selective affinity chromatography of dna polymerases with associated 3' to 5' exonuclease activities.the use of 5'-amp as a ligand for the affinity chromatography of dna polymerases with intrinsic 3' to 5' exonuclease activities was investigated. the basis for this is that 5'-amp would be expected to act as a ligand for the associated 3' to 5' exonuclease. the requirements for binding of escherichia coli dna polymerase i, t4 dna polymerase, and calf thymus dna polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-amp supports with d ...19846377960
cleavage of trna precursors by the rna subunit of e. coli ribonuclease p (m1 rna) is influenced by 3'-proximal cca in the substrates.trna precursor molecules that contain the cca sequence found at the 3' termini of all mature trnas are cleaved in vitro more readily by m1 rna, the catalytic subunit of e. coli rnaase p, than precursors that lack this sequence. the sensitivity to the cca sequence is not apparent when precursors are cleaved by the reconstituted rnaase p holoenzyme that contains both m1 rna and the protein subunit. these results have been obtained with monomeric precursor molecules encoded by the e. coli and human ...19846380759
defining a bacteriophage t4 late promoter: bacteriophage t4 gene 55 protein suffices for directing late promoter recognition.the rna polymerase from bacteriophage t4-infected escherichia coli, which specifically initiates transcription at phage t4 late promoters, is extensively modified by adp-ribosylation of core subunits and by binding several virus-encoded subunits. we show here that one of these subunits, the phage t4 gene 55 protein, designated gp55, alone endows unmodified rna polymerase core enzyme from uninfected e. coli with the ability to selectively initiate transcription at the phage t4 late promoters, wit ...19846382259
[primary structure of the elongation factor g from escherichia coli. vi. structure of peptides of cyanogen bromide cleavage of the g-factor molecule].peptides obtained as a result of cyanogen bromide cleavage of the g-factor have been studied. all 12 peptides embracing the whole structure of fragment t4 have been isolated. for their amino acid sequence determination, cyanogen bromide peptides have been further cleaved with trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease and bnps-skatole. the complete primary structure of 9 from 12 cyanogen bromide peptides has been determined.19836385997
the effect of a bacteriophage t4-induced polypeptide on host rna polymerase interaction with promoters.after infection of escherichia coli with bacteriophage t4, the host rna polymerase acquires several small phage-induced polypeptides (stevens, a. (1974) biochemistry 13, 493-503) and its alpha subunits get adp-ribosylated by a virus-specific enzyme (zillig, w., mailhammer, r., skorko, r., and rohrer, h. (1977) curr. top. cell. regul. 12, 263-271). the modified polymerase displays changed enzymatic properties including sensitivity to increased salt concentration and a higher transition temperatur ...19846386813
molecular organization of the head of bacteriophage teven: underlying design principles.structure and assembly of the bacteriophage t4 head are described as revealed by results obtained in this laboratory. subunit arrangement of the major coat protein, soc and hoc in the head shell has been determined (figs. 2 and 21). two new approaches for studying the assembly pathway are presented: wild type infection at 19 degrees c and gene 23 cold sensitive mutants. we propose an assembly pathway in which the prehead is formed in one direction, starting from the neck and ending at the distal ...19846399817
rapid hydrolysis of deoxynucleoside triphosphates accompanies dna synthesis by t4 dna polymerase and t4 accessory proteins 44/62 and 45. 19826460024
[intracellular neutralization of bacteriophage t4 by antiphagic serum].specific antiserum, introduced into the spheroplasts of escherichia coli b infected with bacteriophage t4, has been shown to neutralize phage particles formed within the cells.19846524168
human t cell antigens involved in cytotoxicity against allogeneic or autologous chemically modified targets. association of the leu 2a/t8 antigen with effector-target cell binding and of the t3/leu 4 antigen with triggering.monoclonal antibodies (mab) recognizing human t cell differentiation antigens were employed to analyze the role of these antigens on t cell-mediated cytotoxicity against autologous 2,4,6-trinitrophenyl (tnp)-modified targets. the okt3/anti-leu 4 and anti-leu 2a/okt8 mab inhibited t cell-mediated cytotoxicity against autologous or unrelated tnp-modified targets, in the absence of complement and at the effector cell level. these cytotoxic effector cells were t3+, t8+, t11+, t4-. to analyze the rol ...19846610558
effect of proofreading and dam-instructed mismatch repair systems on n4-hydroxycytidine-induced mutagenesis.the role of the proofreading (3' leads to 5' exonuclease) function of t4 dna polymerase and the mismatch repair system of e. coli on n4-hydroxycytidine (oh4cyd) induced mutagenesis was investigated. oh4cyd-induced mutation is strongly suppressed when the proofreading activity increases as a result of the presence of tscb87--antimutator polymerase or elevated temperature (43 degrees c vs 30 degrees c). mutagenic activity of oh4cyd, however, is little, if at all, affected by the presence of the ts ...19826750321
characteristics of a bacteriophage t4-induced complex synthesizing deoxyribonucleotides.a preparation of bacteriophage t4-induced deoxyribonucleotide synthetase complex is described. this very large complex of enzymes can be separated by centrifugation at 100,000 x g, by sucrose step gradient centrifugation, or with molecular exclusion columns. by direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following t4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dcmp deaminase, dctp/dutpase, ...19826757252
[functional activity of short fibrils of bacteriophage t4]. 19826759084
surface charge and hydrophobicity of salmonella, e. coli, gonococci in relation to their tendency to associate with animal cells.the surface charge and hydrophobicity of salmonella typhimurium, escherichia coli and neisseria gonorrhoeae bacteria have been compared with their tendency to associate in vitro with human polymorphonuclear leucocytes (pmnl) and hela cells in culture. it was found that lipopolysaccharide mutations in s. typhimurium or e. coli, yielding core-defective mutants, increased the surface hydrophobicity, as assessed by aqueous two-phase partitioning and hydrophobic interaction chromatography, and promot ...19806782659
