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Genes are typically expressed in equal amounts from both parentally inherited chromosomes. However, recent studies have demonstrated that genes can be preferentially transcribed from a locus. Non-random preferential expression of alleles can occur in a parent-of-origin pattern, known as imprinting, where epigenetic factors regulate their transcription. Alternatively, it can occur in a haplotype-specific pattern, where cis-acting polymorphisms in regulatory regions are thought to underlie the phenomenon. Both forms of unequal allelic expression have been associated with human disease. Consequently, it is important to identify genes subject to unequal allelic expression and characterize mechanisms that regulate differential transcription. This thesis presents the results of a screen for unequal allelic expression where approximately 50 murine transcripts homologous to genes on human chromosome 7 were analyzed. Human chromosome 7 was selected due to its association with several human disorders that show parent-of-origin effects. The screen identified non-imprinted preferential allelic expression in numerous transcripts and demonstrated that such patterns can occur in tissue specific patterns. Paraoxonase-1 (Pon1), a gene implicated in arthrosclerosis, was identified as having a dynamic pattern of allelic expression which varies throughout embryonic development. This finding represents the first report of a developmentally regulated pattern of allelic variance. Carboxypeptidase-A4 (Cpa4) was identified as having a tissue-specific imprinted pattern of expression, where the maternal allele was preferentially expressed in all embryonic tissues, with the exception of the brain. The Krüppel-like factor 14 gene (Klf14), a novel imprinted transcript, was found to have ubiquitous maternal expression in all human and murine tissues analyzed. A differentially methylated region, generally associated with imprinted transcripts, was not found in the gene’s CpG island, nor was a differential pattern of histone modifications identified. However, it was determined that maternal methylation regulates the transcript. The data in this thesis contribute to our understanding of the numerous patterns of allelic expression that exist in nature and the diverse mechanisms that regulate them. Ultimately, quantitative analyses of allelic expression patterns and the identification of their underlying genomic DNA sequences will become standard protocol in all biomedical studies.

Allelic expression imbalance, or AEI, is the term given to differences in the expression levels of the two alleles of a gene. AEI has been previously identified in a number of species using various techniques. Here, the genome-wide extent of allelic expression imbalance in the pathogenic yeast species, Candida albicans, was examined through use of RNA sequencing in combination with a novel computational pipeline based around the diploid reference genome. Techniques for validating these results were investigated, and the difficulties surrounding specificity and quantification are discussed. As C. albicans is a highly heterozygous species, it was hypothesised that polymorphisms within alleles lead to differences in allele expression, which are further linked to differences in allele function. The functional consequences of AEI were therefore interrogated through investigation of Gene Ontology, identification of condition specific responses in AEI, and targeted construction and phenotypic screening of heterozygous knockout strains. Together, these results strongly suggest that divergence in allele expression is not linked to differences in allele function. Investigations of the possible control mechanisms behind the differences in allele expression were considered, with a focus upon structural factors such as chromosomal location, GC content, allele length and codon usage. However, issues with establishing causality are present, and difficulties lie in distinguishing between functional differences and consequences of bias in sequencing technologies. This piece of research has advanced the understanding of gene expression mechanisms within a medically important pathogen, paving the way for further investigations into the functional consequences of allelic expression imbalance in Candida albicans.

