Biochim Biophys Acta, 1980 Nov 17, 633(1), 68 - 76 Physical measurements on the lipid-containing bacteriophage phi 6; Berger H et al.; Bacteriophage phi 6 has been studied by small-angle X-ray scattering intensity-fluctuation spectroscopy, analytical ultracentrifugation, and spectroscopy . The sedimentation coefficient (S(0)0,w) is 375 S, the diffusion coefficient (D(0)20, w) is 2.66 x 10(-8) cm 2/s . Using the Svedberg equation and and an estimate of the partial specific volume, the Mr is 1.49 +/- 0.32 x 10(8) . A simple model which describes phi 6, is a central sphere consisting of RNA and protein of radius 330 A and an outer shell of low electron density 40 A thick . The RNA may form five concentric shells in the region r = 140-290 A. Nucleic Acids Res, 1980 Nov 11, 8(21), 5071 - 88 Mapping of promoter sites utilized by T3 RNA polymerase on T3 DNA; Bailey JN et al.; Promoter locations for the T3 RNA polymerase on the physical map of T3 DNa have been determined . Through the use of conditions favoring the synthesis of RNA from the class II region, an agarose-formaldehyde gel system which improves the resolution of high molecular weight RNAs, and template DNA that was cut by one of several restriction endonucleases prior to transcription, seventeen promoter locations for the T3 RNA polymerase have been mapped . Ten promoters have been identified in the class II region and one promotor has been identified in the class II region and one promotor has been identified in the early (class I) region . The locations of previously mapped class III promoters and the internal termination signal for the T3 RNA polymerase have been mapped more precisely than in previous reports . The resulting transcription map demonstrates a striking similarity to the transcription map of bacteriophage T7. Nucleic Acids Res, 1980 Nov 11, 8(21), 5113 - 27 Processive action of T4 endonuclease V on ultraviolet-irradiated DNA; Lloyd RS et al.; The action of the dimer-specific endonuclease V of bacteriophage T4 was studied on UV-irradiated, covalently-closed circular DNa . Form I ColE1 DNA preparations containing average dimer frequencies ranging from 2.5 to 35 pyrimidine dimers per molecule were treated with T4 endonuclease V and analysed by agarose gel electrophoresis . At all dimer frequencies examined, the production of form III DNA was linear with time and the double-strand scissions were made randomly on the ColE1 DNA genome . Since the observed fraction of form III DNA increased with increasing dimer frequency but the initial rate of loss of form I decreased with increasing dimer frequency, it was postulated that multiple single-strand scissions could be produced in a subset of the DNA population while some DNA molecules contained no scissions . When DNA containing an average of 25 dimers per circle was incubated with limiting enzyme concentrations, scissions appeared at most if not all dimmer sites in some molecules before additional strand scissions were produced in other DNA molecules . The results support a processive model for the interaction of T4 endonuclease V with UV-irradiated DNA. J Biol Chem, 1980 Nov 10, 255(21), 10331 - 7 Isolation and characterization of the C and D proteins coded by gene IX and gene VI in the filamentous bacteriophage fl and fd; Lin TC et al.; The C and D proteins from bacteriophage fd and fl have been purified and characterized . Since the DNA sequence is known, the amino acid composition of these purified proteins indicated that they are coded for by the phage, the C protein being the product of Gene IX and the D protein specified by Gene VI . The molecular weights of the C and D proteins were calculated from the DNA sequences to be 3,650 and 12,350, respectively . These values are close to the molecular weights observed after polyacrylamide gel electrophoresis of the proteins in sodium dodecyl sulfate . Since the C and D proteins can be selectively labeled with radioactive cysteine and arginine, it was possible to estimate the number of the C and D protein molecules relative to the number of A protein molecules which had been accurately determined in previous studies . Based on these results, the average phage particle contains 5 A, 5 D, and 10 C protein molecules. J Virol, 1980 Nov, 36(2), 622 - 6 New deletion mutant of bacteriophage T5; Rhoades M et al.; Deletion mutants of bacteriphage T5 can be identified by analyzing the DNA content of single plaques, after alkaline denaturation, by agarose gel electrophoresis . Use of this procedure has resulted in the isolation of a new mutant, designated del2, that lacks a 1.6% segment located 68 to 70% from the left end of T5 DNA . This segment occurs between the early and late gene regions of T5 DNA. Vopr Virusol, 1980 Nov-Dec, (6), 728 - 31 {Physicochemical properties of the Newcastle group bacteriophages}; Sorochkina VV et al.; Physico-chemical properties of dysentery therapeutic-prophylactic Newcastle phages H-I, H-5, H-10, H-22 were studied . The morphology of the phage particles is described; their molecular weight and buoyant density in CsCl were determined by ultracentrifugation . The type of nucleic acids (DNA) was determined . By all the above characteristics and their chemical composition, bacteriophages of the Newcastle group approach T-even phages. Can J Microbiol, 1980 Nov, 26(11), 1312 - 9 The comparison of rhapidosomes and defective bacteriophage particles from Aquaspirillum intersonii; Evers MC et al.; Aquaspirillum itersonii spontaneously produces rhapidosomes . These structures, which are found throughout the cell cycle, are compared with the defective bacteriophage particles that can be induced by mitomycin C treatment (0.5 microgram/mL) . The rhapidosomes are composed of three proteins (MW 63 000, 23 500, and 15 000) . The defective bacteriophage particles are composed of two proteins (MW 50 000 and 17 500) . The defective bacteriophage particles do not react with anti-rhapidosomal protein antiserum . These results indicate that the rhapidosome are not polysheaths of the defective bacteriophage particles inducible in this organism as earlier proposed in the literature . It is concluded that the rhapidosomes of A . itersonii are an assembly of three proteins that are distributed in the cytosol after synthesis and are present at all times in the growth cycle. J Bacteriol, 1980 Nov, 144(2), 524 - 31 Escherichia coli K317, formerly used to define colicin group E2, produces colicin E7, is immune to colicin E2, and carries a bacteriophage-restricting conjugative plasmid; Males BM et al.; Escherichia coli strain CL137, a K-12 derivative made E colicinogenic by contact with Fredericq's strain K317, was unaffected by colicin E2-P9, but K-12 carrying ColE2-P9 was sensitive to the E colicin made by strains CL137 and K317 . This colicin we named E7-K317 because by the test of colicinogenic immunity it differed from colicins E1-K30, E2-P9, and E3-CA38 and from recently recognized colicins termed E4Horak, E5, and E6 . Strain K317 as conjugational donor transmitted E7 colicinogeny; about half the E7-colicinogenic transconjugants were immune to colicin E2-P9 . A spontaneous variant of CL137 retained E7 colicinogeny but was sensitive to E2 colicins . We attribute the E2 immunity of strain CL137 and some E7-coliconogeic transconjugants to a "colicin-immunity plasmid," ColE2imm-K317, from strain K317 . Tra+ E7-colicinogenic transconjugants restricted phage BF23 in the same way as strains carrying ColIb-P9 . We attribute Tra+ and restricting ability to a plasmid, pRES-K317, acquired from strain K317, and related to the ColI plasmids. Proc Natl Acad Sci U S A, 1980 Nov, 77(11), 6511 - 5 Isolation and characterization of the mouse metallothionein-I gene; Durnam DM et al.; Double-stranded cDNA was synthesized from a mouse liver mRNA fraction enriched for metallothionein mRNA activity, ligated to restriction site linkers, inserted into pBR322, and used to transform Escherichia coli chi 1776 . The sequence of the largest plasmid containing DNA that hybridized to metallothionein mRNA was determined and shown to contain a 380-base-pair insert that includes the entire coding region and 3' untranslated region of metallothionein-I . The metallothionein-I insert was nick-translated and used to screen both a mouse myeloma and a mouse embryo DNA library in bacteriophage lambda . A metallothionein-I genomic clone containing 13-15 kilobase pairs of mouse DNA was isolated from each library . Both contain a 3.8-kilobase-pair EcoRI fragment that hybridizes to the metallothionein-I probe . The location, size, and orientation of the metallothionein-I gene within the 3.8-kilobase-pair fragment were determined by heteroduplex and restriction mapping . The gene spans 1.1 kilobase pairs and contains at least two introns. Gene, 1980 Nov, 11(3-4), 197 - 205 DNA sequence analysis of prm-mutations of coliphage lambda; Rosen ED et al.; Nucleotide sequence changes associated with mutation of the prm promoter of bacteriophage lambda have been determined . Prm-mutations have been assigned to two classes . Class I mutations appear to affect the interaction of RNA polymerase with prm; six class I mutations affect four sites, located 14, 33, 38, and 39 bp preceding the prm transcription startpoint . Class II mutations appear to owe their Prm-phenotype to a change in OR, which could prevent activation of prm by repressor . All three class II mutations are in OR 1. Clin Exp Immunol, 1980 Nov, 42(2), 234 - 40 Host immune status in uraemia III . Humoral response to selected antigens in the rat; Nelson J et al.; The humoral immune status of patients with uraemia is controversial . In the present experiments a carefully defined animal model, in which a chronic state of moderate and severe uraemia was induced, has been used to resolve conflicting views . The capacity of the uraemia host to respond to immunogenic stimulation was assessed by challenging uraemic animals with bacterial, viral, T cell-dependent and T cell-independent antigens . The experiments have shown that the immune responsiveness of animals with chronic severe uraemia, immunized with sheep red blood cells, Escherichia coli 075, keyhole limpet haemocyanin and phi X174 bacteriophage is comparable to those found in sham-operated and control groups . The results strongly suggest that uraemia per se does not affect the ability of the host to respond to a wide range of antigenic stimuli . This conclusion is important in that it provides a basis for assessing further the immune status of the uraemic host . If uraemic patients do have an immune deficit, one possible explanation is that uraemia potentiates the immunosuppressive activity of some drugs used in the clinical management of these patients. Proc Natl Acad Sci U S A, 1980 Nov, 77(11), 6381 - 5 In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters; Hawley DK et al.; The in vitro initiation properties of the PR promoter of bacteriophage lambda and of a PR mutant, x3, were compared . Using the abortive initiation reaction, we measured the lags in the approach to a final steady-state rate when dinucleotide synthesis was initiated with RNA polymerase . These lags corresponded to the average times required for the formation of transcriptionally active open complexes . By measuring the lags at different RNA polymerase concentrations, we could separate open complex formation into two steps, based on a simple model in which the initial bimolecular association of free promoter and polymerase in a closed complex is followed by an isomerization to the open complex . The contribution of each step to the overall rate of open complex formation was quantitated for both promoters . We found that the x3 mutation, which is located in the -35 region of PR, resulted in a decrease in the association constant for the initial binding to the closed complex to 5% of its wild-type value and a decrease in the rate of the isomerization to 20% . The lifetimes and abortive initiation characteristics of the mutant and wild-type promoters were similar . We concluded that the main effect of the x3 mutation was to increase the average time of open complex formation and that the functional properties of the open complexes did not differ significantly between the two promoters. J Bacteriol, 1980 Nov, 144(2), 489 - 98 Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli; Baluch J et al.; We investigated the capacity of Escherichia coli mutants defective in the single-strand deoxyribonucleic acid (DNA)-binding protein to amplify the synthesis of the recA protein, induce prophage lambda, and degrade their DNA after treatment with ultraviolet radiation, mitomycin C, or bleomycin . The thermosensitive ssbA1 strain induced recA protein and lambda phage normally at 30 degrees C, but no induction was observed at 42 degrees C when ultraviolet radiation or mitomycin C was used . The lexC113 mutant did not amplify recA protein synthesis or induce phage lambda at either 30 or 42 degrees C with those agents . Bleomycin was able to elicit induction of recA and phage lambda in both mutants at any temperature . After induction with ultraviolet radiation at the elevated temperature, no DNA degradation was observed for 40 min, but at later times there was increased degradation in the lexC113 strain, compared with the wild type, and even greater degradation in the ssbA1 mutant . We discuss the role of single-strand DNA-binding protein in induction and the possibility that the lexC product may exert its influence on recA and lambda induction at the level of the single-strand DNA gap. Biochem J, 1980 Nov 1, 191(2), 593 - 604 The EcoRI restriction endonuclease with bacteriophage lambda DNA . Equilibrium binding studies; Halford SE et al.; The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites . The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites . The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates . The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+ . These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction. Biochem J, 1980 Nov 1, 191(2), 581 - 92 The EcoRI restriction endonuclease with bacteriophage lambda DNA . Kinetic studies; Halford SE et al.; The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site . Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site . But under altered experimental conditions, different reaction rates were observed at each recognition site . These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction . Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme . The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules. Gene, 1980 Nov, 11(3-4), 347 - 57 Bacteriophage Mu-mediated gene transposition and in vitro cloning of the enterochelin gene cluster of Escherichia coli; Laird AJ et al.; Transposition of chromosomal genes using bacteriophage Mu has been used to obtain a partial order of the nine closely linked genes of the enterochelin-dependent iron transport system of Escherichia coli K-12 . Fragments of the ent gene cluster were transposed into the conjugative plasmid RP4 and were characterized by genetic complementation . The partial gene order (entD, fes), entF, fep, entC, ent(ABEG)...lip was derived using six plasmids which carried overlapping parts of the cluster, and the fep mutations were shown to belong to a single complementation group . Two restriction fragments, one carrying ent(ABCEG) and the other carrying fep, were cloned in vitro using one of the RP4::ent plasmids as a source of DNA enriched in enterochelin system genes . A further restriction fragment, carrying the three remaining genes, entD, fes and entF was cloned directly from the chromosome . The three restriction fragments collectively cover a region of the chromosome 29 kg in length, indicating that the genes of the enterochelin system are clustered but not contiguous. Gene, 1980 Nov, 11(3-4), 207 - 18 Construction and characterization of new coliphage M13 cloning vectors; Hines JC et al.; New single-stranded DNA cloning vectors have been constructed by the insertion of additional DNA fragments into a HaeII restriction site in the bacteriophage M13 duplex replicative form (RF) . These inserts into the M13 genome bring a single restriction sites useful for cloning, including PstI, XorII, EcoRI, SstI, XhoI, KpnI, and PvuII . Drug-resistance genes cloned into M13 include the beta-lactamase (bla) gene and the chloramphenicol acetyl transferase (cat) gene . These vectors provide a convenient means of easily obtaining the separated strands of a cloned duplex DNA fragment by cloning the fragment in each of the two possible orientations . Standard cloning techniques commonly applied to double-stranded DNAs can be utilized to insert foreign DNAs into the duplex RF DNAs of these vectors . Cells transformed by chimeric DNAs extrude filamentous phage particles carrying a circular single-stranded copy of the chimeric viral strand . Because M13-infected cells continue to grow and divide, cells can be transformed to yield either plaques or drug-resistant colonies . Specific inserts are readily detected by plaque hybridization techniques using an appropriate probe . Chimeric viral single strands from virus particles in the supernatant of small volumes of infected cultures can be rapidly and sensitively analyzed by agarose gel electrophoresis to determine the size of an insert. Nucleic Acids Res, 1980 Oct 24, 8(20), 4821 - 37 Regulation of promoter selection by the bacteriophage T7 RNA polymerase in vitro; McAllister WT et al.; During bacteriophage T7 infection a phage-specified RNA polymerase transcribes the late phage genes in two temporal classes (class II and class III) . In this report, we show that the purified phage polymerase discriminates between the class II and class III promoters in vitro as a function of variables that alter the stability of the DNA helix . These variables include ionic strength, temperature, and the presence of denaturing agents such as dimethyl sulfoxide . In general, initiation at the class II promoters is preferentially inhibited as helix stability is increased . Conditions required for the establishment of salt-resistant transcription complexes by the T7 RNA polymerase have been determined; the establishment of stable complexes at the class II promoters requires the synthesis of a longer nascent RNA transcript than does formation of such complexes at the class III promoters . A comparison of the nucleotide sequences of several class II and class III promoters suggests certain features that may be responsible for the different responses of these promoters to helix destabilization . The conservation of structural features that are peculiar to the class II or class III promoters indicates that these features are important in regulation of T7 transcription in vivo . Experiments which bear on the physiological significance of these features are discussed. Experientia, 1980 Oct 15, 36(10), 1226 - 7 Blood clearance of MS2 bacteriophage in Salmo trutta: a paradoxon; O'Neill JG; A primary challenge of MS2 bacteriophage was cleared from the blood of the teleost Salmo trutta within 2.5 days . In contrast the clearance of a 2nd challenge required an extra 3.5 days and the induction of the secondary antibody response was delayed, though immune memory was observed later. Biochemistry, 1980 Oct 14, 19(21), 4767 - 72 Repair of neocarzinostatin-induced deoxyribonucleic acid damage in human lymphoblastoid cells: possible involvement of apurinic/apyrimidinic sites as intermediates; Tatsumi K et al.; Neocarzinostatin (NCS) induces repair in a xeroderma pigmentosum lymphoblastoid line deficient in the ability to repair DNA damage induced with (acetoxyacetyl-amino)fluorene . Repair was demonstrated by the induction of repair synthesis and by the disappearance of NCS-induced single-strand breaks and/or alkaline-labile sites in DNA . Estimation of NCS-induced repair patch size, based on the density shift induced in DNA by extensive shear after incubation of treated cells in medium with bromodeoxyuridine or by calculation from the extent of restoration of DNA sedimentation profiles in alkaline sucrose gradients and the amount of repair synthesis measured by the BND cellulose method, indicated that only a few nucleotides were inserted per repaired region . NCS-treated bacteriophage T7 DNA requires incubation with alkaline phosphatase to make it a substrate for DNA polymerase I . NCS-reacted T7 DNA, even after phosphatase treatment, is not a substrate for a DNA polymerase alpha obtained from human lymphoma cells . NCS-treated T7 DNA did serve as a substrate for the DNA polymerase alpha when incubated with an apurinic/apyrimidinic (AP) endonuclease with associated 5'-3'-exonuclease activity . The results suggest that NCS-induced AP sites could be intermediates for the in vivo repair synthesis. Nucleic Acids Res, 1980 Oct 10, 8(19), 4501 - 16 An Escherichia coli endonuclease responsible for primary cleavage of in vitro transcripts of bacteriophage T4 tRNA gene cluster; Goldfarb A et al.; An endonuclease activity was isolated from 100,000 g supernatant fraction of Escherichia coli using in vitro primary transcripts of T4 tRNA gene cluster as assay substrates . The endonuclease cleaves the polycistronic RNA precursors into fragments containing monomeric and dimeric stable RNA sequences . The result strongly suggest that this enzyme participates in the early steps of T4 tRNA maturation pathway preceding the action of RNase P. J Virol, 1980 Oct, 36(1), 264 - 70 Adsorption, capping, and release of a complex bacteriophage by mycoplasma cells; Haberer K et al.; By electron microscopic studies, the adsorption and release of nonlytic, cytocidal mycoplasma virus MVL3, which infects ACholeplasma laidlawii cells, have been examined . The MVL3 virion has a polyhedral head, collar, short tail, and tail fibers and contains linear double-stranded DNA . Adsorbed MVL3 virus showed a temperature-dependent clustering or capping on the mycoplasma cell membrane . During infection, a number of virus-cell membrane-related structures were observed, suggesting a general model in which MVL3-infected cells release progeny virions in membrane vesicles . These vesicles must then break down to release MVL3 particles. J Virol, 1980 Oct, 36(1), 103 - 8 Suppressors of mutations in the bacteriophage T4 gene coding for both RNA ligase and tail fiber attachment activities; Hall DH et al.; The protein product of T4 gene 63 catalyzes both the attachment of tail fibers to fiberless phage particles and the ligation of single-stranded RNA (Snopek at al., Proc . Natl . Acad . Sci . U.S.A . 74:3355-3359, 1977) . To investigate whether the gene 63 product has a role in nucleotide metabolism, we isolated false revertants of amM69 in gene 63 . We screened for revertants that could grow at 30 degrees C but not at 43 degrees C on Escherichia coli OK305 when nucleotides were limiting . These false revertants contained the original mutation in gene 63 and new suppressor mutations . Some of these suppressor mutations caused temperature sensitivity by themselves, allowing single mutants carrying the suppressor to be recognized and isolated . The results of mapping and complementation studies indicated that most of these ts suppressors were in the t gene (lysis), one was in gene 5 (baseplate), and one was in gene 18 (sheath) . The mutation in gene 18, tsDH638, suppressed three different amber mutations in gene 63 but did not suppress amber mutations in several other genes . None of the suppressors that were characterized were in genes with known functions in nucleotide metabolism . However, an intriguing property of these false revertants was that they were very sensitive to hydroxyurea, an inhibitor of nucleotide metabolism. Biophys J, 1980 Oct, 32(1), 531 - 48 Nuclear magnetic resonance of the filamentous bacteriophage fd; Opella SJ et al.; The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy . 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus . There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA . 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations . The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions . The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus . Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings. J Virol, 1980 Oct, 36(1), 220 - 3 Probable localization of the bacteriophage T4 prehead proteinase zymogen in the center of the prehead core; van Driel R et al.; During the assembly of the bacteriophage T4 prehead, a T4-coded protease zymogen (P21) is built into the structure . At a certain stage in head formation, the protease precursor is activated and specifically cleaves most of the prehead proteins . In this paper we show that a correlation existed between the presence of proteinaceous material in the center of the prehead core, observed by electron microscopy, and the availability of P21 during prehead assembly . In the absence of P21, the core enclosed a hold of about 35 nm long and 20 nm wide . We found the same for (i) in vitro-assembled, negatively stained prehead-like structures and (ii) in vivo-formed preheads in thin sections of T4-infected cells . We concluded that P21 was localized in the center of the prehead core. Proc Natl Acad Sci U S A, 1980 Oct, 77(10), 5698 - 702 Pentaribonucleotides of mixed sequence are synthesized and efficiently prime de novo DNA chain starts in the T4 bacteriophage DNA replication system; Liu CC et al.; In the presence of single-stranded DNA, the bacteriophage T4 gene 41 and gene 61 proteins catalyze the synthesis of a group of pentaribonucleotides which are homogeneous in chain length but heterogeneous in nucleotide sequence . When single-stranded T4 DNA is used as template, a unique dinucleoside sequence, pppApC, is found at the 5' end of these pentaribonucleotides with the general sequence pppApCpNpNpN . In the presence of the remaining five T4 replication proteins, the pentaribonucleotides can be utilized with high efficiency to prime de novo DNA chain starts; as a result, the vast majority of them can be detected at the 5' end of newly made DNA molecules in an unaltered form . There are multiple, but specific, sites at which new DNA chains are primed in this way on a natural single-stranded DNA . Because identical RNA primers have been isolated from the 5' end of the Okazaki fragments made in T4-infected cells, we suggest that the T4 gene 41 and gene 61 proteins also make the pentaribonucleotides that prime de novo T4 DNA chain starts in vivo during lagging strand DNA synthesis. Biophys J, 1980 Oct, 32(1), 103 - 38 Movement and self-control in protein assemblies . Quasi-equivalence revisited; Caspar DL; Purposeful switching among different conformational states exerts self-control in the construction and action of protein assemblies . Quasi-equivalence, conceived to explain icosahedral virus structure, arises by differentiation of identical protein subunits into different conformations that conserve essential bonding specificity . Mechanical models designed to represent the energy distribution in the structure, rather than just the arrangement of matter, are used to explore flexibility and self-controlled movements in virus particles . Information about the assembly of bacterial flagella, actin, tobacco mosaic virus and the T4 bacteriophage tail structure show that assembly can be controlled by switching the subunits from an inactive, unsociable form to an active, associable form . Energy to drive this change is provided by the intersubunit bonding in the growing structure; this self-control of assembly by conformational switching is called "autostery", by homology with allostery . A mechanical model of the contractile T4 tail sheath has been constructed to demonstrate how self-controlled activation of a latent bonding potential can drive a purposeful movement . The gradient of quasi-equivalent conformations modelled in the contracting tail sheath has suggested a workable mechanism for self-determination of tail tube length . Concerted action by assemblies of identical proteins may often depend on individually differentiated movements. Proc Natl Acad Sci U S A, 1980 Oct, 77(10), 5794 - 8 Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene; Goldberg DA; The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted . Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA . Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain . This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA . Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains . The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments . Two intervening sequences were identified within Adh by S1 nuclease mapping experiments. Aust J Biol Sci, 1980 Oct, 33(5), 605 - 12 Isolation and characterization of a lambda transducing bacteriophage carrying the cysE gene of Escherichia coli K-12; Ingle CP et al.; A specialized lambda transducing phage carrying the cysE and gpsA genes of E . coli K-12 has been isolated . The transducing phage has been separated from the helper phage on equilibrium gradients and has been shown to be defective . Evidence is presented that the phage kil gene is not expressed. Biophys J, 1980 Oct, 32(1), 403 - 18 On the thermodynamics and kinetics of the cooperative binding of bacteriophage T4-coded gene 32 (helix destabilizing) protein to nucleic acid lattices; Kowalczykowski SC et al.; In this paper we summarize a series of thermodynamic, and preliminary kinetic, studies on the molecular details and specificity of interaction of phage T4-coded gene 32-protein (GP32) with nucleic acid lattices . It is shown that the binding of GP32 to short (l = 2--8 residues) oligonucleotides is essentially independent of base composition and sugar-type, as well as of salt concentration . In contrast, cooperative (continuous) or isolated binding of GP32 to single-stranded polynucleotides is base and sugar composition-dependent (binding is tighter to DNA than to RNA) and highly dependent on salt concentrations . Binding constants (K), cooperativity parameters (w), and binding site sizes (n) are determined for binding to various nucleic acid lattices under a variety of environmental conditions . These results are used to show that GP32 can bind to nucleic acid lattices in two different conformations, and to characterize the molecular details of these binding species . Further insight into the molecular origins of binding cooperativity is obtained by determining these thermodynamic parameters also for the specifically proteolytically degraded GP32 fragments GP32 I (C-terminal peptide removed) and GP32 III (C- and N-terminal peptides removed) . It is also shown that these GP32-nucleic acid binding measurements can be used to provide a quantitative molecular interpretation of the sequential (competitive) binding equilibria involved in the autogenous translational regulation of GP32 synthesis (Lemaire et al., 1978, J . Mol . Biol . 126:73, 1978), and to illustrate some general principles of the development of interactional specificity in cooperatively binding protein-nucleic acid complexes . Preliminary experiments have also been carried out on the kinetics of GP32 association to, and dissociation from, single-stranded nucleic acid lattices . In particular, fluorescence stopped-flow measurements of the dissociation of GP32 from such lattices as a function of lattice saturation (and protein cluster size) can be interpreted to suggest that the protein may translocate ("slide") on the lattice before dissociation, These studies permit an approach to possible rates and mechanisms of such translocation events. Biophys J, 1980 Oct, 32(1), 155 - 73 The structure of a DNA unwinding protein and its complexes with oligodeoxynucleotides by x-ray diffraction; McPherson A et al.; The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been solved to 2.3 A resolution by x-ray diffraction techniques . The molecule contains an extensive cleft region that we have identified as the DNA binding site on the basis of the residues that comprise its surface . The interior of the groove has a rather large number of basic amino acid residues that serve to draw the polynucleotide backbone into the cleft . Arrayed along the external edges of the groove are a number of aromatic amino acid side groups that are in position to stack upon the bases of the DNA and fix it in place . The cleft then acts as an elongated pair of jaws that draws the DNA between them by charge interactions involving the phosphates with the interior lysines and arginines . The jaws then close on the DNA strand through small conformation changes and the rotation of aromatic side-chains into position to stack upon the purines and pyrimidines . Complexes of the gene 5 protein with a variety of oligodeoxynucleotides have been formed and crystallized for x-ray diffraction analysis . The crystallographic parameters of four different unit cells indicate that the fundamental unit of the complex is composed of six gene 5 protein dimers . We believe this aggregate has 622 point group symmetry and is a ring formed by end to end closure of a linear array of six dimers . From our results we have proposed a double helical model for the gene 5 protein-DNA complex in which the protein forms a spindle or core around which the DNA is spooled . 5.0-A x-ray diffraction data from one of the crystalline complexes is currently being analyzed by molecular replacement techniques to obtain what we believe will be the first direct visualization of a protein-deoxyribonucleic acid complex approaching atomic resolution. J Virol, 1980 Oct, 36(1), 254 - 63 Molecular cloning of unintegrated and a portion of integrated moloney murine leukemia viral DNA in bacteriophage lambda; Berns AJ et al.; A covalently closed circular form of unintegrated viral DNA obtained from NIH 3T3 cells freshly infected with Moloney murine leukemia virus (M-MLV) and a port of the endogenous M-MLV from the BALB/Mo mouse strain have been cloned in bacteriophage lambda . The unintegrated viral DNA was cleaved with restriction endonuclease HindIII and inserted into the single HindIII site of lambda phage Charon 21A . Similarly high-molecular-weight DNA from BALB/Mo mice ws cleaved sequentially with restriction endonucleases EcoRI and HindIII and separated on the basis of size, and one of the two fractions which reacted with an M-MLV-specific complementary DNA was inserted into the HindIII site of Charon 21A . Recombinant clones containing M-MLV-reacting DNA were analyzed by restriction endonuclease mapping, heteroduplexing, and infectivity assays . The restriction endonuclease map of the insert derived from unintegrated viral DNA, lambda x MLV-1, was comparable to published maps . Electron microscope analysis of the hybrid formed between lambda x MLV-1 DNA and 35S genomic M-MLV RNA showed a duplex structure . The molecularly cloned lambda x MLV-1 DNA contained only one copy of the long terminal repeat and was not infectious even after end-to-end ligation of the insert DNA . The insert DNA derived from endogenous M-MLV, lambda x MLVint-1, contained a DNA stretch measuring 5.4 kilobase pairs in length, corresponding to the 5' part of the genomic viral RNA, and cellular mouse DNA sequences measuring 3.5 kilobase pairs in length . The viral part of the insert showed the typical restriction pattern of M-MLV DNA except that a single restriction site, PvuII, in the 5' long terminal repeat was missing . Reconstructed genomes containing the 5' half derived from the integrated viral DNA and the 3' half derived from the unintegrated viral DNA were able to induce XC plaques after transfection in uninfected mouse fibroblasts. J Virol, 1980 Oct, 36(1), 1 - 17 Correlation between genetic map and map of cleavage sites for sequence-specific endonucleases SalI, KpnI, BglI, and BamHI in bacteriophage T4 cytosine-containing DNA; Carlson K; Cleavage sites for SalI, KpnI, BglI, and BamHI in cytosine-containing DNA from T4 alc10(alc) nd28(denA) D2a2(denB) amE51x5(56) amN55x5(42) have been mapped relative to each other, and the positions of deletions sa delta 9 (D1-stp), r1589(rII), del(39-56)12, and tk2(rI-tk) relative to these cleavage sites have been determined . Based on these analyses, a physical map of the T4 genome containing 166 kilobase pairs has been constructed. Gene, 1980 Oct, 11(1-2), 129 - 48 Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd; van Wezenbeek PM et al.; The 6407 nucleotide-long sequence of bacteriophage M13 DNA has been determined using both the chemical degradation and chain-termination methods of DNA sequencing . This sequence has been compared with that of the closely related bacteriophage fd (Beck et al., 1978) . M13 DNA appears to be only a single nucleotide shorter than fd DNA . There is an average of 3.0% of nucleotide-sequence differences between the two genomes, but the distribution of these changes is not random; the sequence of some genes is more conserved than of others . In contrast, the nucleotide sequences and positions of the regulatory elements involved in transcription, translation and replication appear to be identical in both filamentous phage DNA genomes. Proc Natl Acad Sci U S A, 1980 Oct, 77(10), 5634 - 8 Rate-limiting steps in RNA chain initiation; McClure WR; Promoter-specific lags in the approach to the steady-state rate of abortive initiation were observed when Escherichia coli RNA polymerase was added to initiate the reaction . The lag times were related to the time required for free enzyme and free promoter to combine and isomerize into a functionally active complex . The lag times measured for several bacteriophage and bacterial promoters differed widely (10 sec to several minutes) and in most cases corresponded to the rate-limiting step in the initiation process . The unique advantage in using the abortive initiation reaction to measure the lags was that the binding and isomerization steps in a simple two-state model could be quantitated separately . The separation of the contributions of both