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MSc., Faculty of Science, University of the Witwatersrand, 2011 / In prokaryotes, transcription and translation are coupled and as a result, the beginning of the messenger RNA is translated by the ribosome while the 3' end is still synthesized. How exactly this occurs is still not clear. One possibility is that RNA polymerase and the ribosomes may be in physical contact with each other at some stage during gene expression or RNA polymerase has a binding site in the ribosomes. Mutational analysis is one method to explore how coordination between these two moieties occurs in bacteria. An Escherichia coli strain with all seven chromosomal ribosomal RNA operons deleted, replaced by a single rrnB plasmid-borne operon, was used to isolate ribosomal RNA mutants with increased rifampicin resistance, two of which were studied further. The altered rrnB operon in pGM1 was obtained by spontaneous whilst in pGM2 by EMS mutagenesis. The mutated rrnB operon in pGM1 conferred resistance to 25μg/ml of rifampicin while in pGM2 resistance of 30μg/ml was observed. A base substitution of T to A at position 355 of the 23S rRNA was detected in pGM1and no nucleotide change was detected in pGM2. The successful isolation of ribosomal RNA mutants with rifampicin resistance is consistent with the hypothesis of interaction between the RNA polymerase and the ribosomes and suggests the part of this interaction is with the large ribosomal subunit.

Thesis advisor: Gabor Marth / While a PhD student in Dr. Gabor Marth's laboratory, I have had primary responsibility for two projects focused on using RNA-Seq to measure differential gene expression. In the first project we used RNA-Seq to identify differentially expressed genes in four yeast species and I analyzed the findings in terms of the evolution of gene expression. In this experiment, gene expression was measured using two biological replicates of each species of yeast. While we had several interesting biological findings, during the analysis we dealt with several statistical issues that were caused by the experiment's low number of replicates. The cost of sequencing has decreased rapidly since this experiment's design and many of these statistical issues can now practically be avoided by sequencing a greater number of samples. However, there is little guidance in the literature as to how to intelligently design an RNA-Seq experiment in terms of the number of replicates that are required and how deeply each replicate must be sequenced. My second project, therefore, was to develop Scotty, a web-based program that allows users to perform power analysis for RNA-Seq experiments. The yeast project resulted in a highly accessed first author publication in BMC Genomics in 2011. I have structured my dissertation as follows: The first chapter, entitled General Issues in RNA-Seq, is intended to synthesize the themes and issues of RNA-Seq statistical analysis that were common to both papers. In this section, I have discussed the main findings from the two papers as they relate to analyzing RNA-Seq data. Like the Scotty application, this section is designed to be "used" by wet-lab biologists who have a limited background in statistics. While some background in statistics would be required to fully understand the following chapters, the essence of this background can be gained by reading this first chapter. The second and third chapters contain the two papers that resulted from the two RNA-Seq projects. Each chapter contains both the original manuscript and original supplementary methods and data section. Finally, I include brief summaries of my contributions to the two papers on which I was a middle author. The first was a functional analysis of the genomic regions affected by mobile element insertions as a part of Chip Stewart's paper with the 1000 Genome Consortium. This paper was published in Plos Genetics. The second was a cluster analysis of microarray gene expression in Toxoplasma gondii, which was included as part of Alexander Lorestani et al.'s paper, Targeted proteomic dissection of Toxoplasma cytoskeleton sub-compartments using MORN1. This paper is currently