binding of ribosomes to linear and circular forms of the 5'-terminal leader fragment of tobacco-mosaic-virus rna.the sequence of the 5'-terminal leader fragment preceding the aug codon in the rna of tobacco mosaic virus (tmv), tomato strain, sps isolate, has been determined. this rna, similarly to the rnas of the u1 and dahlemense strains of tmv [kukla et al. (1979) eur. j. biochem. 98, 61--66] has the 7-methylguanosine(5')triphospho(5')guanosine cap separated from the initiation codon by a long stretch of nucleotides devoid of guanosine residues. the rnase-t1-resistant 73-nucleotide-long leader fragment o ...19816783406
thiol-beta-lactamase: replacement of the active-site serine of rtem beta-lactamase by a cysteine residue.we describe a procedure by which the codon (agc) for the active-site serine-70 of pbr322 beta-lactamase (penicillinase, penicillin amido-beta-lactamhydrolase, ec is altered to that for cysteine (tgc). the pertinent nucleotide bases, a-g-c-a, positions 410-413, of pbr322 are excised by treating a limited hgiai digest of pbr322 with the 3' leads to 5' exonuclease of t4 dna polymerase. the new sequence, t-g-c-a, is inserted in two steps. first, the kpn i molecular linker d(t-g-g-t-a-c-c-a) ...19826818541
recombinational bypass of pyrimidine dimers promoted by the reca protein of escherichia coli.reca protein, in the presence of single-stranded dna binding protein and atp, promotes the complete exchange of strands between circular single-stranded dna containing pyrimidine dimers and a homologous linear duplex, converting the pyrimidine dimer-containing single-stranded dna to a circular duplex. bypass of a pyrimidine dimer during the branch-migration phase of the reaction requires approximately 20 seconds, a rate 1/50th of that in the absence of the dimer. the circular duplex product is s ...19826954468
primary structure of escherichia coli trna uur leu. presence of an unknown adenosine derivative in the first position of the anticodon which recognizes the uu codon series.the primary structure of escherichia coli trna uur le which recognizes the uu series of codons has been determined. the sequence is pg-c-c-c-g-g-a-s4u-g-g-u-g-g-a-a-d-c-gm-c-d-a-g-a-c-a-c-a-a-g-g-g-a-psi-u-n-a-a-ms2i6a-a-psi-c-c-c-c-u-c-g-g-c-g-g-c-g-u-u-c-g-c-g-c-u-g-u-g-c-g-g-g-t-psi-c-a-a-g-u-c-c-c-g-c-u-c-c--g-g-g-u-a-c-c-a. the chain length of trna uur leu is 87 residues, the same as other e. coli trna leu s and t4 phage-coded trna leus. its sequence is especially similar to that of e. coli ...19806986390
effect of bacteriophage t4 nrd mutants on deoxyribonucleotide synthesis in vivo. 19806987228
efficient in vitro replication of double-stranded dna templates by a purified t4 bacteriophage replication system.a wide variety of double-stranded dna templates are replicated extensively in an in vitro dna replication system containing the purified proteins specified by seven t4 bacteriophage dna replication genes (32, 41, 43, 44, 62, 45, and 61). in favorable conditions, this multiprotein system catalyzes the synthesis of several copies of the input dna template in a 30- to 60-min incubation. the replication forks produced in vitro move in a highly processive fashion, at approximately the in vivo rate of ...19806989836
induction of mutations in specific genes of bacteriophage t4 using cloned restriction fragments and marker rescue.saturation of a specific region of a chromosome with conditional lethal mutations becomes increasingly difficult as the genome size of the organism increases. we show that mutagenesis of cloned genes followed by their re-integration into a non-mutagenized organism is a practical way to circumvent this difficulty.19806990202
isolation and characterization of context mutations affecting the suppressibility of nonsense mutations.secondary mutations which increase the efficiency of suppression of nonsense mutations in the riib cistron of bacteriophage t4 have been isolated. these secondary mutations, called context mutations, map at sites very close to the nonsense codon, possibly on the promotor distal side. in context-nonsense double mutants, the amount of suppressed gene product is increased approximately 10-fold. the context mutations examined can act on the uaa (ochre) nonsense allele as well as on the uag (amber) n ...19806991868
transcription termination factor rho and t-even phage development.a functional factor rho is necessary for t-even phage development; phages t2 and t4 require different degrees of rho activity. the rho inactivation by ts-mutations in e. coli causes a reduction of some early protein synthesis and an early formation of some proteins normally typical of a later stage. besides, it weakens the synthesis of some late proteins, impairs the capsid proteins maturation and sharply inhibits phage dna replication in infected cells. however, in the absence of a functional r ...19806991874
a mutant of e. coli that restricts growth of bacteriophage t4 at elevated temperatures.after nitrosoguanidine mutagenesis, a phage host defective (phd) mutant of e. coli hfrh was isolated that supported the growth of t4d wild-type bacteriophage at 30 degrees, but not at 40 degrees or higher. eleven independent spontaneous mutants of t4 (go mutants) were isolated that overcame the growth restriction at high temperature. all of these mutants were located within three percent recombination of a gene 39 amber mutation in the clockwise direction on the standard map. in mixed infections ...19806993283
neighbour and temperature effects on base-analogue-induced mutation in phage t4.the effects of neighbouring base pairs and of temperature on mutation frequencies were measured at nonsense sites in the t4rii region. 2ap-induced at leads to gc transition frequencies are insensitive to nearest-neighbour effects, while 5bu-induced ones are promoted by gc neighbours on the 5' side. the effect of temperature on 2ap- and 5bu-induced mutation frequencies shows no simple dependence on nearest neighbours. these results are incompatible with a unitary mechanism as explanation for the ...19806993847
t4 ribonucleotide reductase. physical and kinetic linkage to other enzymes of deoxyribonucleotide biosynthesis.this laboratory has described a multienzyme aggregate from t4 phage-infected escherichia coli which seems to participate in deoxyribonucleotide biosynthesis and efficient delivery of dna precursors to the replication apparatus. this paper describes improved methodology for isolation of this aggregate, and we present three lines of evidence supporting a role for ribonucleoside diphosphate reductase in functioning of the presumed complex. 1) ribonucleoside diphosphates are readily incorporated int ...19806995453
electrochemical h+ gradient but not phosphate potential is required for escherichia coli infection by phage t4. 19806997076
bacteriophage t4 gene transcription studied by hybridization to cloned restriction fragments. 19806997494
