is located externally on the capsid surface, and it appears to protect the capsid from loss of DNA through the capsid shell . The ability of Psu to specifically bind to the P4 capsid appears not to be dependent on any P4 specific components such as the capsid protein cleavage products h1 and h2, or P4 DNA . We suggest that Psu binds to the P4 capsid as a result of the special structure of the hexamers in the P4 capsid.

Virology, 1993 Jun, 194(2), 570 - 5
Bacteriophage PRD1 proteins: cross-linking and scanning transmission electron microscopy analysis; Luo C et al.; Bacteriophage PRD1, a double-stranded DNA virus infecting Escherichia coli, has a membrane inside the protein capsid . Chemical cross-linking and scanning transmission electron microscopy showed that the multimeric major coat protein (P3) exists in a trimeric form . Cross-linking revealed, in addition, that protein P11, located between the protein coat and the membrane, exists also as a homotrimer . Minor protein P7 was associated with the major coat protein P3 . Under nonreducing conditions the infectivity proteins P16 and P18 formed homomultimeric complexes which were dissociated upon addition of 2-mercaptoethanol.

Virology, 1993 Jun, 194(2), 481 - 90
Protein folding studies in vivo with a bacteriophage T4 expression-packaging-processing vector that delivers encapsidated fusion proteins into bacteria; Hong YR et al.; A cloned phage T4 gene which expresses the nonessential capsid scaffold protein IPIII was modified to permit construction and packaging of protein fusions within the capsid . IPIII deletion phage packaged IPIII-beta-galactosidase, IPIII-beta-globin, and IPIII-beta-globin-beta-galactosidase fusion proteins; the latter protein fusion was specifically processed by the T4 gene 21 head morphogenetic proteinase in vivo at a consensus leu(ile)-P1-glu* cleavage site to regenerate beta-galactosidase . Phage inject IPIII-beta-galactosidase protein into bacteria, but less activity is recovered in infections of Escherichia coli dnaK or groEL mutants, suggesting that these host molecular chaperones are required for beta-galactosidase intracellular folding . This expression-packaging-processing (EPP) vector directs protein fusions into capsids for easy detection and purification and permits study of protein delivery and folding in bacteria.

J Bacteriol, 1993 Jun, 175(11), 3443 - 51
Fine-structure analysis of the P7 plasmid partition site; Hayes F et al.; The par region of bacteriophage P7 is responsible for active partition of the P7 plasmid prophage into daughter cells . The cis-acting partition site was defined precisely as a 75-bp sequence that was necessary and sufficient to promote correct segregation of an unstable vector plasmid when the two P7 partition proteins, ParA and ParB, were supplied in trans . Roughly the same region was necessary to exert partition-mediated incompatibility . The minimal site contains an integration host factor (IHF) protein binding site bracketed by regions containing heptamer repeat sequences that individually bind ParB . An additional sequence forms the left boundary of the site . Site-directed mutations in the latter sequence, as well as the IHF motif and the rightmost ParB box, blocked site function . Although the P7 site shares 55% sequence identity with its counterpart in bacteriophage P1, functional interactions between the partition sites and the Par proteins of the two plasmids were entirely species specific in vivo . The P1 sequence has similar IHF and ParB binding motifs, but the left boundary sequence differs radically and may define a point of species-specific contact with the Par proteins . No evidence was found for the existence of a functional P7 analog of the P1 parS core, a small subregion of the P1 site that, in isolation, acts as an enfeebled partition site with modified incompatibility properties.

