No description available.

No description available.

The project was to clone the gene encoding a nuclear enzyme poly-ADP-ribose polymerase (PARP) involved in the posttranslational modification of nuclear proteins. This modification is important in the regulation of various cellular processes such as cell differentiation, proliferation and in the molecular events involved in the recovery of cells from DNA damage. At the start of this project this enzyme had been well characterized in animal systems but had not as yet been explored in plants. Firstly, I showed the enzyme to be present in higher plant nuclei, by a series of experiments including enzyme assays and western blotting (probed with a polyclonal antibody specific to the protein). Molecular biology approaches were used to isolate the gene encoding the PARP enzyme. These techniques included screening of cDNA libraries, constructed in λZAPII, with both the polyclonal antibody and a gene probe (a restriction digest of a human PARP gene was performed and a 1.4Kb fragment containing the C-terminal region of the gene used as a probe). This identified a number of bacteriophages from which sequence information was obtained (by making and extracting phagemids). Subsequent translation of these nucleotide sequences revealed that one of the sequences (245941) obtained in this manner showed homology to the PARP protein. Degenerate oligonucleotide primers, designed to the conserved C-terminal region of the gene, were also used in an attempt to amplify a 375bp region of the gene thought to contain the PARP signature. PCR products of the expected size (375bp) were obtained from an SST cDNA library. These PCR products hybridized to the gene probe (mentioned earlier). Subsequent subcloning revealed multiple products of 375bp comigrating in the agarose gels. Two different sequences were obtained which were, upon translation, shown not be PARP-like sequence.

Tp1 is a long highly-abundant repetitive element found in the genome of <i>Physarum polycephalum</i>. It predominates in a hypermethylated, high molecular weight HpaII-resistant domain. The structural properties of this element resemble those of eukaryotic retrotransposons such as <i>copia</i> in <i>Drosophila melanogaster</i>. Preliminary evidence has indicated that segments of Tp1 are transcribed in both plasmodia and amoebae. Hence, from the structural analogy with retrotransposons it was supposed that the long terminal repeats (LTRs) of Tp1 may serve as transcriptional promoters. A plasmid (pE1oriCAT) was constructed which contains a CAT reporter gene located 3' to the Tp1 LTR and a <i>Physarum</i> origin of replication. CAT activity observed in cell lysates prepared from pE1oriCAT-transfected amoebae suggested that the Tp1 LTR can function as a transcriptional promoter. Nucleotide sequence information was obtained for the right-hand end of a copy of Tp1. From its predicted amino acid sequence a high degree of homology was found with the reverse transcriptase domain in the C-terminal half of the <i>pol</i> polypeptide in <i>copia</i> from <i>Drosophila melanogaster</i>, Ty912 from <i>Saccharomyces cerevisiae</i> and Ta1 from <i>Arabidopsis thaliana</i>. The high degree of identity between the reverse transcriptase domains in all of the above elements suggests that they may share a common ancestor. A preliminary search for Tp1 virus-like particles (VLPs) was carried out. Fractions collected from sucrose density gradients, prepared from amoebal cell lysates, were examined for VLP structures by transmission electron microscopy. Although putative VLPs were observed, which were morphologically similar to VLPs associated with the yeast retrotransposon Ty, they did not react immunologically with an antibody prepared against Ty VLPs. A member of another transposon-like family, designated Tp2, has also been identified in the genome of <i>Physarum</i>. Tp2 is 1.68kb in length, has 180bp LTR sequences at its termini and is blanked by 5bp direct repeats. Analysis of the predicted amino acid sequence of Tp2 identified an amino acid motif that is homologous to a highly conserved RNA binding domain in the <i>gag</i> polypeptide of retroviruses and retrotransposons.

No description available.

No description available.

Access to abstract restricted until 05/2016. / Access to thesis restricted until 05/2016. / Department of Physics and Astronomy