steps was effected by deriving an equation to describe the rate of formation of the active binary complex . Results from experiments based on the theory showed a linear relationship between the observed lag times and the reciprocal enzyme concentration . The slope and intercept of the equation yielded quantitative estimates of the binding and isomerization steps in initiation . The analysis was applied to the bacteriophage T7 A2 and D promoters to show the bases for the differences in in vitro initiation frequency that have been observed for these promoters. Science, 1980 Sep 19, 209(4463), 1370 - 4 Phase variation: evolution of a controlling element; Simon M et al.; Phase variation in bacteria is regulated by homologous recombination at a specific DNA site . This recombinational event causes the inversion of a 970-base-pair DNA sequence that includes the promoter necessary for transcription of a flagellar gene . The invertible segment is flanked by two sites that are necessary for the inversion and contains a gene (hin) whose product mediates the inversion event . The hin gene shows extensive homology with the TnpR gene carried on the Tn3 transposon . It is also homologous with the gin gene carried on bacteriophage mu . These relationships suggest that the phase variation system may have evolved by the association of a transposon with a resident gene and the subsequent specialization of these elements to regulate flagellar antigen expression. Biochim Biophys Acta, 1980 Sep 18, 601(2), 245 - 59 Structure of the lipid-containing bacteriophage phi 6 . Disruption by Triton X-100 treatment; Bamford DH et al.; The structure of the lipid-containing bacteriophage phi 6 was studied by means of controlled Triton X-100 disruption and subsequent isolation of subviral particles . Rate-zonal centrifugation yielded two fractions, a nucleocapsid fraction with RNA, proteins P1, P2, P4, P7, P8, and about half of the protein P5 and a membrane fraction with associated proteins P3, P6, P9, P10, and the rest of the protein P5 . Following isopycnic sucrose gradient centrifugation, an empty capsid fraction was obtained which lacked RNA but contained a protein composition similar to the nucleocapsid except for the absence of P5 . The membrane fraction isolated after isopycnic centrifugation was morphologically indistinguishable from that isolated after rate-zonal centrifugation but contained only proteins P3, P6, P9 and P10 . By treating phi 6 with Triton X-100 prior to isopycnic sucrose gradient centrifugation the viral membrane was further separated into submembrane structures and the attachment protein, P3, could be isolated in rather pure form. C R Seances Acad Sci D, 1980 Sep 15, 291(2), 199 - 202 {Use of the degeneracy of the genetic code by selective pressure to cut up genes of procaryote genomes}; Rodier F et al.; The DNA sequences of three bacteriophages are analysed in order to localise those parts coding for a protein . A weak stability on the DNA molecule allows us to characterize the beginning and the end of genes . A survey of the codons used shows that the cause for this weak stability is the systematic use of A-T bases in third position, which is made possible by the degeneracy of the genetic code. Am J Trop Med Hyg, 1980 Sep, 29(5 Suppl), 1099 - 106 Site-specific recombination in bacteriophage lambda: structural analyses of reactive DNA sequences; Landy A et al.; Site-specific integrative recombination in bacteriophage lambda involves unequal partners . The minimal phage att site is composed of approximately 240 base pairs and has four distinct Int binding sites that differ in size and response to heparin challenge . There appear to be two size classes of Int binding sites, approximately 30-35 base pairs and 15 base pairs . The sites at the common core and in the P' arm are of the former class . Two sites in the P arm are of the latter class . Thus far, three of the four sites have been shown to be necessary for att site function . In contrast, the minimal sequence required for a phage att site partner (such as the bacterial att site) may not be much larger than the 15 base pair common core . We have suggested a model in which integrative recombination involves two unequal partners; accordingly the phage att site is referred to as the "donor" and the bacterial att site, or its analogue, is referred to as the "recipient." Mol Biol (Mosk), 1980 Sep-Oct, 14(5), 1019 - 22 {Certain physico-chemical properties of bacteriophage phiKZ}; Tiaglov BV et al.; Using UV-spectroscopy and circular dichroism (CD) phi KZ bacteriophage and its DNA were investigated . From the value of optical densities in the 320--400 nm range the size of the phi KZ bacteriophage's head was determined; the diameter of phi KZ bacteriophage head was found to be equal to 1300 +/- 100 A . phi KZ bacteriophage DNA has block structure and the GC-pair content is equal to 43.8 +/- 0.3% . phi KZ bacteriophage CD spectrum has an unusual profile (in comparison with known bacteriophage CD spectra); this spectrum is similar to the CD spectra of DNA in polyethylene glycole solution . To our knowledge CD spectra of such type were not obtained for other bacteriophages. Mol Biol (Mosk), 1980 Sep-Oct, 14(5), 1013 - 8 {Study of fusion of bacteriophage f2 double-stranded RNA, poly(A).poly(U), and poly(G).poly(C) in the presence of tetraethylammonium bromide}; Permogorov VI et al.; The data on the dependence of the melting curve parameters of double-stranded RNA (replicative form of RNA of f2 bacteriophage) poly(A) times poly(U) and poly(G) times poly(C) on the concentration of (C2H5)4NBr were obtained . The RNA melting range width is shown to pass through the minimum value T =2.1+/-0.1degrees at the point of inversion of relative stability of GC and AU pairs that corresponds to 4.0+/-0.1 M concentration of (C2H5)4NBr . Using the melting temperatures of poly(A) times poly(U) and poly(G) times poly(C) the rependence of Tgc-Tau parameter on (C2H5)4NBr concentration was shown . It was concluded from these data that the effect of the double-stranded RNA stacking heterogeneity was negligible in the 0-3 M range of (C2H5)4NBr concentration . Melting curves of RNA were obtained at various values of Tgc-Tau parameter . It was shown that the profile of fine structure of melting curves depends on the value of Tgc-Tau parameter. J Virol, 1980 Sep, 35(3), 748 - 56 Binding of wheat germ ribosomes to fragmented viral mRNA; Kozak M; The specificity of binding of wheat germ ribosomes to mRNA was greatly altered by cleavage of the message . Fragmentation of reovirus mRNA allowed wheat germ ribosomes to bind and protect a variety of internal sequences which were not accessible to ribosomes in the intact message . In experiments using the polycistronic mRNA from bacteriophage R17, wheat germ ribosomes bound preferentially at the beginning of the lysis peptide and synthetase cistrons, and at a third site which may be derived from the C-terminal region of the A protein cistron . This result is similar to that reported previously in a mammalian translational system (J.F . Atkins et al., Cell 18:246-256, 1979) except that, in the present study, limited cleavage of the phage RNA was necessary to activate these sites . More extensive fragmentation of R17 RNA permitted wheat germ ribosomes to bind and protect a great many additional sites . Thus, presence of an (exposed) 5'-terminus on an RNA molecule appears to be necessary and sufficient for attachment of eucaryotic ribosomes. J Virol, 1980 Sep, 35(3), 775 - 89 Role of the host cell in bacteriophage T4 development . II . Characterization of host mutants that have pleiotropic effects on T4 growth; Stitt BL et al.; Mutant host-defective Escherichi coli that fail to propagate bacteriophage T4 and have a pleiotropic effect on T4 development have been isolated and characterized . In phage-infected mutant cells, specific early phage proteins are absent or reduced in amount, phage DNA synthesis is depressed by about 50%, specific structural phage proteins, including some tail and collar components, are deficient or missing, and host-cell lysis is delayed and slow . Almost all phage that can overcome the host block carry mutantions that map in functionally undefined 'nonessential' regions of the T4 genome, most near gene 39 . The mutant host strains are temperature sensitive for growth and show simultaneous reversion of the ts phenotype and the inability to propagate T4+ . The host mutations are cotransduced with ilv (83 min) and may lie in the gene for transcription termination factor rho. J Bacteriol, 1980 Sep, 143(3), 1374 - 83 Pleiotropic mutations rendering Escherichia coli K-12 resistant to bacteriophage TP1; Wandersman C et al.; tpo mutations, located at 74 min on the genetic map, rendered Escherichia coli K-12 resistant to TP1, a phage which can use either the OmpF protein or the LamB protein as its receptor . tpo mutants synthesized decreased amounts of OmpF and LamB proteins but increased amounts of the OmpC product, another outer membrane protein . The effect of the tpo mutations in lam B gene expression was transcriptional . It is one facet of the following effect on the maltose regulon: strong decreases in the syntheses of the LamB protein and the periplasmic MalE protein occurred when the regulon was uninduced; a lesser decrease occurred in the syntheses of the LamB protein the MalE protein, and the cytoplasmic MalQ protein (amylomaltase) when the regulon was induced . The tpo mutants were found to be phenotypically identical to the perA mutant recently described by Wanner et al . (J . Bacteriol . 140:229--239, 1979) and to some of the ompB mutants described by Verhoef et al . (Mol . Gen . Genet . 169:137--146, 1979) . Mapping and complementation analysis suggested that these three types of mutations belong to the same cistron . Our results bring to at least four the number of clearly distinct phenotypes which can result from mutations at, or close to, ompB, a locus which appears increasingly complex. Genetics, 1980 Sep, 96(1), 25 - 41 High negative interference and recombination in bacteriophage T5; Beck BN; The process of close recombinant formation in bacteriophage T5 crosses has been studied by examining the structure of internal heterozygotes (HETs), the immediate products of recombination events . The T5 system was chosen because it permits the study of internal heterozygotes exclusively, thus avoiding the ambiguities inherent in previous studies with T4 . The heterozygotes were obtained by the nonselective screening of progeny phage in a prematurely lysed sample from an eight-factor cross . The molecular structure of each HET was inferred from the strand genotypes displayed among its progeny . This investigation presents unequivocal evidence that both overlap and insertion HETs are intermediates in recombinant formation and that insertion HETs are a significant source of close double recombinants . There is evidence suggesting that mismatch repair of overlap HETs could be the source of close triple exchanges . Thus, a significant part, and perhaps all, of the high negative interference for close-marker recombination observed in this system is a direct consequence of the fine structure of the recombinational intermediates . These findings are compatible with recombination models proposed by others, in which a single branched intermediate can give rise to HETs of both the overlap and insertion types. Genetics, 1980 Sep, 96(1), 1 - 24 Mapability of very close markers of bacteriophage lambda; Gussin GN et al.; Recombinant frequency was compared with nucleotide distance in crosses involving markers in either the PRM or the cy region of phage lambda . For each pair of markers, we performed reciprocal four-factor crosses of the following types: (I) A+m1+m2-B- X A-m1-m2+B+; and (II) A+m1-m2+B- X A-m1+m2-B+ . In crosses of type I, the frequency of A+m1+m2+B+ recombinants among total (selected) A+B+ progeny was directly proportional to nucleotide distance between m1 and m2 in the range from 3 to 160 nucleotides . When less than three nucleotides separated m1 and m2, the measured yields of m1+m2+ recombinants were significantly depressed . We also found that the frequency of A+m1+m2+B+ recombinants among total A+B+ progeny was significantly lower (about 10-fold on the average) in crosses of type II than in the corresponding crosses of type I . Since mismatch correction should yield A+m1+m2+B+ recombinants with approximately equal frequencies in type I and II crosses, we suggest: (1) that most m1+m2+ recombinants produced in type I crosses must arise from the formation of heteroduplex structures with a discontinuity (in the source of genetic information) between sites m1 and m2, and (2) that mismatch correction is not a major pathway for production of recombinants for close markers in normal lambda infection. Gene, 1980 Sep, 10(4), 291 - 300 Purification and characterization of the N gene product of bacteriophage lambda; Ishii S et al.; The N protein (pN) specified by bacteriophage lambda is an antitermination factor and is required for phage development . pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from lambda trp transducing phage bearing N- and fed- mutations is pN dependent (Ishii et al., 1980) . The assay has been used to purify pN . We have observed that pN forms a complex with E . coli protein(s) and is dissociated in the presence of urea . The complex is not formed in host bacteria bearing the nusA-nusB- mutations . pN is a basic protein and heat-stable . Using these characteristics, we have purified pN to virtual homogeneity as judged by polyacrylamide gel electrophoresis in the presence of SDS . pN is a monomeric protein and its mol . wt . is approx . 