Binding of proflavin to T2L bacteriophage and its DNA.

We have measured the binding equilibria of proflavin to T2L bacteriophage, in both “slow” and “fast” sedimenting forms, and to free T2L DNA. Measurements were carried out by difference spectroscopy at 430 nm at temperatures from 13 to 43°C and at pH 5.6 and 7.6. We found no significant difference in the binding parameters of the two phage forms. Also, the fraction of nucleotides available as binding sites for proflavin was the same for both free and intraphage DNA. However, the binding constant is about an order of magnitude lower for encapsulated than for free T2L DNA, due to the decreased exothermicity of the binding reaction within the phage head.     

Partial exclusion of genes of bacteriophage T2 with T4-glucosylated DNA in crosses with bacteriophage T4

A recombinant strain (D41) between phage T2 and T4 was isolated which possessed the T2 region of the genome between genes 32 and 39 and both the T4 genesgt ⁺ andgt ⁺ for glucosyltransferase. D41 was crossed with T4amber mutants in the genes for early functions and in some genes for late funcitions. The progeny of the crosses was examined for the frequency of theam ⁺ markers from D41. Genes 32, 60 and 39 in the T2 region of the recombinant strain were as sensitive to exclusion as those in standard-type T2. The T4 glucosylation of the DNA of these T2 genes did not protect them against partial exclusion by T4. However, genes in the region from gene 56 to 55 in the recombinant were resistent to exclusion. In standard-type T2 this region of the genome is sensitive to partial exclusion by T4. There are at least four exclusion sensitive sites in T2: one near gene 32, one near gene 60, one linked to gene 56 and one between genes 42 and 55.This investigation was carried out partially within the frame of the Association between Euratom and the University of Leiden, contract nr. 052-64-1-BIAN.     

Genetische und physiologische Beziehungen zwischen den Bakteriophagen T1 und D 20

Summary The close relationship between phages T 1 and D 20 was confirmed by genetical crosses. D 20 could be shown to differ from T 1 by at least two genes, both affecting the host range of the phage. The geneDs will render a phage capable of multiplying on the original host of D 20, SF;Ha will enable the phage to lyse B/1. D 20 may thus be formulated as T 1HaDs. Both genes could be localized within the T 1 linkage map, in the vicinity of the geneHr, which is known to control the host range specifity against B/1. The results of genetical, adsorption, and serological studies can be interpreted by the assumption that each gene controls the synthesis of a specific protein, responsible for the adsorption behaviour of the particular phage. A phage will adsorb to SF according to itsDs genotype, to B or its phage resistant mutants according to itsHa genotype. These proteins do not, however, exert their functions independently. Therates of adsorption and neutralization are influenced by the presence or absence of proteins not required for the particular reaction under investigation.

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Studies on bacteriophage T7 DNA synthesis in vitro. II. Reconstitution of the T7 replication system using purified proteins.

Summary DNA synthesis in vitro using intact duplex T7 DNA as template is dependent on a novel group of three phage T7-induced proteins: DNA-priming protein (activity which complements a cell extract lacking the T7 gene 4-protein), T7 DNA polymerase (gene 5-protein plus host factor), and T7 DNA-binding protein. The reaction requires, in addition to the four deoxyribonucleoside triphosphates, all four ribonucleoside triphosphates and is inhibited by low concentrations of actinomycin D. Evidence is presented that the priming protein serves as a novel RNA polymerase to form a priming segment which is subsequently extended by T7 DNA polymerase. T7 RNA polymerase (gene 1-protein) can only partially substitute for the DNA-priming protein. At 30°C, deoxyribonucleotide incorporation proceeds for more than 2 hours and the amount of newly synthesized DNA can exceed the amount of template DNA by 10-fold. The products of synthesis are not covalently attached to the template and sediment as short (12S) DNA chains in alkaline sucrose gradients. Sealing of these fragments into DNA of higher molecular weight requires the presence of E. coli DNA polymerase I and T7 ligase. Examination of the products in the electron microscope reveals many large, forked molecules and a few eye-shaped structures resembling the early replicative intermediates normally observed in vivo.     

