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PubMed1974

Studies on an endonuclease activity associated with bacteriophage T7 DNA-membrane complex.

Infection of Escherichia coli with bacteriophage T7 results in the formation of an endonuclease which is selectively associated with the T7 DNA-membrane complex. A specificity of association with the complex is indicated by the finding that the enzyme is completely resolved from a previously described T7 endonuclease I. When membrane complexes containing (3)H-labeled in vivo synthesized DNA are incubated in the standard reaction mixture a specific cleavage product is formed which is about one-fourth the size of T7 DNA. The endonuclease associated with the complex produces a similar cleavage product after extensive incubation with native T7 DNA or T7 concatemers. Degradation of concatemers occurs by a mechanism in which the DNA is converted to molecules one-half the size of T7. This product is in turn converted to fragments one-fourth the size of mature phage DNA. The endonuclease is not present in membrane complexes from uninfected cells or cells infected with gene 1 mutants. The enzyme activity is, however, present in cells infected with mutants defective in T7 DNA synthesis or maturation.

Cell Membrane↗
PubMed1984

Cloning and analysis of pif, replication and leading regions of the F plasmid.

We describe the molecular cloning of BglII fragments of the hybrid plasmid pRS5 (pSC101 and EcoRI fragments of F; f7, f5, f3 and f6). The clones isolated were examined for the expression of F-specified replication, incompatibility, mobilization and inhibition of T7 bacteriophage multiplication. Proteins directed by the BglII clones were labelled in Escherichia coli K12 maxicells and analyzed by SDS-polyacrylamide gel electrophoresis. The sizes of previously reported proteins, encoded by the replication, incompatibility and leading regions encompassed by these plasmids have been confirmed in this study. In addition, the results demonstrate that a pif gene, which encodes an 80,000 dalton polypeptide essential for the inhibition T7 phage multiplication, is located on the BglII fragment that spans the junction of EcoRI fragments f7 and f5.

Bacterial Proteins↗
PubMed1997

30 years later--a new approach to Sol Spiegelman's and Leslie Orgel's in vitro evolutionary studies. Dedicated to Leslie Orgel on the occasion of his 70th birthday.

The conditions necessary for evolution are amplification, mutagenesis and selection. Here we describe the evolutionary response of an in vitro replicating system to the selection pressure for fast growth and show what happens to the amplified molecules within this replication system. Our emphasis is on methodology, on the monitoring and the automation of experiments in molecular evolution. In order to perform in vitro studies on the evolution of RNA molecules, a modified self-sustained sequence replication (3SR) method was used. In the first step of the 3SR reaction, the RNA template is reversely transcribed by HIV-1 reverse transcriptase, followed by a second strand synthesis and the transcription of the resulting dsDNA by T7 RNA polymerase. The selection pressure (fast growth) was achieved by applying the principle of serial transfer pioneered in the laboratories of Sol Spiegelman and Leslie Orgel. At the end of the exponential growth phase of the 3SR reaction, an aliquot of the reaction mixture is transferred into a new sample containing only buffer, nucleotides and enzymes while RNA template molecules are provided by the transfer. The conditions in the exponential growth phase allow the RNA molecules to be amplified in a constant environment; all enzymes (HIV-1 reverse transcriptase and T7 RNA polymerase) and nucleotides are present in large excess. Therefore, transferring reproducibly within the exponential growth phase is equivalent to selecting for fast growth; those molecules which can replicate faster will displace others after several transfers. The experiments were performed using a serial transfer apparatus (STA) which allows the nucleic acid concentration to be monitored on-line by measuring the laser-induced fluorescence caused by intercalation of thiazole orange monomers into the RNA/DNA amplification products. The serial transfer experiments were carried out with an RNA template (220b RNA) that represents a 220-base segment of the HIV-1 genome and comprises the in vivo primer binding site (PBS) for the HIV-1 reverse transcriptase. It could be shown that after only two serial transfers two RNA species (EP1 and EP2) emerged that were much shorter. EP1 (48b) and EP2 (54b) were formed by deletion mutations within the original 220b RNA template in the very beginning of the serial transfer experiment; due to their higher replication rate (calculated from the growth curves derived on-line) these two deletion mutants displaced the original 220b RNA template in the course of the following thirty transfers. We assume that these two RNA species evolved independently of each other. Their formation was probably induced by a strand-transfer reaction of HIV-1 reverse transcriptase. Sequence analyses of these two evolution products seem to confirm such a presented pathway. 30 years after Spiegelman's experiment, the study described here is another answer to the question he posed: 'How do molecules evolve if the only demand is the biblical injunction: multiply?'. The answer, derived from a modified 3SR amplification system (mimicking a part of the HIV-1 replication cycle in vitro), is the same as thirty years ago: The RNA molecules adapt to the new conditions by throwing away any ballast not needed for fast replication. Clearly, this is only one aspect of molecular evolution; however, it shows that we should be careful in designating unidentified genetic material as 'junk DNA'.

