Proc Natl Acad Sci U S A, 1978 Jan, 75(1), 294 - 7 Mutant RNA polymerase of Escherichia coli terminates transcription in strains making defective rho factor; Guarente LP et al.; We have isolated a rifampicin-resistant mutant of Escherichia coli RNA polymerase that restores transcription termination in strains with a defective rho protein . In such strains, the mutant RNA polymerase terminates transcription at normally rho-dependent sites at the end of the trp operon, in bacteriophage lambda, and within the lac operon . In addition, a strain with this mutant RNA polymerase remains viable with an amber mutation in rho, whereas a strain with wild-type RNA polymerase does not . These results suggest that the mutant RNA polymerase can terminate transcription at normally rho-dependent sites in the absence of rho. Proc Natl Acad Sci U S A, 1978 Jan, 75(1), 136 - 9 Role of the host in virus assembly: cloning of the Escherichia coli groE gene and identification of its protein product; Hendrix RW et al.; Correct assembly of the heads of bacteriophages lambda and T4 requires the function of the groE gene of the Escherichia coli host . We have isolated a transducing derivative of lambda, called lambda gt-Ec.groE, that carries a functional copy of the groE gene . Unlike wild-type lambda, this phage is able to form plaques on hosts with a mutant groE gene . We have isolated an amber mutation in the groE gene carried by the phage, and this has made it possible to identify the groE product as a protein of molecular weight 65,000 . In the phage, the groE gene is under the control of an early phage promoter. Proc Natl Acad Sci U S A, 1978 Jan, 75(1), 131 - 5 Identification of a host protein necessary for bacteriophage morphogenesis (the groE gene product); Georgopoulos CP et al.; Mutations in the groE gene of Escherichia coli, which block the correct assembly of the phage lambda head, have been previously described . Many groE mutations exert pleiotropic effects, such as inability to propagate phages T4 and T5 and inability to form colonies at 43 degrees . With the help of the EcoRI and HindIII restrictionenzymes and the appropriate phage vectors, we have constructed two lambda transducing phages, called W3 and H18, that carry the groE+ bacterial gene . Upon lysogenization by phage H18 the groE bacterial mutants recover their gro+ phenotype for both phage growth and the ability to form colonies at 43 degrees . We have identified the groE+ bacterial gene product as a protein of 65,000 molecular weight . Mutants of the W3 transducing phage that were selected on the basis of their ability to propagate on some groE mutant hosts induce the synthesis of a groE protein with altered electrophoretic mobility. Genetika, 1978, 14(1), 129 - 37 {Radiobiological method of studying the structure of the partially diploid area in T4 bacteriophage}; Matvienko NI et al.; The mechanism of cross-reactivation after UV-irradiation was examined using different deletion mutants as recipients . A model of the rescue mechanism of partly diploid area was suggested . The method of cross-reactivation in demonstrated to permit an easy determination of the orientation of duplicated genes. J Virol, 1978 Jan, 25(1), 433 - 5 Escherichia coli mutant temperature sensitive for group I RNA bacteriophages; Schoulaker R et al.; The temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39, carrying a traD(Ts) mutation, is herein described as also being temperature sensitive for group I RNA phages (MS2, f2, and R17) but not for Q beta . Temperature shift experiments showed that the growth of group I phage MS2 in the mutant could be inhibited by a post-penetration event at high temperature . A possible role for the traD cistron of sex factor F in the intracellular development of MS2 is suggested. Cell, 1978 Jan, 13(1), 65 - 71 Maximizing gene expression on a plasmid using recombination in vitro; Backman K et al.; Recombination in vitro has been used to place one or more copies of a strong promoter, the lac promoter, at varying distances from the cl (repressor) gene of bacteriophage lambda on the E . coli plasmid pMB9 . In all constructions, lambda repressor synthesis is driven wholly or predominantly by the inserted lac promoter . One of our fusions directs the synthesis of very high levels of lambda repressor . In this case, the fused DNA encodes a ribosome binding site which is a "hybrid" of lambda and lac sequences . In principle, this method of construction should elicit high levels of expression in E . coli of any gene, whatever its source . We also described strains with different sequence arrangements that, for reasons not completely understood, produce less repressor. J Bacteriol, 1978 Jan, 133(1), 91 - 100 Identification of the Escherichia coli cell division gene sep and organization of the cell division-cell envelope genes in the sep-mur-ftsA-envA cluster as determined with specialized transducing lambda bacteriophages; Fletcher G et al.; From a lysogen with lambda integrated in the leu operon, specialized transducing phages that carry the cell division, murein biosynthesis, and envelope permeability genes located about 0.5 min to the right of leu were isolated . These phages were used to identify the previously undiscovered cell division gene sep . A genetic map proves that sep is located in the sequence leuA sep murE murF murC ddl ftsA envA . A physical map of this region was prepared by heteroduplex analysis of the phage DNAs . Overlapping segments of host DNA extended rightward for as much as 26.4 kilobase pairs from the prophage insertion point (thought to be in leuA) to include all the genes through envA. J Bacteriol, 1978 Jan, 133(1), 172 - 7 Bacteriophage conversion of heat-labile enterotoxin in Escherichia coli; Takeda Y et al.; A temperate phage designated obeta1 (omicron beta) was mitomycin C induced and isolated from heat-labile enterotoxin (LT)-producing Escherichia coli E2631-C2 . Phage obeta1 infected the nonlysogenic, nontoxigenic, mitomycin C-sensitive strain of E . coli K-12 (CSH38) and converted it to lysogeny and enterotoxigenicity . After the establishment of lysogeny, E . coli CSH38(obeta1) produced produced LT and phage particles at maximal levels following mitomycin C induction . The LT Tox+ character is carried by the temperate phage obeta1. Proc Natl Acad Sci U S A, 1978 Jan, 75(1), 50 - 3 Structure of the orgin of DNA replication of bacteriophage fd; Gray CP et al.; An RNA-polymerase-protected DNA fragment of 125 nucleotides from the origin of single-strand to double-strand replication of bacteriophage fd (ori-DNA) was located on the physical map of the phage genome . A stretch of 187 base pairs of DNA including the ori-DNA was sequenced . This DNA segment contains regions with a highly asymmetric pyrimidine/purine distribution next to regions with 2-fold symmetry that form stable hairpin structures in the viral DNA strand. Proc Natl Acad Sci U S A, 1978 Jan, 75(1), 400 - 4 Determination of capsid size by satellite bacteriophage P4; Shore D et al.; Satellite bacteriophage P4 requires all morphogenic gene products provided by a helper phage, such as coliphage P2, to assemble its own capsid, which is one-third the volume of the larger helper capsid . We have isolated a satellite phage P4 sid (size determination) mutant that is unable to direct the assembly of the small wild-type-size P4 capsid . Instead, this mutant produces P4 plaque-forming units with large P2-size capsids which contain two or three copies of the P4 sid1 genome . P4 sid1 is evidently mutated in a protein that is specifically responsible for determining the precise size and symmetry of the structure into which the helper P2 gene products will assemble . In addition, we have found that the physical size of the genome does not appear to play an essential role in the proper assembly of the icosahedral capsid, since the majority of the P4 sid1 plaque-forming units do not contain a complete capsidful of DNA. Eur J Immunol, 1978 Jan, 8(1), 67 - 71 Specificity of helper T cells for different antigens; Kindred B et al.; BALB/c nude mice have been injected with 10(6) congenic thymus cells, a number which allows some, but not all mice to respond to any particular T-dependent antigen . These mice have been tested for their ability to respond to three bacteriophages, T4, T7 and phiX, sheep and horse erythrocytes, and alloantigens of C3H and C57BL/6 mice . The number of mice able to respond to each of these antigens was of the same order of magnitude . Sheep and horse erythrocytes showed cross-reactivity at the T cell level, i.e . the responses to these two antigens were not independent . The same was observed for C3H and C57BL/6 . Otherwise, the responses were independent showing that the antigens are recognize by different populations of specific helper T cells. Appl Environ Microbiol, 1978 Jan, 35(1), 185 - 91 Isolation and characterization of Arthrobacter bacteriophages and their application to phage typing of soil arthrobacters; Brown DR et al.; Seventeen bacteriophages, active against 19 Arthrobacter soil isolates, were isolated from concentrated samples of river water and sewage . Attempts to isolate Arthrobacter bacteriophages from filtrates of broth cultures of the soil isolates or from ultraviolet light-irradiated cultures were unsuccessful . Bacteriophages were not detected in either concentrated or unconcentrated soil extracts . Electron microscopic studies of 11 phages showed morphologies characteristic of Bradley's groups B (exhibited by 9 phages) and C (exhibited by 2 phages) . Moles percent guanine plus cytosine, calculated from the deoxyribonucleic acid density of three phages, ranged from 60.2 to 65.3 . The phages were characterized by their plague and virion morphology, host range, and serological specificity. Acta Biochim Pol, 1978, 25(3), 207 - 19 On the deacetylase activity of Vi bacteriophage III particles; Beilharz H et al.; 1 . Using the complete phage particles as an enzyme, O-acetyl (1 leads to 4)-alpha-D-galacturonan (acetylated pectic acid) as a substrate, and gas-liquid-chromatography for the determination of the acid liberated, the virus-catalysed deacetylation of the polymer was studied . The activity was found to be stable up to about 50 degrees C, and from pH 4.5 to 9, with an optimum at pH 7.8; it was not affected by EDTA, or by 1,10-phenanthroline . The initial reaction velocity (at 37 degrees C) exhibited a simple hyperbolical dependence on the substrate concentration, with Km = 10.5 mM for O-acetyl (independent of virus concentration), and Vmax = 15 nmoles/min and 10(10) plaque forming units . The reaction was, however, rapidly inhibited by a partially deacetylated product (but neither by acetate, nor by pectic acid itself) . 2 . Using the natural substrate, acetylated (1 leads to 4)-2 amino-2-deoxy-alpha-D-galacturonan (Vi polysaccharide, Vi antigen), and a variety of structural analogues, the following conclusions about the substrate specificity of the Vi phage III deacetylase (acetyl-alpha-1,4-galacturonan acylhydrolase) were reached: (a) acetylated galacturonan is as good a substrate as acetylated aminogalacturonan; (b) of the two substrate diastereomers, acetylated alpha-L-guluronan (also 1 ax leads to 4 ax-linked units, but with axial acetyl residues at C-3), and beta-D-mannuronan (1 eq leads to 4 eq-linkages, and axial acetyl groups at C-2), only the former was acted upon, possibly indicating a specificity for the conformation of the polymer rather than for the configuration of the single residues; (c) all acyl analogues tested, O-monofluoroacetyl, O-propionyl, and O-butyryl galacturonan, were inert, showing a high degree of specificity for O-acetyl; (d) the oligomers, acetylated tri- and digalacturonic acid, as well as methyl-alpha-D-galacturonide, were still deacetylated, although more slowly, demonstrating tolerance of the enzyme of substrate size. J Biol Chem, 1977 Dec 10, 252(23), 8498 - 053 Priming of deoxyribonucleic acid synthesis on phage fd and phiX174 templates by oligoribonucleotides contaminating nucleoside 5'-triphosphates; Masamune Y et al.; Commercial preparations of guanosine 5'-triphosphate and deoxyguanosine 5'-triphosphate are contaminated with oligoribonucleotides 4 to 6 residues in length . The oligoribonucleotides can be separated from the nucleoside 5'-triphosphates by chromatography on DEAE-Sephadex A-25 or by gel filtration through Sephadex G-25 . The oligoribonucleotides are effective primers for the DNA polymerase of bacteriophage T7 on the single-stranded circular DNAs of phage fd and phiX174; they are covalently attached to the 5' terminus of the newly synthesized DNA . The priming activity is specific; the oligoribonucleotides do not serve as primers for DNA polymerase I of Escherichia coli or for the DNA polymerase induced by phage T4. J Biol Chem, 1977 Dec 10, 252(23), 8708 - 12 Mechanism of T5-induced DNA polymerase . II . Characterization of the dead-end complex; Das SK et al.; We have shown that bacteriophage T5-induced DNA polymerase replicates short primer-templates (400 to 600 nucleotides long) at a rapid rate initially, followed by a slower rate sustained for much longer periods (Das, S . K., and Fujimura, R . K . (1977) J . Biol . Chem . 