Marker effects on reversion of T4rII mutants.
The frequencies of 2-aminopurine- and 5-bromouracil-induced A:T leads to G:C transitions were compared at nonsense sites throughout the rII region of bacteriophage T4. These frequencies are influenced both by adjacent base pairs within the nonsense codons and by extracodonic factors. Following 2AP treatment, they are high in amber (UAG) and lower in opal (UGA) codons than in allelic ochre (UAA) codons. In general, 5BU-induced transitions are more frequent in both amber and opal codons than in the allelic ochre codons. 2AP- and 5BU-induced transition frequencies in the first and third positions of opal codons are correlated with those in the corresponding positions of the allelic ochre codons. Similarly, the frequencies of 2AP-induced transition in the first and second positions of amber codons and their ochre alleles are correlated. However, there is little correlation between the frequencies of 5BU-induced transitions in the first and second positions of allelic amber and ochre codons. (+info)
Bacteriophage inactivation at the air-water-solid interface in dynamic batch systems.
Bacteriophages have been widely used as surrogates for human enteric viruses in many studies on virus transport and fate. In this investigation, the fates of three bacteriophages, MS2, R17, and phiX174, were studied in a series of dynamic batch experiments. Both MS2 and R17 readily underwent inactivation in batch experiments where solutions of each phage were percolated through tubes packed with varying ratios of glass and Teflon beads. MS2 and R17 inactivation was the result of exposure to destructive forces at the dynamic air-water-solid interface. phiX174, however, did not undergo inactivation in similar studies, suggesting that this phage does not accumulate at air-water interfaces or is not affected by interfacial forces in the same manner. Other batch experiments showed that MS2 and R17 were increasingly inactivated during mixing in polypropylene tubes as the ionic strength of the solution was raised (phiX174 was not affected). By the addition of Tween 80 to suspensions of MS2 and R17, phage inactivation was prevented. Our data suggest that viral inactivation in simple dynamic batch experiments is dependent upon (i) the presence of a dynamic air-water-solid interface (where the solid is a hydrophobic surface), (ii) the ionic strength of the solution, (iii) the concentration of surface active compounds in the solution, and (iv) the type of virus used. (+info)
End group of naturally terminated and UV lesion terminated T7 in vitro RNA.
The 3' terminal nucleosides of RNA transcribed in vitro by E. coli RNA polymerase from T7 DNA and UV irradiated TN DNA were determined. The 3' terminal nucleoside of naturally terminated (t1 termination site) RNA cytidine. In the case of RNA terminated at UV lesions, it is cytidine in 0 per cent of the molecules and adenosine in the remaining 30 per cent. Cytidine trialcohols are labile in high concentrations of KOH and at high temperature and appear to convert to uridine. (+info)
Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage.
We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage phiC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, phiflu, in the Haemophilus influenzae genome, and two small prophage-like elements, phiRv1 and phiRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage. (+info)
Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product.
Lysogenic bacteriophages are major vehicles for the transfer of genetic information between bacteria, including pathogenicity and/or virulence determinants. In the enteric pathogen Escherichia coli O157:H7, which causes hemorrhagic colitis and hemolytic-uremic syndrome, Shiga toxins 1 and 2 (Stx1 and Stx2) are phage encoded. The sequence and analysis of the Stx2 phage 933W is presented here. We find evidence that the toxin genes are part of a late-phage transcript, suggesting that toxin production may be coupled with, if not dependent upon, phage release during lytic growth. Another phage gene, stk, encodes a product resembling eukaryotic serine/threonine protein kinases. Based on its position in the sequence, Stk may be produced by the prophage in the lysogenic state, and, like the YpkA protein of Yersinia species, it may interfere with the signal transduction pathway of the mammalian host. Three novel tRNA genes present in the phage genome may serve to increase the availability of rare tRNA species associated with efficient expression of pathogenicity determinants: both the Shiga toxin and serine/threonine kinase genes contain rare isoleucine and arginine codons. 933W also has homology to lom, encoding a member of a family of outer membrane proteins associated with virulence by conferring the ability to survive in macrophages, and bor, implicated in serum resistance. (+info)
Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin.