under review. The yeast project was a collaborative effort between Jesse Gray, Michael Springer, and Allen Costa at Harvard Medical School, Jeffery Chuang here at Boston College, and members of the Marth lab. Jesse Gray conceived of the project. While I wrote the draft for the manuscript, many people, particularly Gabor Marth, provided substantial guidance on the actual text. I conceived of and implemented Scotty and wrote its manuscript with only editorial assistance from my co-authors. I produced all figures for the two manuscripts. Chip Stewart provided extensive guidance and mentorship to me on all aspects of my statistical analyses for both projects. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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Micovírus são freqüentemente encontrados infectando vários gêneros de fungos. Em alguns sistemas, bem caracterizados, foi demonstrada a interferência de genes virais com o fenótipo do fungo, sendo o caso melhor documentado a hipovirulência em fungos fitopatogênicos. Em nosso laboratório, seis linhagens de Metarhizium anisopliae, um fungo entomopatogênico usado no controle biológico de pragas importantes em agricultura, foram analisadas para a presença de dsRNA. Todas as linhagens foram isoladas de insetos e três apresentam dsRNA viral (linhagens Al, MS e RJ). Nosso objetivo é analisar a possível interferência dos micovírus (Ma V s) com a entomopatogenicidade de M anisopliae. Neste trabalho foram caracterizados três MaVs através de purificação em gradientes de CsCl, microscopia eletrônica, análise de proteínas, padrão eletroforético dos componentes dsRNA, sorologia e análise da homologia entre os componentes dsRNA individuais de um mesmo Ma V e entre os componentes dsRNA dos diferentes MaVs, utilizando sondas de cDNA. As partículas virais dos MaVs RJ, AI e MS são isométricas, com diâmetro médio de 3S nm e são separados em várias bandas em gradientes de CsCI nas densidades de flotação de 1,32 a 1,41 (MaV-RJ); 1,3S a 1,45 (MaV-Al) e 1,40 a 1,43 g/ml (MaV-MS). Espectros de absorção típicos de nucleoproteínas foram observados em todas estas bandas e nas preparações de micovírus. Após a dissociação das partículas virais foi observada a presença de componentes dsRNA com o mesmo perfil eletroforético obtido em extratos de micélio. Foi demonstrado que o ácido nucléico viral é composto por dsRNA, pela digestão enzimática específica e purificação em colunas de celulose CFll. MaV-RJ apresenta, pelo menos, treze componentes dsRNA, variando de 3,1 a 0,5 kb e um polipeptídeo majoritário do capsídeo de 46 kDa. Ma V-AI apresenta pelo menos, cinco componentes dsRNA variando de 4,1 a 1 kb e um polipeptldeo majoritário do capsídeo de 80 kDa. Ma V -MS é muito semelhante a Ma V -AI em relação ao componente dsRNA de 4,1 kb e polipeptídeo majoritário, entretanto apresenta um número menor de componentes dsRNA pequenos. Durante o desenvolvimento do trabalho, Ma V-AI e MaV-MS apresentaram um padrão instável de componentes dsRNA, progressivamente mais complexo em relação aos componentes dsRNA pequenos. Foi caracterizado um mutante estável (linhagem RJd), derivado espontaneamente da linhagem RJ que não apresenta os componentes dsRNA de maior peso molecular, tendo apenas os nove componentes menores presentes na linhagem parental RJ. Esta linhagem derivada, RJd, apresenta morfologia da colônia alterada, contudo não temos evidências experimentais para associar diretamente estas alterações com o padrão alterado de componentes dsRNA. Anticorpos policlonais foram produzidos contra os três Ma V s e mostramos que estes Ma V s são sorologicamente relacionados. Em testes de dupla difusão MaV-RJ reage com antisoro contra MaV-AL e MaV-M5 indicando relações sorológicas. Em Western, todos os polipeptídeos dos capsídeos dos três Ma V s, reagem com os antisoros contra MaV-Al e MaV-M5. Sondas de cDNA do componente dsRNA de 4,1 kb de MaV-Al hibridizam com os componentes de 4,1 kb de MaV-M5 e com os componentes dsRNA S2, M3 e Ml de MaV-RJ. Quando a sonda de cDNA é preparada a partir do componente dsRNA de 4,1 kb de MaV-M5 também ocorre hibridização com o componente dsRNA de mesmo tamanho de MaV-AL ~com o componente dsRNA