252, 8700-8707) . In order to explain the slower steady rate and the results of polymer-challenge experiments, we conjectured the presence of a "dead-end complex" formed by the enzyme with the primer-template at the end of the primer elongation process . In this communication we present evidence which indicates that the presumed complex shows a first order kinetics of decay with a half-life of 3.5 min at 37 degrees . Energies of activation for the steady phase of synthesis and the decay of the dead-end complex were both found to be about 23 kcal/mol . This indicates that the dissociation of the aforesaid complex might be the rate-limiting step during the steady phase of synthesis . Correlation between the salt-induced reduction in the half-life of the complex and the increase in the steady rate of synthesis is in agreement with the above mentioned possibility. J Biol Chem, 1977 Dec 10, 252(23), 8700 - 7 Mechanism of T5-induced DNA polymerase . I . Replication of short primer templates; Das SK et al.; Bacteriophage T5-induced DNA polymerase shows an initial phase of rapid synthesis, followed by a slower steady rate for much longer periods, with short DNA primer-templates (400 to 600 nucleotides long), in vitro . On extrapolating the line of steady rate back to 0 min, an intercept is obtained on the ordinate . With large DNA primer-templates, such as denatured T5 DNA (average chain length approximately 50,000 bases), the rate of synthesis remains constant and is equal to the initial rate obtained with short primer-templates . The zero time intercept was proportional to the amount of enzyme used and independent of temperature . Polymer challenge experiments indicate that the initial phase of rapid synthesis can be attributed to the processive mode of synthesis by T5 DNA polymerase . After synthesizing a stretch of DNA processively for about 200 nucleotide residues, the enzyme apparently forms a "dead-end complex" with the primer-templates used and must dissociate from the primer-template in order to resume synthesis . The average size of the product made processively, during various phase of synthesis, remains invariant and is in good agreement with the size of the zero time intercept per enzyme molecule. Science, 1977 Dec 9, 198(4321), 1051 - 6 Physical structure of the replication origin of bacteriophage lambda; Denniston-Thompson K et al.; The nucleotide sequence of part of the replication region of wild-type bacteriophage lambda and of four mutants defective in the origin of DNA replication (ori-) has been determined . Three of the ori- mutations are small deletions, and one is a transversion . The sequence of the origin region, defined by these mutations, contains a number of unusual features. Science, 1977 Dec 9, 198(4321), 1046 - 51 Genetic structure of the replication origin of bacteriophage lambda; Furth ME et al.; A fragment of bacteriophage lambda DNA produced by the restriction endonuclease Eco RI and extending from the immunity region to a point inside gene O is found to have a fully functional origin of replication . Seven ori- mutations of lambda cluster in a small region just to the left of the Eco RI cleavage site which defines the right end of this fragment . These mutations lie within gene O. Science, 1977 Dec 9, 198(4321), 1041 - 6 Construction of chimeric phages and plasmids containing the origin of replication of bacteriophage lambda; Moore DD et al.; Segments of the replication control region of bacteriophage lambda (lambda) and lambda mutants defective in replication were attached in vitro to the phi80 phage vector Charon 3 and to the plasmid vector mini Col El (pVH51) . The chimeric phages and plasmids have been used to localize the origin of lambda DNA replication and to facilitate a structural analysis of the lambda replicator. Mol Gen Genet, 1977 Dec 9, 157(3), 333 - 9 Overproducing araC protein with lambda-arabinose transducing phage; Steffen D et al.; Escherichia coli infected with bacteriophage lambda-arabinose transducing phage were tested as sources of araC protein . Infection of cells with such phage produces an intracellular concentration of araC protein up to 100 times that present in wild-type E . coli, apparently resulting from fusion of the araC gene to bacteriophage lambda promoters . Lysates from these phage-infected cells may be fractionated to yield another 100-fold enrichment in araC activity so that the total enrichment is 10,000-fold . A nonsense mutation in araC provided proof of the identification on gel electrophoresis of a band in the purified material . Biologically active araC protein is a dimer with 28,000 M.W . subunits . The araC gene in these phage replaces the int-xis genes but is oriented in the opposite direction . Nonetheless, it appears to be transcribed in this position by the phage promoter pr via transcription the long way around . Furthermore, because araC gene is in this position, we were able to isolate phage on which the araC gene was under phage late gene control by deletion of the late gene transcription stop signals in the b2 region. Biochim Biophys Acta, 1977 Dec 1, 471(2), 169 - 76 Studies of asymmetric membrane assembly; Zwizinski C et al.; The major capsid protein of M13 bacteriophage is incorporated at each stage of infection into the host plasma membrane with its amino terminus exposed on the outer surface . Purified M13 coat protein is incorporated with the same asymmetry into synthetic phosphatidylcholine vesicles formed near the Tm of the lipid by a cholate dilution technique . We now report that the lipid in the pre-dilution mixture exists as mixed micelles of uniform size . Prior to dilution, the coat protein is present in at least two states of aggregation, both of which behave similarly in the model membrane assembly reaction . No detectable lipid-protein interaction occurs prior to dilution . Upon dilution there is rapid production of small closed vesicles and coat protein is converted to a chymotrypsin-resistant form, presumably reflecting its incorporation into these vesicle bilayers . Formation of large (greater than 6000 A diameter) vesicles occurs slowly with preservation of coat protein asymmetry and internal volume . A model for this assembly reaction is proposed. J Immunol, 1977 Dec, 119(6), 2114 - 9 Genetic control of the immune response to phage fd . IV . Complementation between H-2-linked Ir genes; Kolsch E et al.; The immune response to bacteriophage fd has been tested in congenic resistant strains of mice . B10 mice are high responders; B10.BR and B10.D2 mice are low responders . B10.A mice with the recombinant H-2a haplotype as well as heterozygous H-2k/d (B10.BR X B10.D2) F1 mice respond with high antibody titers, indicating the existence of two complementing Ir genes . From the results obtained with recombinant and F1 hybrid mice, Irfdalpha can be localized to the right of the crossover between the IE and IC subregions, Irfdbeta to the left of IB, presumably at IA . Complementation is observed with selected pairs of alleles only, suggesting alpha-beta coupled complementation . In addition to genes in the H-2 complex, there are non-H-2 background genes modulating H-2 regulated anti-fd response . B10.BR mice lack primary IgM and IgG antibodies, yet are able to mount a secondary IgG anti-fd response, giving evidence for intact memory formation in this low responder strain . The combined data are most easily interpreted by assuming that both H- and non-H- genes modulate the effective immunogenic dose of antigen. Nucleic Acids Res, 1977 Dec, 4(12), 4151 - 63 Role of bacteriophage T7 DNA primase in the initiation of DNA strand synthesis; Scherzinger E et al.; Bacteriophage T7 DNA primase (gene-4 protein, 66,000 daltons) enables T7 DNA polymerase to initiate the synthesis of DNA chains on single-stranded templates . An initial step in the process of chain initiation is the formation of an oligoribonucleotide primer by T7 primase . The enzyme, in the presence of natural SS DNA, Mg++ (or Mn++), ATP and CTP (or a mixture of all 4 rNTPs), catalyzes the synthesis of di-, tri-, and tetraribonucleotides all starting at the 5' terminus with pppA . In a subsequent step requiring both T7 DNA polymerase and primase, the short oligoribonucleotides (predominantly pppA-C-C-AOH) are extended by covalent addition of deoxyribonucleotides . With the aid of primase, T7 DNA polymerase can also utilize efficiently a variety of synthetic tri-, tetra-, or pentanucleotides as chain initiators . T7 primase apparently plays an active role in primer extension by stabilizing the short primer segments in a duplex state on the template DNA. Eur J Biochem, 1977 Dec, 81(3), 453 - 63 Studies on DNA unwinding . Proton and phosphorus nuclear-magnetic-resonance studies of gene V protein from bacteriophage M13, interacting with d(pC-G-C-G); Garssen GJ et al.; The interaction of gene V protein from bacteriophage M13 with the self-complementary tetranucleotide d(pC-G-C-G) was studied by 1H and 31P nuclear magnetic resonance . It is shown, using the hydrogen-bonded proton resonances of the Watson-Crick base pairs as a probe, that the protein is able to unwind the small double-helical fragment even at 0 degrees C . Binding of the tetranucleotide causes changes in the aromatic part of the 1H NMR spectrum of the complex, suggesting that aromatic residues, most likely tyrosines, take part in the protein.nucleic-acid interaction . From the 31P NMR spectra of the protein.nucleic-acid complex it follows that the pK value of the 5'-terminal phosphate is lower than for the free nucleic acid species . Moreover, it could be shown that the exchange of the protein between nucleic acid substrates is fast . Combination of these measurements has led us to derive a mechanism of unwinding on the tetranucleotide level . To a large extent the unwinding is determined by fluctuations in the double-helical DNA structure. Mol Biol Rep, 1977 Dec, 3(6), 451 - 7 Restriction endonuclease analysis of the transducing bacteriophage lambda RIF d18; Boros I et al.; The lambda rif d 18 bacteriophage carries essential parts of the E . coli genome which can not be mapped by conventional methods . The phage DNA was analysed with four restriction endonucleases (endo R . BamHI, Sall, Hpal and EcoRI) and a physical map was constructed. J Virol, 1977 Dec, 24(3), 794 - 804 Suppression of gene 49 mutations of bacteriophage T4 by a second mutation in gene X: structure of pseudorevertant DNA; Shah DB et al.; Mutations in gene 49 of bacteriophage T4 were suppressed by a second mutation in gene X . Mapping studies located gene X between genes 41 and 42 . Complementation results indicated that mutations in FdsA gene (a suppressor of gene 49 mutants) were in gene X . The intracellular pseudorevertant DNA was examined for unusual properties which could explain its successful encapsidation . After the in vivo inactivation of a temperature-sensitive gene 32 (DNA unwinding) protein, the intracellular pseudorevertant DNA was converted into DNA pieces of approximately genome size . A similar conversion was observed after in vitro digestion of pseudorevertant DNA with single-strand-specific S1 endonuclease . Appreciable quantities of oligomeric intermediates were not produced during this conversion process . These data indicate that pseudorevertant DNA contains sizable single-stranded gaps and has a conformation similar to that of wild-type DNA . The results further suggest that the suppression of gene 49 mutant abnormal DNA phenotype and the encapsidation defect by a second mutation in gene X is associated with the formation of sizable single-stranded gaps . These studies raise the possibility that single-stranded gaps may be involved directly in the DNA encapsidation process, or may act indirectly as a consequence of their effect on the organization of intracellular DNA. Proc Natl Acad Sci U S A, 1977 Dec, 74(12), 5255 - 9 Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3); Struhl K et al.; A cloned segment of yeast DNA containing the structural gene for imidazoleglycerolphosphate dehydratase (D-erythro-imidazoleglycerolphosphate hydro-lase, EC 4.2.1.19) is transcribed and translated in Escherichia coli with sufficient fidelity to produce functional enzyme . This segment of yeast DNA was isolated as a viable