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At least 991 records · Page 55Linked to original sources
PubMed1979

A method which facilitates the ordering of DNA restriction fragments.

The Southern transfer technique has been used to provide a generally applicable method for ordering DNA restriction fragments. It involves electrophoresis of partially digested DNA, transfer to nitrocellulose filter paper and annealing to a 32P-labelled fragment. Only those partials containing that particular fragment will reanneal to the probe and produce bands on autoradiography. The size of each partial in the labelled set is the sum of the sizes of the fragment used as probe and of one or more adjacent fragments. Thus the size of the adjacent fragments can be determined from the size increments of this set of partials. The method is illustrated by the mapping of certain BamHI sites on coliphage 186 DNA.

Chromosome Mapping↗
PubMed1980

Bacteriophage K7, a double stranded DNA phage that infects strains of Escherichia coli harbouring drug resistance factors of incompatability group W.

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.

Coliphages↗
PubMed1975

Immunological recognition of specific antigenic determinants in UV- and gamma-irradiated phage DNA.

Thermally denatured DNA of coliphage T1 after treatment with uv-light (2537 A) and 60Co-gamma rays acts as a hapten with antigenic determinant groups specific for radiation-induced alterations of the macromolecule. After conjugation to methylated bovine serum albumin the DNA becomes immunogenic in rabbits. Antibodies against irradiated DNA do not react with unirradiated single-stranded DNA. Antigen-antibody complexes were demonstrated by CsCl-density gradient centrifugation. The decrease in buoyant density of the DNA is proportional to the amount of antibody protein bound to the antigen. By this means photoproducts as well as alterations due to ionizing radiation in DNA were detected independent of the type of antigen-antibody complex, i.e. precipitating or soluble aggregate.

Antibody Specificity↗
PubMed1976

A sedimentation velocity method for the separation of complementary strands of DNA.

A method is described for the rapid separation of the complementary strands of homogeneous DNA's. The method takes advantage of a difference in sedimentation coefficients between the complementary strands when these have been dissociated and complexed with poly(U-G). The separation procedure is readily carried out for preparative purposes by zone sedimentation in linear 5--20% (w/v) sucrose gradients containing 2 M NaCl. In analytical band sedimentation experiments, concentrated solutions of CsCl have been used to effect the separation of the complexed complementary strands. Sedimentation coefficients corresponding to corrected values at infinite dilution for the cesium salt of the nucleic acid complexes at 25 degrees C (s 25, w) have been obtained for the complexed complementary strands of coliphage lambdab2b5c DNA and for the uncomplexed single strands of this DNA. The relative values of the parameters affecting s 25, w have been determined. Under these conditions, for which preferential hydration of the sedimenting species is not involved, it was found that the more highly complexed strand has sustained a 24% increase in mass and a 28% decrease in frictional coefficient relative to uncomplexed DNA, while the strand binding the lesser amount of RNA has increased in mass by 11% and changed in frictional coefficient by an insignificant amount relative to the uncomplexed species from that of the uncomplexed DNA do not contribute significantly to the differences in s 25, w between the complexed complementary strands.

Centrifugation, Density Gradient↗
PubMed1983

Coat protein conformation in M13 filaments, I-forms and spheroids.

Circular dichroism studies of the filamentous coliphage M13 were carried out to determine conformational changes in the major capsid protein (the B protein) that occur during contraction of the filaments to I-forms and spheroids. The alpha-helicity of the B protein is somewhat lower in the I-forms than in filaments and much lower in spheroids. This conformational change may explain the increased detergent and lipid solubility of both I forms and spheroids relative to filaments.

Capsid↗
PubMed1975

Intermolecular linking and fragmentation of DNA by beta-propiolactone, a monoalkylating carcinogen.