14 000 . The antiterminating activity of pN appears to be enhanced by complex formation with host-encoded protein(s) depending on the nusA and/or nusB gene function. J Virol, 1980 Sep, 35(3), 619 - 28 Use of lambda pMu bacteriophages to isolate lambda specialized transducing bacteriophages carrying genes for bacterial chemotaxis; Kondoh H et al.; A general method for constructing lambda specialized transducing phages is described . The method, which is potentially applicable to any gene of Escherichia coli, is based on using Mu DNA homology to direct the integration of a lambda pMu phage near the genes whose transduction is desired . With this method we isolated a lambda transducing phage carrying all 10 genes in the che gene cluster (map location, 41.5 to 42.5 min) . The products of the cheA and tar genes were identified by using transducing phages with amber mutations in these genes . It was established that tar codes for methyl-accepting chemotaxis protein II (molecular weight, 62,000) and that cheA codes for two polypeptides (molecular weights, 76,000 and 66,000) . Possible origins of the two cheA polypeptides are discussed. Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5226 - 9 Origin of DNA replication of bacteriophage f1 as the signal for termination; Horiuchi K; Restriction fragments that contain the origin of DNA replication of bacteriophage f1 were inserted in vitro into circular f1 DNA molecules to form genomes that contain two origins . This DNA was used to transfect Escherichia coli . Analyses of the DNA of the progeny phage indicated that one origin and the DNA segment located between the two origins in the infecting DNA molecules had been eliminated . This result is interpreted to mean that the nucleotide sequence of the origin for plus (viral)-strand synthesis also serves as the signal for the termination of DNA synthesis. Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5172 - 6 Novel bacteriophage lambda cloning vector; Karn J et al.; A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage lambda vector, lambda 1059 . The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long . Production of recombinants in lambda 1059 requires deletion of the lambda red and gamma genes . The recombinants are therefore spi- and may be separated from the spi+ vector phages by plating on strains lysogenic for bacteriophage P2 . Random fragments suitable for insertion into lambda 1059 are obtained by partial digestion of high molecular weight eukaryotic DNA with Sau3a . This restriction enzyme cleaves at the sequence G-A-T-C and leaves a 5'-tetranucleotide "sticky end." Because G-A-T-C extensions are also produced by BamHI cleavage, these fragments may be annealed directly to BamHI-cleaved lambda 1059 . By using these methods, a set of clones covering the entire Caenorhabditis elegans genome was constructed . DNA segments which include the unc-54 myosin heavy chain gene have been isolated from this collection. J Bacteriol, 1980 Sep, 143(3), 1519 - 23 Selection of Escherichia coli K-12 chromosomal mutants that prevent expression of F-plasmid functions; Silverman P et al.; Chromosomal mutants of Escherichia coli deficient in the expression of F-plasmid functions were selected by mutagenizing F- cells, introducing an F' plasmid into the mutagenized cells by conjugation, and identifying transconjugants resistant to the donor-specific bacteriophage Q beta by a simple spray test . All but 1 of 25 mutants were defective in an extracellular stage of Q beta infection, suggesting that they fail to elaborate F-pili . At least six of these were also deficient as deoxyribonucleic acid donors . More than half of the mutants appear to be altered in peviously undetected chromosomal genes required for the expression of F-related cellular functions. Nature, 1980 Aug 28, 286(5776), 893 - 5 Biosynthesis of hepatitis B virus surface antigen in Escherichia coli; Charnay P et al.; Hepatitis B is a widespread viral disease . In the absence of cell cultures capable of propagating the virus (HBV) an efficient vaccine has been prepared from viral envelopes isolated from the plasma of chronic carriers . The major polypeptide of the envelope is one of molecular weight 25,000 which carries the surface antigen (HBsAg) . Therefore, the biosynthesis of this polypeptide in Escherichia coli may offer an alternative procedure to produce HbsAg free from human proteins . Recently, the HBV genome has been cloned in E.coli . Determination of its primary structure allowed the localization of the gene (called gene S) coding for HBsAg and the synthesis of the core antigen in E.coli has been reported . We have constructed a derivative of bacteriophage lambda carrying a fusion between the beta-galactosidase gene (lacZ) and the HBsAg coding sequence (lambdalacHBs-1) . Infection of E.coli with lambdalacHBs-1 leads to the biosynthesis of a polypeptide of molecular weitht 138,000 carrying antigenic determinants of HBV surface antigen. Biochim Biophys Acta, 1980 Aug 26, 609(1), 61 - 74 In vitro replication of bacteriophage T7 DNA damaged by ultraviolet radiation; Masker WE et al.; The effect of ultraviolet radiation on DNA replication has been examined with an in vitro system capable of replicating intact chromosomes of T7 DNA from an exogenous template . Exposure of the template DNA to ultraviolet radiation resulted in a sharp drop in the amount of in vitro DNA synthesis . The residual replication detected when irradiated templates were used was found to proceed semiconservatively and to result in the production of pieces of duplex DNA approximately the same size as the average distance between pyrimidine dimers . It was also found that prior irradiation of the template inhibits formation of fast-sedimenting concatemer-like DNA structures normally synthesized in vitro . Hybridization studies demonstrated that the product synthesized in vitro from ultraviolet-irradiated templates includes DNA from both the left and right halves of the T7 chromosome . This may mean that after ultraviolet irradiation more than one origin of replication exists. J Biol Chem, 1980 Aug 25, 255(16), 7965 - 72 Bacteriophage T7 DNA replication in vitro . Electron micrographic analysis of T7 DNA synthesized with purified proteins; Wever GH et al.; Extensive replication of duplex T7 DNA is catalyzed in reactions contining T7 DNA polymerase, T7 gene 4 protein, and T7 RNA polymerase . When the product of this reaction is analyzed in the electron microscope, many eye form and Y form replication intermediates are observed . Replication in vitro is not initiated at a single region of the T7 genome . However, we tentatively conclude that initiation does occur preferentially at a few specific sites along the DNA, and that these sites may be near promoters at which the T7 RNA polymerase initiates transcription. J Biol Chem, 1980 Aug 25, 255(16), 7973 - 7 Purification and characterization of leader (signal) peptidase from Escherichia coli; Zwizinski C et al.; Many membrane proteins and secreted proteins are synthesized in precursor form with 15 to 30 additional NH2-terminal residues . These "leader peptides" (pre-pieces, signal peptides) are removed as these proteins cross or insert into cellular membranes . "Leader peptidase" activities which catalyze this cleavage have been detected in crude extracts and found to be dependent on membrane fractions . We now describe a 6,000-fold purification of a leader peptidase from the membranes of uninfected Escherichia coli . This leader peptidase was assayed by its ability to cleave the 23-residue leader peptide from procoat, the precursor to bacteriophage M13 coat protein . Immunoprecipitation and amino acid sequencing showed that this enzyme cleaved procoat to produce authentic coat protein . No factors other than the leader peptidase were found to be required for the conversion of procoat protein to coat protein. J Biol Chem, 1980 Aug 25, 255(16), 7956 - 64 Bacteriophage T7 DNA replication in vitro . Stimulation of DNA synthesis by T7 RNA polymerase; Fischer H et al.; Four T7 products, DNA polymerase, gene 4 protein, RNA polymerase, and DNA binding protein, have been purified from phage-infected cells . It has been previously shown (Hinkle, D . C., and Richardson, C . C . (1975) J . Biol . Chem . 250, 5523-5529; Kolodner, R., and Richardson, C . C . (1978) J . Biol . Chem . 253, 574-584) that two T7 products, DNA polymerase and gene 4 protein, catalyze extensive synthesis on duplex T7 DNA containing single strand breaks . However, the T7 DNA polymerase purified by our procedure does not efficiently contribute in this reaction, although the preliminary evidence suggests that this enzyme may be the native form of the DNA polymerase . Such inefficient T7 DNA synthesis is greatly augmented by adding the third T7 product, namely T7 RNA polymerase . This DNA synthesis apparently requires transcription, since each of the four rNTPs must be present . The rate of synthesis is increased about 2-fold by the addition of T7 DNA binding protein . In contrast to the results obtained when DNA synthesis is initiated at single strand breaks in a duplex DNA molecule, essentially none of the DNA synthesized in the presence of T7 RNA polymerase is covalently attached to the T7 DNA template . We postulate that in this in vitro system, T7 DNA replication is initiated using an RNA primer synthesized by the T7 RNA polymerase. J Virol, 1980 Aug, 35(2), 451 - 65 Partial replicas of UV-irradiated bacteriophage T4 genomes and their role in multiplicity reactivation; Rayssiguier C et al.; A physicochemical study was made of the replication and transmission of UV-irradiated T4 genomes . The data presented in this paper justify the following conclusions . (i) For both low and high multiplicity of infection there was abundant replication from UV-irradiated parental templates . It exceeded by far the efficiency predicted by the hypothesis that a single lethal hit completely prevents replication of the killed phage DNA: i.e., some dead phage particles must replicate parts of thier DNA . (ii) Replication of the UV-irradiated DNA was repetitive as shown by density reversal experiments . (iii) Newly synthesized progeny DNA originating from UV-irradiated templates appeared as significantly shorter segments of the genomes than progeny DNA produced from non-UV-irradiated templates . A good correlation existed between the number of UV hits and the number of random cuts that would be needed to reduce replication fragments to the length observed . (iv) The contribution of UV-irradiated parental DNA among progeny phage in multiplicity reactivation was disposed in shorter subunits than was the DNA from unirradiated parental phage . It is important to emphasize that it was mainly in the form of replicative hybrid . These conclusions appear to justify excluding interparental recombination as a prerequisite for multiplicity reactivation . They lead directly to some form of partial replica hypothesis for multiplicity reactivation. J Gen Virol, 1980 Aug, 49(2), 433 - 6 Spectrophotometric characteristics of cholera phage phi2 DNA; Chaudhuri K et al.; Purified preparations of cholera bacteriophage phi2 were treated with cold phenol and the nucleic acid examined for its hydroxyapatite chromatographic pattern, thermal denaturation profile, base composition and mol . wt. Biophys J, 1980 Aug, 31(2), 271 - 7 Monte Carlo simulation of particle adsorption rates at high cell concentration; Litwin S; A practical method of simulating Brownian diffusion of small particles and their adsorption by randomly placed cells is used to estimate