molecular hybrid of bacteriophage lambda (lambdagt-Sc2601) which complements a nonrevertible hisB auxotroph of E . coli lacking dehydratase activity . The equivalent segments of DNA cloned from two independent his3 mutants of yeast lacking IGP dehydratase activity do not complement the hisB auxotroph . The two nonfunctional his3 alleles cloned in bacteriophage lambda can be recombined in E . coli to generate a hybrid phage which complements the hisB auxotroph . The dehydratase activity produced in E . coli by the cloned segment of yeast DNA strongly resembles the activity found in yeast. J Virol, 1977 Dec, 24(3), 746 - 60 I protein: bacteriophage T7-coded inhibitor of Escherichia coli RNA polymerase; Hesselbach BA et al.; Bacteriophage T7-coded inhibitor of Escherichia coli RNA polymerase, termed I protein, was purified from an inactive E . coli RNA polymerase-I protein complex isolated from phage T7-infected cells . A molecular weight of about 7,000 to 9,000 was assigned to the purified I protein by acrylamide-sodium dodecyl sulfate gel electrophoresis, Sephadex G-50 gel filtration, and glycerol gradient centrifugation analysis . I protein inhibits initiation of RNA synthesis by directly binding to the RNA polymerase holoenzyme and prevents the binding of the enzyme to the promoter sites on the template T7 DNA . However, once a highly stable transcriptional preinitiation complex between RNA polymerase holoenzyme and T7 DNA is formed at the promoter site on T7 DNA in the absence of nucleoside triphosphates, I protein does not inhibit the initiation of RNA synthesis by this preformed complex upon addition of nucleoside triphosphates . RNA synthesis by the core RNA polymerase and the binding of core RNA polymerase with template DNA are not inhibited by I protein, although a partial association between the core enzyme and I protein can be observed . I protein does not bind to sigma factor or T7 DNA . Therefore, binding of I protein with the RNA polymerase, which results in the inhibition of initiation of RNA synthesis, requires the presence of sigma factor in the RNA polymerase holoenzyme form. J Bacteriol, 1977 Dec, 132(3), 757 - 63 Isolation of a lambdadcys transducing bacteriophage and its use in determining the regulation of cysteine messenger ribonucleic acid synthesis in Escherichia coli K-12; Fimmel AL et al.; A defective specialized lambda transducing phage carrying the cysJ, cysI, cysH, and cysD genes has been isolated from a secondary-site lysogen . Deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization studies utilizing this phage have been carried out to detect cysteine-specific messenger RNA (cys mRNA) synthesized in vivo . A vivo . A 3.5- to 9-fold increase in the rate of synthesis of cys mRNA has been detected in the derepressed wild-type (Cys+) strain grown on glutathione compared with a repressed control grown on cystine . Pleiotropic cysE and cysB mutants grown on glutathione were found to possess rates of synthesis of cys mRNA that were significantly lower than their derepressed isogenic parent . The addition of O-acetyl-L-serine to the cysE strain produced a 5.5-fold increase in the rate of synthesis of cys mRNA . These results indicate that cysteine biosynthesis is controlled at the level of transcription by the inducer O-acetylserine, the cysB protein and cyst(e)ine. Proc Natl Acad Sci U S A, 1977 Dec, 74(12), 5514 - 18 New method for localizing proteins in periodic structures: Fab fragment labeling combined with image processing of electron micrographs; Aebi U et al.; Fab fragments prepared from antisera directed against purified bacteriophage T4 structural proteins and head-related structures were used to label proteins on the surface of T-even giant phage capsids . Optically filtered electron micrographs of the Fab-labeled capsids reveal both the location of specific proteins within the capsomeres and differing conformational states of the protein subunits . We describe parameters affecting the utility of this technique for the study of molecular organization and protein conformation in periodic biological structures. J Virol, 1977 Dec, 24(3), 775 - 85 Slow switchover from host RNA synthesis to bacteriophage RNA synthesis after infection of Escherichia coli with a T4 mutant defective in the bacteriophage T4-induced unfolding of the host nucleoid; Tigges MA et al.; Most, if not all, host RNA synthesis was shut off after infection of Escherichia coli strain B/5 with a bacteriophage T4 multiple mutant defective in the abilities to induce (i) unfolding of the host nucleoid (unf-), (ii) nuclear disruption (ndd-), and (iii) host DNA degradation (denA-, denB-) . The shutoff of host RNA synthesis and turn-on of phage RNA synthesis were slower after infection of E . coli with unf- phage than after infection with unf+ phage . This delay in the switchover from host RNA synthesis to phage RNA synthesis in unf- infections did not result in a measurable delay in the onset of nuclear disruption, deoxyribonucleoside monophosphate kinase synthesis, or DNA synthesis . unf39 did not complement alc (allows late transcription on cytosine-containing DNA) mutants, supporting the proposal of Sirotkin et al . {Nature (London) 265:28-32, 1977} that alc and unf are possibly the same gene. Mol Gen Genet, 1977 Nov 29, 157(2), 139 - 47 Purification and properties of a lambda operator-binding protein which is expected to be autorepressor (tof protein) from E . coli carrying lambdadv plasmid; Murotsu T et al.; In order to study the mode of action of the tof gene product, which is an "autorepressor" of the bacteriophage lambda and plasmid lambdadv, we have purified a DNA-binding protein which is specifically produced in bacteria carrying lambdadv . This protein possesses characteristics expected for the product of the tof gene, since it is produced under conditions where cI-repressor is not made, and since it binds to oL and oR operators on the lambda phage genome . The molecular weight of the native protein is 16,000-17,000 daltons, and the monomeric molecular weight as measured by gel electrophoresis in the presence of sodium dodecyl sulfate is about 10,000 daltons . Denaturation and renaturation experiments demonstrated that the native protein is a dimer of 10,000-dalton monomers . The lambdaDNA-specific binding protein is not produced in cells carrying i21dv or o80dv. J Biol Chem, 1977 Nov 25, 252(22), 8254 - 7 U-G-A suppressor of bacteriophage T4 associated with arginine transfer RNA; Kao SH et al.; A U-G-A suppressor of bacteriophage T4, designated psu4+op, has been isolated and characterized . The transfer RNA species previously shown to have an anticodon sequence complementary to arginine codons is affected by the psu4+op mutation . Wild type and psu4+op arginine tRNAs have the same sequence except for their anticodons, where U-C-U in the wild type species is mutated to U-C-A in the psu4+op species . This mutation is believed to confer U-G-A suppressor activity on the psu4+op arginine tRNA. J Biol Chem, 1977 Nov 25, 252(22), 8245 - 53 Nucleotide sequence of an arginine transfer ribonucleic acid from bacteriophage T4; Mazzara GP et al.; The nucleotide sequence of a phage T4-coded low molecular weight RNA, previously designated polyacrylamide gel band epsilon, has been determined . This RNA can be arranged in the cloverleaf configuration common to tRNAs, with an anticodon sequence, U-C-U, which corresponds to the arginine-specific codons A-G-A and A-G-G; it is therefore assumed to be an arginine tRNA . The complete nucleotide sequence of this RNA species is: pG-U-C-C-C-G-C-U-G-G-U-G-U-A-A-U-Gm2'-G-A-D-A-G-C-A-U-A-C-G-A-U-C-C-U-U-C-U-A-A-G-psi-U-U-G-C-G-G-U-C-C-U-G-G-T-psi-C-G-A-U-C-C-C-A-G-G-G-C-G-G-G-A-U-A-C-C-AOH . The nucleotide sequence was determined by analysis of RNA, uniformly labeled in vivo, according to the conventional techniques . In addition, RNA synthesized in vitro in the presence of alpha-32P-labeled nucleoside triphosphates was analyzed through the use of nearest neighbor sequencing techniques . Although a unique sequence could not be determined by this latter analysis, restrictions on the sequence imposed by nearest neighbor data and secondary structure common to tRNA molecules allowed prediction of the correct nucleotide sequence. Mol Gen Genet, 1977 Nov 14, 156(2), 203 - 14 Molecular cloning of fragments of bacteriophage T4 DNA; Wilson GG et al.; Non-glucosylated T4 DNA was digested with R.EcoRI and the resulting fragments covalently joined to lambda vectors . The genetic content of each lambda-T4 hybrid was determined by marker-rescue tests . The isolation of many recombinants containing partial-digestion products of T4 DNA provided the overlapping sequences necessary to order fragments within the T4 genome . The present analyses include parts of the "early" region between genes 42 and 46, and much of the "late" region between genes 50 and 29 . T4 cytosine-DNA digested to completion by R.EcoRI was used to identify the fragments of DNA within the lambda-T4 recombinants . The T4 cytosine-DNA was also sensitive to R.HindIII and R.Xho but not to R.BamH1. J Biol Chem, 1977 Nov 10, 252(21), 7620 - 4 Biochemical mechanism of uracil uptake regulation in Escherichia coli B . Allosteric effects on uracil phosphoribosyltransferase under stringent conditions; Fast R et al.; The regulation of uracil uptake in bacteria was studied in bacteriophage T4-infected cells, where host-specific, stable RNA synthesis is completely shut-off by phage, and where phage-specific RNA synthesis, which is not stringently regulated, could be followed by a continuous incorporation of uracil . This incorporation into phage RNA was found to be dependent on the allelic state of the rel gene and it was thus severely restricted under stringent conditions . This was not the case with adenine, which was incorported into RNA to almost the same extent under stringent and relaxed conditions, respectively . The inhibition of uracil uptake under proceeding RNA formation, which was furthermore found to be reversed by addition of chloramphenicol, indicated a specific mechanism governing the cellular entry of uracil . This is suggested to involve the allosteric regulation of uracil phosphoribosyltransferase (EC 2.4.2.9.) . The enzyme was partially purified by ammonium sulfate precipitation and gel chromatography . The dependence on GDP and GTP as positive effectors was demonstrated . The stimulatory effect of GTP was abolished in vitro by the addition of guanosine 5'-diphosphate 3-diphosphate, which is known to accumulate during amino acid starvation in stringent bacteria . The reversible inactivation of the enzyme by dilution suggested a subunit structure of uracil phosphoribosyltransferase. Eur J Biochem, 1977 Nov 1, 80(2), 393 - 400 An analysis of the contracted sheath structure of bacteriophage Mu; Cremers AF et al.; Polymers of contracted sheath particles of bacteriophage Mu were imaged by the negative-staining technique and the electron images were analyzed by optical and digital methods . By means of the three-dimensional reconstruction technique of DeRosier and Klug {Nature (Lond.) 217, 130--134 (1968)} an averaged density map of the sheath structure at a resolution of about 2.0 nm was derived . The sheath is known to consist of one type of protein with a molecular weight of about 52000 {Admiraal and Mellema, J . Ultrastr . Res . 