Brief exposure to beta-propiolactone (BPL) increases the sedimentation rate of purified Escherichia coli DNA in neutral and alkaline sucrose gradients. However, when electrophoresed in polyacrylamide-agarose gels, this BPL-treated DNA moves ahead of the control. Longer incubation with BPL gives rise to two new fractions, the first one sedimenting as a heterogeneous material of 6-8S, and the second one of very high sedimentation velocity. In acrylamide-agarose gels, the first fraction is again recovered in the 6-8S area, while the second fraction does not enter the gel at all. The DNA at this stage is hyperchromic in ultraviolet light suggesting that as much as 20% may be denaturated. Coliphage lambda DNA treated briefly with BPL and spread in a protein monolayer appears under the electron microscope as a rigid, extended molecule, up to 15% longer than the control DNA, and usually in compact, folded configurations suggesting intramolecular linking. After longer exposure, localized denaturation associated with single-strand breaks is observed. The single-stranded "whiskers" then interact with other DNA molecules, creating highly complex branched networks of single- and multi-stranded DNA. The possible relevance of these observations to the mechanisms involved in carcinogenesis and mutagenesis is considered.

Alkylating Agents↗
PubMed1985

Conservation of genome form but not sequence in the transcription antitermination determinants of bacteriophages lambda, phi 21 and P22.

Comparisons are made among DNA sequences upstream from terminators in both leftwards and rightwards early operons of related coliphages lambda, phi 21 and P22. These sequences include both left and right determinants of response to phage-coded antitermination proteins, "N", as well as the N structural genes themselves. Despite almost total disparity of DNA sequence, the three genomes can be discerned to include the same elements in the same order and spacing: downstream from the early left promoter are sequentially a site of recognition for host nusA protein, a dyad symmetry "nut" essential for N function in lambda, overlapping sites for processing of the transcript by RNAase III and then the N structural genes; downstream from the cro gene on the right are sites of nusA recognition and nut dyad symmetries homologous to those on the left. Because the N proteins of lambda, phi 21 and P22 do not for the most part complement each other, a specific site of N recognition has been postulated for each N-responding operon. The nut dyad symmetry qualifies as such a site, since the loop of the left dyad in lambda is marked by mutations that block N function leftwards, and since DNA sequences here show close homology between the loops of left and right dyads for each phage, but less if not little homology for different phages.

Adult↗
PubMed1988

Ribonucleoprotein complexes of R17 coat protein and a translational operator analog.

The coat protein of the simple spherical (triangulation no. T = 3) RNA coliphage R17 protects the genomic RNA in the virus particle and acts as a translational repressor of the phage-encoded replicase gene. It has been suggested that these two functions are related and that the translational repression complex serves as a nucleation complex for subsequent assembly of the bacteriophage. We have used a translational operation fragment to examine the relationship between formation of the translational repression complex and the assembly of the protein into T = 3 capsids. In vitro analysis of the aggregation properties of R17 coat protein reveals that binding of the translational operator fragment to the protein dimer triggers polymerization of the protein into T = 3 capsids of well-defined composition. The data further implicate the translational operator in nucleation of assembly and suggest a possible physical-chemical basis of the nucleation step.

Base Sequence↗
PubMed1990

N4 RNA polymerase II sites of transcription initiation.

The RNA polymerase responsible for the synthesis of coliphage N4 middle RNAs, N4 RNA polymerase II, is composed of two subunits of 30,000 and 40,000 molecular weight. It is the smallest DNA-dependent RNA polymerase characterized to date. We have determined the sequences surrounding the sites of in vivo transcription initiation for this enzyme. Two regions of sequence homology are present: a box at +1, 3' AAAT 5', and a box, 3' TTCTGGAC 5' at a variable distance (16 to 24 base-pairs) upstream from +1. Possible mechanisms for recognition of these sequences are discussed.

Base Sequence↗
PubMed1991

Escherichia coli mutations that block transcription termination by phage HK022 Nun protein.