Clin Immunol Immunopathol, 1993 Jun, 67(3 Pt 2), S33 - 40
Regulation of antibody responses: the role of complement and adhesion molecules; Ochs HD et al.; To analyze the importance of cell surface-associated molecules in modulating the immune response by facilitating T/B cell interaction, we used the T cell-dependent antigen, bacteriophage phi X174 . Taking advantage of "experiments of nature", we studied specific antibody synthesis in patients with deficiencies of complement components or of the adhesion molecule CD11/CD18 (leukocyte adhesion defect, LAD) and guinea pigs and dogs with early complement component deficiency . Following intravenous injection of bacteriophage phi X174 into normal subjects or animals, a primary response consisting of IgM, a secondary response consisting of IgM and IgG, and a tertiary, predominantly IgG response can be distinguished . Patients and guinea pigs deficient of early complement component and LAD patients responded to repeated phage immunization with depressed antibody titers, lack of or inadequate amplification, and failure to switch from IgM to IgG, suggesting a defect in generating antigen-specific memory cells . Several mechanisms have to be considered: (i) The complement portion of the antigen-antibody complement complex facilitates the accumulation and trapping of antigen in lymphoid organs, thus improving the response to Ag at low concentrations . (ii) Immune complexes preferentially bind to antigen-specific B cells, cells expressing Fc receptors, or CR2 and CR3, the receptors for C3bi . (iii) The weak binding established between the MHC-II/Ag complex and the TCR complex is strengthened through the binding of several adhesion molecule pairs . (iv) Receptor-ligand binding initiates activation signals . The concept of binding/signaling via interacting molecules is further supported by the observation that mAb 60.3, recognizing the beta chain of CD11/CD18, blocks in vitro synthesis of antibody to bacteriophage by primed PBMC.

J Virol, 1993 Jun, 67(6), 3441 - 5
Juxtaposition between activation and basic domains of human immunodeficiency virus type 1 Tat is required for optimal interactions between Tat and TAR; Luo Y et al.; trans activation of the human immunodeficiency virus type 1 long terminal repeat requires that the viral trans activator Tat interact with the trans-acting responsive region (TAR) RNA . Although the N-terminal 47 amino acids represent an independent activation domain that functions via heterologous nucleic acid-binding proteins, sequences of Tat that are required for interactions between Tat and TAR in cells have not been defined . Although in vitro binding studies suggested that the nine basic amino acids from positions 48 to 57 in Tat bind efficiently to the 5' bulge in the TAR RNA stem-loop, by creating several mutants of Tat and new hybrid proteins between Tat and the coat protein of bacteriophage R17, we determined that this arginine-rich domain is not sufficient for interactions between Tat and TAR in vivo . Rather, the activation domain is also required and must be juxtaposed to the basic domain . Thus, in vitro TAR RNA binding does not translate to function in vivo, which suggests that other proteins are important for specific and productive interactions between Tat and TAR.

J Virol, 1993 Jun, 67(6), 3332 - 7
Interchangeability of the adsorption proteins of bacteriophages Ff and IKe; Endemann H et al.; The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and f1) phage particles . Deletion mutants of the protein, however, are assembled into Ff phage particles . These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function . This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it . Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.

FASEB J, 1993 Jun, 7(9), 760 - 7
Site-specific genetic recombination: hops, flips, and flops; Sadowski PD; Genetic recombination plays a key role in the life of organisms as diverse as bacteriophages and humans . Contrary to our idea that chromosomes are stable structures, studies of recombination over the past few decades have shown that in fact DNA replicons are remarkably plastic, undergoing frequent recombination-induced rearrangements . This review summarizes our recent knowledge of the biochemistry of the two major classes of site-specific recombination: 1) transpositional recombination, and 2) conservative site-specific recombination.

Mol Gen Genet, 1993 Jun, 239(3), 393 - 9
Transactivation of a plasmid-borne bacteriophage T4 late gene; Noguchi T et al.; We examined how a plasmid-borne T4 late gene is activated by infecting T4 phage (transactivation) . A gene fusion system was developed where expression of a late gene promoter fused to the lacZ gene may easily be followed by measuring beta-galactosidase activity . Considerable transactivation can occur, provided that the infecting phage contains a mutation which abolishes the denB-encoded endonuclease, and that the gene 46-encoded exonuclease is functional . The level of transactivation was correlated with the formation of high molecular weight DNA composed of tandem repeats of plasmid DNA . The formation of these molecules and subsequent transactivation depended on DNA replication and homology between phage and plasmid DNAs . Also the capacity of bacteriophage T4, grown on cells containing a plasmid-borne T4 gene, to transduce the plasmid provided indirect evidence of the formation of these tandem-repeat molecules . A good correlation was established between the ability to transduce and the presence of sequence homology between the phage and the plasmid . However, the requirement for phage/plasmid homology is no longer prerequisite if transcription from the plasmid is permitted by introducing an alc mutation into the infecting phage, presumably because this allows DNA replication to start through RNA priming.

