Rabbit antibodies raised against the glutamine-rich castor bean 2S albumin, a heterodimer of 4kDa and 7kDa polypeptides, had previously been shown to immunoprecipitate a 34kDa polypeptide from the total products formed when castor bean endosperm mRNA was translated in vitro. A cDNA library was constructed using maturing castor bean endosperm mRNA as template. Clones containing sequences complementary to albumin mRNA were isolated by hybridisation using as a probe a mixture of synthetic oligonucleotides representing sequences predicted for a peptide present In the 2S albumin large subunit. The nucleotide sequence contained an open reading frame encoding a preproprotein of 258 amino acid residues. The preproprotein included both polypeptides of the previously sequenced 2S albumin. In addition, this precursor Included two further glutamine-rich sequences which, in terms of their size and conserved cysteine residues typically found in seed proteins of the 2S albumin superfamily, possibly represent the small and large polypeptide subunits of a second heterodimeric storage protein. A post-translational processing scheme has been proposed which would result in a single preproprotein generating two distinct heterodimeric 2S albumins. Studies on the protein bodies of castor bean endosperm cells suggested the existence of a second 2S albumin. Genomic Southern blots indicated that at least 4 genes encode Ricinus 2S albumin precursors, and a Northern developmental analysis suggested that expression of such genes is temporally regulated. Genomic clones were isolated that represented the albumin genes and one of these was sequenced fully, in order to study the nature of the promoter control regions further. On comparison with the upstream regulatory regions of other 2S albumin genes, a region of high homology spanning 70 base pairs was found. This included a repetitive domain noted in many seed protein genes and implicated in storage protein promoter specificity. It was also located in ricin found likewise in castor bean endosperm cells.

Because of the potential use of the Bacilli for genetic manipulation, experiments were undertaken to investigate the usefulness of Bacillus subtilis and Bacillus licheniformis strain LO2, as hosts. Attempts at shotgun-cloning directly in B. subtilis met with repeated failure. However, subsequently the Φ3T-thyP3 gene, pC194 chloramphenicol acetyl transferase gene, B. licheniformis 749/C penP gene and the E. coli lacZ gene were expressed in B. subtilis when cloned into the plasmid vector pAB224 or one of its derivatives. Consequently, such plasmids are useful vectors for genetic manipulation in B. subtilis. The properties of the thyP3-containing hybrids were investigated. Of particular interest was the finding that monomeric plasmid DNA, containing the thyP3 gene, was active in the transformation of competent B. subtilis cells. Transformation resulted in integration of the thyP3-containing region of the plasmid into the host chromosome. The secretion of fusion proteins by B. subtilis was investigated employing the B. licheniformis 749/C penicillinase protein signal-peptide. This resulted in the secretion of the E. coli β-galactosidase enzyme from the B. subtilis cell. The thermotolerant Bacillus licheniformis strain LO2 was investigated as a possible host for genetic manipulation. A series of mutant strains were isolated but the induction of competence in two such strains could not be achieved. Additionally, transformation of protoplasts of strain LO2 could not be demonstrated. Thus contrary to previous hopes, at present this strain does not appear to be suitable as a host for genetic manipulation.

Large blocks of DNA repeats are commonly assembled into heterochromatin. Heterochromatic regions impose a transcriptionally repressive environment that can propagate over long distances stochastically silencing native genes as well as reporter genes inserted at these regions independently of DNA sequence. Heterochromatin, associated with repressive histone marks, not only down-regulates transcription but also inhibits recombination at repetitive elements. This epigenetic regulation could be a key regulatory step in organisms, such as microbial pathogens, that have to adapt rapidly to different environments. Here for the first time, we analysed the chromatin state of C. albicans repetitive elements and addressed whether and how this epigenetic state controls C. albicans genome stability. The results show that classic SIR2-dependent heterochromatin is assembled at the rDNA and telomeres. Interestingly, heterochromatin at telomeres is plastic and remodelled upon environmental changes. Pericentromeric regions and MRS (Mayor Repeated Sequences) are assembled into permissive chromatin bearing features of both heterochromatin and euchromatin. Surprisingly, SIR2-dependent heterochromatin does not control recombination at the rDNA. However, it inhibits recombination at the TRE (TLO Recombination Element) sequence associated with some TLO genes at subtelomeric regions. These results show that epigenetic factors promote differential genome stabitlity at different loci.