Norfloxacin (NFLX) caused induction of prophages VT1 and VT2 of enterohemorrhagic Escherichia coli O157 at subinhibitory concentrations. In time course experiments, we observed the following sequential events: upon induction, the phage genomes underwent multiplication; the amount of stx genes increased; and subsequently, large quantities of toxins VT1 and VT2 were produced. Further studies showed that the molecular mechanism of prophage induction is closely related to the RecA system since the prophage VT2 was not induced with NFLX in a recA mutant strain. (+info)
A complex control circuit. Regulation of immunity in temperate bacteriophages.
Temperate bacteriophages can display in a stable way two essentially different behaviours. In the immune state, a gene (cI) produces a repressor which prevents expression of all the other viral genes; in the non-immune state the typically viral functions are expressed. The choice between the two pathways and the establishment of one of them have much in common with cell determination and differentiation. This choice depends on a complex control system, in fact one of the most intricate nets of regulation known in some detail. Our paper provides a formal description and partial analysis of this regulatory net. It is shown that even for relatively simple known models, this kind of analysis uncovers predictions which had previously remained hidden. Some of these predictions were checked experimentally. The experimental part chiefly deals with the efficiency of lysogenization by thermoinducible lambda phage carrying mutations in one or more of the regulatory genes, N, cro and cII. Although N- mutations are widely known for preventing efficient integration, and both N- and cII mutations for preventing efficient establishment of immunity, it is shown that, as predicted by a simple model, both N- and cII- phage efficiently lysogenize at low temperature if they are in addition cro-. In contrast with lambda N- cro+, lambda N- cro- is not propagated as a plasmid at low temperature, precisely because it establishes immunity too efficiently. Genetic control circuits are described in terms of sets of logic equations, which relate the state of expression of genes or of chemical reactions (functions) to input (genetic and environmental) variables and to the presence of gene and reaction products (internal, or memorization varibles). From the set of equations, one derives a matrix which shows the stable stationary states (if any) of the system, and from which one can derive the pathways (temporal sequences of states) consistent with the model. This kind of analysis is complementary to the more widely used analysis based on differential equations; it allows one to analyze in less detail more complex systems. The language might be used as well, mutatis mutandis, in fields very different from genetics. The last part of the discussion deals with the role of positive feedback loops in our specific problem (establishment and maintenance of immunity in temperate bacteriophages) and in developmental genetics in general. As a generalization of an old idea, it is suggested that cell determination (for a given character) depends on a set of genes whose interaction constitutes a positive feedback loop. Such a system has two stable stationary states: which one is chosen will usually depend on additional controls grafted on the loop. (+info)
Filamentous phage replication initiator protein gpII forms a covalent complex with the 5' end of the nick it introduced.
Rolling circle type DNA replication is initiated by introduction of a nick in the leading strand of the origin by the initiator protein, which in most cases binds covalently to the 5' end of the nick. In filamentous phage, however, such a covalent complex has not been detected. Using a suitable substrate and short reaction time, we show that filamentous phage initiator gpII forms a covalent complex with nicked DNA, which rapidly dissociates unless gpII is inactivated. A peptide-DNA complex was isolated from trypsin digest of the complex by ion-exchange column chromatography and gel filtration, and its peptide sequence was determined. The result indicated that gpII was linked to DNA by the tyrosine residue at position 197 from the N-terminus. The mutant protein in which this tyrosine was replaced by phenylalanine did not show any detectable activity to complement gene II amber mutant phage in vivo. In vitro, the mutant protein recognized the origin and bent DNA as well as the wild-type does, but failed to introduce a nick and to relax the superhelicity of cognate DNA. (+info)