"October, 2003" / Bibliography: leaves 130-141. / xvii, 150, [10] leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study describes the silencing of tobacco transgenes carrying TLCV promoters following TLCV infection. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine. 2003

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RNA silencing is a form of post-transcriptional gene regulation, in which a small RNA guides a member of the Argonaute protein family in an effector complex to repress target gene expression. piRNAs, found in germ cells, are the most recently discovered major subset of small RNAs. A key known function of piRNA is to repress the transposable elements in the germline and maintain the germline genome integrity. The defining features of the piRNAs are 1) they are ubiquitously methylated at the 3’-end of the 2’-OH group by methyltransferase Hen1; 2) they associate exclusively with the Piwi subfamily Argonaute proteins. Much is not understood about the biogenesis and the regulation of the piRNA pathway. One of the fundamental questions is how the 3’-end of the piRNA is generated and recognised specifically by Piwi but not by Ago subfamily Argonaute proteins. In this thesis, the high resolution crystal structure of the Aubergine PAZ domain, a domain from a Piwi subfamily Argonaute, bound to a 7 mer single-stranded methylated piRNA ‘mimic’, reveals the mode of recognition for the 3’-end of piRNAs by Piwi subfamily Argonautes. The structure provides the molecular basis for why Piwi but not Ago PAZ domains preferentially bind to RNAs with 2’-O-methylation at the 3’-end, thus conferring substrate specificity. The structural results are confirmed by biochemical studies. Biochemical and biophysical studies on the methyltransferase Hen1 have provided insights into substrate specificity for piRNA 3’-end methylation and revealed a potential regulatory role for the C-terminal region of the protein. Extensive biochemical analysis defined a minimal active Hen1/short RNA complex, though crystallisation screening yielded no crystals for structure determination. Overall, this study provides insights into the generation and molecular recognition of the piRNA in animals.

Gene silencing in Saccharomyces cerevisiae has been conclusively linked to histone deacetylation and the subsequent formation of heterochromatin. The participating histone deacetylases have also been well characterized. In contrast, the opposing histone acetyltransferases (HAT) and their mechanism of action remain elusive. In particular, very little is known about the effects of two of the nine HATs in S. cerevisiae, ESA1 and RTT109. The focus of my research was to test if these HATs influence the silencing of genes at the subtelomeric regions of S.cerevisiae and to assess their mechanism of action. To address these issues I used a panel of recombinant telomeric constructs that harbor the URA3 reporter gene. These constructs were inserted at the VIIL telomere of the mutant strains esa1-414 and ∆rtt109. The level of gene repression of the URA3 reporter in each construct in both of these strains has been assessed by a routine assay measuring the sensitivity of the strain to the drug 5-fluoro-orotic acid. My results indicate than none of these HATs plays a specific major role in gene repression at telomeres. Instead, they show general anti-silencing activity that cannot be linked to any specific sub-telomeric elements. Like many other HATs, Esa1 and Rtt109 seem to operate through global acetylation of histones rather than through specific recruitment. These results provide additional insight in the function of the HATs in gene silencing and suggest that there are multiple mechanisms that we do not yet understand. The information provided here would help in future studies to determine the mode of action of anti-silencers and subtelomeric elements involved in telomeric silencing. These issues are of fundamental significance and will contribute to the dynamic and expanding field of epigenetics. / NSERC 400478

Thesis (Ph. D.)--University of Sydney, 2008. / Title from title screen (viewed April 1, 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular and Microbial Biosciences. Degree awarded 2008; thesis submitted 2007. Includes bibliographical references. Also available in print form.

Written for the Dept. of Chemistry. Title from title page of PDF (viewed 2009/06/25). Includes bibliographical references.

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