the adsorption process rate constant . The ratio of the rate constant to its classical value, 4 pi RD for dilute perfectly adsorbing spheres, is found to be determined by cellular excluded volume . This ratio varies from 1 for dilute solutions of spheres to approximately 40 for spheres in the maximum possible concentration . A function that usefully estimates the rate constant for all possible values of cell concentration, cell radius, and particle diffusion constant is given for random fields of identical spherical cells . The method is also applied to primitive cubic, body centered, and face centered lattices of spheres . At any given excluded volume and concentration the face and body centered lattices have about the same adsorption rate constant whereas the primitive cubic lattices has a smaller one which is, in turn, greater than that for randomly placed spheres . The results will be useful in determining diffusion limited reaction rates under high excluded volume conditions . These include adsorption by red blood cells at normal concentration, the adsorption of molecules by beads in a column, and adsorption of bacteriophage at very high bacterial concentrations. J Bacteriol, 1980 Aug, 143(2), 1054 - 6 Adenosine 5'-triphosphate leakage does not cause abortive infection of bacteriophage T7 in male Escherichia coli; Remes B et al.; galU and rpsL mutations restore plating efficiency of bacteriophage T7 in male Escherichia coli without suppressing leakage of adenosine 5'-triphosphate pools. J Bacteriol, 1980 Aug, 143(2), 1029 - 30 Chromosomal rearrangements induced by temperate bacteriophage D108; Faelen M et al.; As previously shown for mutator phage Mu-1, to which it is closely related, temperate bacteriophage D108 induces chromosomal rearrangements (replicon fusion and transposition of chromosomal segments) in its host genome. Proc Natl Acad Sci U S A, 1980 Aug, 77(8), 4638 - 42 Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13; Cleary JM et al.; The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained within a 507-nucleotide intergenic region of the viral genome . Chimeric plasmids have been constructed by inserting restriction endonuclease fragments of the M13 intergenic region into the plasmid pBR322 . Replication of these hybrid plasmids, under conditions not permissive for the plasmid replicon, depends on specific segments of the M13 origin region and on the presence of M13 helper virus . Thus M13-infected polA- Escherichia coli can be transformed to ampicillin resistance by hybrid plasmids that have a functional M13 origin . Cells transformed to drug resistance by plasmids bearing M13 origin sequences contain the duplex chimeric DNA at high copy number but do not accumulate significant amounts of single-stranded plasmid DNA . Rare transducing phages carrying single-stranded chimeric DNA are produced and can be detected by their ability to transduce cells to ampicillin resistance . Plasmids containing a 270-nucleotide fragment from the gene II-proximal half of the intergenic region produce transformants at high frequency under nonpermissive conditions . A central Hae III fragment, Hae III-G, containing the nucleotide sequence coding for the RNA primer for the complementary strand and the nicking site for gene II protein, is sufficient for plasmid replication in M13-infected polA- cells but not for high frequency transformation . Additional sequence information on the gene II side of the Hae III-G fragment is necessary for efficient transformation by the plasmid DNA. J Bacteriol, 1980 Aug, 143(2), 887 - 96 Effect of ssbA1 and lexC113 mutations on lambda prophage induction, bacteriophage growth, and cell survival; Vales LD et al.; Escherichia coli strains containing mutations (ssbA1 and lexC113) which affect single-strand deoxyribonucleic acid binding protein have been examined . Among the properties studied were: sensitivity to ultraviolet irradiation and methyl methane sulfonate, temperature sensitivity, induction of prophage lambda by ultraviolet light, temperature, and mitomycin C, and deoxyribonucleic acid synthesis . Strains containing the ssbA1 and lexC113 mutations differ significantly in several of these properties. J Bacteriol, 1980 Aug, 143(2), 569 - 81 Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome; Spratt BG et al.; A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated . The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal . These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA . rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2 . The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis . The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis . A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region . Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E . coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified . The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity . Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane. Mutat Res, 1980 Aug, 72(1), 25 - 30 Induction of mutations in bacteriophage T7 by gamma-rays: no influence of the SOS repair pathway; Bleichrodt JF et al.; Under conditions where the reversion of an amber mutant of bacteriophage lambda by gamma-rays is enhanced by subjecting the irradiated phage to SOS repair, gamma-ray-induced reversion of two T7 ambers is not influenced by this error-prone bacterial repair system . The survival of T7 gamma-irradiated under anoxic conditions is somewhat enhanced by SOS repair, whereas the survival of phage irradiated under oxygen is not affected. J Bacteriol, 1980 Aug, 143(2), 731 - 41 Molecular cloning, correlation of genetic and restriction maps, and determination of the direction of transcription of gnd of Escherichia coli; Nasoff MS et al.; Expression of the gene gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, is regulated by growth rate . Using deoxyribonucleic acid from the specialized transducing phage lambda h80 dgnd his as the source of gnd, we cloned restriction fragments carrying the complete gene and portions of it on the plasmid vector pBR322 . A hybrid plasmid carrying a 3.7-megadalton HindIII restriction fragment from the phage was prepared and found to be gnd+ . Through restriction mapping of this fragment and subcloning segments of it, we prepared a gnd+ hybrid plasmid which carried only 1.85 megadaltons of E . coli deoxyribonucleic acid . A cleavage site for the restriction endonuclease PstI was located on the genetic map of gnd by cloning adjacent EcoRI-PstI restriction fragments and crossing the resulting hybrid plasmids with previously mapped gnd deletion and bacteriophage Mu insertion mutants . A maxicell experiment was used to determine the direction of transcription of gnd, to identify which EcoRI-PstI fragment contains the gnd promote, and to localize th beginning of the structural gene to a region about 850 +/- 150 base pairs from the PstI cleavage site . A fine-structure restriction map surrounding the PstI cleavage site was prepared for endonucleases KpnI, HincII, HaeIII, HpaII, and TaqI. Int J Pept Protein Res, 1980 Aug, 16(2), 111 - 23 Protein conformation wheels: cytochromes and lysozymes; Srinivasan AR et al.; Conformation wheels, directly relating to amino acid sequence to the local torsion angles in a protein molecule, are presented for cytochromes c, c2, c550, and c551 and for lysozymes from hen egg-white and T4 bacteriophage . The circular plots for the cytochrome molecules aid in visualizing the common three-dimensional folding ("cytochrome fold") observed in this family of proteins . Conformation wheels for lysozymes from two different species reveal the characteristic differences in their folding patterns . These novel plots are also useful in storing and comparing the several sets of crystallographic data reported for lysozyme. J Virol, 1980 Aug, 35(2), 551 - 4 Bacteriophage T4-related macromolecular synthesis under restriction of plasmid Rts1; Raghavan N et al.; Rts1 is a plasmid which confers upon the host bacteria the capacity to restrict T4 bacteriophage growth at 32 degrees C but not at 42 degrees C . Pulse-labeling of phage-infected cells showed that Rts1 restricts the synthesis of T1 DNA . Despite efficient restriction of T4 phage growth and DNA synthesis, infected Escherichia coli 20SO harboring Rts1 synthesized both early and late T4 phage RNA . Synthesis of early T4 phage RNA under restrictive conditions (32 degrees C) was almost equal to that found under nonrestrictive conditions, and a lesser, but significant, amount of late T4 phage RNA was made in almost complete absence of T4 DNA synthesis . Moreover, very little, if any, T4 phage-coded lysozyme was detected in the infected E . coli 20SO/Rts1 at 32 degrees C, whereas normal amounts of lysozyme were present at 42 degrees C. Gene, 1980 Aug, 10(3), 261 - 71 Bacteriophage lambda cloning vehicles for studies of genetic recombination; Carroll D et al.; A pair of bacteriophage lambda cloning vehicles has been constructed for use in studies of genetic recombination . These phages, lambda rva and lambda rvb, have the following properties: (1) Each vector has a single HindIII site in the immunity region, at which segments of DNA can be inserted . (2) These HindIII sites are flanked by selectable markers with the following phenotypes: Spi+/- (Fec+/-) to the left, and imm lambda or imm434 to the right . (3) There is essentially no sequence homology between the two phages in this region, so recombination of the markers at reasonable frequency depends on the presence of homologous inserts at the HindIII sites . As a consequence, recovered recombinants must have resulted from a crossover event within the insert DNA . Restriction enzyme maps of the vectors have been determined . Variants of the original vectors have been isolated which permit separate examination of the viral (Red) and bacterial (Rec) generalized recombination mechanisms, and which provide a standard interval to which frequencies of recombination in cloned DNAs can be compared. Gene, 1980 Aug, 10(3), 249 - 59 A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes; Loenen WA et al.; Lambda derivatives are described that can be used for cloning DNA fragments of about 20 kilobase pairs (kb) generated by restriction enzymes EcoRi, HindIII, BamHI, MboI and BglII . Recombinants can be selected by their Spi- phenotype and their propagation is facilitated by the presence of a chi site. Gene, 1980 Aug, 10(3), 195 - 203 Cloning of the replication gene O of E . coli bacteriophage lambda and its expression under the control of the lac promoter; Kuypers B et al.; The expression of the replication gene O of bacteriophage lambda was put under the control of the lac promoter-operator region integrated into the pBR322 cloning vehicle . The new plasmid pKK104 was introduced into minicells and the O gene induced by isopropyl-beta-thiogalactoside (IPTG) . The O protein could be identified as a major component in extracts from these cells, in association with the cell membrane fractions . The molecular weight of the O protein in SDS gels is about 33 000, and it is metabolically unstable but apparently stable upon isolation as a membrane-associated fraction. Nucleic Acids Res, 1980 Jul 25, 8(14), 3157 - 73 Restoration of ligase activity in E . coli K12 lig ts7 strain by bacteriophage Mu and cloning of a DNA fragment harbouring the Mu 'lig' gene; Ghelardini P et al.; Restoration of ligase activity has been observed in E . coli K12 ligts7 strain lysogenic for Mu, in presence as well in absence of lysogenic immunity . This restoration consist in phenotypic reversal of temperature sensitivity of E . coli ligts7 which also regain the ability to sustain the complete growth cycle of T4 lig-phages . It is possible to put under the control of the gal operon the expression of the viral gene responsible for the restoration effect . This new gene of Mu has been named 'lig' . A 5 kb fragment responsible for the reported effects and localized between genes gam and lys of Mu genome has been cloned in pBR322 . This recombinant plasmid used for transforming ligts7 strain restores in it normal behaviour for ligation of Okazaki pieces. J Biol Chem, 1980 Jul 25, 255(14), 7040 - 8 Isolation and characterization of a putative bacteriophage T5 transcription.replication enzyme complex from infected Escherichia coli; Ficht TA et al.; A well defined enzyme comples of approximately 5 X 10(6) daltons that contains phage and host cell components known to be required for the processes of phage transcription and DNA replication has been isolated from bacteriophage T5-infected Escherichia coli cells . In addition to the RNA polymerase of the host cell, the complex contains the phage-encoded: gpC2 which has been implicated genetically as a controlling element of late transcription; gpD9, the DNA polymerase required for T5 DNA replication; the proteins gpD5 (DNA-binding protein), and gpD15 (nuclease) which are both known to be essential for T5 DNA replication and for the initiation of late transcription . The viral gpD5 derived from the purified complex is a phosphoprotein . The enzyme complex also contains, protected from the action of nuclease, double-stranded DNA with an approximate molecular weight of 1 to 2 X 10(6) (2 to 3% of the size of the T5 genome) which is derived preferentially from the center of the T5 DNA molecule . The composition of the enzyme complex suggests that the processes of transcription and replication are integrated in T5-infected cells. Nature, 1980 Jul 24, 286(5771), 418 - 20 Transcriptional control of two gene subclusters in the tRNA operon of bacteriophage T4; Goldfarb A et al.; In bacteriophage T4 DNA, transcription units recognized in vitro by host RNA polymerase consist of promotor-proximal 'immediate early' (IE) genes and promotor-distal 'delayed early' (DE) genes separated from each other by rho-dependent transcription terminators . In vivo, the transition from IE to DE transcription requires phage-specific protein synthesis and can be prevented by chloramphenicol (CAM) . Most of the information about IE/DE transition has been obtained by hybridizaton analyses of mixtures of RNA species synthesized simultaneously on several T4 transcription units (for review see ref . 