Browsing by Subject "Viruses"
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Item Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production(Texas Tech University, 1997-05) Colmer, Jane AnnProduction of exotoxin A, the most toxic of the virulence factors produced by the opportunistic pathogen Pseudomonas aeruginosa, is a complicated, highly regulated process involving several genes. In this study, we describe the isolation and characterization of two new regulatory genes for toxA expression, ptxR and ptxS. The presence of extra copies of ptxR in PA01 results in a fivefold increase in exotoxin A activity. Subcloning, complementation, nucleotide sequence analyses, and T7 expression experiments revealed that the ptxR open reading frame (ORE) encodes a 34 kDa protein. Computer analysis for amino acid homology revealed that the product of ptxR (RxR) belongs to the LysR family of transcriptional activators and contains a putative helix-turn-helix DNA binding motif. Transcriptional studies using RNA analysis and a toxAlacZ fusion disclosed that ptxR enhances both toxA and regA transcription. These results suggest ptxR acts through regA to enhance toxA expression. Further studies confirmed that the presence of a 2.1 kbp fragment 5' of ptxR reduced the enhancement in exotoxin A by threefold. Nucleotide sequence analysis and expression experiments revealed that this fragment carries an ORE, ptxS, encoding a 38 kDa protein, RxS. Computer analysis of the amino acid sequence indicated that RxS belongs to the GalR-LacI family of repressors. The presence of a helix-turn-helix motif suggests a DNA binding function for PtxS. Additional experiments, using ptxRlacZ fusions, provided evidence that ptxS affects ptxR expression directly. The presence of three potential GaIR binding sites in the ptxR upstream region suggests that RxS might interfere with pfxP function at the transcriptional level by binding to its upstream region. We have also examined ptxR expression and the effect of ptxR on toxA expression throughout the growth cycle of Pseudomonas aeruginosa strains PA01 and PA103 using toxAlacZ chromosomal integrates and the ptxRlacZ fusion plasmid. These experiments suggest that while the enhancement in toxA transcription by ptxR continues at later stages of the growth cycle, ptxS expression declines; and, that ptxR expression in Pseudomonas aeruginosa is iron-independent.Item Strategies for deciphering the genome(2014-12) Lou, Dianne In-Hye; Sawyer, Sara L.; Press, William H; Paull, Tanya T; Sullivan, Christopher S; Ehrlich, Lauren IR; Miller, Kyle MThe development of highly sophisticated technologies has ushered in the era of the genome. Most importantly, high-throughput sequencing technologies has vastly expanded the number of available genome projects of many different organisms. One of challenges that we now face is in understanding the information encoded within these genomes. Within each chapter of this dissertation, information from existing genome projects are used to answer fundamental biological questions related to human disease and an attempt to further advance new technologies is made. In chapter 2, I describe a novel method that decreases the error rates associated with next-generation sequencing technologies, allowing for the investigation of more complex and heterogenous samples relevant to many biological systems. In chapter 3, I use available primate genome projects to understand the evolutionary trajectory of two DNA repair genes, whose defect increases the development of breast and ovarian cancers. Finally, in chapter 4, wild-type primate alleles are used as tools to uncover novel mechanisms in the lifecycle of viruses. Although seemingly non-overlapping, each of these studies is centered around using the sea of information that is now readily available in order to decipher the many secrets encoded by genome.