Hodgkin lymphoma (HL) is a malignant lymphoma that is diagnosed mostly in young adults, and is the second most common malignancy to affect this age group. This disease is subdivided into two entities with different aetiologies: classical HL (cHL) (~95% of cases) and nodular lymphocyte-predominant HL. In Europe, ~82% of young adults with cHL are non-Epstein-Barr virus associated and epidemiological studies have suggested that a common infectious agent may play a key role in the aetiology of these cases. The molecular biology of HL is not well understood, primarily due to the low number of Hodgkin and Reed-Sternberg (HRS) cells present within these tumours. However, recently developed techniques for the selection and micromanipulation of single HRS cells from tumours, and the development of molecular cytogenetic techniques (i.e. array-comparative genomic hybridisation (CGH)) are overcoming these difficulties. To investigate a potential candidate virus, DNA samples from cHL biopsies were screened for the measles virus (MV) and polyomaviruses (PyV), using immunohistochemistry and highly sensitive PCR assays. Chromosomal imbalances in six well-established cHL-derived cell lines and a cHL case were analysed by array-CGH. To obtain sufficient DNA for array-CGH from the cHL case, single HRS cells were isolated using laser microdissection. DNA was extracted then amplified by degenerate oligonucleotide primer polymerase chain reaction. MV and PyV genomes were not detected within cHL biopsies. Recurrent chromosomal imbalances were confirmed within the cHL-derived cell lines and cHL case, in addition to several novel imbalances. This is the first time that a cHL case has been analysed by array-CGH.

The mitotic cell cycle underlies propagation of eukaryotic cells, continually duplicating and dividing. The past few years have seen major advances in understanding of the regulatory mechanisms that impose on the cell cycle to tightly co-ordinate progression through its individual phases, safeguarding the timing and integrity of its hallmark events, DNA synthesis and mitosis. Transcription is prominent among these processes, manifesting its importance for cell cycle controls by the large number of eukaryotic genes that are expressed at specific cell cycle times. Certain genes are cell cycle regulated in a number of organisms, suggesting that their phase-specific transcription is important for all eukaryotic cells. The budding and fission yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, have been used extensively as model organisms for the study of the eukaryotic cell cycle and cell cycle-regulated transcription, because the cell cycle machinery is conserved among eukaryotes and they are experimentally tractable. Recent microarray analyses have shown that cell cycle-specific expression is a frequent theme in the two yeasts, identifying consecutive, inter-dependent, waves of transcriptional activity, coinciding with the four main cell cycle transitions; G1-S, S, G2-M and M-G1 phases. Each phase-specific transcriptional wave corresponds to at least one group of co-regulated genes, sharing common cis- and trans- acting elements. The work presented in this thesis delves into the regulatory network that drives phase-specific gene expression during late mitosis-early G1 phase in fission yeast. During this late cell cycle stage, fission yeast and, indeed, every eukaryotic cell, undergo major changes; each completes mitosis and cytokinesis, partitioning its duplicated genetic and cytoplasmic material into two progeny cells, which then themselves prepare for a new round of mitotic cell division. Consistent with their periodic pattern of expression, most of the genes transcribed during the M-G1 interval in S. pombe encode proteins that execute important functions during late mitosis and cytokinesis. A DNA sequence promoter motif, the PCB (Pombe cell cycle box), has been identified in fission yeast that confers M-G1 specific transcription, and is bound by the PBF (PCB binding factor) transcription factor complex. PCB promoter motifs are present in several M-G1 transcribed genes, including cdc15+, spo12+, sid2+, fin1+, slp1+, ace2+, mid1+/dmf1+ and plo1+, the latter encoding a Polo-like kinase that also regulates M-G1 gene expression and influences the PCB-dependent binding properties of PBF. Three transcription factors, Sep1p and Fkh2p, both forkhead-like transcription factors, and Mbx1p, a MADS-box protein, have been implicated in M-G1 specific gene expression and are thought to be components of PBF. Consistent with Fkh2p and Sep1p regulating M-G1 specific transcription, forkhead-related sequences are present in the genes’ promoters. Notably, fkh2+ contains both PCB and forkhead promoter sequences and is transcribed during the M-G1 interval, implying that Fkh2p and Plo1p regulate gene transcription during late mitosis and ensuing passage through cytokinesis via feedback loops. This study provides further evidence about transcriptional regulation late in the fission yeast cell cycle, revealing that the PCB sequence is crucial for M-G1 specific transcription, with forkhead-associated DNA motifs playing a parallel but smaller regulatory role. Consistent with this hypothesis, work here and elsewhere shows that both Fkh2p and Sep1p control phase-specific expression of their co-regulated genes through the PCB and forkhead sequences. Notably, data in this thesis reveal that these two forkhead transcription factors associate with each other in vitro and in vivo and bind in vivo to the PCB promoter regions of M-G1 transcribed genes, including cdc15+ and plo1+, in a cell cycle specific manner, consistent with Fkh2p repressing and Sep1p activating transcription. Furthermore, Fkh2p contacts its own promoter, suggesting that it regulates its own expression via a negative feedback mechanism. The Plo1p kinase is shown here to bind in vivo to Mbx1p throughout the cell cycle and in a manner that requires both its kinase and polo-box domains. In agreement with this observation, Plo1p can phosphorylate in vitro Mbx1p, itself known to become phosphorylated during late mitosis. This is the first time that a Polo-like kinase has been shown to bind and phosphorylate a MADS-box protein in any organism. Moreover, in concert with Plo1p binding and phosphorylating Mbx1p, ChIP assays here reveal that this kinase interacts in vivo with the PCB promoter DNA of M-G1 expressed genes, including cdc15+ and fkh2+, in a cell cycle-dependent manner with a timing that coincides with low levels of expression, but follows promoter binding by Fkh2p. Given that Plo1p has previously been shown to positively influence M-G1 dependent transcription, its cell cycle pattern of promoter contact suggests that this Polo-like kinase functions at the genes’ promoters, most-likely via binding and phosphorylation of Mbx1p, to re-stimulate transcription, following repression by Fkh2p. In parallel, these findings suggest that Plo1p regulates its own expression via a positive feedback loop. Overall, the work presented in this thesis unravels crucial regulatory aspects of the transcriptional network that drives M-G1 specific transcription in S. pombe: it suggests an important role for the PCB promoter motif in transcriptional regulation; it proposes that Fkh2p acts as a repressor while Sep1p as an activator of late mitotic transcription; it reveals and proposes novel functions for Plo1p, a conserved Polo-like kinase family member, involving its association with Mbx1p, a MADS box protein, and its cell cycle specific recruitment to PCB promoters of M-G1 transcribed genes. As transcriptional systems, encompassing homologues of most of the components of this S. pombe M-G1 specific transcriptional network operate both in S. cerevisiae and humans, this demonstrates their importance for mitotic cell cycle progression. Thus this work potentially offers new insights into M-G1 specific gene expression in all eukaryotes.