Inactivation of bacteriophage T4 by organic and inorganic tin compounds

Summary Butyltins and inorganic tins inactivated bacteriophage T4. The effect was on the phage and not on its host,Escherichia coli. The order of effectiveness was SnCl₄monobutyltin>dibutyltin>tributyltinSnCl₂. For the butyltins and SnCl₄ this was the reverse of the order of effectiveness usually observed for plants, animals, and microorganisms. This pattern suggests that degradation of tributyltin does not always detoxify it. Monobutyltin (MBT), the most effective organotin, was more effective at pH 4 than at higher pH values and it was more eeffective at low strength. Inactivation proceeded more rapidly at 37°C than at 18°C. The results of experiments in which the ratio of phage to MBT was varied suggests that tin compounds may act by flocculating phage particles. Zinc, which is bound by phage short tail fibers (P12), inhibited phage inactivation by MBT, suggesting that MBT may act upon these tail fibers.     

The terminal redundant regions of bacteriophage T7 DNA

Summary Some aspects of the involvment of the terminal reduntant regions of T7 DNA on phage production have been studied by transfection experiments with T7 DNA after treatment of the molecules with exonuclease or exonuclease plus exonuclease I. It was found that terminal 5 gaps between 0.08 and 6.4% of the total length did not decrease the infectivity of the molecules although such gaps cannot be filled directly by DNA polymerases. Rather, compared to fully native DNA the infectivity of gapped DNA increased up to 20 fold in rec ⁺ spheroplasts and up to 4 fold in recB spheroplasts. This indicates a protective function of the single-stranded termini against the recBC enzyme in rec ⁺ and possibly another unidentified exonuclease present also in recB. The possibility that spontaneous circularization of the gapped molecules in vivo provides protection against exonucleolytic degradation was tested by transfection with T7 DNA circularization in vitro by thermal annealing. Such molecules were separated from linear molecules by neutral sucrose gradient centrifugation. They displayed a 3 to 6 fold higher infectivity in rec ⁺ and recB compared to linear gapped molecules, which shows that T7 phage production may effectively start from circular DNA.When the 3 single-stranded ends from gapped molecules were degraded by treatment with exonuclease I the infectivity of the molecules was largely abolished in rec ⁺ and recB as soon as 40 to 80 base pairs had been removed per end. It is concluded that the terminal regions of T7 DNA molecules are essential for phage production and that the redundancy comprises probably considerably less than 260 base pairs. The results are discussed with respect to the mode of T7 DNA replication.     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

Mutational alteration of the breakage/resealing subunit of bacteriophage T4 DNA topoisomerase confers resistance to antitumor agent m-AMSA

Summary Bacteriophage T4 provides a simple model system in which to examine the mechanism of action of antitumor agents that have been proposed to attack type II DNA topoisomerases. Prior results demonstrated that T4 type II DNA topoisomerase is the target of antitumor agent 4-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in phage-infected Escherichia coli: a point mutation in topoisomerase structural gene 39 was shown to confer both m-AMSA-resistant phage growth and m-AMSA-insensitive topoisomerase activity. We report here that a point mutation in T4 topoisomerase structural gene 52 can also independently render both phage growth and topoisomerase activity resistant to m-AMSA. The DNA relaxation and DNA cleavage activities of this newly isolated mutant topoisomerase were significantly insensitive to m-AMSA. The drug-resistance mutation in gene 52, as well as that in gene 39, alters the DNA cleavage site specificity of wild-type T4 topoisomerase. This fording is consistent with a mechanism of drug action in which both topoisomerase and DNA participate in formation of the drug-binding site.     

Illegitimate recombination mediated by T4 DNA topoisomerase in vitro

Summary Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.     