Genes are typically expressed in equal amounts from both parentally inherited chromosomes. However, recent studies have demonstrated that genes can be preferentially transcribed from a locus. Non-random preferential expression of alleles can occur in a parent-of-origin pattern, known as imprinting, where epigenetic factors regulate their transcription. Alternatively, it can occur in a haplotype-specific pattern, where cis-acting polymorphisms in regulatory regions are thought to underlie the phenomenon. Both forms of unequal allelic expression have been associated with human disease. Consequently, it is important to identify genes subject to unequal allelic expression and characterize mechanisms that regulate differential transcription. This thesis presents the results of a screen for unequal allelic expression where approximately 50 murine transcripts homologous to genes on human chromosome 7 were analyzed. Human chromosome 7 was selected due to its association with several human disorders that show parent-of-origin effects. The screen identified non-imprinted preferential allelic expression in numerous transcripts and demonstrated that such patterns can occur in tissue specific patterns. Paraoxonase-1 (Pon1), a gene implicated in arthrosclerosis, was identified as having a dynamic pattern of allelic expression which varies throughout embryonic development. This finding represents the first report of a developmentally regulated pattern of allelic variance. Carboxypeptidase-A4 (Cpa4) was identified as having a tissue-specific imprinted pattern of expression, where the maternal allele was preferentially expressed in all embryonic tissues, with the exception of the brain. The Krüppel-like factor 14 gene (Klf14), a novel imprinted transcript, was found to have ubiquitous maternal expression in all human and murine tissues analyzed. A differentially methylated region, generally associated with imprinted transcripts, was not found in the gene’s CpG island, nor was a differential pattern of histone modifications identified. However, it was determined that maternal methylation regulates the transcript. The data in this thesis contribute to our understanding of the numerous patterns of allelic expression that exist in nature and the diverse mechanisms that regulate them. Ultimately, quantitative analyses of allelic expression patterns and the identification of their underlying genomic DNA sequences will become standard protocol in all biomedical studies.

Polymorphisms in the gene encoding SORL1, involved in cellular trafficking of APP, have been implicated in late-onset Alzheimer's disease, by a mechanism thought to affect mRNA expression. To search for regulatory polymorphisms, we have measured allele-specific mRNA expression of SORL1 in human autopsy tissues from the prefrontal cortex of 26 Alzheimer's patients, and 51 controls, using two synonymous marker SNPs (rs3824968 in exon 34 (11 heterozygous AD subjects and 16 controls), and rs12364988 in exon 6 (8 heterozygous AD subjects)). Significant allelic expression imbalance (AEI), indicative of the presence of cis-acting regulatory factors, was detected in a single control subject, while allelic ratios were near unity for all other subjects. We genotyped 7 SNPs in two haplotype blocks that had previously been implicated in Alzheimer's disease. Since each of these SNPs was heterozygous in several subjects lacking AEI, this study fails to support a regulatory role for SORL1 polymorphisms in mRNA expression.

Potato (Solanum tuberosum L) is an outcrossing, highly heterozygous cultivated in which the elucidation of the genetic basis of quantitative traits, is more complex than in self-pollinated crops. Both a candidate gene approach and a whole genome SNP genotyping analysis were used to assess allelic variation and to identify loci associated with biosynthesis and accumulation of steroidal glycoalkaloids (SGAs). SGAs are secondary metabolites produced in Solanum species as defense against insects and pathogens. Fragments of genomic DNA coding for regions of five SGA biosynthetic candidate genes were amplified, cloned and sequenced [3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2); 2,3-squalene epoxidase (SQE); solanidine galactosyltransferase (SGT1); and solanidine glucosyltransferase (SGT2)]. A germplasm panel of six wild potato species [Solanum chacoense (chc 80-1), S. commersonii subsp. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, and S. stoloniferum] and a cultivated clone S. tuberosum Group Phureja (phu DH) was used in an allelic variation analysis. A segregating interspecific F2 population phu DH �" chc 80-1 was screened to assess association with SGAs. Sequence diversity analysis showed a tendency of purifying selection and increased frequency of rare alleles in most of the candidate genes. Genes of primary metabolism (HMG1, HMG2 and SQE) had stronger selection constraints than those in secondary metabolism (SGT1 and SGT2). Sequence polymorphism in HMG2, SQE, SGT1 and SGT2 separated either the phu DH clone which produced no SGAs, or chc 80-1, the greatest SGA accumulator, from other accessions in the panel. Segregation analysis of the F2 population revealed that allelic sequences of HMG2 and SGT2 derived from chc 80-1 were significantly associated with the greatest SGA accumulation. In the whole genome analysis, SNP genotyping and cluster analysis based on putative association with SGA accumulation in the germplasm panel, allowed identification of eight informative SNPs that can be used in future studies. In the segregating F2 population, loci located on five pseudochromosomes were associated with SGA synthesis. Loci on pseudochromosomes 1 and 6 explained segregation ratios of synthesis for α-solanine and α-chaconine, the most common SGAs in most potato species. In addition, loci on seven pseudochromosomes were associated with accumulation. New candidate genes, putatively affecting synthesis and accumulation of SGAs, were identified in adjacent genomic regions of significant SNPs. This research demonstrates how the newly available genome sequence of potato and associated biotechnological tools accelerates the identification of genetic factors underling complex traits in a species with a difficult breeding structure. / Ph. D.

