Finally, the fourth part of this thesis demonstrated the potential of plasma RNA integrity for noninvasive clinical diagnosis. Based on previous reports of elevated RNase activities in the circulation of cancer patients, it was hypothesized that plasma RNA integrity might serve as a useful tumor marker. Using nasopharyngeal carcinoma (NPC) as a disease model, it was found that plasma RNA in untreated NPC patients was of lower integrity than that in healthy individuals. Further analysis showed that patients undergoing radiotherapy had increased RNA integrity in the post-treatment plasma samples. These findings hence suggested that measurement of plasma RNA integrity may provide a feasible approach for noninvasive cancer detection. / Much recent interest has been focused on the clinical applications of cell-free circulating RNA for molecular diagnostics. Despite the rapid development of plasma RNA as a potential diagnostic tool, much of the biology of these molecular species remains enigmatic. This thesis aimed to investigate the integrity of cell-free RNA molecules in plasma and to study the effects of different preanalytical factors on this new biological parameter. Moreover, the possibility that plasma RNA integrity might serve as a useful clinical marker was explored. / The first part of this thesis was to develop a quantitative method for analyzing RNA integrity in plasma. One-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify transcript sequences corresponding to multiple regions along a housekeeping gene, glyceraldehyde-3 phosphate dehydrogenase (GAPDH). It was demonstrated that 5' transcript fragments were predominant, when compared with those derived from the middle or 3' region, in the plasma of healthy individuals. This method was validated using two-step RT-PCR and serial dilution assays. / The potential generality of the underrepresentation of 3' mRNA fragments was further studied by using circulating placental RNA as a model system. Seven transcripts were analyzed in the plasma of pregnant women: the beta subunit of human chorionic gonadotropin (betahCG), tissue factor pathway inhibitor 2 (TFPI2), adrenomedullin (ADM), inhibin beta A subunit (INHBA), placenta-specific 1 (PLAC1), pregnancy-associated plasma protein A (PAPPA), and GAPDH. The second part of this thesis showed that for five of the seven genes, there appeared to be a greater abundance of transcript fragments arising from the 5' end than those from the 3' end in maternal plasma, and that for every gene under study, the 5'-specific assay had a higher rate of detection when compared to the 3'-specific one. Apart from biological significance, these data have implications for maximizing the sensitivity of fetal RNA detection in maternal plasma for future diagnostic use. / The results presented in this thesis not only have improved the current understanding of the biological nature of cell-free circulating RNA, but also have provided important information regarding the potential clinical utility of a new parameter, plasma RNA integrity, for the field of medical diagnostics. / To ensure accurate interpretation of the results on plasma RNA integrity, a number of preanalytical issues were investigated. In the third part of this thesis, several findings were described: (1) filtration of plasma samples did not change the observation that 5' end transcript was the predominant species; (2) time delay in the processing of plasma could lead to decreased RNA concentrations despite the lack of variation in plasma RNA integrity; and (3) repeated freezing and thawing of plasma samples, but not extracted RNA, could reduce RNA integrity significantly. / Wong Chi Kwan Blenda. / "August 2006." / Adviser: Yuk Ming Dennis Lo. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1453. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 148-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Three genomic clones encompassing human DNA segments (designated LhX-3, LhX-4, and LhX5) were isolated from an X chromosome-specific library and subjected to analysis by physical mapping and DNA sequencing. It was found that these three clones are very rich in repetitive DNA sequence elements and retropseudogenes.

Errata inserted. Bibliography: leaves 98-120. This study is concerned with the identification of genes related to the very early stages of meiosis when homologous pairing occurs. A cDNA library is prepared at the premeiotic interphase and prophase stages of meioses. Differential screening is used to identify and select clones showing preferential expression in anthers at early meiosis. Two selected clones are chosen for further analysis and to investigate a possible role in chromosome pairing.

Errata inserted. / Bibliography: leaves 98-120. / x, 120, [4] leaves, [13] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study is concerned with the identification of genes related to the very early stages of meiosis when homologous pairing occurs. A cDNA library is prepared at the premeiotic interphase and prophase stages of meioses. Differential screening is used to identify and select clones showing preferential expression in anthers at early meiosis. Two selected clones are chosen for further analysis and to investigate a possible role in chromosome pairing. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Most eukaryotic pre-mRNAs undergo 3′-end cleavage and polyadenylation prior to their export from the nucleus. A large number of proteins in several complexes participate in this 3′-end processing, including cleavage and polyadenylation specificity factor (CPSF) in mammals. The CPSF can be further divided into two sub-complexes: mPSF (mammalian polyadenylation specificity factor) which recognizes the AAUAAA polyadenylation signal (PAS) in the pre- mRNA, and mCF (mammalian cleavage factor) which cleaves the RNA. mPSF consists of CPSF160, CPSF30, WDR33, and hFip1. This thesis shows that AAUAAA PAS is recognized with ∼3 nM affinity by the CPSF160–WDR33–CPSF30 ternary complex, while the proteins alone or the binary complexes do not bind the PAS with high affinity. Furthermore, it is shown that mutations of residues in CPSF30 that have van der Waals interactions with the bases of the PAS lead to a sharp reduction in the affinity. Finally, variations of the AAUAAA or removing the bases downstream also reduce the binding significantly. This thesis goes on to characterize the structure of the CPSF30—hFip1 complex, which was not observed in the previous EM structures of the mPSF. It was known that CPSF30 ZF4–ZF5 recruits the hFip1 subunit of CPSF, although the details of this interaction have not been characterized. Here we report the crystal structure of human CPSF30 ZF4–ZF5 in complex with residues 161–200 of hFip1 at 1.9 Å. Unexpectedly, the structure reveals one hFip1 molecule binding to each ZF4 and ZF5, with a conserved mode of interaction. Mutagenesis studies confirm that the CPSF30–hFip1 complex has 1:2 stoichiometry in vitro. Mutation of each binding site in CPSF30 still allows one copy of hFip1 to bind, while mutation of both sites abrogates binding. Our fluorescence polarization binding assays show that ZF4 has higher affinity for hFip1, with a Kd of 1.8 nM. We also demonstrate that two copies of the catalytic module of poly(A) polymerase (PAP) are recruited by the CPSF30–hFip1 complex in vitro, and both hFip1 binding sites in CPSF30 can support polyadenylation.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5’-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

