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Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2007. / "A dissertation in pharmaceutical science and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed July 16, 2008; title from "catalog record" of the print edition. Includes bibliographical references (leaves 206-220). Online version of the print edition.

In the present study Drosophila melanogaster was used to define the organization and expression of tRNA genes. The three major Drosophila valine tRNAs were isolated and purified by standard chromatographic techniques. Nucleoside analysis indicated that of these tRNAs only tRNA₄Val contained inosine. All three tRNAsVal contained ribothymidine, therefore they resemble yeast tRNAVal in this regard but not the mammalian tRNAsVal which lack ribothymidine. The purified tRNAs were labelled with ¹ ² ⁵I and used to determine the location of the genes for these tRNAs utilizing the technique of in situ hybridization to salivary gland chromosomes. tRNA₄Val hybridized consistently to one site on the right arm of the second chromosome, 56D, which is close to the site of 5S RNA, 56F. tRNA₃bVal hybridized to two sites, 84D and 92B, both on the right arm of the third chromosome. The labelling of site 84D was approximately twice as heavy as that of 92B. Dr. A. Delaney (unpublished) has shown that approximately 13 genes code for tRNA₃bVal per haploid genome. The in situ hybridization data suggests that the 13 genes are divided such that approximately 8 genes are at site 84D and 5 genes are at site 92B. Evidence to support this supposition is derived from measurements on the amount of tRNA₃bVal in mutant flies deficient or duplicated for site 84D on one of their two homologous third chromosomes. tRNA₃bVal amounts, measured relative to the other tRNAVal isoacceptors decrease 31% in the deficiency and increase 30% in the duplication. These results demon- strate a direct relationship of the amount of tRNA₃bVal to gene dosage because the duplication has 8 extra genes, which is a 30% increase and the deletion has 8 fewer genes, a 30% decrease. Finally, it was shown that the amount of total tRNSVal increased by 17% in the duplication but did not decrease in the deletion. This result demonstrates the amount of valine tRNA is under a type of control in which the amount of total valine tRNA is increased to compensate for the deficiency of a single isoacceptor. Also the coding properties of four tRNASer isoacceptors were determined. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The RNA World hypothesis describes a period of time during the origins of life in which RNA molecules performed all catalysis and were the only form of information storage. A great deal of evidence has been obtained in support of this hypothesis, however a few key demonstrations are lacking. The first demonstration is of a molecule capable of self-replication that could have plausibly arisen from the prebiotic soup. Previously in the Lehman Laboratory, a 198-nucleotide RNA was discovered that could be fragmented into as many as four pieces ranging from 39 - 63 nucleotides in length. When these pieces were incubated together in a test tube, they re-formed the necessary covalent bonds to regenerate the full-length 198-nucleotide RNA. Furthermore, the full-length RNAs were catalytically active and made copies of themselves from the remaining pieces in solution, providing a model system of self-replication. I was able to remove >10% of the total length of the RNA, which substantially reduced the catalytic activity of the full-length molecule. I discovered several mutations that restored catalytic activity by improved folding and increased catalytic rates using in vitro selection. A subset of these mutations was found to aid in the assembly of the shortened full-length RNA from smaller fragments than were possible in the original system, enhancing the prebiotic relevance of this system. A second demonstration to bolster the RNA World hypothesis would be showing that RNA is capable of harvesting energy from its environment by performing oxidation and reduction reactions. Again using in vitro selection, I have completed five rounds of selection geared towards identifying a ribozyme that reduces benzoic acid to benzaldehyde using Zn2+ and NADH. Results to date suggest the selection is working and it should be continued for another five to ten generations. Finally, I have discovered an RNA sequence that forms knots during transcription, a phenomenon heretofore undocumented in RNA. This new topology has implications for RNA stability by rendering RNA more resistant to hydrolysis, and could impact catalysis through formation of more complex, knotted active sites. Taken together, these findings have improved our understanding of RNA folding and catalysis, and the plausibility of the RNA World.

