The application of artificial intelligence techniques to a sequencing problem in the biological domain

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Plant plasmalemma structure: an immunological approach

Clonal hybridomas were generated which secreted monoclonal antibodies reactive with a crude membrane preparation from Nicotiana glutinosa suspension culture cells. Antibody secretion was assessed by a radioimmunoassay using such membranes as substrate. A number of the monoclonal antibodies recognised epitopes expressed on the external face of the plasmalemma of N. glutinosa suspension culture derived protoplasts, as assessed by immunofluorescence microscopy and flow cytometry. A second, non-overlapping set of antibodies recognised epitopes on the exterior face of the cell wall of intact cells, whilst a third group showed neither reactivity, and was postulated to recognise epitopes expressed within the cell. Western blotting and immunoprecipitation analyses identified antibodies recognising a number of proteins including several plasmalemma glycoproteins. The recognised epitopes were periodate sensitive, and so probably in carbohydrate moieties. Immunoaffinity chromatography of a detergent extract of plant cells allowed purification of a plasmalemma glycoprotein. This was subjected to amino acid analysis, and used to raise polyclonal antisera and further monoclonal antibodies. Deglycosylation of a partially purified detergent extract of N. glutinosa cells suggested that this plasmalemma glycoprotein consists of a 50 kd molecular weight core protein, which is extensively and heterogeneously glycosylated, raising its apparent molecular weight to 130-230 kd. The plasmalemma glycoprotein was used as an antigenic marker for plasmalemma derived vesicles resolved on sucrose density gradients, and for heterokaryons in protoplast fusions. Similarities between the plasmalemma glycoprotein and arabinogalactan proteins are discussed

Isolation and Structural Analysis of Genomic Variants of Herpes Simplex Virus Type 2

The original aim of the project was to study recombination in HSV-2 strain HG52 using restriction enzyme sites as unselected markers. As no relevant DNA sequence data was available for HSV-2 it was decided to isolate site deletion mutants by enrichment selection of spontaneously occurring variants. The restriction enzyme Xba I was chosen as it makes only four staggered cuts in the HSV-2 genome and a virus, HG52X163X3X53, lacking all four Xba I sites was isolated following three rounds of enrichment selection. However, during the screening procedures involved in the isolation of HG52X163X3X53, a large number of variants with genomic alterations was identified and the work described in this thesis has concentrated on the analysis and characterization of these variants. Of the variants isolated after the initial enrichment selection of HG52 DNA five have been studied in detail. Three (HG52X85/4, HG52X85/5 and HG52X86) have deletions from IRL ranging in size from 2. 5x10e6 daltons to 6x10e6 daltons. Under immediate early conditions VmwIE64 production by HG52X85/5 and HG52X86 in which only the 3' part of IE1 is removed is reduced despite their deletions ending approximately 1kb downstream from the 3' end of UL54 which encodes this polypeptide. HG52X85/4 which also has a deletion ending approximately 1kb downstream of UL54 but in which an entire copy of IE1 is removed makes VmwIE64 in normal amounts. Of the two remaining variants one, HG52X192, has a deletion of approximately 1x10 6 daltons in each copy of RL. The final variant, HG52X19, has a deletion of approximately 9x10e6 daltons which removes the entire internal copy of the long repeat and half of the short repeat. The long segment of the genome is fixed in the prototype orientation whilst the short region inverts inefficiently through the undeleted part of the repeats (the internal copy of the 'a' sequence being deleted). The genome has non-HSV DNA inserted across the deletion. The inserted DNA has been reiterated to give different copy numbers and hence a heterogeneous genome population. It has been demonstrated that the inserted DNA is of bovine origin -presumably the result of recombination with the calf thymus DNA used as a carrier during transfection. To investigate if the enrichment selection procedure was responsible for the production of the unexpectedly high proportion of variants, isolates from untreated HSV stocks were studied. Of the fifty plaques of HG52 analysed, twelve differed significantly from the wild type structure. However, the plaques, five of HG52/5 and seven of HG52/10, represented only two variants with the same genome structures as HG52X86 and HG52X192 respectively. No significant variation was found in the stocks of other HSV strains examined (ie. HSV-2 strains 333 and 186 and HSV-1 strains 17 and KOS or plaque purified HG52 isolates (eg. tsl)). Variation was found in HSV-1 strain McKrae but its relevance was impossible to assess as the history of the stock is unknown. The conclusion has been drawn that variation has arisen in HG52 in the absence of enrichment selection although the structure of HG52X19 must have resulted from the transfection procedure. Three variants, HG52X163X12, HG52X163X14 and HG52X163X21, were isolated following the second round of enrichment selection using as the parent HG52X163 which lacks the 0.7m.c. Xba I site. HG52X163X12 has a deletion removing sequences from 0.94m.c. to 0.99m.c. (ie. part of U S and almost all of TRS) . The isolation of this variant demonstrates that genes US 10, 11, 12 and one copy of IE3 and one copy of oriS are non essential, at least in vitro. The two remaining variants, HG52X163X14 and HG52X163X21, both have deletions removing the same region as from HG52X163X12. However, the deleted sequences are replaced by sequences between 0.83m.c. -0.91m. c. in the inverted orientation thereby effectively deleting US between 0.94-0.96m.c. while extending the short repeats by 6kb. The only differences found between HG52X163X14 and HG52X163X21 were the loss of the 0.91m.c. Xba I site and a small insert containing a Hind III and EcoR I site at the 0.94m.c. Xba I site in the latter. It is possible that the enrichment selection procedure was responsible for the generation of these three variants as the rearrangements appear to be associated with the Xba I sites at 0.91 and 0.94m.c