The ability of certain RNAs to fold into complicated secondary and tertiary structures provides them with the ability to perform a variety of functions in the cell. Since the secondary and tertiary structures formed by certain RNAs in the cell are central to understanding how they function, one of the most active areas of research has been how to accurately and reliably predict RNA secondary structure from sequence; better known as the RNA Folding Problem. This dissertation examines two fundamental areas of research in RNA structure prediction, free energy minimization and comparative analysis. The most popular RNA secondary structure prediction program, Mfold 3.1 predicts RNA secondary structure via free energy minimization using experimentally determined energy parameters. I present an evaluation of the accuracy of Mfold 3.1 using the largest set of phylogenetically diverse, comparatively predicted RNA secondary structures available. This evaluation will show that despite significant revisions to the energy parameters, the prediction accuracy of Mfold 3.1 is not significantly improved when compared to previous versions. In contrast, RNA comparative analysis has repeatedly demonstrated the ability to accurately and reliably predict RNA secondary structure. The downside is that RNA comparative analysis frequently requires an expert systems methodology which is predominately manual in nature. As a result, RNA comparative analysis is not capable of scaling adequately to be useful in the genomic era. Therefore, I developed the Comparative Analysis Toolkit (CAT) which is intended to be the fundamental component of a vertically integrated software infrastructure to facilitate high-throughput RNA comparative analysis using an expert systems methodology.

The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.

The purpose of this experiment was to determine if nucleic acid sequence based amplification (NASBA) is a suitable application for the differentiation of body fluids that might comprise a forensic evidence sample. NASBA is a sensitive RNA transcription based amplification system. NASBA could theorhetically be used for bodily fluid identification based upon amplification of tissue-specific mRNA transcripts present in a given forensic sample.Amplification of both Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Matrix Metalloproteinase 1 1 (MMPmRNA transcripts were used to determine that NASBA could amplify body fluid transcripts and whether it could distinguish between menstrual and non-menstrual blood, respectively. GAPDH is a housekeeping gene that is constituently expressed and its mRNA transcripts could therefore be used to determine whether non-menstrual blood could be amplified using the NASBA procedure. MMP 11 is a menstrual cycle-specific gene associated with endometrial breakdown. Using the mRNA transcripts from MMP 11, NASBA could be utilized for menstrual blood identification. In this study, non-menstrual and menstrual blood samples were analyzed with NASBA both in the presence and absence of chemical contamination. Contaminants utilized ranged from commercial automotive wax, transmission fluid, brake fluid, artificial tears, hand soap, 10% bleach, and the luminol blood detecting reagent. Non-menstrual blood was aliquoted onto a 1 cm x 1 cm cotton cloth for contamination, while menstrual blood was provided on a 1 cm x 1 cm area of sterile menstrual pad. All samples underwent Tri reagent extraction to obtain RNA samples for NASBA amplification.With respect to NASBA amplification data, non-menstrual blood data (from extracted RNA and unextracted blood samples) revealed the highest levels of amplification as shown in relative fluorescence units (RFU). Uncontaminated menstrual blood revealed the second highest amplification data. In the presence of chemical contamination, high levels of amplification were observed when samples were contaminated with brake fluid and commercial hand soap. Moderately low amplification was observed with samples contaminated with transmission fluid, 10% bleach, and artificial tears. NASBA amplification was completely inhibited in the presence of automotive wax and luminol. Cycle threshold (CO values for each amplification result were also obtained from each reaction. Smaller Ct values correspond to a higher NASBAreaction efficiency and therefore larger amplification values. The Ct values obtained for each amplified sample correlate strongly with the amount of amplification observed from reaction. Based upon the results of this experiment, NASBA should be considered as a novel tool for forensic evidence analysis. / Department of Biology

Background Within eukaryotes there is a complex cascade of RNA-based macromolecules that process other RNA molecules, especially mRNA, tRNA and rRNA. A simple example is the RNase MRP processing of ribosomal RNA (rRNA) in ribosome biogenesis. One hypothesis is that this complexity was present early in eukaryotic evolution; an alternative is that an initial simplified network later gained complexity by gene duplication in lineages that led to animals, fungi and plants. Recently there has been a rapid increase in support for the complexity-early theory because the vast majority of these RNA-processing reactions are found throughout eukaryotes, and thus were likely to be present in the last common ancestor of living eukaryotes, named here as the Eukaryotic Ancestor. Results We present an overview of the RNA processing cascade in the Eukaryotic Ancestor and investigate in particular, RNase MRP which was previously thought to have evolved later in eukaryotes due to its apparent limited distribution in fungi and animals and plants. Recent publications, as well as our own genomic searches have uncovered previously unknown RNase MRP RNAs, indicating that RNase MRP has a wide distribution in eukaryotes. Combining secondary structure and promoter region analysis of new and previously discovered RNase MRP RNAs along with analysis of the primary substrate (rRNA), allows us to discuss this distribution in the light of eukaryotic evolution. Conclusions We conclude that RNase MRP can now be placed in the RNA-processing cascade present in the Eukaryotic Ancestor. This highlights the complexity of RNAprocessing in early eukaryotes.