Biophys J, 1993 Jun, 64(6), 1861 - 8
Analysis of 31P nuclear magnetic resonance lineshapes and transversal relaxation of bacteriophage M13 and tobacco mosaic virus; Magusin PC et al.; The experimentally observed 31P lineshapes and transversal relaxation of 15% (wt/wt) M13, 30% M13, and 30% tobacco mosaic virus (TMV) are compared with lineshapes and relaxation curves that are simulated for various types of rotational diffusion using the models discussed previously (Magusin, P . C . M . M., and M . A . Hemminga . 1993 . Biophys . J . 64:1851-1860) . It is found that isotropic diffusion cannot explain the observed lineshape effects . A rigid rod diffusion model is only successful in describing the experimental data obtained for 15% M13 . For 30% M13 the experimental lineshape and relaxation curve cannot be interpreted consistently and the TMV lineshape cannot even be simulated alone, indicating that the rigid rod diffusion model does not generally apply . A combined diffusion model with fast isolated motions of the encapsulated nucleic acid dominating the lineshape and a slow overall rotation of the virion as a whole, which mainly is reflected in the transversal relaxation, is able to provide a consistent picture for the 15 and 30% M13 samples, but not for TMV . Strongly improved lineshape fits for TMV are obtained assuming that there are three binding sites with different mobilities . The presence of three binding sites is consistent with previous models of TMV . The best lineshapes are simulated for a combination of one mobile and two static sites . Although less markedly, the assumption that two fractions of DNA with different mobilities exist within M13 also improves the simulated lineshapes . The possible existence of two 31P fractions in M13 sheds new light on the nonintegral ratio 2.4:1 between the number of nucleotides and protein coat subunits in the phage: 83% of the viral DNA is less mobile, suggesting that the binding of the DNA molecule to the protein coat actually occurs at the integral ratio of two nucleotides per protein subunit.

Biophys J, 1993 Jun, 64(6), 1851 - 60
A theoretical study of rotational diffusion models for rod-shaped viruses . The influence of motion on 31P nuclear magnetic resonance lineshapes and transversal relaxation; Magusin PC et al.; Information about the interaction between nucleic acids and coat proteins in intact virus particles may be obtained by studying the restricted backbone dynamics of the incapsulated nucleic acids using 31P nuclear magnetic resonance (NMR) spectroscopy . In this article, simulations are carried out to investigate how reorientation of a rod-shaped virus particle as a whole and isolated nucleic acid motions within the virion influence the 31P NMR lineshape and transversal relaxation dominated by the phosphorus chemical shift anisotropy . Two opposite cases are considered on a theoretical level . First, isotropic rotational diffusion is used as a model for mobile nucleic acids that are loosely or partially bound to the protein coat . The effect of this type of diffusion on lineshape and transversal relaxation is calculated by solving the stochastic Liouville equation by an expansion in spherical functions . Next, uniaxial rotational diffusion is assumed to represent the mobility of phosphorus in a virion that rotates as a rigid rod about its length axis . This type of diffusion is approximated by an exchange process among discrete sites . As turns out from these simulations, the amplitude and the frequency of the motion can only be unequivocally determined from experimental data by a combined analysis of the lineshape and the transversal relaxation . In the fast motional region both the isotropic and the uniaxial diffusion model predict the same transversal relaxation as the Redfield theory . For very slow motion, transversal relaxation resembles the nonexponential relaxation as observed for water molecules undergoing translational diffusion in a magnetic field gradient . In this frequency region T2e is inversely proportional to the cube root of the diffusion coefficient . In addition to the isotropic and uniaxial diffusion models, a third model is presented, in which fast restricted nucleic acid backbone motions dominating the lineshape are superimposed on a slow rotation of the virion about its length axis, dominating transversal relaxation . In an accompanying article the models are applied to the 31P NMR results obtained for bacteriophage M13 and tobacco mosaic virus.

