Analysis of the HSV-1 UL2 and UL1 Genes by Insertional Mutagenesis

Insertional mutagenesis has been used to investigate the functional role of the HSV-1 UL1 and UL2 gene products in the lytic cycle of the virus. The genes were mutagenised by disruption of the protein coding sequences, which was achieved by inserting a segment of foreign DNA into a plasmid-cloned copy of the target gene. The insert contained a marker gene, the Escherichia coli LacZ gene under the direction of the SV40 early promoter, and was tranferred into the wt genome by homologous recombination of the flanking sequences. Recombinant progeny were isolated by the addition of X-gal, a chromogenic substrate for B-galactosidase (the product of the LacZ gene) to an agar overlay. Plaques of recombinant virus, which appear blue using this screening technique, were subjected to plaque purification. This technique was used in attempts to construct three different recombinants. In all three cases, the cotransfection gave rise to blue plaques, and in two cases the recombinants were purified. The first of these (designated in1601) carried the insert in the coding region of the gene UL2 as verified by restriction endonuclease analysis. Earlier mapping studies had indicated a possible function for the product of this gene, namely uracil-DNA giycosylase, a DNA repair enzyme which appears to be ubiquitous. Enzyme assays performed on extracts of tissue culture cells infected with inl601 were consistent with this assignation, as they demonstrated that in1601-infected extracts had only mock-infected levels of uracil-DNA glycosylase activity, but wt levels of three other virus-induced enzymes. Final confirmation came with comparison between the amino acid sequence of the UL2 gene product and the amino acid sequences translated from other known uracil-DNA glycosylase genes (E. coli, Saccharomyces cerevisiae and human). Immunoblotting analysis, using antisera raised against a synthetic oligopeptide representing the carboxy terminus of the protein, showed a reaction with a protein of approximate Mr 39,000, which is similar to enzymes from other sources. The mutant was readily isolated and possesses growth characteristics very similar to wt, indicating that the enzyme does not appear to be required for lytic growth, at least in the tissue culture system employed for growth and maintenance of the recombinant. This is in contrast with the observations that a high degree of amino acid conservation exists between the enzymes from different sources, and that the gene is found in all of the herpesviruses sequenced to date. The latter would tend to imply an important role in vivo for this enzyme. It is accepted that this in vivo function is the removal of uracil from DNA, brought about by deamination of cytosine. This is a potentially mutagenic event, and if left uncorrected, would result in a G.C to A.T transition mutation. The second recombinant which was purified was designated in1602 and was constructed by cotransfection of the mutated plasmid-borne copy of the UL2 gene with a virus containing an insertion in the gene encoding the virus induced dUTPase. This enzyme is thought to be responsible for minimising the misincorporation of dUTP into DNA during replication, by lowering the pool of dUTP. Although this also results in the presence of uracil in the DNA, it will not result in mutagenesis if left uncorrected, as subsequent replication will not alter the sequence of the DNA. Misincorporation of dUTP into DNA and in situ deamination of cytosine are the two major mutagenic events which result in the presence of uracil in DNA. Therefore the recombinant in1602 is deficient in both of these genes, as confirmed by restriction analysis, and in both of these activities, as confirmed by enzyme assays. The third recombinant, whose construction was attempted unsuccessfully, represented insertion of the LacZ sequences into the coding sequence of the gene UL1. Although the initial co-transfection also yielded blue plaques, these failed to grow through subsequent rounds of plaque purification. The co-transfection was repeated twice and repeated attempts were made to titrate the blue plaques obtained from the co-transfection, all without success. Control experiments done in tandem with these titrations indicate that the procedure was functioning normally. The two most likely explanations for this phenomenon are: (a) The recombinant was unstable due to other, unpredicted alterations to the genome. This has been observed in the construction of other recombinant viruses, but usually when the construction has involved duplication of virus DNA sequences, (b) There is a stringent requirement for the UL1 gene product in the replicative cycle of the virus. At present there is no direct evidence which would support either of these explanations at the expense of the other. Searches of amino acid sequence databases failed to identify any sequences which could be homologues of this protein, so there is no further functional information about this protein