56, 48--64 (1976)} . The interpretation of the map has given information about packing and shape of the protein subunits . One way to describe the structure is by a set of annular rings with 6-fold symmetry . The height of these rings is about 1.8 nm and successive rings in the structure change by about 33 degrees in azimuth . The protein subunit which occupies more than one ring in the polymer, is elongated . The long axis of the protein subunit is at an angle of about 20 degrees with the plane normal to the polymer axis . These data are discussed in relation to changes in the sheath molecules upon contraction. J Virol, 1977 Nov, 24(2), 716 - 9 DNA injection and genetic recombination of alkylated bacteriophage T7 in the presence of nalidixic acid; Karska-Wysocki B et al.; Marker rescue experiments with alkylated T7 bacteriophage carried out in the presence and in the absence of nalidixic acid suggest that the gradient in rescue is due to two alkylation-induced causes: a DNA injection defect and an interference with DNA synthesis. J Virol, 1977 Nov, 24(2), 709 - 11 Superinfection exclusion by bacteriophage T7; McAllister WT et al.; Only two of the early genes of bacteriophage T7 were found to play a significant role in exclusion of superinfecting bacteriophage T3 particles; genes 0.3 and 1 . Protein synthesis by the preinfecting phage particle was not required for efficient exclusion . These findings are discussed with regard to the known functions of these genes during T7 development. J Virol, 1977 Nov, 24(2), 673 - 84 Electron microscopic studies of bacteriophage M13 DNA replication; Allison DP et al.; Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation . The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis . In addition to single-stranded circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (theta, rolling circles), and, occasionally, RF DNAs with theta structures . The tails in theta molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of theta to other RF molecules does not change significantly with time after infection . The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion . This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map. J Virol, 1977 Nov, 24(2), 436 - 43 Distinctive protein requirements of replication-dependent and -uncoupled bacteriophage T4 late gene expression; Wu R et al.; This paper further explores the relationship of viral DNA replication to bacteriophage T4 late gene expression . It is shown that replication coupled and -independent late transcription make different qualitative or quantitative demands on phage protein synthesis . In further analysis of these different protein synthesis requirements, experiments were performed with a temperature-sensitive mutant in T4 gene 55 (ts553) . It is known that the gene 55 product regulates T4 late gene expression and binds to RNA polymerase . In the experiments presented here, it is shown that the temperature sensitivity of the ts553 gene 55 protein depends on whether it is involved in replication-coupled or -independent T4 late transcription . This is evidence that the proteins constituting the transcription apparatus interact differently with late transcription units in T4 DNA, depending on whether late transcription is replication coupled or independent. Nucleic Acids Res, 1977 Nov, 4(11), 3715 - 26 Mapping and cloning of Eco RI-fragments of bacteriophage T5+ DNA; Nichols BP et al.; The Eco RI-fragments of bacteriophage T5 DNA were mapped using a technique which involves primarily length measurements of molecules observed in the electron microscope . Since Eco RI cleavage generates termini with 4-nucleotide long cohesive ends, fragments of complete and partial Eco RI digests were covalently circularized with DNA ligase at dilute DNA concentrations before measuring relative to internal length standards . This established the order of the internal Eco RI fragments . The two external Eco RI fragments, which had only one Eco RI terminus, were positioned relative to the internal fragments by identifying the location of some of the naturally-occurring nicks in partially denatured linear Eco RI fragments . An attempt was made to clone each of the internal Eco RI-fragments of T5 DNA via transformation into E . coli after ligation in vitro with the plasmid pMB 9 . Only one fragment could be cloned and this fragment did not specify any new polypeptides in minicells of either the E . coli EK1 host, X1411, or the EK 2 host, X1776. Mutat Res, 1977 Nov, 45(2), 161 - 7 Phage yield during W-reactivation of bacteriophage; Caillet-Fauquet P et al.; Phage production in liquid medium during W-reactivation parallels the extent of W-reactivation of infective centres on plates . The mean burst size is independent of W-reactivation; thus the reactivated phage yields a normal burst . As 8 plates, the lex- mutant shows no W-reactivation in liquid medium . It is concluded that W-reactivation is a consequence of an induced DNA repair which reactivates the damaged parental phage DNA to its full biological activity. J Virol, 1977 Nov, 24(2), 557 - 63 In vivo functional interaction between DNA polymerase and dCMP-hydroxymethylase of bacteriophage T4; Chao J et al.; Some mutations in the structural gene for T4 DNA polymerase (gene 43) behave as suppressors of a deficiency in T4 dCMP-hydroxymethylase (gene 42) . The suppression appears to involve a functional interaction between the two enzymes at the level of DNA replication . The hydroxymethylase deficiency caused DNA structural abnormalities in replication, and DNA polymerase lesions appeared to partially reverse these abnormalities . The results do not necessarily imply protein-protein interactions between the two enzymes, although both enzymes appear to play roles in controlling the fidelity of phage DNA replication. J Virol, 1977 Nov, 24(2), 590 - 601 Changes in the capsid structure and stability of defective particles of bacteriophage R17; Iglewski WJ; Serological and chemical methods were used to compare the capsid structure and stability of R17 phage and amA31 defective particles . Immunodiffusion analysis demonstrated identity between intact R17 and amA31 capside and between dissociated subunits of both R17 and amA31 and purified coat protein . Radioimmunoassays detected an antibody in R17 antisera that binds to intact R17 but could not be absorbed from R17 antisera with amA31 . The R17 antibody remaining in amA31-absorbed sera did not neutralize infectivity of R17 phage . Differences between the surface composition of R17 and amA31 capsids were also detected by iodination . Capsids of R17 bound approximately four times more 125I than amA31, which was accounted for by a decreased 125I labeling of coat protein . Finally, amA31 capsids dissociated under milder conditions of sodium dodecyl sulfate treatment than R17 capsids . The sodium dodecyl sulfate dissociation of both R17 and amA31 capsids resulted in the formation of a transient 38,000-dalton intermediate, which subsequently dissociated to coat protein monomers . Preparations of dissociated R17 capsids also contained assembly protein was also found in preparations of dissociated amA31 capsids. Nucleic Acids Res, 1977 Nov, 4(11), 3743 - 52 In vitro transcription of E . coli tRNA genes; Grimberg JI et al.; Transcription of tRNA genes carried by transducing bacteriophages phi80psu3+ (tRNA1Tyr) and lambdah80T (tRNA2Tyr, tRNA2Glysu36+, tRNA3Thr) was studied in vitro in a system consisting of whole bacteriophage DNA and purified RNA polymerase . In contrast to unusual requirements for tRNA1Tyr gene transcription from DNA fragments, the transcription on whole bacteriophage DNA was found to be relatively not salt sensitive, did not require glycerol and rifampicin-resistant complexes with RNA polymerase were formed in the absence of nucleoside triphosphates . Termination factor rho stimulated the transcription of the tRNA genes as well as that of 4S RNA on lambdah80T DNA template . The stimulatory effect of rho was abolished by rifampicin and seems to be due to the release of RNA polymerase and reinitiation of transcription. Proc Natl Acad Sci U S A, 1977 Nov, 74(11), 4891 - 4 Ribonuclease III cleavage of bacteriophage T3RNA polymerase transcripts to late T3 mRNAs; Majumder HK et al.; In vitro transcription of T3 DNA by T3 phage-induced RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) yields eight discrete RNAs (designated I-VIII) with molecular weights of approximately 6.2, 4.7, 4, 2.8, 1.8, 0.9, 0.52, and 0.21 X 10(6), respectively . Comparison of the size of in vitro T3 RNA polymerase transcripts with in vivo late T3 mRNAs indicates that several late RNAs produced in T3-infected cells do not correspond to any of the in vitro RNAs, and no RNAs as large as the three largest in vitro RNA species, I, II, and III, are observed . Escherichia coli RNase III cleaves these three high molecular weight T3 RNA polymerase transcripts to discrete RNAs that comigrate in polyacrylamide gel electrophoresis with some of the late T3 RNAs. J Virol, 1977 Nov, 24(2), 642 - 50 Isolation and characterization of a bacteriophage T5 mutant deficient in deoxynucleoside 5'-monophosphatase activity; Mozer TJ et al.; A bacteriophage T5 mutant has been isolated that is completely deficient in the induction of deoxynucleoside 5'-monophosphatase activity during infection of Escherichia coli F . The mutant bacteriophage has been shown to be deficient in the excretion of the final products of DNA degradation during infection of E . coli F, and about 30% of the host DNA's thymine residues were reinocorporated into phage DNA . During infection with this mutant, host DNA degradation to trichloroacetic acid-soluble products was normal, host DNA synthesis was shut off normally, and second-step transfer was not delayed . However, induction of early phage enzymes and production of DNA and phage were delayed by 5 to 15 min but eventually reached normal levels . The mutant's phenotype strongly suggests that the enzyme's role is to act at the final stage in the T5-induced system of host DNA degradation by hydrolyzing deoxynucleoside 5'-monophosphates to deoxynucleosides and free phosphate; failure to do this may delay expression of the second-step-transfer DNA. J Bacteriol, 1977 Nov, 132(2), 740 - 3 Physical mapping of genes on the F plasmid of Escherichia coli responsible for inhibition of growth of female-specific bacteriophages; Palchaudhuri S et al.; By correlating the resistance or sensitivity to female-specific phages of strains carrying F plasmids with deletions for part of the region 32.6 to 42.9 F, cloned F fragments, and other plasmids, it was shown that the pif loci are located near and clockwise to a point on F with coordinate 38.3 F. Eur J Immunol, 1977 Nov, 7(11), 769 - 75 Suppressor T cells in low zone tolerance . II . Characterization of suppressor T and amplifier cells by physical and serological methods; Heuer J et al.; Suppressor cells involved in low zone tolerance to bacteriophage fd have been characterized by serological and physical methods . Suppressor T cells are peripheral T2 cells which are Ly-1-, Ly-2+ and Ia+ . They are of high electrophoretic mobility, sediment 5 mm/h at 1 x g in a linear Ficoll 70 gradient, but are not restricted to a distinct band in a discontinuous bovine serum albumin gradient . Adherent phagocytic cells of low electrophoretic mobility can act as amplifier cells with a much shorter half-life than suppressor T cells. Proc Natl Acad Sci U S A, 1977 Nov, 74(11), 4806 - 10 2-Aminopurine-induced mutagenesis in T4 bacteriophage: a model relating mutation frequency to 2-aminopurine incorporation in DNA; Goodman MF et al.; We measured the in vivo incorporation of 2-aminopurine into DNA of T4 bacteriophage allelic for gene 43 (DNA polymerase), mutator (L56), 43+, and antimutator (L141) . The magnitude of incorporation (mol/mol of Thy) was 1/1500 in L56, 1/1600 in 43+, and 1/8900 in L141 . The incorporation ratio L56:43+:L141 in vivo was equal to that mediated by the purified DNA polymerases of these allelic phages in vitro . A model for 2-aminopurine-induced A-T in equilibrium G-C transitions is discussed . The model is used to predict the magnitudes of replication errors (C mispairing with a template 2-aminopurine) and incorporation errors (2-aminopurine mispairing with a template C) per round of replication and to investigate the asymmetry in 2-aminopurine-induced transitions favoring the A-T leads to G-C pathway over G-C leads to A-T . We suggest that the fidelity of L56 and L141 DNA polymerases exemplifies one-step and two-step editing, respectively. Proc Natl Acad Sci U S A, 1977 Nov, 74(11), 4839 - 42 Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase; England TE et al.; RNA ligase isolated from bacteriophage T4-infected Escherichia coli will utilize a number of different compounds with the general structure Ado-5'PP-X as substrates in an ATP-independent reaction . The P-X portions of these molecules are transferred to the 3'-hydroxyl of an oligoribonucleotide to form a phosphodiester bond, and the Ado-5'P (AMP) portion is released . AMP, CMP, GMP, UMP, dTMP, NMN, alphaNMN, reduced NMN, FMN, Rib-5P, phosphopantetheine, and cyanoethylphosphate all have been added to {Cyd-3H}(Ap)3C from their corresponding AMP adducts . Contrary to the relative lack of specificity of RNA ligase for the P-X -group added, the failure of NADP+, deamino-NAD+, epsilonNCD+, epsilon NAD and CoA to react indicates that the enzyme shows a high degree of selectivity for the AMP portion of the substrate . The diversity of chemical groups that can be efficiently added suggests that this reaction of RNA ligase will prove useful for the modification of the 3' ends of RNA molecules. J Virol, 1977 Nov, 24(2), 635 - 41 Properties of deoxynucleoside 5'-monophosphatase induced by bacteriophage T5 after infection of Escherichia coli; Mozer TJ et al.; Bacteriophage T5 induced a deoxynucleoside 5'-monophosphatase during its infection of Escherichia coli . The enzyme was purified about 100-fold . It was clearly distinct from the host 5'-nucleotidase activity in its physical characteristics and substrate specificity . The enzyme was active on deoxynucleoside 5'-monophosphates but was not active as a phosphatase on ribonucleotides, deoxynucleoside 5'-triphosphates, deoxynucleoside 3'-monophosphates, or deoxyoligonucleotides . Furthermore, it did not have oligonucleotidase or exonuclease activity . The enzyme could exist in multimeric form but had a monomer molecular weight of about 25,000. Mol Gen Genet, 1977 Oct 24, 155(3), 347 - 9 Repressor and int synthesis of bacteriophage lambda in the E . coli host mutant ER437; Belfort M et al.; Analysis of lambda phage infection of the host mutant ER437 by SDS polyacrylamide gel electrophoresis and autoradiography has revealed altered expression of repressor and integration function (Int) . We show that in this host Int as well as repressor synthesis is not dependent upon the lambdacIII gene product in the usual manner, nor is their synthesis turned off in the normal way. Mol Gen Genet, 1977 Oct 20, 155(2), 231 - 4 Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant; Caillet-Fauquet P et al.; Preincubation at 42 degrees, before infection at permissive temperature by phage lambda, of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage . Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair . This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating lambda DNA, probably due to induction of diffusible products. Biochemistry, 1977 Oct 18, 16(21), 4550 - 6 Amino acid sequence of the small core protein from bacteriophage phiX174; Freymeyer DK 2nd et al.; The amino acid sequence of small core protein of bacteriophage phiX174 has been determined by a combination of automated Edman degradation of the intact polypeptide and by analysis of tryptic and thermolytic peptides . The six lysyl and six arginyl residues of this 37-residue polypeptide are concentrated in two structurally homologous 12-residue segments of the sequence . The hydrophobic residues of valine, tryptophan, tyrosine, and phenylalanine are contained in the carboxyl-terminal nine residues of the protein, together with one of the two leucyl residues and two of the three glutaminyl residues . The single free carboxyl group in the protein is the alpha-COOH of the C-terminal phenylalanyl residue . The overall sequence of this small core protein suggests that it may function as a DNA-condensing protein . The protein sequence presented here corresponds exactly to the DNA base sequence of the cistron J region of the phiX174 genome determined in another laboratory. Biochemistry, 1977 Oct 18, 16(21), 4545 - 9 Isolation and characterization of the four major proteins in the virion of bacteriophage phiX174; Shank PR et al.; A preparative method is described for the isolation of the major protein species from the virion of bacteriophage phiX174 . Two proteins, the cistron G and H products, are located in the virion spikes . After removal of the spikes, the capsid contains the cistron F product as well as a small protein which is the product of cistron J and the majority of the DNA . During the removal of the spikes, a precipitate containing the F and G proteins is formed . The proteins from the spike, capsid, or precipitate can be isolated on the basis of size by gel-filtration chromatography . The cistron G protein has an aminoterminal methionine, while the small J protein has an amino-terminal serine . Amino acid compositions as well as peptide maps indicate each species is unique and that, in sum, they account for over half the coding capacity of the viral genome. Eur J Biochem, 1977 Oct 17, 80(1), 73 - 7 Endonucleolytic cleavage of parental DNA and T4 late-gene expression: distribution analysis of single-strand and double-strand breaks; Grossi G et al.; In order to investigate the dependency of late transcription on concurrent DNA replication during bacteriophage T4 development, we analyzed the endonucleolytic cleavage kinetics of the DNA of a T4 mutant lacking DNA polymerase, DNA ligase and exonuclease by using the sucrose gradient sedimentation technique . Our results can be summarized as follows . 1 . The single-strand endonucleolytic cleavage of the T4 mutant DNA is not a random process . 2 . The number of single-strand nicks reaches a plateau level of 10--12 nicks/molecule . 3 . The occurrence of a double-strand break is delayed and their number is at any time lower than the number of single-strand nicks . 4 . The circular permutation T4 genome, as computer-simulated by the Monte Carlo method, produces a smoothing of the discrete distribution which would be expected if nicks were localized in the promoter sites of late transcription units . We conclude that our findings support the model which relates single-strand DNA nicks to the late transcription initiation sites. J Biol Chem, 1977 Oct 10, 252(19), 6646 - 50 Analysis of the structure of T4 bacteriophage-modified valyl-tRNA synthetase by limited proteolysis and isoelectric focusing; Muller UR et al.; The new form of valyl-tRNA synthetase (EC 6.1.1.9) that appears immediately after infection of Escherichia coli with bacteriophage T4 was purified and subjected to mild proteolysis using five different proteases . The inactivation of aminoacylation activity was both more extensive and rapid than that obtained with valyl-tRNA synthetase purified from uninfected E . coli . The addition of bulk tRNA from E . coli B protected the phage-specific form of valyl-tRNA synthetase from proteolysis, but ATP and valine did not exhibit a similar protective effect . The characteristic property of phage-modified valyl-tRNA synthetase, resistance to denaturation by 4 M urea, remained unaffected during treatment with trypsin . This suggested that the phage-specific factor tau, known to be associated with the synthetase in phage-infected cells, was protected from proteolysis in the synthetase-tau complex . Comparison by isoelectric focusing of normal valyl-tRNA synthetase, the phage-specific form of this enzyme, and phage enzyme from which tau had been removed, revealed no differences in the isoelectric points of these three molecules . Based on these results a model was drawn for the structural changes occurring in valyl-tRNA synthetase after association with the phage factor tau. J Biol Chem, 1977 Oct 10, 252(19), 6640 - 5 Purification and properties of a T4 bacteriophage factor that modifies valyl-tRNA synthetase of Escherichia coli; Muller UR et al.; After T4 bacteriophage infects Escherichia coli, a peptide tau, produced under the control of a phage gene, binds to the host valyl transfer ribonucleic acid synthetase (EC 6.1.1.9) and thereby changes several of its physicochemical properties . The interaction of tau with the host enzyme was investigated in vitro after extensively purifying the factor from T4-infected E . coli using a rapid purification procedure . The tau preparation migrated as a single, protein-staining band with a molecular weight of 11,000 during sodium dodecyl sulfate-gel electrophoresis . The purified peptide completely converted partially purified valyl-tRNA synthetase from uninfected E . coli into the form present in cell-free extracts prepared from virus-infected bacteria . The enzyme modified in vitro also exhibited the enhanced affinity for tRNA characteristic of the viral form of valyl-tRNA synthetase . The addition of bulk tRNA from E . coli B, tRNAVal, or tRNA1Val to enzyme modified in vitro increased its sedimentation rate to that of enzyme prepared from phage-infected cells . Amino acid analysis of the purified tau peptide revealed a relatively high concentration of the amino acids lysine and alanine, and a lack of detectable proline, tyrosine, phenylalanine, and methionine. Biochemistry, 1977 Oct 4, 16(20), 4497 - 503 Synthesis in vitro of ribosomal protein S20 and its precursor; Mackie GA; I have purified and characterized two products synthesized in vitro in a system for coupled transcription and translation programmed by DNA from a transducing bacteriophage carrying the gene for ribosomal protein S20 . One of these polypeptides appears to be identical with authentic S20 by several criteria, including its electrophoretic and chromatographic mobilities, and its ability to bind to 16S RNA . The second polypeptide is less basic than S20, but exhibits all the structural and functional properties of a precursor to S20, including the presence of an additional methionine residue, apparently as N-formylmethionine . Moreover, it is converted, albeit slowly, to S20 in cell-free extracts . The persistence of the precursor form of S40 may be functionally significant as well. J Clin Microbiol, 1977 Oct, 6(4), 420 - 4 Isolation of bacteriophage from Thermoactinomyces; Treuhaft MW; Bacteriophages were isolated from strains of Thermoactinomyces vulgaris, T . candidus, and T . sacchari used to produce antigen for hypersensitivity pneumonitis screening at the Marshfield Medical Foundation . Whereas the one phage isolated from T . sacchari and two phages from T . vulgaris were species specific, three other phages isolated from T . vulgaris and the two phages isolated from T . candidus were infectious for both T . vulgaris and T . candidus, thus indicating a close relationship between these two species . A simple reproducible scheme for classification of newly isolated T . vulgaris-T . candidus phages into seven groups on the basis of host range is presented . Examination of plaque morphology of the T . vulgaris-T . candidus phages supported the host range classification scheme . The ease of isolation of phages from cultures of Thermoactinomyces suggests that they are commonly associated with this genus. J Virol, 1977 Oct, 24(1), 303 - 13 Assembly of bacteriophage phi X174: identification of a virion capsid precursor and proposal of a model for the functions of bacteriophage gene products during morphogenesis; Fujisawa H et al.; A capsomeric structure sedimenting with an S value of 108 in sucrose gradients was isolated from Escherichia coli infected with bacteriophage phi X174 . The 108S material contained viral proteins F, G, H, and D, and the relative amounts of these proteins in the 108S material were similar to those in the infectious 132S particle, which has previously been described as a possible intermediate in the assembly of 114S phage particles . Electron micrographs indicated that the size and shape of the 108S material resemble those of the 132S particle . The 108S material contained no DNA, and its formation occurred independently of DNA synthesis . The 108S material accumulated in infected cells when viral DNA replication was prevented either by mutation in phage genes A or C or by removal of thymidine from a culture infected with wild-type phage or with a lysis gene E mutant . Upon restoration of thymidine to cells infected with the lysis gene E mutant and then starved of thymidine, the accumulated 108S material was converted to 132S particles and to 114S phage particles, implying that the 108S material is a precursor of phage particles . A model that proposes possible functions for the products of phi X174 genes A, B, C, D, F, and G during viral replication and phage maturation is described. J Virol, 1977 Oct, 24(1), 28 - 40 Properties of the nonlethal recombinational repair x and y mutants of bacteriophage T4 . II . DNA synthesis; Melamede RJ et al.; The bacteriophage T4 recombination-deficient mutants x and y exhibited decreased rates of DNA synthesis as compared to wild-type T4 . Mutant-induced DNA synthesis was more sensitive to mitomycin C than was wild-type synthesis . However, DNA synthesis in mutant- and wild-type-infected cells exhibited the same sensitivity to UV light and X-irradiation . When high-specific-activity label was administered at various times postinfection, mutant DNA synthesis resembled that of wild type for 12 min . after which time mutant-induced incorporation was greatly decreased and sensitive to mitomycin C as compared to that of the wild type . Rifampin and chloramphenicol studies indicated that the gene products necessary for synthesis measured at 15 min postinfection, including those of x+ and y+ were transcribed within 2 min and translated within 8 min postinfection . Administration of chloramphenicol to mutant x- or mutant y-infected cells exactly 8 min postinfection, however, allowed for increased synthesis at 15 min that was sensitive to mitomycin C . Cells coinfected with T4+ and T4x or T4x and T4y retained a reduced mutant-type synthesis, whereas cells coinfected with T4+ and T4y exhibited a synthesis more closely resembling that of wild type. J Virol, 1977 Oct, 24(1), 249 - 60 Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI; Hamlett NV et al.; A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments) . The following techniques were used to order the fragments . (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region . (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with lambda exonuclease before restriction endonuclease cleavage . (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases . (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme . (v) Treatment of restriction digests with lambda exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites . (vi) Exonucleases III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions . (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments. J Virol, 1977 Oct, 24(1), 177 - 93 Role of the T5 gene D15 nuclease in the generation of nicked bacteriophage T5 DNA; Moyer RW et al.; The processing of newly replicated concatameric T5 DNA into both single stranded DNA changed of unit length and single-stranded fragments of sizes comparable to those found in mature T5 virion DNA occurs in the absence of late T5 protein synthesis . The formation of unit-length, single-stranded DNA chains does not require the early T5 gene D15 nuclease: however, the subsequent formation of the single-stranded fragments does require that the D15 nuclease be functional . A reexamination of the properties of the purified D15 nuclease under a variety of conditions showed that, in addition to functioning as a 5' leads to 3' exonuclease, the enzyme can also introduce endonucleolytic scissions into mature T5 DNA in a reaction that requires duplex T5 DNA and preexisting, single-stranded interruptions. J Virol, 1977 Oct, 24(1), 121 - 34 Localization of minor protein components of the head of bacteriophage T4; Muller-Salamin L et al.; The bacteriophage T4 capsid contains a number of minor proteins that are required for head assembly but whose detailed function and position in the head are unknown . We have found that by systematically varying the conditions of extraction, some of these minor proteins can be removed while the main capsid structure is left substantially intact . Electron microscopic examination of the residual capsids showed that the extraction of the product of gene 20 is correlated with the loss of a plug that distinguishes one vertex position (presumably the tail attachment site) from the others . Extraction of the product of gene 24 is correlated with the loss of the other 11 (nonproximal) vertexes of the capsid . We further show that antibody to P24 binds specifically to the nonproximal vertexes of both T4 preheads and T4 phages . On the basis of our findings, we suggest that P20 is located at or near the tail attachment site of the capsid, whereas P24 forms the 11 nonproximal vertexes of preheads and P24 forms the nonproximal vertexes of the mature head. Isr J Med Sci, 1977 Oct, 13(10), 1022 - 7 Viroimmunoassay utilizing a synthetic peptide: a test equivalent to the carcinoembryonic antigen radioimmunoassay; Arnon R et al.; A recently developed immunoassay which utilizes the synthetic fragment CEA(1-11), corresponding to the N-terminal segment of carcinoembryonic antigen (CEA), was used for the evaluation of human sera . The various sera were tested for their capacity to inhibit the inactivation of the modified bacteriophage preparation CEA(1-11)-T4 by antiserum prepared against the bovine serum albumin (BSA) conjugate CEA(1-11)-BSA . In this immunological system both the free synthetic peptide and a semipurified preparation of intact CEA serve as inhibitors . Sera from a large proportion (85%) of patients with adenocarcinomas of the digestive tract, including the pancrease, gave 50 to 88% inhibition . Sera from patients with other cancers, particularly of breast and ovary, also caused inhibition, although it was less marked in both incidence and level . Most normal sera gave less than 40% inhibition, which was considered as the cutoff point . This assay, like the CEA radioimmunoassay, is not suitable for mass screening nor can it be the primary criterion for diagnosis of cancer, but it might be of value as a follow-up procedure for postoperative diagnosis and prognosis. J Bacteriol, 1977 Oct, 132(1), 60 - 6 Use of argA-lac fusions to generate lambda argA-lac bacteriophages and to determine the direction of argA transcription in Escherichia coli; Eckhardt T; Fusions of lac genes to the argA operator were constructed, and lambda phages carrying these fusions were isolated and characterized . With the aid of a lambda phage carrying an argA-lac fusion, the direction of argA transcription on the Escherichia coli chromosome was determined to be clockwise. Eur J Immunol, 1977 Oct, 7(10), 749 - 51 The "patchy" immunodeficiency of CBA/N mice; Quintans J; The in vivo responses of CBA/N (which have an X-linked defect of B lymphocyte differentiation) and CBA/J normal mice to 2,4,6-trinitrophenylated (TNP) bacteriophage T4 and Diplococcus pneumoniae CS variant have been compared . Both antigens are thymus-independent (TI), and TNP-CS additionally contains the phosphorylcholine (PC) epitope, CBA/N and CBA/J mice give TNP plaque-forming cell responses similar in magnitude and avidity although only CBA/J give a PC response when challenged with TNP-CS . These results indicate that CBA/N mice have a "patchy" defect of antigen-induced humoral responses . This defect is manifested only in certain subpopulations of B cells and is not restricted to TI responses. J Gen Microbiol, 1977 Oct, 102(2), 349 - 63 Characterization of pili determined by drug resistance plasmids R711b and R778b; Bradley DE; The bacterial drug resistance plasmids R711b and R778b, at present classified in the X incompatibility group, determine pili (designated 711) that resemble F pili morphologically . Like F pili, 711 pili adsorb F-specific filamentous bacteriophages to their tips, though more often in pairs, than singly . However, F-specific RNA-containing bacteriophages are not adsorbed to their sides, and strains carrying the plasmids are resistant to these phages . Pili determined by the only IncFV plasmid Folac are similar to 711 pili in their phage adsorption properties, but they are serologically different, as are F pili . It is concluded that F, Folac and 711 pili have basic differences in spite of a morphological resemblance. J Gen Microbiol, 1977 Oct, 102(2), 319 - 26 Bacteriophage-resistant mutants of Escherichia coli K12 with altered lipopolysaccharide . Studies with concanavalin A; Picken RN et al.; Three classes of mutants of Escherichia coli K12, isolated by selection for resistance to lipopolysaccharide-specific bacteriophages, were agglutinated by Concanavalin A which is presumed to interact with the lipopolysaccharide component of the outer membrane . Wheat germ and soy bean agglutinins did not agglutinate the parent or mutant strains . The adsorption of certain bacteriophages was also inhibited by Concanavalin A . The pattern of inhibition of adsorption of bacteriophages suggests that non-specific masking of receptors may occur, as well as specific masking of terminal glucose residues . Although bacteria were agglutinated by Concanavalin A, the permeability of the outer membrane seemed unaffected. J Gen Microbiol, 1977 Oct, 102(2), 305 - 18 Bacteriophage-resistant mutants of Escherichia coli K12 . Location of receptors within the lipopolysaccharide; Picken RN et al.; A series of mutants of Escherichia coli K12 resistant to lipopolysaccharide (LPS)-specific bacteriophages were isolated, and examined with regard to their general properties, phage typing, chemical analysis of their LPS, and genetic analysis . Fourteen classes of mutants were distinguished on the basis of phage typing and sensitivity to bile salts . Three of the mutant classes are sensitive to phages to which the parent is resistant . Mutants which are sensitive to bile salts generally lack heptose in their LPS, but two mutant classes are exceptions to this rule . Analyses of the sugars in the purified LPS of all mutant classes indicated that mutants were obtained which are blocked at most stages in core polysaccharide synthesis . On the basis of the chemical analysis, in conjunction with phage typing data and other known properties of the mutants, it is deduced which residue(s) is involved as a receptor for each of the phages used and which residues hinder these receptors . Some of the mutant classes do not seem to be changed in their LPS structure . Many of the mutations map in or near the rfa locus, but some are far removed from this region. J Bacteriol, 1977 Oct, 132(1), 359 - 61 Localized mutagenesis with bacteriophage Mu: method for increasing the frequency of specific bacterial mutants; Tabor H et al.; A method is described for markedly enriching a bacterial population for cells containing any given Mu insertion mutation . The method involves the transfer of a small piece of deoxyribonucleic acid from a Mu-infected Hfr donor donor strain to a suitable F- strain and a subsequent selection of those recombinant organisms that have received a Mu prophage from the donor . The method is particularly usefule for isolating mutants whose selection requires "brute-force" assay, since only a few hundred colonies have to be screened. J Bacteriol, 1977 Oct, 132(1), 270 - 81 Tryptophan-transducing bacteriophages: in vitro studies with restriction endonucleases HindII + III and Escherichia coli ribonucleic acid polymerase; Jones BB et al.; The HindII + III restriction enzyme fragmentation pattern of various lambda-phi80trp deoxyribonucleic acid molecules is presented . An analysis of deoxyribonucleic acid molecules carrying deletions ending within the trp regulatory elements and a deoxyribonucleic acid molecule carrying a deletion within trpE indicates that a fragment of 8.3 X 10(5) daltons contains at least part of the trp promoter, the entire trp leader region, and part of the trpE gene . The observation that ribonucleic acid polymerase, when present in the HindII + III digestion mixture, results in the fusion of this 8.3 X 10(5)-dalton fragment to the preceding bacterial fragment suggests that HindII + III cuts within trpP. J Virol, 1977 Oct, 24(1), 419 - 21 Inactivation of receptors for bacteriophage T5 during infection of Escherichia coli B; Dunn GB et al.; During infection of Escherichia coli by bacteriophage T5, the cell surface receptors for the phage were inactivated so that they could not be isolated from the infected cells . A mutant of T5 that could only inject 8% of the T5 DNA did not cause the inactivation. Proc Natl Acad Sci U S A, 1977 Oct, 74(10), 4406 - 10 Cloning specific segments of the mammalian genome: bacteriophage lambda containing mouse globin and surrounding gene sequences; Tilghman SM et al.; We have developed a general approach to the cloning of specific segments of the mammalian genome that involves a two-step purification of EcoRI fragments of mammalian DNA and their in vitro insertion into a suitably constructed EK2 derivative of bacteriophage lambda . The combination of fragment purification, exclusion of parental-type recombinants, and simple phage screening techniques permits the isolation of virtually any gene segment for which there is an identifying hybridization probe . We illustrate the approach by describing the cloning of an approximately 7000-base-long segment of mouse DNA containing globin and surrounding gene sequences. Proc Natl Acad Sci U S A, 1977 Oct, 74(10), 4219 - 22 Nucleotide sequences near the origin of replication of bacteriophage f1; Ravetch JV et al.; The nucleotide sequence of a region related to the initiation of the reaction in which single-stranded DNA gives rise to the replicative form (complementary strand synthesis) in bacteriophage f1 has been determined . The sequence can be drawn in an extensively base-paired structure, i.e., a single hairpin-helix 55 bases long. Nucleic Acids Res, 1977 Oct, 4(10), 3483 - 96 Studies on the biological role of DNA methylation: III Role in excision of one-genome long single-stranded phi X 174 DNA; Friedman J et al.; Accumulation of replicative intermediates of the bacteriophage phi X174 was observed in E . coli C infected cells when phage DNA methylation has been inhibited by nicotinamide or when cells were infected with a temperature-sensitive mutant in gene A . Analysis of the accumulating replicative intermediates by electron microscopy revealed that these molecules are composed of double-stranded DNA rings with multiple-genome length single-stranded "tails" . These results suggest that the single 5-methylcytosine residue present in the phage DNA serves as a recognition site for the gene A protein mediating the excision of one-genome long phage DNA . This excision process is oligatory for the final maturation of the phage. J Virol, 1977 Oct, 24(1), 201 - 10 Properties of "diplophage": a lipid-containing bacteriophage; Lopez R et al.; We describe the purification and