The nun gene product of the lambdoid coliphage HK022 provokes premature transcription termination at, or near, the phage lambda nut sites. Termination by Nun and antitermination by lambda N protein both require the nut sites and Escherichia coli NusA, NusB and NusE proteins. To characterize further the host requirements for Nun termination, we selected host mutations that blocked termination at lambda nutR. In addition to mutations in nusA, nusB and nusE, we obtained mutations in rpoC, encoding the RNA polymerase beta' subunit. The nusA and rpoC mutations suppressed Nun termination but not antitermination by lambda N function. The mutations antagonized Nun only at lambda nutR; termination at lambda nutL occurred in all the mutant strains. Thus, nutL is not functionally equivalent to nutR. We conclude that the host requirements for Nun termination overlap but are not identical with those for N antitermination, and, in particular, that the beta' subunit of RNP may be Nun-specific.

Bacteriophage lambda↗
PubMed1975

Study of genetic effects of high energy radiations with different ionizing capacities on extracellular phages.

The inactivating and mutagenic action of high-energy radiations with different ionizing capacities (gamma-rays, protons, alpha-particles and accelerated ions of 12C and 20Ne) was studied by using coliphages lambda11 and SD as subjects. In particular the role of irradiation conditions (broth suspension, pure buffer, dry samples) and of the host functions recA, exrA and polA was investigated. The dose-response curve of induced mutagenesis was studied by measuring the yield of vir mutants in lambda11 and plaque mutants in SD. The following results were obtained. (1) The inactivation kinetics of phages under the action of gamma-rays and protons was first order to a survival of 10(-7). Heavy ions also showed exponential inactivation kinetics to a survival of 10(-4). At higher doses of 20Ne ion bombardment some deviation from one-hit kinetics was observed. For dry samples of phages the dimensions of targets for all types of radiation were approximately proportional to the molecular weights of phage DNA's. For densely ionizing radiation (heavy ions) the inactivating action was 3-5 times weaker than for gamma-rays and protons. (2) Mutagenesis was observed for all types of radiation, but heavy ions were 1-5-2 times less efficient than gamma-rays. For both phages studied the dose-response curve of mutagenesis was non-linear. The dependence on the dose was near to parabolic for lambda11. For SD a plateau or maximum of mutagenesis was observed for the relative number of mutants at a survival of about 10(-4). (3) Host-cell functions recA and exrA were practically indifferent for survival of gamma-irradiated phage lambda11, but indispensable for mutagenesis. Mutation recAI3 abolished induced vir mutations totally and exrA- reduced them significantly. The absence of the function polA had a considerable influence on phage survival, but no effect on vir mutation yield (if compared at the same survival level). (4) In conditions of indirect action of gamma-rays no vir mutations were induced. This is regarded as evidence that the single-strand breaks formed under indirect action conditions cannot serve as pre-mutational damage in DNA.

Alpha Particles↗
PubMed1984

Identification of the phage gene for host receptor specificity by analyzing hybrid phages of T5 and BF23.

The closely related coliphages T5 and BF23 differ in the receptor used for adsorption to cells. In order to identify phage genes encoding host receptor specificity, hybrid phages were isolated from crosses of T5 and BF23. These hybrid phages were analyzed for their protein composition, kinetics of adsorption to various bacterial strains, and DNA restriction maps. Analyses of the protein compositions of purified tails suggested that only one tail protein was involved in the expression of host specificity. In T5 tails this protein was identified as a minor tail protein of an apparent molecular weight of 67K. A corresponding BF23 protein could not be detected. The DNA region encoding structural proteins was analyzed by digestion with the restriction enzymes BamHI, HindIII, and PstI. The distribution of restriction sites in these recombinants implied that the region affecting host specificity was located at ca. 90% of the T5 genome length. Evidence is presented that this gene is identical to the oad gene previously described (K.J. Heller and D. Bryniok (1984), J. Virol. 49, 20-25). The location of other genes encoding tail proteins is discussed.

Chromosome Mapping↗
PubMed1988

Cloning and generation of a genetic map of bacteriophage N4 DNA.