Book Review: \u27An Historian\u27s Approach to Religion\u27 by Arnold J. Toynbee

A review of the book, An Historian\u27s Approach to Religion, which was published by Oxford University Press in 1956 and authored by Arnold J. Toynbee

Book Review: \u27Judaism and Modern Man\u27 by Will Herberg

A review of the book, Judaism and Modern Man, which was published by Farrar, Straus and Young in 1951 and authored by Will Herberg

Monitoring techniques for the manufacture of tapered optical fibers

The use of a range of optical techniques to monitor the process of fabricating optical fiber tapers is investigated. Thermal imaging was used to optimize the alignment of the optical system; the transmission spectrum of the fiber was monitored to confirm that the tapers had the required optical properties and the strain induced in the fiber during tapering was monitored using in-line optical fiber Bragg gratings. Tapers were fabricated with diameters down to 5 μm and with waist lengths of 20 mm using single-mode SMF-28 fiber

Ammonia sensing using lossy mode resonances in a tapered optical fibre coated with porphyrin-incorporated titanium dioxide

The development of an ammonia sensor, formed by the deposition of a functionalised titanium dioxide film onto a tapered optical fibre is presented. The titanium dioxide coating allows the coupling of light from the fundamental core mode to a lossy mode supported by the coating, thus creating lossy mode resonance (LMR) in the transmission spectrum. The porphyrin compound that was used to functionalise the coating was removed from the titanium dioxide coating upon exposure to ammonia, causing a change in the refractive index of the coating and a concomitant shift in the central wavelength of the lossy mode resonance. Concentrations of ammonia as small as 1ppm was detected with a response time of less than 1min. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only

An ammonia sensor based on Lossy Mode Resonances on a tapered optical fibre coated with porphyrin-incorporated titanium dioxide

The development of a highly sensitive ammonia sensor is described. The sensor is formed by deposition of a nanoscale coating of titanium dioxide, containing a porphyrin as a functional material, onto a tapered optical fibre. The titanium dioxide coating allows coupling of light from the fundamental core mode to a lossy mode supported by the coating, thus creating a Lossy Mode Resonance (LMR) in the transmission spectrum. A change in the refractive index of the coating caused by the interaction of the porphyrin with ammonia causes a change in the centre wavelength of the LMR, allowing concentrations of ammonia in water as low as 0.1 ppm to be detected, with a response time of less than 30 s

Novel highly sensitive protein sensors based on tapered optical fibres modified with Au-based nanocoatings

Novel protein sensors based on tapered optical fibres modified with Au coatings deposited using two different procedures are proposed. Au-based coatings are deposited onto a nonadiabatic tapered optical fibre using (i) a novel facile method composed of layer-by-layer deposition consisting of polycation (poly(allylamine hydrochloride), PAH) and negatively charged SiO₂ nanoparticles (NPs) followed by the deposition of the charged Au NPs and (ii) the sputtering technique.The Au NPs and Au thin film surfaces are then modified with biotin in order to bind streptavidin (SV) molecules and detect them. The sensing principle is based on the sensitivity of the transmission spectrum of the device to changes in the refractive index of the coatings induced by the SV binding to the biotin. Both sensors showed high sensitivity to SV, with the lowest measured concentration levels below 2.5 nM. The calculated binding constant for the biotin-SV pair was 2.2×10‾¹¹ M‾¹ when a tapered fibre modified with the LbL method was used, with a limit of detection (LoD) of 271 pM. The sensor formed using sputtering had a binding constant of 1.01 × 10‾¹⁰ M‾¹ with a LoD of 806 pM. These new structures and their simple fabrication technique could be used to develop other biosensors