3) . A useful model for the study of T4 gene expression at the level of primary transcripts and individual gene products is provided by the T4 tRNA operon, a cluster of genes coding for eight T4-specific transfer RNAs and two stable RNAs (species 1 and 2) of unknown function (Fig . 1) . The 10 genes of the tRNA operon are arranged in two subclusters (I and II) with a promotor located about 1 kilobase pair upstream . The primary transcripts and the final gene products of this region have been identified and isolated . Moreover, this genetic region was recently cloned and a part of it sequenced . We describe here the expression of T4 tRNA genes in vivo and in vitro in terms of the IE/DE concept and demonstrate that the two subclusters of the tRNA operon are subject to different modes of control. Biochemistry, 1980 Jul 22, 19(15), 3504 - 15 Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter-containing fragment: evaluation of promoter binding constants as a function of solution conditions; Strauss HS et al.; In this paper we obtain thermodynamic and molecular information about the specific complexes formed between Escherichia coli RNA polymerase holoenzyme and a restriction fragment of T7 D111 DNA carrying the A1 and D promoters . Specific binding was observed at both 0 and 37 degrees C over a side range of pH values and ion concentrations {Strauss, H . S., Burgess, R . R., & Record, M . T., Jr . (1980) Biochemistry (first paper of four in this issue)} . The specific complexes formed at these two temperatures may correspond to the closed and open promoter complexes discussed by Chamberlin {Chamberlin, M . J . (1976) RNA Polymerase (Losick, R., & Chamberlin, M., Eds.) pp 159-161, Cold Spring Harbor Laboratory, cold Spring Harbor, NY} . Promoter binding constants KobsdRP are obtained from competition filter binding data by using a statistical analysis and previously determined values of the nonspecific holoenzyme-DNA binding constant KobsdRD . From the magnitudes of KobsdRP at 0 and 37 degrees C, and the dependences of these binding constants on pH and ion concentrations, we conclude that, under physiological ionic conditions, both the 0 and the 37 degrees C complexes are stabilized to a large extent by the formation of ionic interactions and the accompanying release of counterions and that one or two protonation events (pK approximately 7.4) are required for complex formation in both cases . However, the 0 and 37 degrees C complexes differ in their sensitivity to ion concentrations as well as in the magnitude of KobsdRP, and we conclude that the two complexes are distinct . (More counterion release accompanies formation of the 37 degrees C complex) . Comparisons of the two complexes with one another and with nonspecific holoenzyme-DNA complexes are drawn from the binding data . We have also examined the equilibrium selectivity ratio (KobsdRP/DobsdRD) and find it to be a sensitive function of temperature and ionic conditions . Selectivity of holoenzyme for promoter sites on the promoter-containing fragment is higher at 37 degrees C than at 0 degrees C under the conditions investigated . Selectivity at either temperature is increased by reducing the pH (in the range 6.1-8.6) . At 37 degrees C, selectivity is increased by reducing the salt concentration . Under approximately physiological conditions (0.2 M NaCl and 0.003 M MgCl2, pH 7.4, 37 degrees C), the equilibrium selectivity ratio is found to be of order of magnitude 10(4). Biochemistry, 1980 Jul 22, 19(15), 3516 - 22 Use of difference boundary sedimentation velocity to investigate nonspecific protein-nucleic acid interactions; Lohman TM et al.; The difference boundary sedimentation velocity technique of Schachman and co-workers is demonstrated to be applicalbe to the measurement of binding constants (Kobsd) in the range 10(2)-10(5) M(-1) for the nonspecific interactions of proteins with DNA . The difference technique can reproducibly detect a 2% change in the sedimentation coefficient of the DNA upon binding ligands, corresponding to average extents of association as low as 10 molecules of protein (in the cases of Escherichia coli lac repressor and E . coli RNA polymerase) per molecule of bacteriophage T7 DNA . At these low binding densities, it is plausible to assume that the primary effect of ligand binding is on the buoyant mass of the complex and not on the frictional coefficient of the flexible DNA coil . Binding constants calculated by using this assumption agree well with literature values for the nonspecific interactions of RNase and lac repressor proteins with double-stranded DNA . Advantages of the method are that it is relatively rapid, requires the optical detection of the DNA only, and can be performed on small amounts of sample . The method appears useful for surveying (to an accuracy of +/-50% in Kobsd or +/-10% in log Kobsd) the effects of solution variables on Kobsd of protein-DNA interactions . Applications of the method to the nonspecific interactions of RNA polymerase core and holoenzymes with T7 DNA are discussed. Biochemistry, 1980 Jul 22, 19(15), 3496 - 504 Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter-containing fragment: selectivity exists over a wide range of solution conditions; Strauss HS et al.; The selectivity of binding of Escherichia coli RNA polymerase holoenzyme to a promoter-containing fragment of T7 DNA has been investigated over a range of solution conditions by using a double-label nitrocellulose filter binding assay . A 32P-labeled HaeIII restriction fragment of T7 D111 DNA containing the A1 and D promoters for the E . coli enzyme and a 3H-labeled nonpromoter HaeIII fragment of comparable size were incubated with sigma-saturated holoenzyme and filtered through a nitrocellulose membrane filter . We find that the extent of binding of polymerase to the promoter-containing fragment decreases dramatically with increasing salt concentrations and with increasing pH and increases moderately with increasing temperature in the range 0-37 degrees C . By contrast, the nonspecific interaction of polymerase with the nonpromoter fragment is known to be relatively insensitive to pH and temperature, though a strong function of salt concentration {deHaseth, O . L., Lohman, T . M., Burgess, R . R., & Record, M . T., Jr . (1978) Biochemistry 17, 1612-1622} . Selectivity of binding of RNA polymerase in our assay is demonstrated by a greater fractional retention of the promoter-containing fragment than of the nonpromoter fragment on the filter . We observe selective binding over the temperature range from 0 to 37 degrees C near neutral pH and over a wide range of Na+ concentrations, in the presence or absence of Mg2+ . Because of the different dependences of promoter and nonpromoter binding on pH and temperature, the extent of selectivity increases with increasing temperature and decreases with increasing pH . Quantitative treatment of these binding data {Strauss, H . S., Burgess, R . R., & Record, M . t., Jr . (1980) Biochemistry (second paper of four in this issue)} confirms these conclusions and shows that selectivity is a function of ion concentration as well. Nature, 1980 Jul 17, 286(5770), 218 - 22 Invertible DNA determines host specificity of bacteriophage mu; van de Putte P et al.; The function of the invertible G region of bacteriophage Mu is apparently to confer different host specificities on Mu . Two products of genes S and U, situated in the G region are not needed for the infectivity of Mu G(-) particles . In the Mu G(-) phage the S gene product and the 21-K polypeptide, presumably the product of gene U, are missing . Instead, two other polypeptides with different molecular weights are observed. Nucleic Acids Res, 1980 Jul 11, 8(13), 3043 - 53 The role of bacteriophage T7 gene 2 protein in DNA replication; Mooney PQ et al.; The in vivo function of the gene 2 protein of bacteriophage T7 has been examined . The gene 2 protein appears to modulate the activity of the gene 3 endonuclease in order to prevent the premature degradation of any newly-formed DNA concatemers . This modulation is not however a direct interacton between the two proteins . In single-burst experiments rifamycin can substitute for the gene 2 protein, allowing formation of fast-sedimenting replicative DNA intermediates and progeny phage production . This suggests that the sole function of the gene 2 protein is inhibition of the host RNA polymerase and that the latter enzyme directs or promotes the endonucleolytic action of the gene 3 protein. Science, 1980 Jul 11, 209(4453), 294 - 5 Mutations in a nonessential viral gene permit bacteriophage T4 to form plaques on Escherichia coli valS ts relA; Marchin GL; During viral development bacteriophage T4 modifies the valyl-transfer RNA synthetase of its host Escherichia coli, but the function of the modification has remained elusive . A strain of Escherichia coli has now been identified which is nonpermissive for wild-type bacteriophage T4, but permissive for bacteriophage mutants impaired in the modification reaction . A comparison with other bacteria suggests that nonpermissiveness is due to synthesis of a thermolabile valyl-transfer RNA synthetase and relaxed control of RNA accumulation. Nature, 1980 Jul 10, 286(5769), 182 - 5 A pyrimidine dimer-DNA glycosylase activity associated with the v gene product of bacterophage T4; Radany EH et al.; Mutations in the v gene of bacteriophage T4 are associated with a marked increase in sensitivity to killing by UV radiation at 254 nm, but not to a variety of other forms of base damage to DNA . Early studies from this laboratory provided evidence for a role of the v gene in the excision of pyrimidine dimers (PD) from DNA . Specifically, it was shown that extracts of T4v+-infected Escherichia coli catalyse the formation of single-strand breaks (nicks) and/or alkali-labile sites in UIV-irradiated duplex DNA . Comparable hydrolysis of phosphodiester bonds is not observed with extracts of E . coli infected with the mutant T4v1 (ref . 