molecular basis for substitution mutations. effect of primer terminal and template residues on nucleotide selection by phage t4 dna polymerase in vitro.the dna-dependent conversion of incorrect deoxynucleoside triphosphate precursors to monophosphates (turnover) by bacteriophage t4 dna polymerase was determined using either poly(da) x (dt) or poly(dg) x (dc) homopolymer templates. competition between correct and incorrect triphosphates for incorporation into dna, and the use of chain-terminating dideoxynucleoside triphosphates enabled us to determine the amount of turnover occurring at the end of each strand of the homopolymer duplex (e.g. amou ...19807002928
effects of growth conditions and mutations in rna polymerase on translational activity in vitro in escherichia coli.the translational capacity in vitro in escherichia coli, using rna from phage r17 or q beta as messenger, is several times higher if the extracts are prepared from cells harvested in early exponential phase or grown under conditions of good aeration compared to if extracts are prepared from cells harvested in a later growth phase or grown under semi-aerobic conditions. in low activity extracts the production of phage replicase protein is preferentially affected. growth of a wild type strain unde ...19807003312
in vitro thermal inactivation of a temperature-sensitive sigma subunit mutant (rpod800) of escherichia coli rna polymerase proceeds by aggregation.a temperature-sensitive mutant sigma subunit (rpod800) purified from escherichia coli was inactivated in vitro by temperatures in excess of 37 degrees c whereas wild type sigma remained stable up to 49 degrees c. both temperature-sensitive and wild type sigma formed multimeric aggregates upon thermal inactivation which were visualized by electron microscopy as polymeric chains. conditions favoring sigma monomer (low sigma concentration and binding to core polymerase) protected temperature-sensit ...19817007376
[serologic affinity and specificity of action of pseudotuberculosis and coli-dysentery phages].the study of serological properties, specificity and the range of action has revealed affinity between y. pseudotuberculosis phages (pst, 3m, kotlyarova, 2344, 2391), some coliphages (t2, t3, t4) and sh. dysenteriae phage (dd iv). the existence of serovar iii of y. pseudotuberculosis phages has been established; to this serovar phage pst belongs. newly isolated 2344 and 2391 belong to serovar i. the problem of the existence of y. pseudotuberculosis phages as an independent group is discussed.19817023155
visualization of dna in various phages (t4, chi, t7, phi 29) by ethidium bromide epi-fluorescent microscopy. 19817028505
control of promoter utilization by bacteriophage t4-induced modification of rna polymerase alpha subunit.after infection of escherichia coli cells, bacteriophage t4 induces several changes in the host dna-dependent rna polymerase. a well-characterized chemical change is a two-step adp-ribosylation of the enzyme's alpha subunit (1). in order to investigate the effect of this change on rna polymerase transcriptional properties in an in vitro system, we have reconstituted the enzyme from separated individual subunits which were obtained from normal or t4-modified rna polymerases. it is demonstrated th ...19817031602
fine mapping of secondary structures of fd phage dna in the region of the replication origin.a synthetic heptaribonucleotide, gaccccc, which is complementary to a unique site on fd bacteriophage dna, primes dna synthesis of fd by t4 bacteriophage dna polymerase. the rate of the gaccccc-primed dna synthesis was not uniform as reflected by the appearance of discrete dna fragments as replication intermediates on an alkaline agarose gel. after 10 minutes of synthesis a significant fraction of the dna product ran as a single band with a length of about 1960 nucleotides. we have isolated this ...19817031605
t4 head assembly and high temperature. 19817036182
assembly of bacteriophage t4 tail fibers: identification and characterization of the nonstructural protein gp57.formation of both the tail fiber and the baseplate of bacteriophage t4 depends on the product of t4 gene 57. a single amber mutation in that gene causes loss of two t4-specific proteins. their molecular weights are 18,000 and about 6,000, respectively, based on their electrophoretic mobilities in sds-polyacrylamide gels. e. coli carrying a cloned t4 dna fragment of about 700 basepairs, which directs the synthesis of the smaller protein only, specifically supports the growth of gene 57 amber muta ...19817038383
caffeine inhibits dna polymerase i from escherichia coli: studies in vitro.caffeine inhibits the activity of dna polymerase i (e. coli) and its proteolytic large fragment in in vitro dna replication system. dna polymerase from micrococcus luteus is also equally inhibited by caffeine. the extent of inhibition was more with the activated adenovirus, t4 and calf thymus dna than with synthetic dna template-primers. results obtained from time-course studies indicated that caffeine inhibition reached maximum by 30 min of incubation. enzyme kinetic studies showed that inhibit ...19827039855
polyamine biosynthesis in escherichia coli: construction of polyamine-deficient mutants.previous work is summarized on the biosynthetic pathway for polyamines in escherichia coli. deletion mutants have been obtained in the various biosynthetic steps, resulting in cells with no polyamines. these mutants grow at one-third the rate of polyamine-supplemented cultures and can serve as suitable hosts for bacteriophages t4, t7, q beta, and f2. the major effects of polyamine deficiency in these polyamine-deficient strains are: (i) these cells do not serve as hosts for bacteriophage gamma a ...19817040834
sealing of gaps in duplex dna by t4 dna ligase.single-strand gaps in dna molecules were found to be a substrate for t4 dna ligase. sealing of the gaps was optimal at the same conditions as ligation of blunt-ended dna molecules. spermidine at a concentration of 2 mm stimulated the ligation of gaps, as well as the joining of dna molecules with cohesive and blunt ends. in addition, spermidine reduced the optimal atp concentration. the ligation of single-stranded gaps was a slow process, reaching a plateau after several hours at 25 degrees c. ap ...19827041091
two alternative mechanisms for initiation of dna replication forks in bacteriophage t4: priming by rna polymerase and by recombination.we show that bacteriophage t4 has two alternative mechanisms to initiate dna replication; one dependent on escherichia coli rna polymerase (rna nucleotidyltransferase, ec, and one dependent on general recombination. continued dna synthesis under recombination-defective conditions was sensitive to rifampin, an inhibitor of rna polymerase. on the other hand, dna synthesis accelerated in spite of the present of rifampin if recombination occurred.19827041114