fruitless (fru) is a pleiotropic gene which produces an array of variant transcription factor isoforms to fulfil a range of developmental and behavioural roles, from the regulation of axonal pathfinding during embryogenesis, all the way to the precise orchestration of individual steps of the Drosophila melanogaster male courtship ritual. Much of the transcriptional differentiation is achieved through the use of multiple promoters, sex-specific splicing and variant C-termini. Alternative splicing at the 3′ end enables generation of transcripts containing one of at least three zinc finger (Zn-F) domains (A, B or C). Due to the close proximity of these Zn-F–encoding exons at the fru locus, almost all extant fru mutants reduce or eliminate expression of all isoforms from a given promoter(s). This has prevented genetic dissection of their individual roles, and limited functional assignment to the zinc finger triumvirate as a whole. Using gene targeting by homologous recombination, this project set out to generate precise null mutations for type-A and -B Fru isoforms in an attempt to determine which aspects of fru function are conferred by these isoforms. Isoform-specifc antibodies were also generated to confirm the loss of individual isoforms within the generated mutants, and to investigate the expression of different isoforms throughout development. Generation of such an antibody to FruB proteins enabled the developmental expression pattern of this isoform to be assessed for the first time, and expression in the male-specific serotonergic neurons of the abdominal ganglion suggested a possible role for male-specifc FruB isoforms in male fertility. Investigation of the developmental expression patterns of FruC revealed a novel immunostaining pattern for this isoform in a group of coalescing cells which appear towards the end of embryonic development. Isolation of specific-isoform mutants was achieved, giving rise to multiple mutant phenotypes, varying in severity between mutant lines. Analysis of mutants lacking type-A and -B Fru isoforms demonstrated the importance male-specific type-A and/or type-B isoforms in establishing male fertility, and suggested essential roles for sex-non-specifc type-A and/or type-B isoforms in the viability and morphology of both sexes.

