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Identifying the genes important for fetal growth will help to understand common, serious complications of pregnancy such as intrauterine growth restriction (IUGR). Of particular interest are imprinted genes, which show monoallelic, parent-of-origin specific expression. Genes that are paternally derived tend to enhance fetal growth whereas maternally expressed genes suppress growth. A significant correlation between lower birth weight and increased expression of maternally expressed, pleckstrin homology-like domain, family A, member 2 (PHLDA2) in term placenta, without loss of imprinting, had been reported previously. We have identified a novel copy number variant (CNV1) in the PHLDA2 promoter which reduced the promoter efficiency in a luciferase reporter gene assay. Meta-analysis of CNV1 genotype data obtained from three independent white European normal birth cohorts (total n = 9,433) showed that maternal inheritance of CNV1 resulted in a 93 g increase in birth weight (P = 0.01). Moreover, when the mother was homozygous for CNV1, the influence on birth weight was 155 g (P = 0.04), a similar magnitude to the reduction caused by maternal smoking. The expression levels of paternally expressed gene 3 (PEG3), delta-like 1 homolog (DLK1) and maternally expressed gene 3 (MEG3) measured by real-time PCR in >110 normal term placentas from a white European cohort did not show correlation with fetal birth size measurements, except for a trend of positive association observed for DLK1 expression and birth weight (P = 0.072). Paternal inheritance of the type 1 diabetes (T1D) protective G allele of rs941576 SNP located in the DLK1-MEG3 locus was shown to be associated with a 132 g (P = 0.011) and 0.5 cm (P = 0.011) reduction in birth weight and head circumference respectively. This newly described PHLDA2 promoter CNV and rs941576 SNP, alongside their expressions, may provide useful genetic biomarkers/indicators for predicting birth size.

If information is power, then the battle of the sexes has been won for many a year by males, led by SRY and an army of male factors. While many key female-specific genes are known to both promote female development and antagonise these male factors, no one-leader stands out as a sole “switch” governess. Foxl2 is a strong candidate for an ovary-determining and/or anti-testis gene in mammals. Mutations in the gene are associated with premature ovarian failure in women (as part of the Blepharophimosis Ptosis Epicanthus Inversus (BPES) syndrome) and XX male sex reversal in goats with the polled intersex syndrome (PIS). However, despite it being expressed from 12.0 dpc specifically in granulosa cells in the early ovary, no gonadal phenotype is seen in XX mice homozygous for null mutations in Foxl2 until after birth. Wnt signalling is also implicated in repressing testis development, but ovary development still occurs with mutations affecting both Foxl2 and Wnt signalling, suggesting that they are part of a redundant system, with one or more critical additional genes or pathways still to be found. Sox9, together with its close homologues within the SoxE subfamily, Sox8 and 10, are required for Sertoli cell differentiation, typical of the testis, and must be repressed to allow granulosa cell and ovary development. Through examining how these SoxE genes are expressed during development of XX gonads mutant in Foxl2 and/or Wnt signalling, additional complex stage-specific requirements for anti-testis gene activity have been revealed. Two approaches have also been taken to begin to define the regulatory regions driving Foxl2 expression in the ovary, with the long-term aim of unravelling a critical enhancer(s), as a female equivalent of the Sertoli cell-specific Sox9 element TESCO, and identifying the factors that bind to this, which may include missing ovary-determinants. First, a strategy was used that makes no prior assumptions, where BAC sequences flanking Foxl2 were used to drive reporter gene expression in transgenic mice. Preliminary data suggests that a 110 kb BAC can replicate aspects of Foxl2 expression, in ovaries and the pituitary. Secondly, an approach based on genomic studies was employed, which assumes that mutations associated with Foxl2 that lay outside the coding region may directly affect tissue-specific regulatory sequences. The caveat with this approach is that these may be associated with levels of expression, or as chromatin organisers, rather than tissue-specific enhancers. D’haene et al (2009) showed a de novo deletion as small as 7.4 kb, located 283 kb 5’ to FOXL2, was capable of causing BPES in humans, while a 11.7 kb DNA element, reported to affect the transcription of Caprine Foxl2, was deleted in PIS from a region about 200 Kb upstream (Pailhoux et al., 2001). These regions contain many Conserved Non-Coding sequences (CNCs), which, in other loci, have sometimes been implicated in the regulation of gene expression. A 6 kb mouse sequence homologous to the 7.4Kb human region was cloned into EYFP, LacZ and Luciferase reporter vectors, and assayed in cell culture and transgenic mice, where it was shown to be able to drive expression in vivo. The results are discussed with reference to the complex genomic architecture of the Foxl2 locus.