Analysis of coliphage N4 development has been hindered by the lack of a genetic map. Conventional methods of complementation and recombination have been inadequate because N4 shows very high levels of recombination, which are independent of the host recombination system. We have cloned restriction fragments of the 72-kb genome of N4 and have used these clones to rescue a collection of suppressor-sensitive and temperature-sensitive mutants. After mutations were localized to a small region by marker rescue, complementation groups were defined. In this way several functions essential for transcription and replication have been mapped. Finally, a nonessential 6-kb region of the N4 genome has been identified by characterizing phage deletions isolated after heat and citrate treatment of virions.

Chromosome Deletion↗
PubMed1981

Membrane assembly from purified components. I. Isolated M13 procoat does not require ribosomes or soluble proteins for processing by membranes.

The coat protein of coliphage M13 is an integral protein of the host-cell cytoplasmic membrane prior to its assembly into virions. It is initially synthesized as procoat, a soluble precursor with a 23 amino acid leader sequence at its amino terminus. 35S-labeled procoat accumulates during an in vitro translation reaction that contains 35S-methionine and RNA from M13-infected cells. Radiochemically pure procoat has been isolated from in vitro translation reactions by extraction into an organic solvent and gel filtration through Sephadex LH-60. Radiochemically pure procoat can be used as substrate in rapid and quantitative assays for leader peptidase and for leader peptide hydrolase, an enzyme that degrades the leader peptide after its release from procoat. Procoat solubility, digestion by leader peptidase and processing by membranes are affected by the presence of Mg2+ ion. Isolated procoat is soluble in water at low ionic strength and mildly alkaline pH as well as in detergent solutions. It is cleaved to coat protein by purified E. coli leader peptidase and by inverted E. coli inner-membrane vesicles. These properties of the purified procoat mirror those of the procoat in crude extracts. This suggests that there are no other soluble components that are necessary for the assembly of procoat into the membrane and its conversion to coat; specifically, it provides powerful evidence that protein synthesis is not involved.

Cell Membrane↗
PubMed1981

Membrane assembly from purified components. II. Assembly of M13 procoat into liposomes reconstituted with purified leader peptidase.

The major coat protein of coliphage M13 is an integral protein of the E. coli plasma membrane prior to its assembly into new virus particles. It is generated from its precursor, procoat, by a membrane-bound leader peptidase. We now describe the reconstitution of a highly purified preparation of this enzyme into vesicles of E. coli phospholipids. These vesicles bind procoat made in vitro and procoat isolated from in vitro synthesis. Both the crude and the purified substrates were converted post-translationally to coat protein. A significant proportion of the coat protein becomes inserted into the vesicle bilayer, with the N terminus facing the vesicle interior and the C terminus exposed to the external medium. These results strongly suggest that highly purified leader peptidase from E. coli and phospholipids are the only components necessary to mediate the binding, processing and insertion of this integral membrane protein.

Coliphages↗
PubMed1980

Concentration of bacteriophage lysates by filter chromatography.

High-titered phage stocks are required for carrying out genetic and physicochemical studies on bacteriophages. This study describes a simple method for the concentration of coliphages MS-2 and T2 by charge-modified filters. Phage lysates were first clarified by filtration through serum-coated membrane filters. The clarified lysate was adjusted to pH 6 and passed through a Zeta-plus filter (30 S size). Greater than 99% virus adsorption occurred under these conditions. Adsorbed viruses were successfully eluted by small volumes of 3% beef extract (pH 10) which was subsequently neutralized with 3% beef extract (pH2). A concentration factor as great as 126 could be achieved by this method, with recoveries ranging from 54% to 97% (average = 60%).

Adsorption↗
PubMed1986

A comparison of Selas and membrane filters for the sterilization of bacteriophage preparations.

Lysates of three different coliphage were sterilized by filtration through Selas, Millipore GVWP, and Millipore GS filters. Phage titers were comparable when either the Selas or Millipore GVWP (hydrophilic) filters were used; however, the GVWP filters were faster and could accommodate more lysate before the filters clogged. The Millipore GS (hydrophobic) filters were unsatisfactory.

Coliphages↗