properties of Dp-1, a bacteriophage isolated from Diplococcus pneumoniae . The phage was sensitive to the organic solvents deoxycholate and Sarkosyl, and its infectivity was reduced by treatment with phospholipase C . Electron microscopy indicated the presence of a double-layered coat around the phage particles . Purified phage preparations contained lipid amounting to about 8.5% of the dry weight of the phage, and thin-layer chromatography resolved the lipids into four components . The phage had a buoyant density in CsCl of 1.47 g/cm3, and a sedimentation constant in 0.1 M NaCl of 313S . Analysis in acrylamide gel electrophoresis indicated the presence of three major proteins . Dp-1 DNA shows a density of 1.681 g/cm3 . Neutralizing antisera against the phage have a low potency (K less than 120/min). Mol Gen Genet, 1977 Sep 21, 155(1), 1 - 5 Stimulation of cell division by T4 infection in Escherichia coli dnaEts at nonpermissive temperature; Sato K et al.; Filamentous cells resulting from growth of a dnaEts mutant of Escherichia coli at high temperature were stimulated to divide by infection with bacteriophage T4 . The effect appears to be related to T4 DNA synthesis; no increase in cell number took place in chloramphenicol-treated . T4-infected cells nor in cells infected with DNA synthesis-less mutants of T4 . The ability of cells to divide after T4 infection was dependent on the length of time that the cells had been grown at 42 degrees C, indicating that a potential for cell division accumulates during preincubation. Biochemistry, 1977 Sep 20, 16(19), 4209 - 17 Synthesis and biological activity of a lambda pseudo operator; Kawashima E et al.; The chemical and enzymatic syntheses of bacteriophage lambda pseudo operator DNA are described . The 17 base-paired duplex contains the DNA which has been proposed as the binding site for cI repressor protein . The synthetic duplex is twofold symmetric and represents the best possible nucleotide summation of the six proposed operator sites in the leftward and rightward operators . However, it does not correspond exactly to any single proposed operator sequence . The chemical synthesis includes the deoxyoligonucleotides d(T-A-T-C-A-C), d(C-G-C-C-G-G-T-G-A-T-A), d(T-A-T-C-A-C-C), and d(G-G-C-G-G-T-G-A-T-A) . These deoxyoligonucleotides were joined with T4 DNA ligase to form d(T-A-T-C-A-C-C-G-C-C-G-G-T-G-A-T-A) and d(T-A-T-C-A-C-C-G-G-C-G-G-T-G-A-T-A) . The cI repressor protein was found to bind to the duplex formed from these two segments. Eur J Biochem, 1977 Sep 15, 79(1), 309 - 17 Investigation of the binding of Escherichia coli RNA polymerase to DNA from bacteriophages T2 and T7 by kinetic formaldehyde method and electron microscopy; Cherny DI et al.; The complexes of T2 DNA with RNA polymerase of Escherichia coli were studied by two methods: kinetic formaldehyde method with preliminary fixation of complexes with low formaldehyde concentrations, and electron microscopy . For electron-microscopic investigations the effect of different conditions of formaldehyde fixation for DNA-RNA-polymerase complexes was studied and optimal fixation conditions were found . The suggested fixation method for DNA-RNA-polymerase complexes allows investigation of RNA polymerase molecule distribution on DNA in a wide range of conditions (ionic strength of the solution, weight ration of enzyme to DNA etc.) . The comparison of the concentration of RNA polymerase molecules bound to DNA, determined by electron microscopy, and the concentration of defects in DNA as determined by the kinetic formaldehyde method, showed their coincidence . The electron-microscopic procedure was used to make maps of RNA polymerase distribution on T7 DNA . A correlation between the binding regions of the enzyme and the genetic map of early DNA T7 region was found. J Biol Chem, 1977 Sep 10, 252(17), 6177 - 83 Cro regulatory protein specified by bacteriophage lambda . Structure, DNA-binding, and repression of RNA synthesis; Takeda Y et al.; The Cro protein specified by bacteriophage lambda is a repressor of the genes expressed early in phage development and is required for a normal late stage of lytic growth . We have purified Cro protein to virtual homogeneity and analyzed its structure and properties as a DNA-binding protein and repressor of RNA synthesis . To confirm that the protein is the product of the cro gene, we have also shown that a missense mutation in the cro gene leads to a product that is more temperature- and salt-sensitive in its DNA-binding property . As purified, Cro protein is a dimer of identical subunits of molecular weight 8600 . The purified protein binds to lambda-DNA carrying the specific binding sites (operators oL and oR) with an estimated dissociation constant of 10(-10) M to 10(-11) M; there is also weaker binding to other sites on DNA, as found for other DNA-binding regulatory proteins . In a purified transcription system, the Cro protein is an effective and specific repressor of RNA synthesis from the N and cro genes; thus Cro is an autorepressor which regulates its own synthesis . A comparison of the properties of the two lambda repressor proteins, cI and Cro, indicates that cI is a "strong repressor" specialized for complete turnoff of lytic functions needed for the maintenance of lysogeny, whereas Cro is a "weak repressor" specialized for a gradual turnoff of early viral genes that potentiates the late stage of lytic development. Mol Gen Genet, 1977 Sep 9, 154(3), 319 - 26 Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes; Mattson T et al.; Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56, denA and denB . This DNA can be cut by a number of restriction endonucleases . Fragments obtained by digestion of this DNA with EcoRI have been cloned using the vector plasmid pCR1 . Clones containing T4 DNA were identified by hybridization with radioactive early and late T4 RNA . A simple marker rescue technique is described for the genetic identification of the cloned T4 fragments . Some of the T4-hybrid plasmids which contain entire T4 genes can complement temperature sensitive and amber mutants of T4. Mol Gen Genet, 1977 Sep 9, 154(3), 231 - 48 Mapping of restriction sites in the attachment site region of bacteriophage lambda; Kamp D et al.; A find structure map of the EcoRI fragment containing the lambda attachment-site region has been constructed . 38 different restriction endonucleases have been employed and 170 sites located in this fragment . In addition, sites in adjacent regions have been determined for several enzymes . Complete cleavage maps of the entire lambda genome have been obtained for endonucleases BglII, BluI, KpnI, SacI, SacII, SalI and XbaI . The strategy employed for mapping included comparison of deletion and substitution mutants, analysis of mixed digests, and detailed analysis of subfragments. Gene, 1977 Sep, 2(1), 33 - 54 HindII, HindIII, and HpaI restriction fragment maps of the left arm of bacteriophage lambda DNA; Robinson LH et al.; The sites on the left arm of bacteriophage lambda DNA cleaved by the restriction endonucleases isolated from Hemophilus influenzae strain Rc (HincII) and Rd (HindII + III), and Hemophilus parainfluenzae (HpaI) were localized on the lambda physical map, and the fragments resulting from these cleavages were identified by gel electrophoresis . The restriction sites within the b2 region of lambda were mapped by analysis of the digestion profiles of deletion and substitution derivatives of lambda, as well as by digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease . The restriction sites of the lambda genome between the left vegetative end and the b2 region were mapped entirely by succesive digestion experiments . The restriction fragment map for the right arm of lambda may be found in the accompanying paper (Robinson and Landy, 1977). Gene, 1977 Sep, 2(1), 1 - 31 HindII, HindIII, and HpaI restriction fragment maps of bacteriophage lambda DNA; Robinson LH et al.; The site-specific restriction endonucleases isolated from Hemophilus influenzae strains Rc (HincII) and Rd (HindII + III), and Hemophilus parainfluenzae (HpaI) were used to digest bacteriophage lambda DNA into 34, 40, and 15 specific fragments, respectively . The sites cleaved by each of these enzymes were localized on the lambda physical map and the fragments resulting from these cleavages were electrophoretically identified on gels by (1) analysis of the digestion profiles of deletion and transducing derivatives of lambda; and (2) digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease . This paper presents the HindII, HindIII, and HpaI restriction fragment maps for the entire lambda genome, and the data used to derive these maps for the region of the lambda genome between the attachment site (at 57.3% lambda) and the right vegetative end (100% lambda) . The data for mapping the left arm of lambda may be found in the accompanying paper (Robinson and Landy, 1977). Biophys J, 1977 Sep, 19(3), 299 - 306 The effects of temperature and ultraviolet irradiation on multiplication of bacteriophage phi29; Larcom LL et al.; The effects of temperature and of ultraviolet radiation on the multiplication of bacteriophage phi29 were studied . Samples of phi29 that had been irradiated to surviving fractions of 0.44 or 0.10 were propagated at 37 degrees C, 42 degrees C and 43.5 degrees C . Latent periods and burst sizes were obtained from one-step growth curves . At a particular temperature, as the dose delivered to the virus was increased, the latent period was extended and the burst size was decreased . For unirradiated virus, the burst size was the same at 42 degrees C as at 37 degrees C, but decreased dramatically at 43.5 degrees C . For virus subjected to a particular dose, the burst size decreased as the temperature was raised . A statistical technique for improving the reliability of parameters obtained from one-step growth curves is presented. Proc Natl Acad Sci U S A, 1977 Sep, 74(9), 3740 - 4 Electron microscope studies of transient complexes formed between Escherichia coli RNA polymerase holoenzyme and T7 DNA; Williams RC et al.; Electron microscopy was used to study the formation of random complexes between Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a promoterless fragment (Mbo I-C) of bacteriophage T7 DNA, and to determine the location of the polymerase molecules bound at 3 degrees to the promoter-containing (Hinf)1100 fragment of the same DNA . The value of the Ka of random binding is about 3 times 10(4)M-1 when the enzyme is slowly diluted from its storage condition and is incubated with DNA for up to 2 min at 37 degrees . If dilution is rapid and occurs in a single step, or if incubation extends beyond 5 min, a substantial portion of RNA polymerase is converted to a form that binds randomly with a much greater affinity (about 10(8)M-1) . Hence true random binding by RNA polymerase holoenzyme is much weaker than previously thought . However, great caution is required in assessing the extent of random binding where damage to the enzyme may occur . When RNApolymerase holoenzyme is incubated at 0 degrees with promoter-containing fragment (Hinf)1100, complexes form at the same promoter sites utilized at 37 degrees, although the highly stable "open" promoter complex is not formed under these conditions . However, the extent of binding is reduced as compared to promoter complexes formed at 37 degrees . This gives direct evidence for formation of complexes with promoter sites that have properties of the hypothetical "closed"complexes formed between RNA polymerase and duplex DNA. Nucleic Acids Res, 1977 Sep, 4(9), 3175 - 86 Bacteriophage T4 RNA ligase: preparation of a physically homogeneous, nuclease-free enzyme from hyperproducing infected cells; Higgins NP et al.; Infection of Escherichia coli by a bacteriophage T4 regA, gene 44 double mutant leads to about a 7-fold increase in the amount of RNA ligase obtained after infection by wild-type phage . Using cells infected by the double mutant, RNA ligase was purified to homogeneity with a 20% yield . Unlike previous preparations of this enzyme, the ligase is free of contaminating nuclease and is therefore suitable for intermolecular ligation of DNA substrates . In the course of these studies it was discovered that adenylalation of the enzyme--a step in the reaction pathway--markedly decreased the electrophoretic mobility of RNA ligase through polyacrylamide gels containing sodium dodecyl sulfate . This behavior allows identification of RNA ligase among a mixture of proteins and was used to demonstrate that virtually all of the purified protein is enzymatically active. Can J Microbiol, 1977 Sep, 23(9), 1151 - 3 Phage typing of Escherichia coli isolated from chickens; Bhatia TR; Ten different