A coupled in vitro system for the formation and packaging of concatemeric phage T1 DNA

Summary Extracts derived from E. coli cells infected non-permissively with phage T1 amber mutants were used in an in vitro system to investigate the packaging of T1 DNA into phage heads. The standard extract used infections with amber mutants in genes 1 and 2 (g1^-g2^-) which are defective in T1 DNA synthesis but can synthesis the proteins required for particle morphogenesis. g1^-g2^- extracts packaged T1⁺ virion DNA molecules with an efficiency of 3×10⁵ pfu/g DNA. Extracts from cells infected with phage also defective in DNA synthesis but carrying additional mutations in genes 3.5 or 4 which are required for concatemer formation in vivo (g1^-g3.5^- and g1^-g4^- extracts) package T1 virion DNA at substantially lower efficiencies.Analysis of the DNA products from these in vitro reaction showed that concatemeric DNA is formed very efficiently by g1^-g2^- extracts but not by g1^-g3.5^- or g1^-g4^- extracts. These results are interpreted as evidence that the T1 in vitro DNA packaging system primarily operates in a similar manner to the in vivo headful mechanism. This is achieved in vitro by the highly efficient conversion of T1 virion DNA into concatemers which are then packaged with a much lower efficiency into heads to form infectious particles. A secondary pathway for packaging T1 DNA into heads and unrelated to the headful mechanism may also exist.     

In vitro studies on the role of phage T7 gene 4 product in DNA replication

Summary A soluble enzyme fraction prepared from T7-infected E. coli is able to initiate DNA synthesis on circular single-stranded phage DNA. The product synthesized in vitro is a full-length linear complementary strand as judged by alkaline sucrose gradient analysis. DNA synthesis requires the products of the phage genes 4 and 5, Mg⁺⁺, dNTPs and rNTPs; however, ATP by itself can almost completely satisfy the rNTP requirement. The gene 4 product is essential for DNA chain initiation on unprimed single-stranded DNA, but is dispensable for the replication of a X174 DNA-RNA hybrid. The enzyme system from T7-infected cells does not discriminate between the DNA templates from phages X174, M13 or fd and is also capable of replicating native T7 DNA. However, a striking difference with regard to the template DNA is revealed by complementation analysis. Extracts of T7 mutant-infected cells complement each other only with T7 DNA but not with X174 DNA as template.Abbreviations rNTP ribonucleoside triphosphate - dNTP deoxyribonucleoside triphosphate - BSA bovine serum albumin     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.     

Flow dichroism of capsid DNA phages. II. Effect of DNA deletions and intercalating dyes

The flow linear dichroism of bacteriophage λ and its deletion mutants, λ b2 and λ b221, was determined. The hydrodynamic behavior of the three phages differed slightly, but the magnitude of the dichroism was substantially the same with 〈cos²θ_μp〉 = 0.364, 0.368, and 0.372, respectively. The dichroism of intercalating dyes combined with bacteriophage was used as a further probe of phage structure. The reduced dichroism from proflavin with T4 showed no change with time during the reaction, but the interpretation of the ligand dichroism is complicated by an alteration of the hydrodynamic behavior of the phage–dye complex relative to the phage alone. Ethidium with λ also produced a stable reduced dichroism, but the signal indicated an average orientation of intercalated dye that is different from the average base orientation. The reduced dichroism of ethidium changes with time as it penetrates λ b2, eventually approaching the dichroism of the nucleotide bases. The implication of these findings on the plausibility of various simple DNA packing models is discussed.     

Host-dependent modification of bacteriophage T7 and SAMase-negative T3 derivatives affecting their adsorption ability

Summary When passaging phage T7 and SAMase-negative T3 mutants betweenE. coli strains with identical (EcoB) or without (EcoO) DNA host specificity, phenotypically a host-controlled modification and restriction is observed. This phenomenon is not due to classical modification and restriction of the bacteriophage DNA but depends on the reversibly altered adsorption capacity of the phages on the different host strains.     

