Thesis (Ph. D.)--University of Georgia, 2004. / Directed by E.W. Taylor. Includes articles submitted to Journal of RNA, Journal of nucleic acid research, and Journal of AIDS research and human retrovirology. Includes bibliographical references.

Thesis (Ph. D.) -- University of Maryland, College Park, 2006. / Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

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The RNAs of many viruses contain a frameshift stimulatory element (FSE) that grants access to an alternate reading frame via −1 programmed ribosomal frameshifting (PRF). This −1PRF is essential for effective viral replication. The −1PRF efficiency relies on the presence of conserved RNA elements within the FSE, such as a slippery sequence, spacer, and a downstream secondary structure – often a hairpin or a pseudoknot. The PRF efficiency is also affected by trans-acting factors such as proteins, miRNAs and metabolites. The interactions of these factors with the RNA and the translation machinery have not yet been completely understood. Traditional ensemble methods used previously to study these events focus on the whole population of molecular species. This results in innate averaging of the molecular behavior and a loss of heterogeneity information. Here, we first established the experimental workflow to study the RNA structures and the effect of potential trans-acting factors using single-molecule force spectroscopy technique, optical tweezers. Additionally, to streamline the data analysis, we developed an algorithm for automatized data processing. Next, we harnessed this knowledge to study viral RNA elements responsible for stimulation of PRF and how the presence of trans-acting factors affects the RNA behavior. We further complemented these single-molecule structural data with ensemble functional assays to gain a complex view on the dynamics behind the programmed ribosomal frameshifting. Specifically, two different viral RNA elements have been studied in the presented work. First, the dynamics of SARS-CoV-2 FSE and the role of extended sequences have been explored. Then, the mode of action of the host-encoded trans-acting factor ZAP-S inhibition of SARS-CoV-2 PRF has been examined. Finally, the mechanism of the trans-acting viral factor induced PRF in Encephalomyocarditis virus (EMCV) has been uncovered. / Die RNAs vieler Viren enthalten ein Lese-Rasterverschiebung-stimulierendes Element (FSE), das über die −1 programmierte ribosomale Rasterverschiebung (PRF) Zugriff auf einen alternativen Leserahmen gewährt. Dieser −1PRF ist für eine effektive Virusreplikation unerlässlich. Die −1PRF-Effizienz beruht auf dem Vorhandensein konservierter RNA-Elemente innerhalb des FSE, wie z.B. einer Slippery-Sequenz, einem Platzhalter und einer nachgelagerten Sekundärstruktur – oft eine Haarnadel oder ein Pseudoknoten. Die −1PRF-Effizienz wird auch durch trans-aktive Faktoren wie Proteine, miRNAs und Metaboliten beeinflusst. Die Wechselwirkungen dieser Faktoren mit der RNA und der Translationsmaschinerie sind noch nicht vollständig verstanden. Traditionelle Ensemble-Methoden, die früher zur Untersuchung dieser Ereignisse verwendet wurden, konzentrieren sich auf die gesamte Population molekularer Spezies. Dies führt zu einer inhärenten Durchschnittsbildung des molekularen Verhaltens und einem Verlust von Heterogenitätsinformationen. Hier haben wir zunächst den experimentellen Arbeitsablauf zur Untersuchung der RNA-Strukturen und der Wirkung potenzieller trans-aktiver Faktoren mithilfe der Einzelmolekül-Kraftspektroskopietechnik Optischer Pinzetten etabliert. Um die Datenanalyse zu optimieren, haben wir außerdem einen Algorithmus zur automatisierten Datenverarbeitung entwickelt. Als nächstes nutzten wir dieses Wissen, um virale RNA-Elemente zu untersuchen, die für die Stimulierung von −1PRF verantwortlich sind, und wie sich das Vorhandensein trans-aktiver Faktoren auf das Verhalten der RNA auswirkt. Wir haben diese Einzelmolekülstrukturdaten weiter durch Ensemble-Funktionsassays ergänzt, um einen komplexen Überblick über die Dynamik hinter der programmierten ribosomalen Rasterverschiebung zu erhalten. Konkret wurden in der vorgestellten Arbeit zwei verschiedene virale RNA-Elemente untersucht. Zunächst wurden die Dynamik des SARS-CoV-2-FSE und die Rolle erweiterter Sequenzen untersucht. Anschließend wurde die hemmende Wirkungsweise des vom Wirt kodierten trans-wirkenden Faktors ZAP-S auf SARS-CoV-2-PRF untersucht. Schließlich wurde der Mechanismus der, durch den trans-aktiven Virusfaktor induzierten PRF beim Enzephalomyokarditis-Virus (EMCV), entschlüsselt