Biokhimiia, 1993 Jun, 58(6), 857 - 65
{Substrate specificity of T4 RNA-ligase: the role of a purine nucleotide base in forming a covalent AMP-RNA-ligase complex}; Iuodka BA et al.; The NTP binding site of bacteriophage T4 RNA-ligase (EC 6.5.1.3) was studied using several ATP analogs modified in the purine moiety of the nucleotide at positions 1, 2, 6 and 8: adenosine-N'-oxide 5'-triphosphate (I), 6-chloropurine riboside 5'-triphosphate (II), 6-mercaptopurine riboside 5'-triphosphate (III), 1,N6-ethenoadenosine 5'-triphosphate (IV), 8-sulphoadenosine 5'-triphosphate (V), 8-bromoadenosine 5'-triphosphate (VI), inosine 5'-triphosphate (VII) and guanosine 5'-triphosphate (VIII) . For all of the ATP analogs under study a reversible inhibition was demonstrated . These analogs appeared to be competitive inhibitors of the T4 RNA-ligase adenylation reaction and only V, VI and VII were efficient as substrates . The kinetic parameters for the competitive inhibition, (I50, Ki) were determined . A putative structure for the T4 RNA-ligase active site was proposed.

J Gen Microbiol, 1993 Jun, 139 ( Pt 6), 1283 - 90
Sensitivity of naturally occurring coliphages to type I and type II restriction and modification; Korona R et al.; Protection against lethal infections by bacteriophage may seem the most likely role of restriction-modification (R-M) systems in bacteria and the reason for their evolution . There are, however, phenomena which question this phage-mediated selection hypothesis for the maintenance of extant R-M systems . Most prominent among these are the mechanisms phage have to avoid or otherwise limit the effects of the restriction endonucleases produced by their host bacteria . To evaluate the importance of these antirestriction mechanisms in Escherichia coli, we have examined the sensitivity of coliphage from natural and laboratory sources to a series of type I and II R-M systems . The results of our study indicate that, in vivo, restriction endonucleases have no effect on a substantial fraction of naturally occurring coliphage . The absence of restriction sites appears to be the most common reason why these phage are unaffected by type II restriction endonucleases, but other antirestriction mechanisms also operate . On the other hand, the frequency of naturally occurring coliphage sensitive to restriction appears sufficiently great for phage-mediated selection to be a viable hypothesis for the maintenance of R-M in E . coli and its accessory elements.

Antiviral Res, 1993 Jun, 21(2), 93 - 102
Influence of template primary structure on 3'-azido-3'-deoxythymidine triphosphate incorporation into DNA; Bridges EG et al.; In the present study, templates containing a specific segment of the G gamma-globin gene were constructed and incorporation of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZT-TP) or 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) into these templates was compared to that observed in M13 bacteriophage DNA . Investigations on the intrinsic fidelity of T7 DNA polymerase in reactions with AZT-TP and without deoxythymidine 5'-triphosphate, resulted in DNA synthesis beyond the first T site, suggesting that other normal deoxynucleotides misincorporated at these T sites . Modified T7 DNA polymerase incorporated AZT-TP into T sites of elongating DNA strands . Chain termination at noncomplementary sites was also observed with AZT-TP when a genomic DNA template was used and interestingly, this phenomenon was not detected in the presence of a M13 DNA template . These DNA template-dependent effects were not detected with either ddTTP, 2',3'-dideoxycytidine 5'-triphosphate, or 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate (D4T-TP) . A variation in the extent of chain termination at T sites was observed with D4T-TP suggesting that each 2',3'-dideoxynucleoside may exhibit unique chain termination patterns along with the template sequence.

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