The biochemical genetics of two natural populations of house sparrows, Passer domesticus, at sites 20km apart in Nottinghamshire, England, were investigated. Seven polymorphic protein loci were sampled non-destructively by taking blood samples from over 1500 individually marked birds. A detailed investigation of the genetics of these loci was conducted for 124 clutches containing 357 nestlings where the parents were also sampled. Segregations at four loci (6PGD, PEPD2, PEPD3 and lDHC) agreed with a simple Mendelian model of codominant inheritance. One locus (EST2) contained null alleles. Two loci (PEPD3 and GP1) showed segregation distortion in all sex, site and year classes. This distortion was not attributable to the misinterpretation of gel patterns; possible causes involving the operation of natural selection were discussed. Linkage analyses were conducted, and no significant evidence was obtained for linkage between any combination of loci. Of the nestling genotypes, 12.9% were interpreted as being genetically incompatible with those of their parents. Exclusion probabilities were calculated as 43-51% for nonpaternity and 59-67% for nonparentage. The applicability of these estimated probabilities was tested by the random reassortment and comparison of observed parental genotypes among observed sibship genotypes. Significantly fewer nestlings were excluded in these simulations than expected from calculated exclusion probabilities, though the distribution of multiple mismatches did not differ from expectation. A deficiency of multiple mismatches was found in the field data, implying the occurrence of errors; the possible sources of error were considered. The most parsimonious interpretation of those mismatches that did not appear to be due to errors was that they resulted from a rate of nonpaternity of about 6%. No heterogeneity in the rate of mismatches was observed within or among breeding seasons or sites. Genotype and allele frequencies were presented for each locus in each age, sex and sampling year class at each study site. The samples were not found to depart from Hardy Weinberg equilibrium, and there was no evidence for significant inbreeding within sites. There were no differences in allelic distributions between the sexes or among years for adults within the populations. No differences were found among age groups or nestling year classes when allowance was made for sib correlations. Heterozygosities were higher at Brackenhurst than at Sutton Bonington for most loci, and the overall difference was significant. There was a particularly large difference in allele frequencies between nestlings in each population for GP1. Digenic gametic disequilibria were investigated. A detailed analysis of the mating types was made. No evidence was obtained for any departure from random mating at the protein loci. There was a significant tendency amongst the loci and samples for the inbreeding coefficients of the successful breeders to be negative. Significant assortative mating was found with respect to weight and tail-length in one population.

This study concerns a population of the Pied Flycatcher (Ficedula hypoleuca) living in nestboxes in an area of woodland in Mid-Wales. The occupants of 180 nestboxes were monitored during 1988 and 1989. In addition to behavioural observations and records of breeding performance, individual adults and pulli were caught and measured, and a blood sample taken. In the Pied Flycatcher, polygyny is a common mating strategy in which the two or more females mated to a single male nest in discrete territories up to 500m apart This behaviour has been interpreted in two ways, firstly as the result of female choice for the quality of the male or his territory, and secondly, as a consequence of male deception, by which already-mated males attract secondary females who suffer reduced breeding success as a result. In this population polygyny was a rare occurrence; only 3 of 240 breeding males were recognised to be polygynous. These males defended two adjacent nestboxes. The breeding success of the three secondary females was not unusually low. These results suggest that a model of male- or territory quality might better explain the situation in this population. The occurrence of extra-pair mating has being noted in a number of species, including the Pied Flycatcher. In this study it was found to account for 2.7% of the offspring screened by genetic fingerprinting. Another common method for detecting extra-pair paternity uses the heritability of a skeletal measurement.The results from the two methods are shown to be incompatible. A number of weaknesses with the heritability method are described and discussed. The increasing number of studies on the Pied Flycatcher throughout Europe reveal that the frequency of mating strategies such as polygyny and extra-pair mating differ from area to area This suggests that environmental factors may play a major part in determining the costs and benefits of such strategies.