demonstration of pyrimidine dimer-dna glycosylase activity in vivo: bacteriophage t4-infected escherichia coli as a model approach to the detection of pyrimidine dimer-dna glycosylase activity in living cells is presented. mutants of escherichia coli defective in uvr functions required for incision of uv-irradiated dna were infected with phage t4 denv+ or denv- (defective in the t4 pyrimidine dimer-dna glycosylase activity). in the former case the denv gene product catalyzed the incision of uv-irradiated host dna, facilitating the subsequent excision of thymine-containing pyrimidine dimers. isolation of these di ...19827045391
in vitro transcription of bacteriophage t4 trna gene cluster from two different promoters.analysis of primary transcripts made by escherichia coli rna polymerase on t4 dna containing an intact or partially deleted trna gene cluster demonstrates that the t4 trna genes are transcribed from two promoters differing in their strength. the stronger (p1) and the weaker (p2) promoters are located at distances of 1 kb and 1.5 kb from the trna genes, respectively. selective initiation of individual transcripts with dinucleotides shows that p1 and p2 promoters contain the sequences tat and cac ...19817220346
the catalytic domain of a bacterial lytic transglycosylase defines a novel class of lysozymes.the 70-kda soluble lytic transglycosylase (slt70) from escherichia coli is a bacterial exo-muramidase that cleaves the cell wall peptidoglycan, producing 1,6-anhydro-muropeptides. the x-ray structure of slt70 showed that one of its domains is structurally related to lysozyme, although there is no obvious similarity in amino acid sequence. to relate discrete structural features to differences in reaction mechanism and substrate/product specificity, we compared the three-dimensional structure of t ...19957479697
dependence of frequency of homologous recombination on the homology length.the frequency of homologous recombination is believed to be a linear function of the length (n bp) of homology between dnas. here, the n intercept is believed to be determined by a threshold length below which some physical constraint is effective. in the mammalian gene targeting systems, however, the frequency depends more steeply than linearly on the homology length. to explain both the linear dependence and the steeper dependence, we propose a model where the branch point of a reaction interm ...19957498755
synthesis of circular rna in bacteria and yeast using rna cyclase ribozymes derived from a group i intron of phage t4.studies on the function of circular rna and rna topology in vivo have been limited by the difficulty in expressing circular rna of desired sequence. to overcome this, the group i intron from the phage t4 td gene was split in a peripheral loop (l6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. the group i splicing reactions excise the internal exon rna as a circle (rna cyclase ribozyme activity). ...19947512723
vaccinia virus gene a18r encodes an essential dna helicase.the vaccinia virus a18r protein is a dna-dependent atpase that contains the canonical sequence motifs associated with the dexh group of dna and rna helicases. investigation of a18r protein function during infection indicated it functions in the early and late phases of vaccinia virus transcription. the a18r protein shares sequence similarity with the mammalian dna helicase ercc3. the ercc3 protein has a dual function: it is a component of the transcription factor tfiih and is an essential partic ...19957545242
protection from proteolysis using a t4::t7-rnap phage expression-packaging-processing system.dna coding for bacteriophage t7 rna polymerase (t7-rnap) was inserted into a positive selection-vector form of the t4 genome, placing it under the control of bacteriophage t4 ipiii promoters. the recombinant t4::t7-rnap fusion phage retained infectivity and produced t7-rnap in infected cells. fusion genes were constructed by insertion into a plasmid containing an ipiii (encoding internal protein iii) target portion and a bacteriophage t7 promoter region. when escherichia coli cells containing th ...19957557416
recombination-dependent dna replication stimulated by double-strand breaks in bacteriophage t4.we analyzed the mechanism of recombination-dependent dna replication in bacteriophage t4-infected escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. consistent with prior studies, a pbr322 plasmid, initially resident in the infected host cell, does not replicate following infection by t4. however, the resident plasmid can be induced to replicate when an integrated copy of pbr322 vector is present in the phage chromosome. as expected for recombination-d ...19957592477
[does taxis exist in bacterial viruses?].by way of example of interaction of t4 bacteriophage with e. coli bacterium the scenario of enhancement of sorption of phages on bacteria through the remote mechanism of their cyclic interaction has been considered. an enlargement of the typical phage size, when its fibrillae are opened, underlies this mechanism. modification of the structure of phages also occurs through the medium by release of products of bacterial metabolism into it. allied questions related to investigation of physicochemic ...19957703277
lytic infection of escherichia coli biofilms by bacteriophage t4.escherichia coli 3000 xiii formed biofilms on the surface of polyvinylchloride coupons in a modified robbins device. bacteriophage t4d+ infected cells in the biofilm and replicated. it is commonly held that bacteriophage cannot infect surface-attached bacteria (biofilms) because such bacteria are protected by an exopolymeric matrix that binds macromolecules and prevents their diffusion into the biofilm. to our knowledge this is the first observation that a bacteriophage can infect and multiply w ...19957728652
phage t4 dna [n6-adenine]methyltransferase. overexpression, purification, and characterization.the bacteriophage t4 dam gene, encoding the dam dna [n6-adenine]methyltransferase (mtase), has been subcloned into the plasmid expression vector, pjw2. in this construct, designated pint4dam, transcription is from the regulatable phage lambda pr and pl promoters, arranged in tandem. a two-step purification scheme using deae-cellulose and phosphocellulose columns in series, followed by hydroxyapatite chromatography, was developed to purify the enzyme to near homogeneity. the yield of purified pro ...19957782299
the cooh-terminal domain of the rna polymerase alpha subunit in transcriptional enhancement and deactivation at the bacteriophage t4 late promoter.many activator proteins generate their positive control of transcription through interactions with the cooh-terminal domain of the escherichia coli rna polymerase alpha subunit. we have examined the participation of this alpha-domain in transcriptional enhancement and suppression at bacteriophage t4 late promoters. enhancement is generated by the t4 gene 45 protein, which is the dna-tracking processivity factor of viral dna replication; suppression of unenhanced transcription is generated by the ...19957797594