S2 de MaV-RJ. Se conclui que os três micovírus são relacionados entre si em relação a pelo menos um componente dsRNA. Além disso, a análise de hibridização mostrou que existe homologia entre todos os componentes de Ma V-RJ mas sondas a partir do componente dsRNA de 4,1 kb de MaV-Al não hibridizam com nenhum outro componente da mesma linhagem. Nossas tentativas de transferir micovírus ou dsRNA a linhagens "livres de micovírus" utilizando biobalística falharam. / Micovirus are frequently found infecting severa! genus of fungi in nature. In some, well characterised systems, the interference of vira! genes with the fungai phenotype was demonstrated, as is the case of hypovirulence mediated by viral dsRNA in phytopathogenic fungi. In our laboratory, six strains of Metarhizium anisopliae, an entomopathogenic fungus used in biological control of agronomic important pests, were screened for the presence of dsRNA. The strains analysed were isolated from insects and three have vira! dsRNA (strains AI, M5 and RJ). We aim to access the possibility of interference o f micovirus (Ma V s) with the entomopathogenicity o f M anisopliae. In this work the Ma V s were characterised through purification by CsCl gradients, eletron microscopy, protein analysis, dsRNA pattem, serology and homology between individual dsRNA components within the same Ma V and among the Ma V s. The Ma V s parti eles are isometric, with an average diameter o f ca. 3 5 nm and are resolved into severa! bands in CsCl gradients at buoyant densities o f 1.32 to 1.41 (Ma V -RJ); 1.35 to 1.45 (MaV-Al) and 1.40 to 1.43 g/ml (MaV-M5). Absorbance spectra, typical of nucleoproteins, waS detected in ali vira! preparations. The vira! nucleic acid was shown to be dsRNA, by specific enzymatic digestions and celiulose-CF11 chromatography. Upon dissociation, the particles were shown to contain dsRNAs, with the same eletrophoretic pattem of dsRNA components as extracted from mycelium. MaV-RJ have, at least, thirteen dsRNA components, ranging from 3.1 to 0.5 kb and one major capsid polypeptide o f 46 kDa. Ma V -AI have, at least, five dsRNA components, ranging from 4.1 to 1 kb and one major capsid polypeptide of 80 kDa. MaV-M5 is very similar to MaV-Al, in respect to the dsRNA component of 4.1 kb and major polypeptide, however, has fewer smalier dsRNA components. During the development of the work, Ma V -Al and Ma V -MS displayed an unstable dsRNA pattem, progressively more complex in relation to the smaller components. A stable mutant (strain RJd), derived spontaneously from strain RJ, was characterised and it lacks some of the large dsRNA components and has only nine of the smali dsRNA components present in the parental strain RJ. This derived strain displays altered colony morphology, nevertheless we have no experimental data to associate these alterations directly to the altered dsRNA pattem. Polyclonal antibodies were produced against the three Ma V s and we show that the Ma V s are serologicaliy related to each other. In double-diffusion serological test Ma VRJ reacted with antiserum against MaV-Al and MaV-M5, indicating serological relationships. In Westem blot, ali capsid polypeptides of the three MaVs, reacted with both antiserum against MaV-Al and against MaV-M5. cDNA probes from the 4.1 kbdsRNA component of Ma V -Al hybridised with the 4.1 kb-dsRNA component from MaV-M5 and the S2, M3 and Ml-dsRNA components from MaV-RJ. Moreover, a cDNA probe from 4.1 kb-dsRNA component from MaV-M5 hybridised with both the 4.1 kb-dsRNA component from MaV-Al and S2-dsRNA component from MaV-RJ. lt was concluded that the three viroses are related to each other, at least in one dsRNA component. In addition, hybridisation analysis revealed sequence homology between all dsRNA components of MaV-RJ but probes from the 4.1 kb-dsRNA component from MaV-Al do not hybridise with any other dsRNA components of the same strain. Our attempts to transfer the Ma V s to different "free o f vírus" strains of M. anisopliae by biobalistic, using both micovirus and naked dsRNA, have failed.

Thesis (Ph.D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (February 27, 2007). Vita. Includes bibliographical references.

