Theses and Dissertations from UMD
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Item PERPETUATION OF NON-GENETIC CHANGES AT A TRANSGENE LOCUS IN C. ELEGANS(2018) Devanapally, Sindhuja; Jose, Antony M; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Development of a multicellular organism from a single cell or from a few cells in every generation relies on the reproducible expression pattern of genes. At the beginning of every generation in every organism, at least a single progenitor cell is necessary to produce the organism. This cell is subjected to reprogramming mechanisms that erase epigenetic information transmitted from the previous generation and as it develops, the organism goes through experiences that affect gene expression. Despite this, developmental processes give rise to nearly the same organism in the next generation, suggesting that the components in the cells are similarly regulated in every generation. How a complex organism can develop and reproduce its gene expression pattern using only the information present within the progenitor cell is not understood. Here, we describe an engineered genetic locus in the nematode worm C. elegans that shows robust transgenerational expression like many loci in the genome but, unlike other tested loci, can be uniquely susceptible to transgenerational silencing by one of two distinct processes. This locus could be silenced by double-stranded RNA (dsRNA) transported from neurons and could also be silenced when inherited solely from the male parent in a genetic cross. Each process could initiate transgenerational silencing within the germline that lasted for >25 generations. The two processes depended on distinct mechanisms to initiate silencing – while neuronal dsRNA required the conserved dsRNA importer SID-1 and the Argonaute RDE-1, mating-induced silencing required the Piwi-interacting Argonaute PRG-1. Both processes engaged the same germline Argonaute HRDE-1 to maintain heritable silencing suggesting that both processes trigger silencing independently but converge on the same pathway for maintenance. No other locus that was tested showed such indefinite silencing by either mechanism, suggesting that most loci are resistant to changes in gene expression. Thus, the discovery of a locus that is susceptible to transgenerational change provides us with the first instance of how a single change at a gene sequence can be used to explain evolution of gene regulation.Item Characterization of the TrxSR Two-Component Signal Transduction System of Streptococcus pyogenes and its Role in Virulence Regulation(2011) Gold, Kathryn; McIver, Kevin; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Gram-positive group A streptococcus (GAS) is a strict human pathogen, which causes a wide variety of infections, ranging in severity from minor to life threatening. In order to cause such a diverse array of diseases, GAS utilizes two-component signal transduction systems (TCS) to coordinately regulate sets of virulence genes in response to changing host conditions. The present study investigates the role of the TrxSR TCS in the regulation of virulence of the GAS. Using an insertional inactivation mutation in TrxR in serotype M1 MGAS5005, transcriptome studies established that TrxR activates transcription of Mga-regulated virulence genes, a separate non-TCS regulatory pathway controlling factors important for immune evasion and colonization. Transcriptional reporter fusions of Pmga to firefly luciferase revealed that the TrxR regulation occurs through the Pmga promoter. Additionally, electrophoretic mobility shift assays using purified His-MBP tagged TrxR established specific binding of TrxR to Pmga, although the interaction appeared to be transient. To determine the importance of signal transduction for TrxR-mediated regulation of the Mga regulon and virulence, an in vitro reconstitution assay was performed with purified TrxR and TrxS. Using both wild type and mutated forms of the TrxSR proteins, we demonstrated that TrxSR is a functional two-component phosphorelay system. Interestingly, phosphorylation of TrxR did not appear to be critical for DNA binding and regulation, since a TrxR D55A mutation did not change the expression of TrxR regulated genes in GAS based on EMSA and qPCR. In order to investigate whether there is a functional conservation of TrxR's involvement in GAS virulence regulation, mutations were made in serotype M4 and M49 strains representing either throat only or generalist strains. We have determined that TrxR regulates mga and Mga-regulated genes (emm, arp) in the M4 and M49 backgrounds, suggesting conservation of TrxR's role in virulence regulation. Overall, TrxSR represents a functional TCS that appears to directly regulate the Mga virulence regulon independent of phosphorelay. Furthermore, the functional conservation of TrxR regulation of Mga in other serotypes suggests a conserved role for its involvement in virulence regulation in GAS.Item EVOLUTION, DEVELOPMENT, AND GENETICS OF OPSIN GENE EXPRESSION IN AFRICAN CICHLID FISHES(2011) O'Quin, Kelly E; Carleton, Karen L; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The molecular genetic mechanisms that underlie phenotypic evolution include mutations within protein-coding, cis-regulatory, and trans-regulatory factors. Although many studies have examined how these mutations individually contribute to phenotypic divergence and the formation of new species, none have examined how they may do so collectively. In this study, I examine how these molecular genetic mutations collectively contribute to the evolution of color vision among African cichlid fishes. I show that phenotypic divergence in cichlid color vision is achieved by mutations affecting the coding sequence and expression of seven opsin genes. After contrasting the roles of these two mechanisms, I use bioinformatic-, association-, and experimental genetic analyses to determine what role mutations in cis- and trans-regulatory DNA play in the evolution of cichlid opsin expression. Specifically, I demonstrate that: (1) Protein-coding mutations primarily affect cichlid opsins sensitive to the ends of the visible light spectrum (SWS1 [ultraviolet-sensitive] and LWS [red-sensitive]). (2) Changes in opsin gene expression contribute to large differences in color vision among closely related species. These analyses also reveal that the expression of the SWS1 and SWS2B opsins have diverged among closely related cichlids in association with foraging preferences and ambient light intensity, suggesting that their expression has evolved due to natural selection. Ancestral state reconstructions reveal that changes in opsin expression have evolved repeatedly among cichlids in Lakes Tanganyika and Malawi; further, I find that this repeated evolution has likely been achieved by repeated changes to cichlid development. (3) Bioinformatic analyses suggest that cichlids have diverged in multiple cis-regulatory sequences surrounding the opsin genes, and association mapping identified three putative single nucleotide polymorphisms upstream of the SWS2A (blue), RH2B (blue-green), and LWS (red) opsins that may contribute to cichlid opsin expression differences in cis. (4) Genetic mapping in experimental crosses suggests that divergence in multiple trans-regulatory factors also contribute to the evolution of SWS2B (violet), RH2A (green), and LWS (red) opsin expression. The contribution of these trans-regulatory factors to the evolution of cichlid opsin expression may outweigh those in cis. These results reveal that multiple molecular genetic mechanisms can contribute to phenotypic evolution among closely related species.