Telomeres are specialised nucleoprotein structures present at the ends of each chromatid that function to maintain genome stability. It is well established that a gradual decline in telomere length is associated with the process of cellular ageing, and thereby to the pathobiology of age-related diseases. In addition, the localisation of the telomere at the nuclear periphery plays an important role in the spatio-temporal organisation of the genome and in ensuring faithful segregation of chromosomes during meiosis. The aims of this thesis were to investigate telomere localisation in the nucleus, and telomere length in three hitherto early stages of development, gametogenesis, preimplantation embryogenesis and the neonatal period. Specifically: 1. To test the hypothesis that telomeres localised at the nuclear periphery in sperm cells and that this organisation was altered in sub-fertile men 2. To optimise a means of assessing average telomere length using DNA from small sample sizes and using whole genome amplified DNA from single cells 3. To investigate the role of telomere length in reproductive ageing and aneuploidy generation in women by testing the hypothesis that telomere length is significantly shorter in the first polar bodies and cleavage stage embryos of older women 4. To test the hypothesis that “preterm at term” babies (i.e. premature babies assessed at the time of their due date) displayed genetic signs of premature ageing (as manifested by significantly shorter telomeres than their term born counterparts) alongside the already established clinical signs (characterised by hypertension, diabetes and altered body fat distribution) Results confirmed the peripheral distribution of telomeres in the sperm heads of normally fertile males (using both 2D and 3D imaging) plus the novel finding that telomere distribution patterns are altered in the sperm heads of infertile males. Secondly, a reliable means of measuring telomere length was optimised in order to assess average telomere length using DNA from small sample volumes (down to single cells). Using this technology, average telomere length analysis in polar bodies and embryos found no evidence to support the hypothesis that telomere length is associated with either advanced maternal age or aneuploidy generation. Similarly, results suggest that telomere length is not significantly shorter in “preterm at term” infants compared to term born controls, thus providing no evidence that telomere attrition is involved in the pathobiology of the ‘aged phenotype’ observed in preterm infants. Taken together, results from this thesis provide some novel insights into the function of these highly important features of the genome, but also highlight that a great deal remains to be uncovered in the complex molecular mechanisms that contribute to the regulation of telomere length and nuclear distribution.

Steroids play a major role in the development of the CNS with those brain areas involved in sexual behaviour having been the focus of most neuroendocrine studies to date e.g. the hypothalamus and pituitary gland (Feist and Schreck, 1996; Mong, et al., 1999; Toran-Allerand, et al., 1980). This occurs via intracellular and cell-surface receptors that regulate changes in protein synthesis (Mensah-Nyagan, et al., 1999) to modify events related to neuronal survival and synapse formation (Breedlove, 1992). Many steroidogenesis-associated enzymes have been described in the cerebellum and cortex, but the impact of steroids on their development has yet to be investigated. A steroidal fetal programming model was utilised to investigate how gene expression in these two brain regions is affected by steroid exposure during development, and examine how this may lead to a change in brain architecture and function. The main aims of this study were to identify changes in the cerebellar expression of genetic markers of steroid metabolism, using quantitative real-time polymerase chain reaction (qRT-PCR), that result from exposure to testosterone propionate (TP) during development in male sheep. The cerebellum follows a clear developmental trajectory and contains an established cytoarchitecture that enabled us to readily identify the effects of TP treatment on the developing brain by utilising histological analyses. Further alterations of gene expression in the developing male ovine cortex were also identified using microarray analysis. The results provide novel findings in regards to androgen-sensitive gene expression in the developing ovine cerebellum and cortex but perhaps the most striking result was that androgen over-exposure delays cerebellar development, which may have consequences in later life on motor and/or cognitive function.

In 1993, a mutant strain, AS/AGU arose spontaneously in an enclosed colony of the Albino Swiss (AS) strain of rat. AS/AGU animals exhibit a set of locomotor abnormalities. They display a general instability and whole body tremor, are slow at initiating movement, show reductions in purposeful action, and perform poorly at locomotor tests such as mid-air righting. L-dopa administration or fetal midbrain transplants reverse the majority of the symptoms, resembling the observations made on Parkinson's disease patients. These features make the AS/AGU strain a useful model for movement disorders due in significant part to failure of the dopaminergic transmission system. Crosses of AS/AGU to other laboratory rat strains point to a single recessive mutation with essentially complete penetrance (agu/agu) as the cause of the abnormal phenotype. There is no evidence of sex linkage or maternal inheritance. In the absence of any evidence of the function of the agu gene product, positional cloning of this locus was begun. The first step was the establishment of a genetic map location for the agu locus. A large series of microsatellite markers were analysed and used to identify which of the strains PVG, BN, and F344 differed to a greater extent from AS/AGU. Differences at 43%, 62% and 47% of the loci were recorded, respectively. BN and F344 were therefore selected as the reference strains in backcrosses to AS/AGU, in an attempt to maximise the number of informative markers which could be used to type the progeny.

Heat stress has a significant impact on the productivity and yield of crops grown in the hot, arid zones of the world. There is mounting evidence that what has classically been termed ‘drought stress’ may in some cases be caused not by water stress per se, but rather by the uncontrolled elevation in leaf temperature that occurs when a plant loses its capacity for transpirational cooling. A previous screen of activation-tagged Arabidopsis thaliana seeds for novel halotolerant mutants implicated elevated levels of the transcription factor MYB64 in mediating improved survival on high salt growth medium, and subsequent transcript profiling of this activation-tagged halotolerant line (HT5) revealed the upregulation of several members of the heat shock protein family. Based on these preliminary findings, expression of two of the small heat shock proteins reported to be among the most highly upregulated in the HT5 line was investigated under various stress conditions in wild type Arabidopsis. Transcript and protein levels were measured in response to heat; their subcellular localisation was observed; and the phenotype of various knockout mutants was recorded. These studies have contributed to an understanding of how these might function in relation to one another and to the rest of the heat shock protein family. This thesis also reports on the investigations of a transgenic line created to constitutively overexpress the MYB64 transcription factor. Transcript profiling produced a list of ‘upregulated’ sequences, of which a significant proportion were previously shown to play key roles in abiotic (and, to an extent, biotic) stress responses. The robustness of these responses in the transgenic lines was investigated by qPCR under heat stress, and the phenotype of the plants was characterised in response to various stress regimes. The findings implicate MYB64 in the regulation of a wide range of stress responses, and as plants are unlikely to encounter stress factors individually outside of the controlled conditions of a laboratory, these findings highlight the importance of considering such stresses in concert rather than isolation.