No description available.

The functions of many cellular RNAs require that they fold into specific three-dimensional native structures, which typically involves arranging secondary structure elements and stabilizing the folded structure with tertiary contacts. However, RNA folding is inherently complex, as most RNAs fold along pathways containing multiple intermediates, including some misfolded intermediates that can accumulate and persist. Our understanding of the origins and structures of misfolded forms and the resolution of misfolding remains limited. Here, we investigate folding of the Tetrahymena intron, an extensively studied RNA folding model system since its initial discovery decades ago. The ribozyme variant predominantly misfolds, and slow refolding to the native state requires extensive structural disruption. Paradoxically, the misfolded conformation contains extensive native structure and lacks incorrect secondary and tertiary contacts despite requiring displacement of a native helix, termed P3, with incorrect secondary structure to misfold. We propose a model for a new origin of RNA misfolding to resolve this paradox, wherein misfolded ribozyme contains within its core incorrect arrangement of two single-stranded segments, i.e. altered topology. This model predicts a requirement for P3 disruption to exchange the misfolded and native topologies. We mutated P3 to modulate its stability and used the ribozyme's catalytic activity to show that P3 is disrupted during the refolding transition. Furthermore, we demonstrate that unfolding of the peripheral tertiary contacts precedes disruption of P3 to allow the necessary structural transitions. We then explored the influence of topology on the pathways leading to the misfolded and native states. Our results suggest that P3 exists in an earlier pathway intermediate that resembles the misfolded conformation, and that P3 unfolds to allow a small yet significant fraction of ribozyme to avoid misfolding. Despite being on a path to misfolding, the decision to misfold depends upon the probability of disrupting P3 and exchanging topology at this intermediate. Additionally, we show that having a stable P3 in the unfolded ribozyme allows almost complete avoidance of misfolding. Together, these studies lead to a physical model for folding and misfolding of a large RNA that is unprecedented in its scope and detail. / text

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [87]-96).

The production of fully mature protein-coding transcripts is an intricate process that involves numerous regulation steps. The complexity of these steps provides the means for multilayered control of gene expression. Comprehensive understanding of gene expression regulation is essential for interpreting the role of gene expression programs in tissue specificity, development and disease. In this thesis, we aim to provide a better global view of the human transcriptome, focusing on its content, synthesis, processing and regulation using next-generation sequencing as a read-out. In Paper I, we show that sequencing of total RNA provides unique insights into RNA processing. Our results revealed that co-transcriptional splicing is a widespread mechanism in human and chimpanzee brain tissues. We also found a correlation between slowly removed introns and alternative splicing. In Paper II, we explore the benefits of exome capture approaches in combination with RNA-sequencing to detect transcripts expressed at low-levels. Based on our results, we demonstrate that this approach increases the sensitivity for detecting low level transcripts and leads to the identification of novel exons and splice isoforms. In Paper III, we highlight the advantages of performing RNA-sequencing on separate cytoplasmic and nuclear RNA fractions. In comparison with conventional poly(A) RNA, cytoplasmic RNA contained a significantly higher fraction of exonic sequence, providing increased sensitivity for splice junction detection and for improved de novo assembly. Conversely, the nuclear fraction showed an enrichment of unprocessed RNA compared to when sequencing total RNA, making it suitable for analysis of RNA processing dynamics. In Paper IV, we used exome sequencing to sequence the DNA of a patient with unexplained intellectual disability and identified a de novo mutation in BAZ1A, which encodes the chromatin-remodeling factor ACF1. Functional studies indicated that the mutation influences the expression of genes involved in extracellular matrix organization, synaptic function and vitamin D3 metabolism. The differential expression of CYP24A, SYNGAP1 and COL1A2 correlated with the patient’s clinical diagnosis. The findings presented in this thesis contribute towards an improved understanding of the human transcriptome in health and disease, and highlight the advantages of developing novel methods to obtain global and comprehensive views of the transcriptome.