bacteriophages were isolated from untreated city sewage water . These phages were stable at 57 degrees C for 40 min . A modified agar layer technique was used to obtain high titre phages . Ninety-four of a stock of 101 cultures of Escherichia coli, which were isolated from inflamed portions of intestines of chickens, were lysed by one or more of these phages . The E . coli of a known serological grouping were phage typed. J Virol, 1977 Sep, 23(3), 825 - 6 DNA modification of bacteriophage Mu-1 requires both host and bacteriophage functions; Toussaint A; It was previously shown that resistance of phage Mu-1 to several restriction enzymes is due to a modification function (called mom) encoded by the phage . More recent studies emphasized that modification of Mu requires not only an active mom function, but also an active dam function supplied by the Escherichia coli host. J Virol, 1977 Sep, 23(3), 725 - 36 Localization of single-chain interruptions in bacteriophage T5 DNA I . Electron microscopic studies; Scheible PP et al.; Bacteriophage T5 DNA was examined in an electron microscope after limited digestion with exonuclease III from Escherichia coli . The effect of the exonuclease treatment was to convert each naturally occurring single-chain interruption in T5 DNA into a short segment of single-stranded DNA . The locations of these segments were determined for T5st(+) DNA, T5st(0) DNA, and fragments of T5st(0) DNA generated by EcoRI restriction endonuclease . The results indicate that single-chain interruptions occurr in a variable, but nonrandom, manner in T5 DNA . T5st(+) DNA has four principal interruptions located at sites approximately 7.9, 18.5, 32.6, and 64.8% from one end of the molecule . Interruptions occur at these sites in 80 to 90% of the population . A large number of additional sites, located primarily at the ends of the DNA, contain interruptions at lower frequencies . The average number of interruptions per genome, as determined by this method, is 8 . A similar distribution of breaks occurs in T5st(0) DNA, except that the 32.6% site is missing . At least one of the principal interruptions is reproducibly located within an interval of 0.2% of the entire DNA. J Virol, 1977 Sep, 23(3), 476 - 82 Maintenance of bacteriophage P1 plasmid; Jaffe-Brachet A et al.; Three mutants of bacteriophage P1 affected in their ability to maintain the lysogenic state stably are described here . These mutants were normal in lytic growth, but lysogenic derivatives segregated nonlysogens at abnormally high rates (1 to 30% per division) . Cells harboring these mutant prophages were elongated or filamentous . The mutations responsible for this prophage instability fell into two classes on the bases of their genetic location, their effect on the ability to lysogenize recA bacteria, and their suppressibility by ant mutations eliminating antirepressor activity . The two mutants that were able to form recA lysogens showed the same prophage instability and partial inhibition of cell division in recA as in rec+ lysogens . The fact that plasmid-linked mutations can cause prophage instability suggests that P1 codes for at least some of the functions determining its own autonomy and segregation. J Virol, 1977 Sep, 23(3), 467 - 75 Effects of mutations in the immunity system of bacteriophage P1; D'Ari R; A mutant of bacteriophage P1 that made an altered c1 repressor is described . The mutant c1 product had two configurations: in lysogens, at high temperatures, it permitted constitutive expression of the normally repressed DNA replication function ban and was insensitive to the action of ant, a product expressed by the virulent mutant P1virs and by the heteroimmune phage P7 (formerly phiamp+) and normally able to overcome c1 repression; in mutant lysogens at low temperatures, the mutant repressor was apparently normal (able to repress ban and sensitive to ant action) . Genetic studies of this mutant led to the isolation of a derivative that formed unstable lysogens . These studies suggested that the ban product was normally under c1 control; they further showed that ant overcame c1 repression by inactivating c1 rather than by creating a bypass of repressor activity. J Bacteriol, 1977 Sep, 131(3), 821 - 9 Major proteins of the Escherichia coli outer cell envelope membrane as bacteriophage receptors; Datta DB et al.; Three Escherichia coli phages, TuIa, TuIb, and TuII, were isolated from local sewage . We present evidence that they use the major outer membrane proteins Ia, Ib, and II, respectively, as receptors . In all cases the proteins, under the experimental conditions used, required lipopolysaccharide to exhibit their receptor activity . For proteins Ia and II, an approximately two- to eightfold molar excess of lipopolysaccharide (based on one diglucosamine unit) was necessary to reach maximal receptor activity . Lipopolysaccharide did not appear to possess phage-binding sites . It seemed that the lipopolysaccharide requirement reflected a protein-lipopolysaccharide interaction in vivo, and lipopolysaccharide may thus cause the specific localization of these proteins . Inactivation of phage TuII by a protein II-lipopolysaccharide complex was reversible as long as the complex was in solution . Precipitation of the complex with Mg2+ led to irreversible phage inactivation with an inactivation constant (37 degrees C)K = 7 X 10-2 ml/min per microgram . With phages TuIa and TuIb and their respective protein-lipopolysaccharide complexes, only irreversible inactivation was found at 37 degrees C . The activity of the three proteins as phage receptors shows that part of them must be located at the cells surface . In addition, the association of proteins Ia and Ib with the murein layer of the cell envelope makes this pair trans-membrane proteins. Proc Natl Acad Sci U S A, 1977 Sep, 74(9), 3652 - 6 DNA packaging and the pathway of bacteriophage T4 head assembly; Hsiao CL et al.; A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA . Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature . It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation) . The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing . Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging . Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume . Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction. Z Naturforsch {C}, 1977 Sep-Oct, 32(9-10), 850 - 4 Genetic information analysis of bacteriophage phiX 174; Figueroa R et al.; The genetic information of phix 174 genome (genes and intermediate segments) is analyzed in terms of its independent (D1 index) and dependent information (D2 and D3 Markovian indexes), as well as of its ability to generate secondary structure . Genes B and E, enclosed in A and D respectively, have: 1) values of D1 and D3 indexes closer to the theoretical random distribution curves than those of (A-B) and (D-E) gene fractions, and 2) in the ability for secondary structure generation minor differences with genes A and D . F leads to G and mRNA start leads to A intermediate segments differ from randomness in their D1 and D2 indexes, but not so much in the D3 values . All these data point out the use of code degeneracy for increasing the genetic information density of the virus. J Biol Chem, 1977 Aug 25, 252(16), 5911 - 5 Envelope composition and antibiotic hypersensitivity of Escherichia coli mutants defective in phosphatidylserine synthetase; Raetz CR et al.; Mutants of Escherichia coli K12, defective in phosphatidylserine synthetase (pss), can be isolated as temperature-sensitive, conditional lethals . When cultivated at intermediate temperatures (30 degrees), such mutants contain approximately 3 times more phosphatidylglycerol plus cardiolipin (and less phosphatidylethanolamine) than normal . We now wish to report that, under these conditions, the pss-8 mutant is hypersensitive to certain antibiotics, especially to streptomycin, kanamycin, and gentamicin, although also to ampicillin and novobiocin . At 30 degrees, the membrane protein and fatty acid composition of pss-8 is nearly normal, i.e . identical with an isogenic pss+ organism . Radiochemical labeling and bacteriophage growth studies show that lipopolysaccharide is also unaltered . Therefore, the antibiotic hypersensitivity of pss-8 differs from previously reported hypersensitivities, associated with lipopolysaccharide defects . These results suggest that the polar phospholipid headgroups may play an important role in maintaining the barrier function of the outer gramnegative membrane and that putative inhibitors of the phosphatidylserine synthetase might potentiate the action of numerous antibiotics currently in clinical use. Science, 1977 Aug 19, 197(4305), 763 - 4 Water-to-air transfer of virus; Baylor ER et al.; Bubbles rising through suspensions of the bacteriophages T2 and T4 and of Escherichia coli adsorb and eject these particles in droplets that are formed when the bubbles burst . The concentration of the viruses in ejected droplets, determined from electron microscopy, exceeded the suspension concentration by 50 times . Similar results were obtained for Escherichia coli . The viability of some of the adsorbed particles was established by biological counts. J Biol Chem, 1977 Aug 10, 252(15), 5177 - 9 Escherichia coli ribosomal protein S1 recognizes two sites in bacteriophage Qbeta RNA; Goelz S et al.; As a component of bacteriophage Qbeta replicase, S1 is required both for initiation of Qbeta minus strand RNA synthesis and for translational repression, which has been traced to the ability of the enzyme to bind to an internal site in the Qbeta RNA molecule . Previously, Senear and Steitz (Senear, A . W., and Steitz, J . A . (1976) J . Biol . Chem . 251, 1902-1912) found that isolated S1 protein binds specifically to an oligonucleotide spanning residues -38 to -63 from the 3' terminus of Qbeta RNA . Here we report that S1 also interacts strongly with a second oligonucleotide in Qbeta RNA, which is derived from the region recognized by replicase just 5' to the Qbeta coat protein cistron . Both sequences exhibit pyrimidine-rich regions. J Virol, 1977 Aug, 23(2), 439 - 42 Two infectious forms of bacteriophage phi X 174; Fujisawa H et al.; Infectious particles with S values of 114 and 132 were isolated from cells infected with bacteriophage phi chi 174 . Electron micrographs of the 132S particle revealed a spherical structure with a diameter of about 40 nm . The 114S particle had spikelike projections and a diameter of about 32 nm . The 132S particles could be converted to 114S particles in vitro . However, pulse and pulse-chase experiments indicated no precursor-product relationship between these two particles in vivo. Proc Natl Acad Sci U S A, 1977 Aug, 74(8), 3355 - 9 Bacteriophage T4 RNA ligase is gene 63 product, the protein that promotes tail fiber attachment to the baseplate; Snopek TJ et al.; RNA ligase and tail fiber attachment activities, normally induced following bacteriophage T4 infection of Escherichia coli, are not induced when gene 63 amber mutants of T4 infect nonpermissive host cells . Both activities are induced when these mutants infect permissive hosts, or when revertants of these mutants infect nonpermissive hosts . When one of these mutants infects a host that carries supF, both activities are more than normally heat labile . RNA ligase, purified to homogeneity, promotes the tail fiber attachment reaction in vitro with a specific activity similar to that of the most highly purified preparations of gene 63 product isolated on the basis of tail fiber attachment activity . We conclude that T4 RNA ligase is gene 63 product . The RNA ligase and tail fiber attachment reactions differ in requirements and in response to some inhibitors, suggesting that the two activities of the gene 63 product may be mechanistically unrelated. Proc Natl Acad Sci U S A, 1977 Aug, 74(8), 3259 - 63 Packaging recombinant DNA molecules into bacteriophage particles in vitro; Hohn B et al.; Recombinant phage genomes made in reactions with purified enzymes may be recovered directly by packaging into phage heads in vitro . The process is efficient and nonselective and offers containment in initial stages of handling recombinant DNA . Ligase {poly(deoxyribonucleotide):poly-(deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1} reaction products can recombine with endogenous phage DNA during packaging, but UV-irradiation eliminates the biological activity of the endogenous DNA. Mutat Res, 1977 Aug, 44(2), 165 - 76 Conditional lethal mutants of bacteriophage T4 unable to grow on a streptomycin resistant mutant of Escherichia coli; Childs JD; Sixteen conditional lethal mutants of bacteriophage T4D have been isolated which grow on Escherichia coli CR63