Thiamine pyrophosphate (TPP) serves as a cofactor in universal metabolic pathways, including glycolysis, the pentose phosphate pathway (PPP) and the tricarboxylic acid cycle; moreover, it is essential for the proper functioning of all organisms. Recently, several steps of the plant thiamine biosynthetic pathway have been characterised, and a mechanism of feedback regulation for thiamine biosynthesis via riboswitching has been unravelled. In plants, thiamine is made in the chloroplasts and then transferred to the cytosol to generate the active form of thiamine, TPP. The mitochondria and chloroplasts must import TPP from the cytosol because both organelles contain TPP-dependent enzymes. In Arabidopsis, two members of the mitochondrial carrier family (MCF), AtTpc1 and AtTpc2, export TPP to the mitochondria, but the chloroplast TPP carrier is still unknown. This project aims to identify thiamine chloroplast transporter(s) and investigate mitochondrial thiamine transporters in Arabidopsis. The approaches used to achieve the aims of this project include phylogenetic analysis, mutant analysis, polymerase chain reaction techniques and gene construct techniques. AtTpc1 and AtTpc2 have a limited effect on plant growth under normal conditions, at least in terms of stem length and secondary stem length. Moreover, phylogenetic analysis showed that AtFolt1 (Arabidopsis folate transporter) and CTT (AT3G51870) are putative chloroplast TPP transporters. The inhibition of AtFolt1 by about 50% and CTT by about 80% was associated with reduced TPP levels in leaves. Thus, AtFolt1 and CTT may be chloroplast TPP transporters.

Altered maternal nutrition during pregnancy induces persistent phenotypic changes in offspring, which can be transmitted between generations and are associated with differences in disease risk. Epigenetic processes have been shown to underlie the effects of the early life environment on phenotype. It is not known if epigenetic marks are transferred directly between generations or established de novo in each generation. The extent to which induced changes in phenotype and epigenotype are affected by different nutrients and the timing of nutritional change, and the stability of such effects during the life course are also not known. This thesis describes a series of experiments to address these issues. To investigate the mechanism by which induced phenotypic and epigenetic traits are transmitted between generations, dietary energy was increased 25% at conception in FO female rats and maintained at this level to the F3 generation. Body weight, energy intake, glucose and lipid homeostasis and the expression of specific hepatic genes in the pregnant dams differed between generations. In offspring, fasting glucose and fatty acid homeostasis was dysregulated in the F1 generation, but showed progressively improved metabolic control in subsequent generations despite continued exposure to the high energy diet. The effects on plasma glucose concentration were associated with altered mRNA expression and promoter methylation of phosphoenolpyruvate carboxykinase (PEPCK). Altered DNA methylation was associated with changes in the epigenetic regulation of DNA methyltransferase (DNMT) 3a2. Protein restriction of the maternal diet modified the trajectory of changes in maternal and offspring phenotype, gene expression and epigenome between generations. These findings suggest that the changes in signals from mother to the fetus, induced by differences in the interaction between maternal phenotype and nutrition in each generation, provided a stimulus for modification of the phenotype of the offspring. Such effects appeared to produce beneficial adjustments in metabolic control via altered epigenetic control of specific genes. The effects of different maternal nutritional exposures on the epigenetic regulation of specific genes was investigated by comparing differences in gene regulation following feeding of a protein restricted, 7 and 21 % safflower oil and fish oil diets during pregnancy. No offspring showed altered methylation of the PEPCK promoter, whereas feeding altered fat diets lead to significant changes in the promoter methylation of fads2. This suggests that alterations in the epigenetic regulation of transcription of specific genes are related to the nature of the nutritional challenge. In order to determine whether induced changes in the epigenetic regulation of genes are influenced by the timing of the nutritional challenge during the life course, folic acid supplementation was provided during pregnancy or to the offspring during their juvenile-pubertal (JP) period. Compared to prenatal folic acid supplementation, JP supplementation led to increased PEPCK and DNMT3a2 mRNA expression, and altered promoter methylation contingent on sex. This may be due to specific windows of plasticity in the epigenome. Differences in methylation of PEPCK and DNMT3a2 induced during development were found to change with increasing age. This suggests continued plasticity of the epigenome beyond the immediate developmental period. Overall, the findings of these studies show that the nature of induced phenotypes involves signals produced by the interaction of the mother and her environment, and the timing and nature of the nutritional challenge acting via the epigenome. Such induced traits may confer harm or benefit both within and between generations according to the nature of the nutritional exposure.

No description available.