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Thesis (Ph. D.)--Michigan State University. Microbiology and Molecular Genetics, 2008. / Title from PDF t.p. (Proquest, viewed on Aug. 20, 2009) Includes bibliographical references. Also issued in print.

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

Thesis (Ph.D.)--Murdoch University, 2005. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 184-207.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 90-99).

Transposons constitute much of the animal genome. While many transposons are ancient and inactivated, numerous others are intact and must be actively repressed. Uncontrolled transposons can cause genomic instability through DNA damage or mutations and must be carefully silenced in the germline or risk sterility or mutations that are passed on to offspring. In Drosophila melanogaster, 23–30 nt long piRNAs direct transposon silencing by serving as guides for Aubergine, Argonaute3, and Piwi, the three fly PIWI proteins. piRNAs derive from piRNA clusters—large heterochromatic DNA loci comprising transposons and transposon fragments. piRNAs are loaded into PIWI proteins via the ping-pong cycle which serves to amplify guide piRNAs. Loaded Piwi then enters the nucleus to transcriptionally repress transposons by establishing heterochromatin. Therefore, to silence transposons, transposon sequences must also be expressed. To bypass this paradox, the HP1 homolog Rhino (Rhi) allows non-canonical, promoter-independent, transcription of transposons embedded in heterochromatin. Transposon RNAs produced in this manner are “incoherent” and have little risk of being translated into transposon-encoded proteins required for transposition. This thesis focuses on understanding how piRNA clusters permit non-canonical transcription yet restrict canonical transcription. We found that although Rhi promotes non-canonical transcription in piRNA clusters, it also creates a transcriptionally permissive environment that is amenable to canonical transcription. In addition, we discovered that the conserved protein, Maelstrom, is required to repress promoter-driven transcription of individual, potentially active, transposons within piRNA clusters and allows Rhi to transcribe such transposon sequences into incoherent piRNA precursors.

RNA interference (RNAi) is one of the key mechanisms that are involved in many biological processes such as control of plant gene expression, influence on chromatin arrangement or providing protection against invasive DNA or RNA transposons, viruses and transgenes. In plants, RNAi is triggered by double stranded RNA (dsRNA) that is cleaved by DICER LIKE (DCL) proteins to small RNAs (sRNAs). The size of these sRNAs is in range of 21 - 24 nucleotides (nt). Small RNA acts in the place of origin and they are also a mobile signal which in plants can move to a short distance through plasmodesmata and to a long distance trough phloem. sRNA and Argonaute (AGO) protein form RNA-induced silencing complex (RISC). Together, they recognize the target RNA molecule and contribute to an efficient RNAi phase which may be exhibited by gene silencing at posttranscriptional level (PTGS) or transcriptional level (TGS). The purpose of this study was to compare the effects of silencing constructs, witch in a controlled way differently trigger RNAi directed against the expression of the GFP reporter gene in the model organism Arabidopsis thaliana. Silencing constructs were placed under an inducible promoter activated by the presence of 17-β-estradiol (XVE system). They differed in the way of the dsRNA formation and in the...

Genome sequencing has yielded unparalleled insights into fundamental biological processes and the genetics that guide them. In contrast to expectations that protein-coding genes would be the primary output of eukaryotic genomes, however, it is now clear that the vast majority of transcription is devoted to noncoding RNAs (ncRNAs). Although originally regarded as 'transcriptional noise', it is now clear that these transcripts are essential regulators of genetic activity. In this thesis I build upon the hypothesis that the genomes of eukaryotes encode a regulatory 'RNA machine' dominated by ncRNAs. In the Introduction (Chapter 1) I discuss how prior gene models may have inadvertently prevented a full understanding of ncRNAs, review the transcriptional landscape of eukaryotes, and examine the biogenesis and function of small regulatory RNAs. In support of a role for ncRNAs in complex metazoa, Chapter 2 presents data showing a positive correlation between the proportion of non-protein-coding DNA and biological complexity, suggesting that the evolutionary trajectory of intricate developmental phenotypes may have been facilitated by ncRNAs. In the following chapters two more 'gears' are added to the RNA machine. Chapter 3 details the discovery of snoRNA-derived RNAs - an evolutionarily ancient class of Argonaute-assocaited RNA whose biogenesis overlaps with microRNAs (miRNAs) and silencing RNAs (siRNAs). Likewise, Chapter 4 reports a new class of ~18 nt transcription initiation RNAs (tiRNAs) derived from regions proximal to transcription start sites. tiRNAs are enriched at GC-rich promoters and regions of active transcription, implicating them in transcriptional regulation. Chapter 5 presents evidence that tiRNAs are restricted to metazoa, and describes a model of RNA Polymerase II dependent tiRNA biogenesis. This thesis concludes with a general discussion of the implications of these findings, and the potential development of RNA therapeutics. Gathering evidence suggests that eukaryotic genomes are driven by a complex and interwoven network of RNA regulatory feedback loops. This thesis takes a small step towards developing a complete picture of this system.

Genome sequencing has yielded unparalleled insights into fundamental biological processes and the genetics that guide them. In contrast to expectations that protein-coding genes would be the primary output of eukaryotic genomes, however, it is now clear that the vast majority of transcription is devoted to noncoding RNAs (ncRNAs). Although originally regarded as 'transcriptional noise', it is now clear that these transcripts are essential regulators of genetic activity. In this thesis I build upon the hypothesis that the genomes of eukaryotes encode a regulatory 'RNA machine' dominated by ncRNAs. In the Introduction (Chapter 1) I discuss how prior gene models may have inadvertently prevented a full understanding of ncRNAs, review the transcriptional landscape of eukaryotes, and examine the biogenesis and function of small regulatory RNAs. In support of a role for ncRNAs in complex metazoa, Chapter 2 presents data showing a positive correlation between the proportion of non-protein-coding DNA and biological complexity, suggesting that the evolutionary trajectory of intricate developmental phenotypes may have been facilitated by ncRNAs. In the following chapters two more 'gears' are added to the RNA machine. Chapter 3 details the discovery of snoRNA-derived RNAs - an evolutionarily ancient class of Argonaute-assocaited RNA whose biogenesis overlaps with microRNAs (miRNAs) and silencing RNAs (siRNAs). Likewise, Chapter 4 reports a new class of ~18 nt transcription initiation RNAs (tiRNAs) derived from regions proximal to transcription start sites. tiRNAs are enriched at GC-rich promoters and regions of active transcription, implicating them in transcriptional regulation. Chapter 5 presents evidence that tiRNAs are restricted to metazoa, and describes a model of RNA Polymerase II dependent tiRNA biogenesis. This thesis concludes with a general discussion of the implications of these findings, and the potential development of RNA therapeutics. Gathering evidence suggests that eukaryotic genomes are driven by a complex and interwoven network of RNA regulatory feedback loops. This thesis takes a small step towards developing a complete picture of this system.

Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions. A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated. The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos. The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV. In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.