the pseudodisaccharides: a novel class of group i intron splicing inhibitors.lysinomicin, a naturally-occurring pseudodisaccharide, inhibits translation in prokaryotes. we report that lysinomicin (and three related compounds) are able to inhibit the self-splicing of group i introns, thus identifying pseudodisaccharides as a novel class of group i intron splicing inhibitors. lysinomicin inhibited the self-splicing of the suny intron of phage t4 with a ki of 8.5 microm (+/- 5 microm) and was active against other group i introns. inhibition was found to be competitive with ...19947800490
crystal structure of thymidylate synthase from t4 phage: component of a deoxynucleoside triphosphate-synthesizing complex.thymidylate synthase from phage t4 (t4ts) is part of a complex of several enzymes required for coordinate dna synthesis in infected escherichia coli cells. it has been proposed that similar complexes of enzymes related to dna synthesis are also functional in eukaryotes [pardee, a. b. (1989) science 246, 603-608]. to delineate the role of structure in the function of this complex, we have solved the structure of t4ts as a basis for mapping the complex by mutagenesis. the 3.1 a structure of the un ...19947803410
binding of the junction-resolving enzyme bacteriophage t7 endonuclease i to dna: separation of binding and catalysis by mutation.bacteriophage t7 endonuclease i is a resolving enzyme that selectively cleaves four-way dna junctions, and related branched species. we have isolated mutants of this protein that retain full structural selectivity of binding to four-way junctions, but which are completely inactive as nucleases. this is consistent with a divisibility of structure-selective binding and catalysis. the mutations that inactivate endonuclease i as a nuclease are clustered into the second quarter of the primary sequenc ...19957853409
head-on collision between a dna replication apparatus and rna polymerase transcription in vitro system reconstituted from purified proteins has been used to examine what happens when the dna replication apparatus of bacteriophage t4 collides with an escherichia coli rna polymerase ternary transcription complex that is poised to move in the direction opposite to that of the moving replication fork. in the absence of a dna helicase, the replication fork stalls for many minutes after its encounter with the rna polymerase. however, when the t4 gene 41 dna helicase is present, the r ...19957855590
t4 endonuclease v protects the dna strand opposite a thymine dimer from cleavage by the footprinting reagents dnase i and 1,10-phenanthroline-copper.the glycosylase/abasic lyase t4 endonuclease v initiates the repair of ultraviolet light-induced pyrimidine dimers. this enzyme forms an imino intermediate between its n-terminal alpha-nh2 group and c-1' of the 5'-residue within the dimer. sodium borohydride was used to covalently trap endonuclease v to a 49-base pair oligodeoxynucleotide containing a site-specific cyclobutane thymine dimer. the bound and free oligonucleotides were then subjected to nuclease protection assays using dnase i and a ...19957876117
sliding clamps of dna polymerases.the determination of the structure of the processivity factor (beta subunit) of escherichia coli dna polymerase iii holoenzyme showed that this protein acts to clamp the polymerase onto dna by forming a closed circular structure that can encircle duplex dna (x.-p. kong, r. onrust, m. o'donnell & j. kuriyan. (1992). cell, 69, 425-437). in this review we describe the features of the beta subunit that allow it to be linked tightly but non-specifically to dna, and discuss the surprisingly symmetrica ...19937903401
fibritin encoded by bacteriophage t4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure.the bacteriophage t4 late gene wac (whisker's antigen control) encodes a fibrous protein which forms a collar/whiskers complex. whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions. in this work we show that expression of the cloned wac gene in escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac t4 partic ...19947932704
escherichia coli proteins, including ribosomal protein s12, facilitate in vitro splicing of phage t4 introns by acting as rna address the effect of host proteins on the self-splicing properties of the group i introns of bacteriophage t4, we have purified an activity from escherichia coli extracts that facilitates both trans- and cis-splicing of the t4 introns in vitro. the activity is attributable to a number of proteins, several of which are ribosomal proteins. although these proteins have variable abilities to stimulate splicing, ribosomal protein s12 is the most effective. the activity mitigates the negative effe ...19947958841
a single stranded dna binding protein isolated from hela cells facilitates ni2+ activation of dna polymerases in vitro.the divalent nickel ion (ni2+) is one of several metal ions that can substitute for mg2+ in the activation of dna polymerases in vitro, but usually with very low efficiency. we have purified and partially characterized a ni(2+)-binding protein (p40) from hela cell extracts that can specifically enhance the polymerase activity of dna polymerase alpha (pol alpha) and other dna polymerases in response to ni2+. this protein, with a molecular mass of 40 kda, is a single stranded dna binding protein t ...19947999774
sequence-specific cleavage of oligoribonucleotide capable of forming a stem and loop structure.the precursor of an rna molecule from t4-infected escherichia coli cells (p2sp1 rna) has the capacity to cleave itself at specific positions ((upa (137-138) and cpa (170-171)) within a possible loop and stem structure. this specific cleavage requires at least a monovalent cation and nonionic detergents. to confirm that the sequence-specific cleavage occurs autolytically, we studied the selective hydrolysis of rna using a chemically synthesized 13-mer (guuucguacaaac) having sequences correspondin ...19948051096
intron-containing t4 bacteriophage gene suny encodes an anaerobic ribonucleotide reductase.the function of the suny protein, encoded by an intron-containing gene of bacteriophage t4, has remained hitherto unknown in contrast to the extensively studied self-splicing reaction of the suny intron. here we show that anaerobic t4 infections of escherichia coli induce a ribonucleoside triphosphate reductase activity that is 10-30-fold higher than the bacterial host level of the corresponding enzyme. inactivation of the t4 suny gene (in this communication renamed nrdd) significantly decreased ...19948051113
site-specific incorporation of biophysical probes into proteins.biophysical probes which can detect structural changes in proteins and the interaction of proteins with other macromolecules are important tools in studying protein function. many difficulties remain, however, in introducing probes into proteins site-specifically. here we report the successful site-specific incorporation of a spin-labeled, a fluorescent, and a photoactivatible amino acid into a variety of surface and internal sites in bacteriophage t4 lysozyme by using unnatural amino acid mutag ...19948159678