A C. perfringens type A genomic library was constructed in E. coli by banking overlapping 6-10 kbp Hind III fragments of chromosomal DNA from the enterotoxin (CPE) positive strain NCTC 8239 into the pUC derived vector pHG165. The library was screened by colony hybridization with a degenerate 26 bp oligonucleotide probe, derived from the amino acid sequence CPE9_17A. complex mixture of plasmid DNA was isolated from the only hybridization positive clone. A second round of screening picked out a single plasmid, with an apparently altered copy number, pLWl, that carried the CPE gene, cpe, on a 6.8 kbp insert. A sequence deduced primer strategy for direct plasmid sequencing was initiated using a primer deduced in a similar manner to the 26 bp probe, obviating the need for prior mapping and subcloning of the insert. The amino acid sequence for the conceptual gene product of the single open reading frame differed only slightly from the known CPE sequence but lacked the C terminal residues. The biased cpe codon usage reflected the low %G+C content of the DNA. The %G+C content was even lower in the upstream region and possessed properties characteristic of bent DNA. The region 5' to the ATG translational start codon contained a Shine-Dalgarno sequence and several sequences with significant homology to the putative transcriptional control regions for the tetanus toxin gene. The N-terminal coding region contained a direct repeat of an upstream sequence that shared considerable homologies with the crossover point in site 1 of the Tn3 res region. Southern blot analyses of chromosomal and plasmid DNAs from several isolates indicated that the majority of strains were cpe-. The chromosomal location and architecture of cpe appeared identical in all cpe+ strains. A second copy, pLW2, of the 5' end of cpe, on a 4.5 kbp Pst I/Eco RI restriction fragment, was cloned during one of many unsuccessful attempts to clone the 3' end. A separate re-cloning experiment isolated several different clones that contained the 0.6 kbp Hind III located = 2.5 kbp 5' to the ATG codon of both cloned copies of cpe but none of them carried the CPE gene. The fragment was used as a DNA probe to show that it was present in high copy number in some strains of C. perfringens but completely absent from others. An hypothesis describing the possible involvement of a mobile genetic element in C. perfringens enterotoxin production offers explanations for the cloning of a complex mixture of plasmids, the apparent alteration in plasmid copy number, the identification of putative DNA crossover points, the failure to clone the 3' end of cpe and the isolation of a novel DNA fragment.

The studies reported in this thesis aimed to identify the pattern of variation across the CYP11B1/CYP11B2 locus in order to determine the genetic variation responsible for the observed impaired 11ß-hydroxylation and its link with aldosterone levels and hypertension. In Chapter 3, an attempt was made to identify polymorphisms in CYP11B1 that associate with hypertension and a raised aldesterone-to-renin ratio (ARR) and therefore might contribute to the genetic component of these phenotypes. The study in Chapter 4 aimed to analyze the pattern of variation across the CYP11B2/B1 locus in detail. In Chapter 5, the frequency and phenotypic associations of the newly identified CYP11B1 5’UTR polymorphisms -1889 G/T and -1859 A/G were investigated in a hypertensive population. The frequencies were found to be -1889 G-0.53, T- 0.47; -1859 A-0.5, G-0.5. Interestingly, the -1889 G/T and -1859 A/G polymorphisms were found to be associated with impaired 11ß-hydroxylase efficiency in a hypertensive population. The THS/total F ratio (index of 11ß-hydroxylase activity) was significantly higher in -1889 TT homozygotes than in GG homozygotes (p=0.025). A similar pattern was seen with the -1859 SNP, with GG homozygotes tending to have a higher ratio than AA homozygotes (p=0.056). These steroid data were supported by the results from Chapter 6 that examined the effects of these polymorphisms in vitro using luciferase reporter gene constructs transfected into Y1 mouse adrenal cells. In summary, these studies have confirmed that the CYP11B2 and CYP11B1 genes contain a high frequency of genetic variation. Many of the variants identified in CYP11B1 have been shown to alter activity significantly. There is strong evidence to suggest that the impaired 11ß-hydroxylase efficiency previously associated with the -344 C/T and IC variants in CYP11B2 is due to linkage with the -1889 G/T and -1859 A/G polymorphisms upstream of CYP11B1.