The hammerhead ribozyme (HHRz) catalyzes a site-specific phosphodiester bond cleavage reaction that is enhanced by the presence of metal cations. Metal cations are thought to aid in the folding and possibly the catalytic mechanism of this ribozyme. The goal of this research is to characterize the activities and metal interactions of minimal and extended HHRz constructs using kinetic and spectroscopic studies. Metal binding to the cleavage site of the HHRz was probed using 31P NMR to monitor Cd2+ titrations of HHRzs with a phosphorothioate modification at the cleavage site. Either a 2'-F or a 2'-NH2 substitution at the nucleophile position was used to block cleavage. With a 2'-F, no metal binding to the cleavage site phosphate was observed. However, with a 2'-NH2 substitution, a large change in 31P chemical shift of the phosphorothioate peak suggests Cd2+ binding. A 2'-NH2 is a potential metal ligand, but a 2'-F is not. This suggests that a metal ion binds to the cleavage site phosphate when the 2' nucleophile position also provides a ligand. Minimal HHRzs with only one stem loop structure show little activity in presence of physiologically relevant concentrations of divalent cations. A kinetic and thermodynamic characterization of an extended HHRz derived from Schistosoma mansoni with loops in stems I and II was performed. High catalytic activity was observed with low concentrations of divalent cations, and loss of potential loop-loop interactions resulted in a large decrease in activity. An electrostatic surface plot of a HHRz crystal structure revealed an area of high negative electrostatic potential in the cleft between stems I and II with contributions from nucleotides U7, A6, and C17 of the HHRz that could serve to trap metal ions. To probe this putative metal site, kinetic studies of HHRz constructs with phosphorothioate substitutions 5' to U7 or C17 or with an A6 2'- OMe substitution were performed. Results of these studies suggest that a metal interaction at this site would include direct coordination to A6 2'-OH, but indirect interactions with the phosphates.

CHOP, or Gadd153, is a 29 kDa protein that plays a pivotal role in the mediation of cellular stress-induced cell death. The expression of the gene encoding CHOP/Gadd153 is regulated at both the transcriptional and post-transcriptional levels. Compared to transcription, the regulation of Chop gene expression at the post-transcriptional level is much less understood. In this study, the role played by the 3’-untranslated region of CHOP mRNA (3UTRChop) in mediating mRNA stability was examined. A reporter plasmid was constructed so that the mRNA expressed has 3UTRChop as its 3’-untranslated region. Partial 5’ deletions, or deletion of short internal (~30 nucleotides) sequences, or the deletion of a putative AU-rich element (ARE) within 3UTRChop, all resulted in the elevation of the steady state levels of mRNA and the encoded reporter protein, EGFP. Deletion of the ARE or sequences remote from ARE resulted in the reduced rate of mRNA degradation. Such data suggested that 3UTRChop is closely related to mRNA stability. The effect of cellular stress on the functioning of 3UTRChop was studied by examining the change in mRNA level in cells treated with arsenic trioxide (ATO). The presence of arsenic stress stimulated a marked increase in the steady state level of not only the reporter mRNA, but also control mRNAs that did not have 3UTRChop as the 3’-untranslated region. A non-specific effect of arsenic stress on mRNA levels was therefore suggested. Consistent with the increase in the level of reporter mRNA, the expression of EGFP protein was also increased. Arsenic stress and partial deletion of 3UTRChop produce additive increase in mRNA level and EGFP protein level implying that the mRNA destabilizing function of 3UTRChop is unlikely to be stress-regulated. The contribution of 3UTRChop in mRNA translation was then examined using reporter constructs that expressed EGFP mRNA having its original 5’ and 3’-untranslated regions replaced with 5UTRChop and 3UTRChop respectively. In the presence of wild-type 3UTRChop, the abolition of the translation repressor functions of 5UTRChop produced only mild increase in EGFP expression. However, the additional partial deletion of 3UTRChop resulted in massive increase in EGFP expression. In the presence of wild-type 5UTRChop, the partial deletion of 3UTRChop resulted in only a small increase in EGFP expression. Such data demonstrated a complementary relationship between 5UTRChop and 3UTRChop in the regulation of Chop expression in unstressed cells. EGFP mRNA having 5UTRChop and 3UTRChop as the 5’ and 3’-untranslated region respectively expressed significant EGFP protein only in the presence of ATO. The expression of EGFP was not significantly affected with swopping of 3UTRChop with another 3UTR. 3UTRChop is therefore not essential for the mediation of ATO-stimulated expression of EGFP. The present study demonstrated that full length 3UTRChop may have constitutive mRNA destabilizing effect that is not alleviated by cellular stress. The evaluation of the relative contributions of 5UTRChop and 3UTRChop in mRNA translation suggested a model for Chop gene expression whereby the eventual protein level of Chop is determined by 5UTRChop-mediated translation as well as by 3UTRChop-mediated destabilization of mRNA template. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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