Neisseria meningitidis (Nm) is an opportunistic pathogen that asymptomatically colonises the human upper respiratory tract (nasopharynx) in a state known as carriage. Antigenic or phase variation (PV) of outer membrane proteins (OMP) contributes to adaptability of Nm during carriage. PV of five Nm OMPs was investigated in multiple isolates from 21 carriers persistently colonised with a single strain. PV of these genes was mediated by simple sequence repeats (SSRs). Changes in SSRs were detected using GeneScan assays and correlated with expression states by Western blotting. Mutations in SSR loci occurred at a rate of 0.07 mutations/gene/month. These mutations caused reductions in gene expression of fetA and combinations of OMPs during persistent carriage. Levels of anti-FetA IgG-antibodies were measured in the sera of the 21 carriers using multiplex immunoassays containing four allelic-variant FetA proteins coupled to fluorescent microspheres. High levels of FetA-antibodies were detected in multiple carrier sera with some allele-specific reactivity. Evidence was found for reductions in FetA expression to be associated with escape of anti-FetA IgG antibodies. The fetA gene exhibited a high in vitro PV frequency of 3.9 X 10-3 for switching from an 8C to 9C tract. In addition to PV-mediated expression and fur regulation, a putative binding site for a second regulator of fetA gene expression was detected 451 bp upstream of the fetA core promoter. Utilisation of ferric-enterobactin via FetA was limited to serogroup B strains as neither serogroup Y nor E strains exhibited an ability to utilise ferric-enterobactin as the sole iron source. Utilisation of siderophores-produced by four other bacterial species was not detected. In summary, this study shows that PV-mediated reductions in gene expression occur during long-term meningococcal carriage. These reductions in gene expression are posited to be driven by an active immune response and thus PV mediates immune escape of Nm in vivo.

Telomeres are essential for maintaining the integrity of the genome. Shortening and dysfunction of telomeres initiates cellular senescence, halting further cell division, and instigating alterations in biological processes which contribute to ageing. Progeroid syndromes are disorders of ageing. Patients exhibit not only an early external appearance of old age, but several age-related diseases including osteoporosis, muscle wasting and cancer. Laminopathies are diseases caused by mutations in LMNA, the gene encoding the key nuclear matrix component lamin A. Some mutations result in progeroid phenotypes that are associated with elevated levels of senescent cells. The rate of telomere shortening in progeria cells was found to be accelerated relative to healthy controls, confirming a role for lamin A function in telomere biology. In order for a cell to bypass the protective mechanism of senescence, it must initiate a telomere maintenance mechanism (TMM), resulting in cancer. Several biological changes accompanying the alternative lengthening of telomeres (ALT) TMM overlap with mechanisms affected by changes in lamin biology. As such, lamins and related nuclear proteins were investigated in ALT cells and found to differ from tumour-derived cells which activate the telomerase-based TMM. It was found that although nuclear levels of lamins differed between cells of the two TMMs, the interaction of lamin A with chromatin was not altered. Localisation studies of lamin A revealed different distribution of lamins in the nucleus. An excess of intranuclear lamin observed in ALT cells is proposed to be permissive for recombination-based processes required for telomere elongation through ALT, whereas laminopathy lines exhibit a reduced intranuclear lamin, and an accompanying DNA damage repair deficiency. These findings reinforce the importance of lamin organisation and intranuclear network stability in chromatin maintenance, and highlight a new role for lamins in ALT cancers.