direct pcr sequencing of the ndd gene of bacteriophage t4: identification of a product involved in bacterial nucleoid disruption.the rapid disruption of the escherichia coli nucleoid after t4 infection requires the activity of the phage-encoded ndd gene. we have genetically identified the sequence encoding ndd. determination of the sequence of a 2.5-kb segment including ndd closed the last significant gap in the sequence of the t4 genome. this analysis was performed on pcr-amplified fragments that were purified by gel-exclusion chromatography and then submitted to linear amplification cycle sequencing. this technology per ...19948163181
a tightly regulated system for overproduction of bacteriophage t4 lysozyme in escherichia coli.bacteriophage t4 lysozyme has been purified using the ni-chelate affinity chromatography technique from overexpressing escherichia coli cells by fusion to an n-terminal 6x his tail. regulation of the lysozyme gene expression has been found to be critical during growth phase of the bacteria by comparing different plasmid constructions. whereas a tac-promoter fusion construct alone did not lead to efficient production of t4 lysozyme because of early cell lysis, an improved repressor sequence and c ...19938251753
genetic and biochemical studies of bacteriophage t4 dna polymerase 3'-->5'-exonuclease activity.dna polymerase exonucleolytic proofreading is important in attaining high fidelity dna replication. one of the most well characterized proofreading activities is the 3'-->5'-exonuclease activity of bacteriophage t4 dna polymerase. we have used genetic analyses and protein sequence comparisons to escherichia coli dna polymerase i to identify amino acids in the n-terminal region of t4 dna polymerase that are required for exonucleolytic proofreading. mutant dna polymerases with amino acid substitut ...19938262948
from the double-helix to novel approaches to the sequencing of large genomes.elucidation of the structure of dna by watson and crick [nature 171 (1953) 737-738] has led to many crucial molecular experiments, including studies on dna replication, transcription, physical mapping, and most recently to serious attempts directed toward the sequencing of large genomes [watson, science 248 (1990) 44-49]. i am totally convinced of the great importance of the human genome project, and toward achieving this goal i strongly favor 'top-down' approaches consisting of the physical map ...19938276270
enzyme interactions involving t4 phage-coded thymidylate synthase and deoxycytidylate hydroxymethylase. 19938304181
dna conformation induced by the bacteriophage t4 uvsx protein appears identical to the conformation induced by the escherichia coli reca protein.the study of homologous genetic recombination has been dominated by the reca protein of escherichia coli, which is involved in dna recombination and repair, as well as phage induction, in vivo. the active form of the reca protein is a helical filament formed on dna in the presence of atp, and within this filament, the dna is extensively stretched to about 5.1 a rise per base-pair and untwisted to about 19 base-pairs per turn. the bacteriophage t4 uvsx protein is only weakly homologous to reca, b ...19938331653
identification of unusual rna folding patterns encoded by bacteriophage t4 gene 60.a 50-nucleotide (nt) untranslated region (coding gap sequence) that interrupts the amino acid coding sequence in t4 gene 60, plus an additional 5 nt upstream and another 3 nt downstream from the gap sequence, shows unusual folding patterns according to rna structure prediction. a predicted highly stable and significant hairpin structure in the 5' half of the gap sequence and a plausible tertiary structural element computed in the 3' part of the gap sequence seem significant by statistical tests ...19938382655
a comparative study of scatchard-type and linear lattice models for the analysis of epr competition experiments with spin-labeled nucleic acids and single-strand binding epr competition formalism is developed which provides relative affinities of proteins for nucleic acids. two models for analyzing protein-nucleic acid interactions, one assuming independent binding sites (model 1) and the other considering site overlap (model 2), are examined with respect to their validity and limitations. the models are employed to derive affinity ratio relationships which are used to calculate the relative affinities of gene 32, gene 5, and ssb proteins for various nucleic ...19938382967
mapping transcription start points with t4 dna polymerase. 19938386296
structure/function analysis of the ala116-->lys121 region of endonuclease v by random targeted mutagenesis.endonuclease v is the product of the denv gene of bacteriophage t4 and is responsible for the recognition and repair of pyrimidine dimers due to uv irradiation of dna. this is accomplished by a two-step mechanism involving incision at the site of the lesion followed by cleavage of the phosphate backbone. in order to better understand this molecule, and to validate our new mutagenesis procedure, we have constructed a series of random mutations within the region ala116-->lys121 using a random targ ...19938441681
introduction of ca(2+)-binding amino-acid sequence into the t4 lysozyme.the 51-62 loop of t4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical ef-hand motif. a ca(2+)-binding site was designed and created by replacing both gly-51 and asn-53 with aspartic acid. the mutant t4 lysozyme (g51d/n53d) was expressed in escherichia coli. the activity of the g51d/n53d-mutant was about 60% of that of the wild-type protein. this mutant can bind ca2+ ions specifically, while the effective dissociation constant was essentially gre ...19938448199
assembly of a functional replication complex without atp hydrolysis: a direct interaction of bacteriophage t4 gp45 with t4 dna polymerase.the seven-protein bacteriophage t4 dna replication complex can be manipulated in vitro to study mechanistic aspects of the elongation phase of dna replication. under physiological conditions, the processivity of dna synthesis catalyzed by the t4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the t4 single-stranded dna binding protein (gp32). the assembly of this t4 holoenzyme requires hydrolysis of atp by the gp44/62 co ...19938475061
concentration evaluation of chromatin in unstained resin-embedded sections by means of low-dose ratio-contrast imaging in stem.quantitative stem with the imaging mode of ratio-contrast was investigated in order to evaluate the local concentration of dna in situ for different kinds of dna plasms in terms of intracellular packing densities (p.d.). the ability of ratio imaging to suppress thickness variations provided the basis to use unstained sections from cryofixed and freeze-substituted material. the dna p.d. within the nucleoid of e. coli was determined to be about 100 mg ml-1. quantitative data concerning the p.d. of ...19938475602