Analytical applications of ion selective devices

PhD ThesisIon selective electrodes, ISEs, and ion sensitive field effect transistors, ISFETs, are small, relatively simple to operate and easily automated sensors and, therefore, have a wide range of uses e. g. for field measurements in portable detectors, for on-line measurements in industrial flow systems and in clinical work. Several flow systems were studied for use with ion selective devices. New design ISE flow cells, designed at Newcastle, were found to minimise dead space and carry-over of sample solutions, allowing more rapid sample throughput. An ISFET flow cell studied, however, was found to have serious design faults. The constant volume dilution method of calibration and selectivity determination was shown to be a simple easy-touse method but must be implemented with caution. The selectivity of sensors to the primary ion was determined, where applicable, and the optical sensitivity of ISFETs was examined. Potassium concentrations in fertilizers were determined, using ISEs, in both flow systems described above; more accurate results were obtained using the newer flow-cells. Failure of ISEs after prolonged use in fertilizer solutions is believed to be have been caused by Donnan Breakdown due to HPO 2- ions. A computer controlled titration system was developed which can be used for volumetric or coulometric titrations. Coulometry, an absolute method, is particularly suitable for titration of sub micro-litre samples and for chemically labile species as sample manipulation is minimised and avoids addition of solution reagent, obviating CO 2 contamination of hydroxide. The advantages of coulometry were exploited in work to confirm the second dissociation constant for hydrogen sulphide. Aerial oxidation and sample carbon dioxide uptake are common problems associated with sulphide solutions. Using degassed water for sample preparation, keeping all solutions under nitrogen and using a sulphide anti-oxidant buffer it was possible to reduce sulphide oxidation. Coulometry was used to generate hydrogen ions and potentiometric measurements of the pH and sulphide ion concentrations, made simultaneously, were used to calculate the pK 2d of hydrogen sulphide for a range of 50 gl sodium sulphide solutions. A non-linear least squares programme, SUPERQUAD, was used to obtain a better value for pK 2d. Though a coulometric option exists in SUPERQUAD, it is not often implemented. ISE titration results have not been used much with SUPERQUAD; this work examined the potential of expanding the application of SUPERQUAD. Values of pK 2d of 12.08 ± 1.0 and 11.83 ± 0.4 were obtained by visual inspection and SUPERQUAD refinement, respectively. These values agree well with the text-book value, of 11.96, and demonstrate the accuracy of coulometry. The auto-titration system developed has advantages in many areas, particularly in clinical chemistry where determinations of available species in sub micro-litre samples, delivered in a flow system are required

Cloning and characterisation of the human carbonic anhydrase I gene and mapping of the carbonic anhydrase gene cluster on chromosome 8

The carbonic anhydrase I gene (CA1) is part of a multigene family, the protein products of which are enzymes characterised by their ability to catalyse the reversible hydration of CO2. It exhibits a tissue specific pattern of expression, notably being expressed at high levels in erythroid cells in humans. It also shows regulation at a developmental level, with the CA1 protein at very low concentration in the blood of the foetus till a few weeks before birth. cDNA and genomic clones encoding CA1 were isolated and charachterised. Analysis of cDNA clones showed the presence of an occasional exon in the 5' leader of the transcript, while at the 3'-end two polyadenylation sites could be used, while analysis of genomic clones showed that CAI is atypical amongst the carbonic anhydrases in having a large intron of 36 kb separating the erythroid specific promoter from the coding region, making the entire gene some 50 kb in length. Pulsed-field gel electrophoresis was used in the analysis of the physical linkage relationship between CA1 and the CA2 and CA3 genes. Both of these genes lie 5' to CA1 and are transcribed away from it. CA3 lies centrally in this cluster separated from the 5' end of CA1 by 80 kb, while the 5'-end of CA2 lies some 20 kb downstream of the 3' end of CA3. The DNA methylation state of the gene in several erythroid and non-erythroid cell lines was examined. This showed that in the majority of these cell lines, which do not express the CA1 gene, extensive regions around CA1 were largely demethylated. In contrast, DNA from the only CA1 expressing cell line, HEL, appeared highly methylated at all HpaII sites tested apart for one site at the erythroid promoter and another at the 3'-end of the gene. High levels of methylation of the CA1 gene were also found in DNA from untransformed cells. The possible implications of this are discussed

Nanosatellites for quantum science and technology

Bringing quantum science and technology to the space frontier offers exciting prospects for both fundamental physics and applications such as long-range secure communication and space-borne quantum probes for inertial sensing with enhanced accuracy and sensitivity. But despite important terrestrial pathfinding precursors on common microgravity platforms and promising proposals to exploit the significant advantages of space quantum missions, large-scale quantum testbeds in space are yet to be realized due to the high costs and leadtimes of traditional “Big Space” satellite development. But the “small space” revolution, spearheaded by the rise of nanosatellites such as CubeSats, is an opportunity to greatly accelerate the progress of quantum space missions by providing easy and affordable access to space and encouraging agile development. We review space quantum science and technology, CubeSats and their rapidly developing capabilities, and how they can be used to advance quantum satellite systems