When an organism migrates from one area to another it comes into contact with many boundaries to its survival and fitness. The fruitfly Drosophila melanogaster has migrated from Africa, into Europe and colonised much of the rest of the world. The subject of this thesis is to better understand how Drosophila has adapted to survive the temperate climates in Europe. The variability of temperature and light from one season to the next makes adaptation of the circadian clock and life history strategy all the more important. Drosophila appear to have adjusted to the new conditions by exhibiting diapause in low temperatures when the nights are long and by altering several other characteristics of its circadian clock that may be related to diapause. One of these is a novel European single nucleotide polymorphism in the timeless gene that allows flies to maintain a more robust diapause than flies carrying the ancestral allele. This variant exists in all European populations and winter simulation experiments reveal that it maintains its diapause for longer than the ancestral variant. These experiments also supported the possibility that Drosophila diapause can be maintained for much longer periods than previous studies have indicated. Experiments with ancestral African D. melanogaster lines alongside several closely related species indicates that diapause may not be a recent adaptation, but an ancient response to stressful conditions that has adapted in Europe to be more sensitive to low temperatures and short photoperiods. I also discover that a splice variant of the period gene has a dramatic, further cementing the controversial relationship between clock genes and diapause. In addition I have performed a study of putative functional polymorphisms in this untranslated region around this splice site from European populations. Finally, a study of putative clock genes reported in Chapter 3 provides a cautionary tale as to the dangers of using RNAi.

Human population history has been described by analysing different genetic markers. Much attention has focused on haploid markers (such as mtDNA and Y chromosome), but this study addresses the X chromosome (haploid in males, and with a female bias in its history) in order to describe genetic diversity and to infer human population history in Western Europe. HapMap Phase I/II data reveal haplotype blocks on the X chromosome, called PHAXs (Phylogeographically informative Haplotypes on Autosomes and X chromosome), in which recombination is historically absent and mutations are likely to be the only source of genetic variation. Three such regions were chosen to meet several criteria such that mutations in these loci are putatively neutral. These loci have been deep sequenced in 240 samples from Europe, the Middle East and Africa using the Ion Torrent PGM next-generation sequencing (NGS) platform. Short-tandem repeats (STRs) were also typed in order to provide: i) greater discrimination, and ii) a test of different software tools able to call STRs from NGS data. This dataset was validated against a standard STR typing procedure using capillary electrophoresis. PHAXs were confirmed to be largely non-recombining across European samples. All three loci show a remarkably homogeneous pattern across Europe and high Fst values with the African sample. Overall, 30% of singleton variants are European-specific, and this excess of rare alleles is consistent with signals of expansion in Europe, with the Middle East and Africa showing a weaker pattern. Additional samples from the Complete Genomics dataset confirmed these patterns showing European- and Middle Eastern-specific haplotypes. Bayesian skyline plots suggest a European-specific expansion around 20 KYA with a more recent peak around 10 KYA. This study demonstrates the value of the use of haplotype blocks, and the accurate ascertainment of rare variants to infer human demographic history.

The diploid wild grass Thinopyrum bessarabicum (2n = 2x = 14, JJ or EbEb) is a rich source of important genes for bread wheat (2n = 6x = 42) improvement because of its salinity tolerance and disease resistance. Development of wheat–Th. bessarabicum translocation lines by backcrossing amphiploids in the absence of the Ph1 gene (allowing intergenomic recombination) enables its practical utilization in wheat improvement. Using genomic in situ hybridization (GISH) and repetitive probes for fluorescent in situ hybridization (FISH), six novel wheat–Th. bessarabicum translocation lines involving different chromosome segments (T4BS.4BL-4JL, T6BS.6BL-6JL, T5AS.5AL-5JL, T5DL.5DS-5JS, T2BS.2BL-2JL, and the whole arm translocation T1AL.1JS) were identified and characterized in this study. No background translocations between wheat genomes were observed. The involvement of 5 of the 7 chromosomes, and small terminal segments of the Th. bessarabicum chromosome arm were important, contributing to both reduced linkage drag of the derived lines by minimizing agronomically deleterious genes from the alien species, and high stability including transmission of the alien segment. All three wheat genomes were involved in the translocations with the alien chromosome, and GISH showed the Th. bessarabicum genome was more closely related to the D genome in wheat. All the introgression lines were disomic, stable and with good morphological characters. The work also generated a high-resolution karyotype of two accessions of Th. bessarabicum using multiple repetitive DNA probes for chromosome identification. A complete CS-Th. bessarabicum amphiploid (2n=8x=56, AABBDDJJ) was used and each individual Jgenome unambiguously identified. The established karyotype will be useful for the rapid identification of potential donor chromosomes in wheat improvement programs, allowing appropriate alien-chromosome transfer. Genotyping-by-sequencing (GBS) data was collected from the wheat-Th. bessarabicum introgression lines, but the complexity of the wheat genome and need for further development of data analysis pathways limited interpretation.