laser-induced protein-dna cross-links via psoralen furanside monoadducts.we have developed a technique for cross-linking dna binding proteins to dna using psoralen furanside monoadducts as photoaffinity probes and a continuous-wave argon ion laser (366 nm) as a light source. several dna binding proteins (t7 rna polymerase, uvrb, single-stranded dna binding protein of escherichia coli, t4 gp32, and reca of e. coli) are shown to cross-link to single-stranded psoralen monoadducted dna oligos differing in length and sequence. increasing fluences of laser light on a fixed ...19938504073
identification, isolation, and characterization of the structural gene encoding the delta' subunit of escherichia coli dna polymerase iii holoenzyme.the gene encoding the delta' subunit of dna polymerase iii holoenzyme, designated holb, was cloned by a strategy in which peptide sequence was used to derive a dna hybridization probe. the gene maps to 24.95 centisomes of the chromosome. sequencing of holb revealed a 1,002-bp open reading frame predicted to produce a 36,936-da protein. the gene has a ribosome-binding site and promoter that are highly similar to the consensus sequences and is flanked by two potential open reading frames. protein ...19938509334
comparison of the rate of excision of major uv photoproducts in the strands of the human hprt gene of normal and xeroderma pigmentosum variant cells.xeroderma pigmentosum (xp) variant patients are genetically predisposed to sunlight-induced skin cancer. fibroblasts from such patients are extremely sensitive to mutations induced by uv radiation, and the spectrum of mutations induced in their hypoxanthine phosphoribosyltransferase (hprt) gene differs significantly from that seen in normal cells. to determine if this uv hypermutability reflects abnormally slow excision repair of cyclobutane pyrimidine dimers (cpd) or 6-4 pyrimidine-pyrimidones ...19968538650
distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of escherichia shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in t4 lysozyme, a protein of known structure. to evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of escherichia coli, a paradigm for po ...19958618889
preliminary crystallographic studies of bacteriophage t4 fibritin confirm a trimeric coiled-coil structure.fibritin, a 52-kda product of gene wac of bacteriophage t4, forms fibrous "whiskers" that connect to the phage tail and facilitate the later stages of phage assembly. preliminary experiments suggest that fibritin is a trimer, and its predominant central part has a parallel alpha-helical coiled-coil structure. to investigate the oligomerization and function of fibritin, we have designed and studied two related deletion mutants, denoted m and e, that consist of its last 75 and 120 amino acids, res ...19968623529
t4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex.after t4 bacteriophage infection of escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call t4 deoxyribonucleoside triphosphate (dntp) synthetase. at least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mda complex. the complex may shuttle dntps to dna replication sites, because replication draws from small pools, which are probably highly localized. several specific protein-protein contacts within the ...19968626661
carboranyl oligonucleotides. 3. biochemical properties of oligonucleotides containing 5-(o-carboranyl-1-yl)-2'-deoxyuridine.boronated oligonucleotides are potential candidates for boron neutron capture therapy, antisense technology, and as tools in molecular biology. the biological properties of dodecathymidylic acids containing one or more 5-(o-carboran-1-yl)-2'-deoxyuridine residues at different locations within the oligonucleotide chain were studied. 5-(o-carboran-1-yl)-2'-deoxyuridine containing oligonucleotides manifested marked increased lipophilicity and resistance to 3'- or 5'-phosphodiesterases compared to t ...19968639534
kinetic mechanism of dna binding and dna-induced dimerization of the escherichia coli rep helicase.the monomeric escherichia coli rep protein undergoes a dna-induced dimerization upon binding either single-stranded (ss) or duplex dna with the dimer being the active form of the rep helicase. using stopped-flow fluorescence, we have determined a minimal kinetic mechanism for this reaction in which rep monomer (p) binds to ss oligodeoxynucleotides (dn(pn)15) (s) by a two-step mechanism to form ps*, which can then dimerize with p to form p2s as indicated: [reaction in text]. this minimal mechanis ...19968652567
identification of a second functional glutaredoxin encoded by the bacteriophage t4 genome.thioredoxins and glutaredoxins are small ubiquitous redox proteins that were discovered as hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. some organisms encode more than one redox protein. in this study, we demonstrate that an open reading frame in the bacteriophage t4 genome, reported earlier and designated as y55.7 (tomaschewski, j., and rüger, w. (1987) nucleic acids res. 15, 3632-3633), encodes a second functional redox protein. gene y55.7 ...19968662944
acute ethanol intoxication and endotoxemia after determine actions of acute intoxication on pathophysiologic responses to trauma, anesthetized and ventilated mongrel pigs received a 20% solution of ethanol (etoh) by an intravenous (iv group; 2 g/kg, n = 8) or an oral (po group; 3 g/kg, n = 12 x 60 minutes) route of administration, or the lactated ringer's vehicle (lr group; n = 12). after 60 minutes, all were subjected to soft tissue injury and 30 to 35% hemorrhage, 60-minute shock, and then resuscitation, with shed blood plus supplemental ...19968676425
in vitro characterization of transcription termination factor rho from escherichia coli rho(nusd) mutants.escherichia coli nusd strains are bacteria that carry mutations in rho, the gene for transcription termination factor rho, that block the growth of phages t4 and lambdar32. we have identified the rho mutation in six independent nusd strains, and although five of the strains have different mutations, with one exception the mutations are in the proposed rna-binding domain of rho. we overexpressed, purified, and characterized the five different mutant rho proteins. all show substantial rna-dependen ...19968757797
prokaryotic dna ligases unwind superhelical dna.we have studied the effect on dna topology of binding of prokaryotic dna ligases (t4 and e. coli) to superhelical or nicked circular dna. performing topoisomerase i-mediated relaxation in the presence of increasing amounts of t4 ligase led to a shift in the topoisomer distribution to increasingly more negative values. this result suggested that t4 ligase unwound the dna and was further substantiated by ligation of nicked circular molecules by e. coli dna ligase in the presence of increasing amou ...19968806663