Caffeine as a comutagen for ethylmethanesulfonate in strains of phage T4

Summary The mutation frequency of DNA polymerase mutants of phage T4 treated with ethyl methanesulfonate (EMS) then incubated in the presence and absence of caffeine was studied using an rII reversion system. The DNA polymerase mutation is shown to be antimutagenic for EMS induction of reversions which occur by a GC to AT transition. Caffeine acts as a comutagen for the induction by EMS of mutant phages and produces a significant increase in the frequency of reversions from rII to r⁺. Caffeine is slightly mutagenic for the phage strain carrying the wild type polymerase and inhibits the action of the 35 exonuclease function of T₄ DNA polymerase as measured in vitro. These findings suggest that caffeine acts by directly influencing nucleotide selection or the editing function of the DNA polymerase.     

Genetic analysis of two bacterial RNA polymerase mutants that inhibit the growth of bacteriophage T7

Summary The Escherichia coli mutants 7009 and BR3 are defective in the growth of bacteriophage T7. We have previously shown that both of these mutant hosts produce an altered RNA polymerase which is resistant to inhibition by the T7 gene 2 protein (De Wyngaert and Hinkle 1979). In both strains, the mutation which prevents T7 growth is closely linked to rifA (rpoB). Both mutants are complemented by transformation with a multicopy plasmid carrying rpoB and rpoC but not by a plasmid carrying only rpoB. This indicates that the mutations reside in rpoC, the structural gene for the subunit of RNA polymerase. When a single copy of the wildtype rpoC allele is introduced into the mutant using the transducing phage drif ^d18, the mutant allele is dominant over wildtype. The drif ^d18 transductant also remains unable to support the growth of T7 in the presence of rifampin. This supports our conclusion that the mutation is in rpoC. We have measured the growth of T7 phage, the kinetics of phage DNA synthesis, and the structure of replicative DNA intermediates in several transductants, and compared these results with those obtained in the original mutant strains.     

Mutants of phage HK239 defective in excluding phages lambda T4rII, P1, and P2

Summary Escherichia coli cells lysogenic for temperate phage HK239 exclude phages , HK022, P1 vir, P2, and rII mutants of phage T4. After mutagenic treatment, four isolates were obtained for their inability to exclude T4rII. It is shown that this mutation, designated exc, is located in the prophage HK239, and that, it also abolishes the exclusion of phages , HK022, P1 vir, and P2.     

Kinetics of ethidium's intercalation in packaged bacteriophage T7 DNA: effects of DNA packing density

The kinetics of ethidium's intercalative binding to DNA packaged in bacteriophage T7 and two T7 deletion mutants have been determined, using enhancement of fluorescence to quantitate binding. At a constant ethidium concentration, the results can be described as first-order binding with two different rate constants, k (= k₁ + k_?1) and k (= k₂ + k_?2). The larger rate constant (k) was at least four orders of magnitude smaller than the comparable first-order forward rate constant for binding to DNA released from its capsid. At 25°C values of k decreased as the amount of DNA packaged per internal volume increased. This latter observation indicates that the rate of ethidium's binding to packaged T7 DNA is limited by an event that occurs inside of the DNA-containing region of T7, not by the crossing of T7 capsid's outer shell. Arrhenius plots of kM are biphasic, indicating a transition for packaged DNA at a temperature of 20°C. The data indicate that k s are limited by either sieving of ethidium during its passage through the packaged DNA or subsequent hindered intercalation.     

Dinitrogen fixation (Acetylene reduction) and nitrogen accumulation in peas cultivated under the standard growth conditions

InPisum sativum cultivated under standard growth conditions the extent of N₂ fixation with time estimated by the acetylene reduction assay (PN₂F) and rates of the actual nitrogen accumulation of plant biomass (ANA) were calculated from six independent growth experiments. In the plants inoculated with indigenous soilRhizobium populations and cultivated on 0.63 mmol/L nitrate level the percentage PN₂F:ANA ratios ranged from 25.7 to 61.5%. In peas inoculated with the inoculant strain the PN₂F:ANA ratios were markedly higher, ranging from 59.8 to 65.1%. The plants cultivated on N-free nutrient solutions showed both PN₂F:ANA and C₂H₄N₂ ratios to be somewhat higher compared with the 0.63 mmol/L nitrate cultivated plants.