DNA topoisomerase II is an essential enzyme in eukaryotes and is required for many aspects of DNA metabolism including DNA replication, recombination, chromosome segregation and chromosome condensation. It is also a major component of the nuclear scaffold. Topoisomerase II from lower eukaryotes has been shown to be phosphorylated in vivo and this phosphorylation leads to a modulation of activity. However, unlike these lower eukaryotes, human topoisomerase II exists as two closely related, but genetically distinct, isozymes which have markedly different expression and localization patterns. Topoisomerase IIα is a 170kDa protein and topoisomerase IIβ is 180kDa. This study set out to analyse the phosphorylation of these specific isozymes and understand how this leads to the regulation of their distinct biological functions. In order to undertake this study, two polyclonal anti-topoisomerase II antibodies were generated and a series of other polyclonal and monoclonal antibodies characterized. Furthermore, the α isozyme of human topoisomerase II was purified to near homogeneity from cultured HeLa cells. A kinase activity with the biochemical characteristics of casein kinase II co-purified with and could phosphorylate the purified topoisomerase Hot protein. The α and β isozymes of human topoisomerase II were both shown to be phosphoproteins in vivo. The α isozyme is phosphorylated predominantly on serine residues but with a minor proportion of phosphothreonine. Both the α isozyme and a stable ISOkDa fragment of the β isozyme are phosphorylated in vitro by casein kinase II and the catalytic subunit of PKA (cAMP-dependent protein kinase). The α isozyme can also be phosphorylated in vitro by Ca²⁺-dependent and -independent isozymes of protein kinase C and the cell cycle-regulated p34^cdc2 kinase. Two-dimensional tryptic phosphopeptide mapping suggested that the pattern of phosphorylation of human topoisomerase Ha protein in vivo is complex with phosphorylation occurring on multiple residues. Comparison with in vitro maps suggested that casein kinase II and PKA could account for most of the phosphorylation seen in vivo. Using a one- dimensional phosphopeptide mapping approach, a major site of phosphorylation in vivo appeared to be within the C-terminal 20kDa, and that casein kinase II, PKA and PKC may all phosphorylate this region. Phosphorylation of human topoisomerase Hoc protein by casein kinase II, PKA and PKC all led to a stimulation of activity as measured by plasmid relaxation and decatenation. In contrast, dephosphorylation led to a marked decrease in activity of the enzyme. The dephosphorylated enzyme could be reactivated by casein kinase II but not PKA phosphorylation. These data suggest that phosphorylation plays a crucial role in the control of DNA tertiary structure in human cells via regulation of the activity of topoisomerase II proteins.

At the base of the hair follicle epidermal matrix is a population of germinative epidermal (GE) cells that is in close communication with the dermal papilla. These GE cells are at the core of activities that comprise the fundamental processes of cell signalling and differentiation in the hair follicle. Since it is in the germinative region that the signals that produce hair are being received and transcribed, identification of genes expressed in the GE cells will be important for our understanding of hair growth control and the molecular mechanisms operating at the site of epidermal proliferation and differentiation. This study describes the production of a series of cDNA libraries, both by conventional means from rat vibrissa follicles and follicle end bulbs, and by PGR from the GE cells and the tissues of the upper end bulb. These libraries were then used for a variety of screening approaches to isolate cDNA clones, firstly for molecules which are known to be involved in the control of hair growth, and secondly for molecules which are differentially expressed in the follicular germinative epidermis. In order to identify such preferentially expressed genes, a dual labelling differential screen of the vibrissa follicle end bulb cDNA library was performed, using probes derived from the germinative epidermal and upper end bulb PGR generated libraries. Nine putative differentially expressed clones were isolated and sequenced. RNase protection analysis and non radioactive in situ hybridisation was then performed to confirm that these clones were expressed in the germinative epidermis of rat vibrissa follicles. Further characterisation by northern blotting revealed that several of the clones were expressed in multiple tissues. Nucleotide sequence analysis revealed that six of the clones had a concensus BG1 repeat sequence at the end of their 3'UTR. This has been implicated in post-transcriptional control of intracellular mRNA localisation. Three of these clones were related to genes implicated in induction and vesicle trafficking. These clones may therefore be involved in the signal transduction pathways operating in the germinative epidermis in response to primary signalling molecules received from the dermal papilla.

Enantiopure A and A lanthanide complexes, bearing a phenanthridinium or a dipyridoquinoxaline chromophore as a sensidser, have been designed with the aim of developing structural and reactive probes for nucleic acids. Their interaction with DNA was studied using various spectroscopic techniques. A certain degree of stereoselectivity in DNA binding was discerned. The A enantiomers of the Eu tetramide and of the EuPh(_3)dpq complexes interacted with nucleic acids in a predominantly intercalative binding mode, by inserting their planar aromatic chromophore between the base-pairs. The former showed a preference for C-G sites, while the latter bound preferentially to A-T base-pairs. A rather different binding mode, probably involving the minor groove, was revealed in the interaction of the A enantiomer of the EuPh(_3)dpq complex with nucleic acids, with a higher affinity for C and G bases. In the presence of nucleic acid, a charge transfer process occurred in each case, which quenched the singlet excited state of the phenanthridinium moiety or the lanthanide excited state (in Ph(_3)dpq complexes). In the unique case of the EuNp(_3)dpq complexes, the interaction resulted in an increase in the metal emission intensity and lifetime, as a consequence of the protection of the molecule, probably accommodated in the DNA minor groove, from a quenching process. This light switch' effect can be exploited in the development of spectroscopic probes. The TbNp(_3)dpq, on the other hand, was found to generate singlet oxygen efficiently and could therefore act as a reactive probe. Moreover, the EuPh(_3)dpq and TbPh(_3)dpq complexes showed extraordinarily high emission quantum yields in aqueous media, due to the favourable photophysical properties of the dpq antenna as well as the nonadentate nature of the Ph(_3)dpq ligand. This makes them valuable luminescent probes.