Bacteriophage T7↗
PubMed1992

In vitro DNA replication implicates O2-ethyldeoxythymidine in transversion mutagenesis by ethylating agents.

A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized. The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT. At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). DNA synthesis past the lesion was low (6%). Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM. Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions. DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis. dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo. These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.

Base Sequence↗
PubMed1989

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae.

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.

Cations, Divalent↗
PubMed2005

The plastidic DNA replication enzyme complex of Plasmodium falciparum.

The replication and repair of organellar genomes in the malaria parasite Plasmodium falciparum is poorly understood. We have assessed the properties of an open reading frame Pfprex (formerly known as pom1) and confirm that it specifies a multi-domain polypeptide with DNA primase, DNA helicase, DNA polymerase and 3'-5' exonuclease activities. The sequence of the primase/helicase domain is phylogenetically related to the T7-bacteriophage gene 4 product and mammalian mitochondrial helicase, Twinkle. Despite that, the N-terminal sequence of this multi-domain polypeptide directs a green fluorescent protein reporter specifically to the P. falciparum apicoplast and not to the mitochondrion. Phylogenetic analysis placed the DNA polymerase sequence with the family A bacterial polymerases, most closely to those of the thermophilic Aquifex species. Notably, the malarial enzyme was optimally active at 75 degrees C. Pfprex is the first example of a gene encoding contiguous DNA polymerase, DNA primase and DNA helicase components. We propose it has a key role in replication of the malarial plastid genome, a validated drug target.

Animals↗
PubMed2003

The complete sequence of marine bacteriophage VpV262 infecting vibrio parahaemolyticus indicates that an ancestral component of a T7 viral supergroup is widespread in the marine environment.

The 46,012-bp sequence of the marine bacteriophage VpV262 infecting the bacterium Vibrio parahaemolyticus is reported. The VpV262 sequence reveals that it is a distant relative of marine Roseophage SIO1, and an even more distant relative of coliphage T7. VpV262 and SIO1 appear to represent a widespread marine phage group that lacks an RNA polymerase gene and is ancestral to the T7-like phages. We propose that this group together with the T7-like phages be designated as the T7 supergroup. The ancestral head structure gene module for the T7 supergroup was reconstructed by using sensitive biased Psi-blast searches supplemented by statistical support derived from gene order. In the early and replicative segments, these phages have participated in extensive interchange with the viral gene pool. VpV262 carries a different replicative module than SIO1 and the T7-like phages.

Bacteriophage T7↗
PubMed1982

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein.