<p>Allelic imbalance, in which two alleles at a given locus exhibit differences in gene expression, chromatin composition and/or protein binding, is a widespread phenomenon in the human and other complex genomes. Most examples concern individual loci located more or less randomly around the genome and thus imply local and gene-specific mechanisms. However, genomic or chromosomal basis for allelic imbalance is supported by multi-locus examples such as those exemplified by domains of imprinted genes, spanning ~1-2 Mb, or by X chromosome inactivation, involving much of an entire chromosome. Recent studies have shown that genes on the two female X chromosomes exhibit a breadth of expression patterns ranging from complete silencing of one allele to fully balanced biallelic expression. Although evidence for heritability of allele-specific chromatin and expression patterns exists at individual loci, it is unknown whether heritability is also reflected in the chromosome-wide patterns of X inactivation.</p><p>The aim of this thesis is to elucidate the extent to which the widespread variable patterns of allelic imbalance on the human X chromosome in females are under genetic control and how access of the transcription machinery to the human inactive X chromosome in females is determined at a genomic level. For the set of variable genes examined in this study, the absence or presence of expression appears to be stochastic with respect to the population rather than abiding by strict genetic rules. Furthermore, variable gene expression that I have detected even among multiple clonal cell lines derived from a single individual suggests fluctuation in transcriptional machinery engagement. I find that, although expression at most genes on the human inactive X chromosome is repressed as a result of X inactivation, a number of loci are accessible to the transcriptional machinery. It appears that RNA Polymerase II is present at alleles on the inactive X even at the promoters of several silenced genes, indicating a potential for expression. </p><p>This thesis embodies a transition in the field of human X chromosome inactivation from gene by gene approaches used in the past to utilizing high-throughput technologies and applying follow-up analytic techniques to draw upon the vast data publicly available from large consortia projects.</p> / Dissertation

The mop2 gene is required for epigenetic silencing; it was originally defined as a mutation, Mop2-1, which when dominant prevented paramutation at b1. Paramutation is an allele communication that causes a mitotically and meiotically heritable change in gene expression. Mop2-1 was subsequently shown to be involved in maintaining the silenced paramutant state and to prevent dsRNA-mediated transcriptional gene silencing (activities revealed only when the mutation is homozygous). Understanding the product encoded by mop2 will help dissect the underlying mechanisms involved in paramutation and dsRNA-mediated transcriptional silencing. This dissertation describes map-based cloning and candidate gene approaches directed toward the eventual goal of identification of mop2.Initial mapping of mop2 placed it within a region delineated by the markers umc1823 and eks1. On the maize physical map this region contains 21 BAC (Bacteria Artificial Chromosome) clones, representing 2.9 Mb. Skim sequencing identified additional markers for mapping and revealed the gene content. Extensive candidate gene examinations, including gene sequencing, expression profiling with microarrays and RT-PCR, and complementation tests with mutant alleles did not identify any of the four chromatin and RNAi-related genes as mop2.The new markers developed from the skim sequence enabled further mapping and molecular genotyping, which revealed that the Mop2-1 mutation was unstable. Approxi¬mately 10% of phenotypic heterozygous plants were actually genotypic homozygous. Further mapping using only Mop2-1 homozygous plants reduced the mop2 interval to a region of nine BACs, containing 57 genes.The mop2 region is highly syntenic to a rice region of 1.25 Mb on chromosome 4. The gene alignment and repetitive sequence analyses between the syntenic regions in these two species revealed both syntenic and non-syntenic blocks of sequences. Analyses suggested several potential mechanisms for the collinearity breakage, including, but not limited to, tandem duplications of genes in one species but not the other and the presence of gene fragments in maize, but not in rice.The research described herein provides the basis for continued efforts to clone mop2. Fine-structure mapping with new markers and a larger population, as well as candidate gene sequencing in the Mop2-1 BAC library, should be pursued to clone mop2.