an n-terminal mutation in the bacteriophage t4 mota gene yields a protein that binds dna but is defective for activation of transcription.the bacteriophage t4 mota protein is a transcriptional activator of t4-modified host rna polymerase and is required for activation of the middle class of t4 promoters. mota alone binds to the -30 region of t4 middle promoters, a region that contains the mota box consensus sequence [(t/a)(t/a)tgctt(t/c)a]. we report the isolation and characterization of a protein designated mot21, in which the first 8 codons of the wild-type mota sequence have been replaced with 11 different codons. in gel retard ...19968892810
domain organization of the escherichia coli rna polymerase sigma 70 subunit.we used limited trypsin digestion to determine the domain organization of the escherichia coli rna polymerase sigma 70 subunit. trypsin-resistant fragments containing sigma 70 conserved region 2 (sigma 70(2)), and carboxy-terminal fragments containing conserved regions 3 and 4 (sigma 70(3-4)) were identified by a combination of amino acid sequencing and mass spectrometry. the domains were studied for partial biochemical functions of sigma 70.sigma 70(2) bound core rna polymerase competitively wi ...19968947564
a common mechanism of action for the n-glycosylase activity of dna n-glycosylase/ap lyases from e. coli and t4.duplex oligonucleotides containing the base lesion analogs, o-methylhydroxylamine- and o-benzylhydroxylamine-modified abasic (ap) sites, were substrates for the dna n-glycosylases endonuclease iii, formamidopyrimidine dna n-glycosylase and t4 endonuclease v. these n-glycosylases are known to have associated ap lyase activities. in contrast, uracil dna n-glycosylase, a simple n-glycosylase which does not have an associated ap lyase activity, was unable to recognize the modified ap sites. endonucl ...19968960131
tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes.phages t4 and e79 were fluorescently-labeled with rhodamine isothiocyanate (ritc), fluoroscein isothiocyanate (fitc), and by the addition of 4'6-diamidino-2-phenylindole (dapi) to phage-infected host cells of escherichia coli and pseudomonas aeruginosa. comparisons of electron micrographs with scanning confocal laser microscope (sclm) images indicated that single ritc-labeled phage particles could be visualized. biofilms of each bacterium were infected by labeled phage. sclm and epifluorescence ...19968987490
cloning and characterization of bacteriophage-like dna from haemophilus somnus homologous to phages p2 and an attempt to identify and characterize components of a heme uptake system of haemophilus somnus, an escherichia coli cosmid library of h. somnus genomic dna was screened for the ability to bind hemin (hmb+). the hmb+ phenotype was associated with a 7,814-bp hindiii fragment of h. somnus dna that was subcloned and sequenced. thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the hmb+ phe ...19979068631
bacteriophage t4 uvsw protein is a helicase involved in recombination, repair and the regulation of dna replication origins.bacteriophage t4 uvsw protein is involved in phage recombination, repair and the regulation of replication origins. here, we provide evidence that uvsw functions as a helicase. first, expression of uvsw allows growth of an (otherwise inviable) escherichia coli recg rnha double mutant, consistent with uvsw being a functional analog of the recg helicase. second, uvsw contains helicase sequence motifs, and a substitution (k141r) in the walker 'a' motif prevents growth of the e.coli recg rnha double ...19979233823
cloning of linear dnas in vivo by overexpressed t4 dna ligase: construction of a t4 phage hoc gene display vector.a method was developed to clone linear dnas by overexpressing t4 phage dna ligase in vivo, based upon recombination deficient e. coli derivatives that carry a plasmid containing an inducible t4 dna ligase gene. integration of this ligase-plasmid into the chromosome of such e. coli allows standard plasmid isolation following linear dna transformation of the strains containing high levels of t4 dna ligase. intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end t ...19979305776
the role of base flipping in damage recognition and catalysis by t4 endonuclease v.the process of moving a dna base extrahelical (base flipping) has been shown in the co-crystal structure of a uv-induced pyrimidine dimer-specific glycosylase, t4 endonuclease v, with its substrate dna. compared with other enzymes known to use base flipping, endonuclease v is unique in that it moves the base opposite the target site extrahelical, rather than moving the target base itself. utilizing substrate analogs and catalytically inactive mutants of t4 endonuclease v, this study investigates ...19979341165
an e. coli b mutation, rpob5081, that prevents growth of phage t4 strains defective in host dna e. coli b tab strain, em121, was isolated that restricts t4 dena (dna endonuclease ii) mutants at 37 degrees c and above, but is permissive for wild-type t4 at all temperatures examined. at 42 degrees c, other mutants affected in nucleic acid metabolism (t4 dexa, rega and uvsw strains) are also restricted. genetic analysis revealed that one mutation (rpob5081) in the rna polymerase beta subunit gene is sufficient for restricting all dena mutants. rpob5081, together with a second linked mutati ...19979418245
role of the acidic carboxyl-terminal domain of the single-stranded dna-binding protein of bacteriophage t7 in specific protein-protein interactions.the gene 2.5 single-stranded dna (ssdna) binding protein of bacteriophage t7 is essential for t7 dna replication and recombination. earlier studies have shown that the cooh-terminal 21 amino acids of the gene 2.5 protein are essential for specific protein-protein interaction with t7 dna polymerase and t7 dna helicase/primase. a truncated gene 2.5 protein, in which the acidic cooh-terminal 21 amino acid residues are deleted no longer supports t7 growth, forms dimers, or interacts with either t7 d ...19989497392
specific peptide-activated proteolytic cleavage of escherichia coli elongation factor tu.phage exclusion is a form of programmed cell death in prokaryotes in which death is triggered by infection with phage, a seemingly altruistic response that limits multiplication of the phage and its spread through the population. one of the best-characterized examples of phage exclusion is the exclusion of t-even phages such as t4 by the e14-encoded lit protein in many escherichia coli k-12 strains. in this exclusion system, transcription and translation of a short region of the major head coat ...19989501186
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