5) . The product of the v gene has been extensively purified in a number of laboratories; however, convincing evidence of purification to physical homogeneity has not yet been presented. Mol Biol (Mosk), 1980 Jul-Aug, 14(4), 814 - 9 {Circular dichroism study of the reaction between the protein product of bacteriophage f1 gene 5 and synthetic polynucleotides}; Tiaglov BV et al.; The complexes of gene 5 protein (phage f1) with single-stranded and double-stranded polynucleotides were investigated by circular dichroism (CD) . In our experiments the concentration of the protein varied accordingly to polynucleotide . A decrease of the CD amplitude for polynucleotide-protein complexes was shown in all the regions of wavelength studied . The data indicate that the protein is bounded to the polynucleotide . This protein bounds differently to double-stranded polynucleotides containing ribo-ribo, ribo-deoxyribo and deoxyribo-deoxyribo chains . This difference is explained by differences in the form of the secondary structure of polynucleotides, containing ribo-ribo, ribo-deoxyribo and deoxyribo-deoxyribo chains. J Virol, 1980 Jul, 35(1), 249 - 51 In vitro translation of the three bacteriophage phi 6 RNAs; Cuppels DA et al.; In vitro translation of the three single-stranded RNAs transcribed in vitro by bacteriophage phi 6 RNA polymerase revealed that the large RNA codes for phage proteins P1, P2, P4, and P7, the medium RNA codes for P3, P6, and P10, and the smaller RNA for P5, P8, and P9. Arch Microbiol, 1980 Jul, 126(3), 271 - 5 Bacteriophage K7, a double stranded DNA phage that infects strains of Escherichia coli harbouring drug resistance factors of incompatability group W; Morris DW et al.; Bacteriophage K7 is specific for Escherichia coli strains harbouring R factors of incompatability group W, including hybrid coliphage P1-Myxococcus virescens plasmids . The phage has an unusual morphology with an isometric head and long tail of variable length . The tail lengths appear to fall into classes corresoonsing to simple multimers of a unit length . Partially purified lysates of the phage include material that may represent phage particles in the process of biogenesis and other material demonstrating attachment of phage to cell envelope . Newly released phage DNA contains single standed ends . In the course of work . E . coli strains that harbour R factor Sa were found to be apparently restrictive. Proc Natl Acad Sci U S A, 1980 Jul, 77(7), 3908 - 11 Isolation and sequence determination of 5'-terminal oligonucleotide fragments of RNA transcripts synthesized by bacteriophage T3-induced RNA polymerase from T3 DNA; Maitra U et al.; The nucleotide sequence of the 5'-terminal oligonucleotides produced by pancreatic RNase digestion of bacteriophage T3 RNA polymerase (EC 2.7.7.6) transcripts of T3 DNA has been determined . The sequence determination is based upon a simple isolation procedure for the 5'-terminal oligonucleotides . This procedure involves treatment of pancreatic RNase digests of alpha 32P-labeled T3 RNA polymerase transcripts with bovine brain exoribonuclease to remove oligonucleotides with free 5'-hydroxyl termini and then chromatographing the products on hydroxylapatite to resolve the remaining oligonucleotides having 5'-phosphate termini . By application of standard two-dimensional separation and sequence techniques, the major 5'-end sequences deduced were pppGpGpGpApGpApGpApY(Y = pyrimidine nucleoside) and pppGpGpGpApGpApCp . In addition, the sequences of other minor 5'-terminal oligonucleotides observed on homochromatograms were also determined . The sequences of these 5'-oligonucleotides were pppGpGpGpApApCpY, pppGpGpGpApApUpY, pppGpGp(2-4 Gp, 2-3 ApGp)..., and pppGpGpGp... . These results demonstrate that T3 phage-induced RNA polymerase possesses a high degree of specificity in the initiation of RNA chains. J Gen Virol, 1980 Jul, 49(1), 41 - 50 Growth of bacteriophage phiX-174 at elevated temperatures; Dowell CE; The replication of bacteriophage phiX-174 is impaired at temperatures above 40 degrees C . Mutants (ht) that replicate at high temperature were isolated and partially characterized . Wild-type phiX fails to grow at high temperature because, unlike the mutants, it does not make appreciable amounts of single-stranded (ss)DNA . An unusual form of ssDNA, not found in complete virions, is described. J Virol, 1980 Jul, 35(1), 93 - 104 Exclusion of bacteriophage T1 by bacteriophage lambda . II . Synthesis of T1-specific macromolecules under N-mediated excluding conditions; Gawron MC et al.; The results of experiments investigating T1 macromolecular synthesis under N-mediated excluding conditions failed to demonstrate a substantial alteration in the T1 mRNA production in excluding cultures at any stage in the T1 infectious cycle . The number of T1 DNA sequences in the excluding culture was found to be one-third to one-half that found in T1-infected cultures . The most severe reduction in T1-specific macromolecules was seen in protein synthesis . Total incorporation of labeled amino acids was reduced sixfold, and gel experiments confirmed that the T1-specific proteins capable of detection are reduced in excluding cells. J Virol, 1980 Jul, 35(1), 194 - 202 Shutoff of lambda gene expression by bacteriophage T4: role of the T4 alc gene; Pearson RE et al.; Bacteriophage T4 normally contains 5-hydroxymethylcytosine instead of cytosine in its DNA . Multiple mutants of T4 which synthesize DNA with cytosine do not transcribe their late genes due to the action of the T4 alc gene (Snyder et al., Proc . Natl . Acad . Sci . U.S.A . 73:3098--3102, 1976), which is also responsible for unfolding the host nucleoid after T4 infection (Sirotkin et al., Nature {London} 265:28--32, 1977; Tigges et al., J . Virol . 24:775--785, 1977) . It seems reasonable that T4 alc function plays a role in shutting off host transcription, and the observation that some of the RNA made after infection with a T4 alc mutant hybridizes to Escherichia coli DNA (Sirotkin et al., Nature {London} 265:28--32, 1977; Tigges et al., J . Virol . 24:775--785, 1977) supports this hypothesis . Although it is likely that the roles of the alc function in the blocking of some types of transcription and in the unfolding of the host nucleoid are related, it is not known how these effects are achieved or, in fact, whether all types of transcription are affected equally by the alc function . In an attempt to answer these questions, we studied the effect of T4 alc function on bacteriophage lambda transcription and on the structure of intracellular lambda DNA . We found that the alc function is responsible for the shutoff of lambda late transcription but probably not for the shutoff of lambda early transcription . We also found that alc does not block lambda transcription by directly removing the supercoils from circular lambda DNA via either a nicking or topoisomerase activity . Furthermore, we conclude that T4 infection also prevents the translation of non-T4 mRNA because late lambda mRNA's were made after superinfection by a T4 alcs mutant and were of normal length but were not translated into lambda late proteins. Proc Natl Acad Sci U S A, 1980 Jul, 77(7), 4229 - 33 Cloning and characterization of DNA sequences surrounding the human gamma-, delta-, and beta-globin genes; Kaufman RE et al.; The human gamma-, delta-, and beta-globin genes are located within a 30-kilobase (kb) region of DNA, of which only 20% represents the globin genes . We have attempted to define the nature of flanking and intergenic sequences by isolating recombinants containing the human epsilon, both gamma-, or the 3' end of the beta-globin gene from a bacteriophage library of cloned human DNA . Comparison of these recombinants and a recombinant containing the delta- and beta-globin genes (H beta G1) has provided the following results . The epsilon-globin gene is located 14 kb 5' to the G gamma gene . DNA sequence homology between the region containing the two G gamma genes and the delta nd beta gene region is limited to only a few hundred nucleotides which include the globin coding sequences . Repetitive DNA sequences have been found in the region 3' to the beta-globin gene . Sequences located adjacent to the beta-globin gene are repeated in the globin gene region . A repetitive DNA sequence more than 3.2 kb long is repeated frequently in the human genome but is not repeated in the globin gene region in the clones examined. Proc Natl Acad Sci U S A, 1980 Jul, 77(7), 3917 - 21 Nucleotide sequence of the primary origin of bacteriophage T7 DNA replication: relationship to adjacent genes and regulatory elements; Saito H et al.; The 682-base-pair nucleotide sequence between positions 14.45 and 16.15 on the bacteriophage T7 DNA molecule has been determined . We can identify not only the sequence of the primary origin of DNA replication but also the termination of gene 1, all of genes 1.1 and 1.2, the start of gene 1.3, and a number of regulatory sequences . The endpoints of four deletion mutations that extend into this region have been determined . These mutations are inferred to have arisen by recombination between short homologous sequences, three of which ar T7 RNA polymerase promoters . The base changes of four point mutations in gene 1.2 have been identified . The sequence essential for initiation at the primary origin is located between the left endpoints of the two deletions D2 and D303 . Sequence analysis of these mutants assigns the primary origin to a 129-base-pair segment between positions 14.73 and 15.05 . This intergenic segment is A+T-rich (75%) and contains a single T7 gene 4 protein recognition site; it is preceded by two tandem T7 RNA polymerase promoters . A model for initiation of T7 DNA replication is presented. Cell, 1980 Jul, 20(3), 711 - 9 An E . coli gene product required for lambda site-specific recombination; Miller HI et al.; We report characteristics of himA mutations of E . coli, selected for their inability to support the site-specific recombination reaction involved in the formation of lysogens by bacteriophage lambda . The himA allele lies at minute 38 on the chromosome . Three noncomplementing and closely linked mutations define the himA locus; one is a nonsense mutation which shows that the gene product is a protein . HimA mutations reduce both lambda integrative and excisive site-specific recombination . Since dominance tests demonstrate that himA mutations are recessive, it is probable that the himA protein is either a necessary component for site-specific recombination or, alternatively, regulates the expression of such a function . HimA mutations exhibit pleiotropic effects . They reduce integration of phages that have different attachment specificities from lambda and inhibit the growth of phage mu . In addition, himA mutations reduce precise excision of integrated phage mu as well as Tn elements . This pleiotropy suggests that the role of himA protein is nonspecific . Since all of the processes affected by himA mutations ultimately rely on protein-DNA interactions, we suggest that himA protein may act in an auxillary manner to facilitate these interactions. J Virol, 1980 Jul, 35(1), 176 - 83 Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA; Bailey JN et al.; The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases . AvaI, XbaI, BglII, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites . The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards . As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases . The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA . The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7. Gene, 1980 Jul, 10(2), 167 - 75 Location of ribosome-binding sites in the nin5 region of bacteriophage lambda; Chiang T et al.; Seven ribosome-binding sites on DNA have been located within the region defined by the nin5 deletion as well as several ribosome-binding sites on each side of the nin5 region . These were mapped by electron microscopy relative to the end points of the nin5 deletion and two Tn903 transposons, one inserted into gene Rz and another inserted near gene Q . These ribosomes binding sites within the nin5 region may correspond to polypeptide initiation sites for up to seven new dispensible lambda genes. Nucleic Acids Res, 1980 Jun 11, 8(11), 2517 - 25 Melting fine structure of filamentous fungus nuclear DNA; Szecsi A et al.; Melting fine structure of the nuclear DNA isolated from the filamentous fungus Fusarium graminearum Schwabe is presented . Optical melting profiles of nuclear DNA were analyzed by using a combination of curve fitting and derivative techniques . The "melting components" were obtained from the derivative curve by a simple decomposition technique . Differential optical melting curves of unsheared nuclear DNA indicate the presence of 15 "melting components" in filamentous fungus nuclear genome . It should be emphasized that the "melting components" observed here are different from the "thermalites" which can be observed in bacteriophage DNA . The "melting components" reported here represent the separately melting of large "blocks" of fungus nuclear DNA. Biophys Chem, 1980 Jun, 11(3-4), 339 - 43 Polyelectrolyte effects in DNA condensation by polyamines; Bloomfield VA et al.; The conditions required for the counterion induced collapse of T7 bacteriophage DNA are briefly reviewed . Using Manning's counterion condensation theory we calculate a striking unity among collapse conditions: collapse occurs when from 89% to 90% of the DNA phosphate charges are neutralized by condensed counterions . The forces involved in collapsed DNA are investigated with emphasis on electrostatic repulsion . It is concluded that polyelectro