The microflora of two acidic environments was investigated using analysis of 16S rDNA amplified by the polymerase chain reaction (PCR) from environmental DNA. These environments had different chemical characteristics from most of the acidic environments studied by others. The first sample site, a coal spoil (Birch Coppice, Warwickshire), might have been expected to produce niches enriched in humic matter. The second, comprising geothermal vents on the Island of Vulcano, was unusual for natural acidic environments since it was saline. Three vent regions of different temperatures (30°C, 45°C and 80°C) were examined. Prior to the 16S rDNA analysis of the sites, a brief investigation into selection of a suitable method of DNA extraction was carried out. A bead-beating method and a chemical lysis/freeze-thaw method were compared. With regard to clone types found via each method, there was little qualitative difference. DNA was extracted from the two sites and 16S rRNA genes were amplified by PCR. PCR products were ligated and competent E. coli cells were transformed to produce clone libraries. Restriction fragment length polymorphisms (RFLPs) were examined and representatives of each RFLP type were sequenced and analysed with reference to RNA gene sequence data bases. The coal spoil clone library was dominated by sequences related to those from uncultured actinobacteria, particularly those found previously in peat bogs and various soils. Representatives of some well-known acidophiles were also found (e.g. Leptospirillum species). The clone bank from the saline, geothermal site DNA comprised sequences from acidophiles capable of growth at the respective temperatures of different samples. The lowest temperature samples produced sequences from a novel Acidithiobacillus species and also indicated a novel species probably related to Thiobacillus prosperus (which was isolated previously from Vulcano). A high temperature sample gave sequences from archaeal acidophiles, Acidianus brierleyi and, previously isolated from Vulcano, Acidianus infernus and Thermoplasma volcanium. Where the clone banks revealed the presence of novel organisms, attempts were made to isolate and characterise them. The novel actinobacteria did not appear to grow in laboratory enrichment cultures. The novel Acidithiobacillus species and two novel Thiobacillus prosperus-like species were characterised.

The rotaviruses are a recently defined ubiquitous group of viruses responsible for causing acute-gastroenteritis in human infants and young animals. Biochemical studies have shown that the rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA). This thesis concerns an investigation of the nature and extent of genomic variation in rotaviruses. A rapid and sensitive method for analyzing the genome profiles of rotavirus field isolates was developed. This is based on the direct extraction of dsRNA from faecal samples followed by radiolabelling with [³²P] pCp using T4 RNA ligase. This procedure has been further developed to produce a method for generating diagnostic fingerprints from individual species of dsRNA. A detailed structural study making use of this fingerprinting method has been undertaken on bovine, porcine and human rotavirus isolates. These analyses show that genome segment mobility variations are always associated with detectable changes in nucleotide sequence. They also show that corresponding genome segments with no mobility variation can have sequence-changes at least as great as those found in segments showing electrophoretic mobility variation. These results also revealed evidence for genome segment specific regions of terminal sequence conservation. Evidence for the occurrence of genome segment reassortment between viruses in the field was obtained. Finally evidence for the existence of a 'new' porcine rotavirus which is antigenically unrelated to previously described rotaviruses and has an unusual pattern for its 11 genome segments is presented.

Studies on the molecular organisation and properties of the first three enzymes of pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPTase), aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase), in various organisms have been reviewed. The molecular organisation of these three enzymes has been investigated in wheat using gel filtration chromatography. CPSase activity could not be detected in gel filtered extracts and in crude extracts from wheat seedlings was shown to be highly labile. ATCase and DHOase activity was detected and the molecular weights of these enzymes were estimated to be 1.03 x 105 ( 1.4 x 10^4) and 8.6 x 10^4 (6 x 103), respectively. At no time during these investigations were high molecular weight species (consistent with the presence of a multifunctional complex containing these enzymes) detected. During the course of these investigations, a protease was detected which was shown to co-migrate with ATCase and DHOase activities. This protease was shown to be insensitive to the serine protease inhibitor PMSF, but was partially inactivated by iodoacetamide, consistent with the protease being a member of the cysteinyl protease family. Inclusion of iodoacetamide during chromatography also failed to reveal high molecular weight species of these enzymes. Antisera were raised against purified wheat ATCase and were characterised by their ability to inactivate the enzyme. These antisera were then used to probe western blots of crude extracts from wheat seedlings and screen a wheat cDNA expression library to ATCase sequences. Western blotting failed to show any immunoreactive species in extracts prepared under conditions which suppressed protease activity (SDS, -mercaptoethanol), although a low molecular weight (approximately 3.7 x 10^4) ATCase could be detected in samples obtained after gel filtration chromatography. Antisera also showed very little cross-reactivity with the ATCase from E.coli a result consistent with studies on the enzyme from B. subtilis.