Bacteriophage T7 codes for a single-stranded DNA binding protein. This protein is the product of gene 2.5 and has been found previously to stimulate specifically the activity of the phage-coded DNA polymerase. We report here that the T7 DNA binding protein also stimulates the activity of the phage-coded exonuclease. The gene 6 exonuclease is a double-stranded DNA specific 5'-exonuclease that has been implicated in destruction of bacterial DNA, removal of RNA primers during DNA replication, genetic recombination, and DNA maturation. The enzyme is markedly inhibited by physiological concentrations of NaCl. This inhibition, which is due to a marked reduction in the Vmax of the enzyme, can be largely overcome by the phage-coded DNA binding protein. This stimulation is specific since the Escherichia coli DNA binding protein is without effect. The stimulation by the binding protein is apparently not due to its coating of the 3' single-stranded tails generated during the digestion. Kinetic studies show that the stimulation is due to a combined effect on both the Km and Vmax of the exonuclease. These studies are consistent with a loose binding of the binding protein to either the DNA or the exonuclease.

DNA Helicases↗
PubMed1974

Packaging and maturation of DNA of bacteriophage T7 in vitro.

We have developed an in vitro complementation assay to demonstrate packaging and maturation of DNA of phage T7. Cells of Escherichia coli B infected with an appropriate T7 amber mutant are concentrated 200-fold and lysed by freezing and thawing. Two extracts from cells infected with different amber mutants are mixed and incubated at 30 degrees . Positive complementation results in a 100-fold increase in phage titer. Using this assay we have demonstrated the packaging of phage DNA from an extract that contains no phage heads (gene 9(-), 10(-)), within head structures present in an extract that contains no phage DNA (gene 5(-)). We have also demonstrated an activity in extracts that contain no phage DNA or heads (gene 5(-), 9(-), 10(-)), which complements gene 19(-)-infected cells. We have proven that this activity is due to the gene-19 product by showing that the activity is temperature-sensitive if the extract is made from cells infected with a mutant having a temperature-sensitive mutation in gene 19. This assay should be useful in elucidating the mechanism of packaging and maturation of DNA of phage T7.

Cell-Free System↗
PubMed1973

Growth of bacteriophage H on male and female strains of Escherichia coli.

Phage H propagated on Yersinia pestis was reported by Molnar and Lawton to be rapidly adsorbed to female but not to male strains of Escherichia coli. In contrast, we find phage H adsorbs to all E. coli strains tested (both male and female) and forms plaques on a wide variety of male strains. Phage H appears to be related to the T3-T7 group of coliphages.

Adsorption↗
PubMed1992

Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA.

Using the unique sequence organization of copy-back defective interfering (DI) RNAs of paramyxoviruses, Sendai virus (SV), and measles virus copy-back DI RNAs were PCR amplified and cloned, without having to separate them from their helper nondefective genomes. The cloning was designed so that T7 polymerase transcription of the plasmids would generate DI RNAs with the exact 5' and 3' ends. The SV DI clone, transcribed from the plasmid in BHK cells using T7 polymerase produced by a vaccinia virus recombinant, was encapsidated and replicated by the SV-L, P/C, and NP proteins expressed from cloned genes. Such experiments open the possibility of examining the cis-acting sequences involved in viral multiplication directly, without using indirect markers such as CAT activity.

Animals↗
PubMed2005

The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli.

Bacteriophage K1F specifically infects Escherichia coli strains that produce the K1 polysaccharide capsule. Like several other K1 capsule-specific phages, K1F encodes an endo-neuraminidase (endosialidase) that is part of the tail structure which allows the phage to recognize and degrade the polysaccharide capsule. The complete nucleotide sequence of the K1F genome reveals that it is closely related to bacteriophage T7 in both genome organization and sequence similarity. The most striking difference between the two phages is that K1F encodes the endosialidase in the analogous position to the T7 tail fiber gene. This is in contrast with bacteriophage K1-5, another K1-specific phage, which encodes a very similar endosialidase which is part of a tail gene "module" at the end of the phage genome. It appears that diverse phages have acquired endosialidase genes by horizontal gene transfer and that these genes or gene products have adapted to different genome and virion architectures.

Bacteriophage T7↗