The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution.

X-chromosome inactivation achieves dosage compensation of X-linked genes between male (XY) and female (XX) mammals. This process involves the down-regulation of most, but not all genes on one of the two X-chromosomes in the nucleus of each female somatic cell. The mechanism of X-inactivation has yet to be elucidated in full, but is known to involve the noncoding transcript of theXist gene, DNA methylation, histone hypo-acetylation and the condensation of higher order chromatin. Recent studies have established mechanisms linking methylation to repressive chromatin structures through methyl-binding proteins and histone deacetylase complexes. In order to better understand the role of methylation in X-inactivation, the promoters of the human Pyruvate dehydrogenase El a (PDHA1) and the human and murine Norrie disease protein (NDP/Ndp) genes were subjected to direct methylation sequencing, allowing the definition of methylation profiles at nucleotide resolution. The promoter of the PDHA1 gene was found to be hyper-methylated on the inactive X-chromosome and hypo-methylated on the active X-chromosome in agreement with studies at the promoters of other X-linked housekeeping genes. Methylation at the promoters of the NDP/Ndp genes was extensively investigated in a range of primary tissues and cell lines. The Ndp promoter was found to be methylated on both active and inactive X-chromosomes, but hypo-methylated in the proximal promoter exclusively in tissues that expressed the Ndp gene. The NDP promoter was found to be unmethylated on the active X-chromosome and hyper-methylated across the proximal promoter on the inactive X-chromosome in expressing cell lines and human retinal tissues. The novel promoter sequences of the human and murine SMCX/Smcx genes were isolated for comparative analysis and to provide a future resource for studying methylation at the promoters of genes which escape the X-inactivation process. Promoter sequences of the PDHA1, NDPI Ndp and SMCX/Smcx genes were screened for putative transcription factor binding sites and for conserved CpG-dinucleotide content. Promoter-reporter gene constructs for these genes were transfected into mammalian cells establishing that the sequences studied were functional promoters. Artificial methylation of these constructs was shown to repress their promoter activities.

Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 205-233).

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2008.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.