<p>Preserving genome stability is critical to ensure the faithful transmission of intact genetic material through each cell division. One of the key components of this preservation is maintaining low levels of mutagenesis. Most mutations arise during replication of the genome, either as polymerase errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many different DNA repair proteins act both prior to and during replication to prevent the occurrence of these mutations. Although the mechanisms by which mutations occur and the various repair proteins that act to suppress mutagenesis are conserved throughout all species, they are best characterized in the yeast <italic>Saccharomyces cerevisiae</italic>. In this work, we have used this model system to study two types of spontaneous mutagenesis: oxidative mutagenesis and frameshift mutagenesis. In the first part of this work, we have examined mutagenesis that arises due to one of the most common oxidative lesions in the cell, 7,8-dihydro-8-oxoguanine or GO. When present during replication, these GO lesions generate characteristic transversion events that are accurately repaired by the mismatch repair pathway. We provide the first evidence that a second pathway involving the translesion synthesis polymerase Pol&eta acts independently of the mismatch repair pathway to suppress GO-associated mutagenesis. We have also examined how differences in replication timing during S phase contribute to variations in the rate of these mutations across the genome. In the second part of this work, we have examined how spontaneous frameshift mutations are generated during replication. While most frameshift mutations occur in regions of repetitive DNA, we have designed a system to examine frameshifts that occur in very short repeats (< 4 nucleotides) and noniterated sequences. We have examined the patterns of frameshifts at these sites and how the mismatch repair pathway acts to suppress these mutations. Together, the experiments presented here provide further insight into the different mechanisms that suppress and/or influence rates of oxidative mutagenesis and describe a system in which we have begun to characterize how frameshift mutations are generated at very short repeats and non-repetitive DNA.</p> / Dissertation

Many substances in the plant kingdom and in man's diet occur as glycosides. Recent studies have indicated that many glycosides that are not mutagenic in tests such as the Salmonella/microsome test become mutagenic upon hydrolysis of the glycosidic linkages. The Salmonella/microsome test utilizes a liver homogenate to approximate mammalian metabolism but does not provide a source of the enzymes present in intestinal bacterial flora that hydrolyze the wide variety of glycosides present in nature. This investigation was designed to study the effect of stable cell-free extracts from enteric bacteria of human feces, fecalase, which was shown to contain glycosidases which bioactivate in vitro many natural diet glycosides to compounds which are mutagenic in the Salmonella/ liver homogenate test. Cantaloupe (Cucumis melo); Raspberry (Rubus idaeus), Red and Yellow Onion (Allium cepa) varieties all contain quercetin which presumably forms mutagenic flavonol glycosides in the gut. The White Onion does not contain quercetin. Flavonol extract of Cantaloupe, Raspberry, Red and Yellow varieties of onion were mutagenic in the test when fecalase was added. Frameshift mutagenicity (TA 1537, TA 98, and TA 97) among the flavonoid extracts tests was mainly confined to the flavonols (flavon-3-ols). The base-pair mutants (TA 1535, TA 100) did not show mutagenic activity upon testing the flavonoid extracts of the samples investigated in this study. Since the flavonols are probably the single largest group of flavonoids, and the mutagenic agent detected, quercetin, is the most common flavonol aglycone, plant breeding has been suggested to reduce the amount of flavonoids present in the food we eat. / Graduation date: 1983

Caveolin-1 (CAV1) is an essential protein for the formation of caveolae, invaginations found at the plasma membrane. Caveolae are abundant in many cell types and numerous functions have been identified, including modulating cell signaling and buffering cells from mechanical stress. However, exactly how newly identified mutant forms of CAV1 impact caveolae formation and function remains to be elucidated. Previous work has highlighted the importance of the C-terminus of CAV1 in the assembly of CAV1 complexes, and trafficking to the plasma membrane. Investigation of the frameshift mutant CAV1-P158 and the truncation mutant CAV1-F160X revealed that alteration of the amino acid sequence, or truncation of the C-terminus of CAV1 had little effect on caveolae assembly. Interestingly, incorporation of these mutant proteins into hybrid caveolae with wild type CAV1 led to the formation of caveolae with altered biochemical and molecular properties, indicating that an intact C-terminus may also be important for normal caveolae function. Thus, the incorporation of CAV1 proteins with C-terminal mutations into caveolae is a critical factor associated with human diseases.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1999. / Vita. / Includes bibliographical references (leaves 143-154). / by Li Su. / Ph.D.

Human immunodeficiency virus type I (HIV-1) is a global health issue affecting approximately 35 million people worldwide. Despite the success of highly active antiretroviral therapy (HAART), drug resistant viruses and adverse drug reactions raise the need for new classes of antiretroviral drugs. During HIV-1 replication, the viral frameshifting element orchestrates expression of a specific ratio of the essential Gag and Gag-Pol polyproteins from overlapping reading frames. This process is vital for viral assembly and maturation, since altering the ratio of Gag and Gag-Pol polyproteins through HIV-1 frameshifting, perturbs HIV-1 replication. To identify modulators of HIV-1 frameshifting, a bi-cistronic fluorescent construct was designed to establish a clonally-derived cell culture model of HIV-1 Gag:Gag-Pol frameshifting. Following assay development and validation of screening parameters, a primary diversity screen with 92,071 small molecules was conducted. The initial screening triage of hit compounds led to the selection of several false positives due to auto-fluorescent compound properties. I subsequently re-designed the screening cascade to incorporate a counter-screen that was effective at eliminating false positives. Ultimately, no hit compounds were identified capable of activating or inhibiting HIV-1 frameshifting. However, compound DDD00049811 was fortuitously discovered to inhibit the spread of HIV-1 infection, but not through the desired target of HIV-1 frameshifting. The high concentration required to elicit an antiviral response, the minimal effect in a single-cycle of HIV-1 replication and the slight cellular viability defect suggest that DDD00049811 is not a good anti-HIV-1 candidate drug. My work indicates that HIV-1 frameshifting is a challenging drug target; however, addressing limitations in the strategy used in this study could allow this critical function to be drugged successfully.