No description available.

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.

Cancer is a leading cause of death worldwide, accounting for around 13% of all death [1]. Traditional cancer therapeutics usually require careful selection of one or more intervention, such as surgery, radiotherapy, and chemotherapy, which have made momentous progress, but have ample limitations [2]. The next generation of cancer therapeutics will specifically target processes responsible for the growth and survival of cancer cells. Among the most promising of these molecularly-targeted therapeutics are short interfering RNAs (siRNAs). These siRNAs serve as the effectors of RNA interference, a naturally occurring and highly specific mechanism for regulating gene expression through sequence-specific degradation of messenger RNA. However, the native structure of RNA is plagued with undesirable chemical properties. For example, the sugar-phosphate backbone contains a negative charge which hinders its ability to cross the negatively charged lipid bilayer. Furthermore, the phosphodiester backbone is a substrate for nucleases, which catalytically cleaves the phosphate-oxygen bond, thus degrading the native RNA [3]. As such, there is widespread interest in chemically modifying the backbone of siRNAs in order to overcome some of the inherent problems with its native structure. There have been only two reports that have employed amide-bond linkages as phosphate replacements within siRNAs [4, 5]. In both of these studies, the amide bond containing monomer units were placed at the 3’-overhangs and not within the internal Watson-Crick region of the double stranded siRNA due to the limitation of standard solid-phase oligonucleotide synthesis. In this thesis, we proposed to utilize phosphoramidite chemistry to localize internal amide-bond modifications [6]. A practical synthesis of a peptide nucleic acid unit combined with an RNA nucleoside (PNA-RNA dimer, UaU) is reported [7]. Using this PNA-RNA dimer phosphoramidite allows us to control the site-specific location of the internal amide-bond modification throughout the desired RNA strand. Polyacrylamide gel (PAGE) and mass spectrometry analysis were performed to ensure the formation of full-length modified siRNA molecules. The effects of these modifications were explored with respect to the biophysical and biological properties of the modified siRNAs. The techniques used in this work included hybridization affinity assays (melting temperature), secondary structure determination (circular dichroism), cell-based luciferase assays, and nuclease stability assays. Melting temperature experiment reveals that localizing a UaU dimer unit within the RNA oligonucleotides has an overall destabilizing effect, whereas UaU modifications at the 3’-overhang positions show little change in thermal stability. Circular dichroism experimental results illustrate that all chemically modified siRNAs exhibit the standard A-form helix. In cell-based luciferase assays, we utilized two different target sequences and our results highlight the compatibility of utilizing a neutral amide-bond backbone within siRNAs. Specifically, the internal amide-bond modification is compatible within the RNAi machinery when placed at 3’-overhang position in the sense strand of the double-stranded siRNA. However, poor efficacy is observed when this unit is placed adjacent the Ago 2 cleavage site on the antisense strand. The nuclease stability assays reveal that the introduction of a PNA-RNA dimer at the 3’-end of the siRNA where the exonuclease cleaves the terminal nucleotide, increased markedly the resistance to serum-derived nucleases. To the best of our knowledge, this is the first report that involves amide-bonds as phosphate backbone replacements within the internal regions of siRNAs and thus opens the future possibility for examining and utilizing this modification in studying new structure-function relationships. / UOIT

No description available.

No description available.

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.