Browsing by Subject "Transcriptome"
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Item Genetic basis for ichthyotoxicity and osmoregulation in the euryhaline haptophyte, Prymnesium parvum N. Carter(2014-05) Talarski, Aimee Elizabeth; La Claire, John W., 1951-There is limited information currently available regarding the underlying physiological responses and molecular mechanisms of osmoregulation, acetate metabolism [in relation to the synthesis of glycerolipids, polyunsaturated fatty acids (PUFA), and ichthyotoxins], and transport in Prymnesium parvum N. Carter, a microalga that causes devastating harmful algal blooms (HAB) worldwide. This dissertation examines gene expression under environmental conditions that are associated with HAB formation, including phosphate limitation and low salinity, using microarrays and RNA sequencing (RNA-Seq). A comparative fatty acid methyl ester (FAME) analysis at 30 vs. 5 practical salinity units (psu) was performed to gain additional insight into acetate metabolism. The RNA-Seq analysis included a de novo assembly of the P. parvum transcriptome, generating 47,289 transcripts, of which 35.4% were identifiable. This permitted the evaluation of the expression of many more genes compared with the microarray analysis, which examined ~3,500 genes. Relevant candidate genes identified included those whose products are involved in osmolyte production, salinity stress, and ion transport. With respect to the putative synthesis of polyketide ichthyotoxins, 32 different polyketide synthase (PKS) transcripts were identified in the transcriptome assembly, none of which were differentially expressed. Hemolysin and monogalactosyldiacylglycerol synthase were downregulated at 30 vs. 5 psu, suggesting the increased presence of additional ichthyotoxins at the lower salinity. Evidence for several PUFA synthesis pathways was also revealed. Fatty acid compositions were largely similar at the two salinities, containing relatively prominent quantities of the PUFA stearidonic acid, but compositions varied among strains. The transcription of genes whose products are associated with vesicular transport was elevated, and higher levels of extracellular prymnesins were observed in HAB-forming conditions. Thus, with regard to acetate metabolism, I have revealed evidence for the post-transcriptional regulation of the production of prymnesins and the contributory effects of hemolysin, monogalactosyldiacylglycerol, and PUFA towards ichthyotoxicity. Further, I propose that toxin transport is triggered in HAB-forming conditions, in which the toxins are actively being excreted. Collectively, these data shed light on the transcriptional responses that occur following alterations in phosphate availability and salinity, including those associated with the synthesis and delivery of a number of potential ichthyotoxins from P. parvum.Item Genomic Insights into Sexual Selection and the Evolution of Reproductive Genes in Teleost Fishes(2012-10-19) Small, ClaytonSexual selection has long been a working explanation for the elaboration of appreciable traits in plants and animals, but the idea that it is an equally potent agent of change at the level of individual molecules is relatively recent. Indications that genes associated with reproductive biology evolve especially rapidly planted this notion, but many details about the genomics of sex remain elusive. Numerous studies have characterized rapid sequence and expression divergence of sex-related molecules, but few if any have demonstrated convincingly that these patterns exist as a result of sexual selection. This dissertation describes several genome-scale studies related to reproduction and the sexes in teleost fishes, a group of animals underexploited in regard to this topic. Using commercial microarrays I measured the extent of sexually dimorphic gene expression in the zebrafish, Danio rerio. Sex-biased patterns of gene expression in this species are similar to those described in other animals. A number of genes expressed at high levels in ovaries and testes relative to the body were identified as a product of the study, and these data may be useful for future studies of reproductive genes in Danio fishes. In a second study, the recent advent of high throughput cDNA pyrosequencing was leveraged to characterize the relationships between tissue-, sex-, and species-specific expression patterns of genes and rates of sequence evolution in swordtail fishes (Xiphophorus). I discovered ample evidence for expression biases of all three types, and a generally positive but idiosyncratic relationship between the magnitude of expression bias and rates of protein-coding sequence evolution. Pyrosequencing of cDNA was also used to explore the possibility that postcopulatory sexual selection drives the rapid evolution of male pregnancy genes, a novel class of reproductive molecules unique to syngnathid fishes (seahorses and pipefishes). Genes differentially expressed in the male brooding tissues as a function of pregnancy status evolve more rapidly at the amino acid level than genes exhibiting static expression. Brooding tissue genes expressed during male pregnancy have evolved especially rapidly in polyandrous lineages, a finding that supports the hypothesized relationship between postcopulatory sexual selection and the adaptive evolution of reproductive molecules.Item Systematic analysis of transcriptome and proteome in opportunistic bacterium Pseudomonas aeruginosa(2012-05) Kwon, Taejoon; Marcotte, Edward M.Transcription and translation are the two most important central mechanisms to control gene regulation in living organism. Although these two mechanisms have been studied intensively for last several decades, it is still not clear how all the information encoded on genomic DNA is converted to mRNA and proteins, the molecular functional components that change the characteristics of cells, depending on their needs. Here, I investigated the gene regulation of opportunistic bacterium Pseudomonas aeruginosa, using recently developed high-throughput techniques, microarray for transcriptomics and LC-MS/MS for proteomics. By analyzing transcriptome of 17 strains isolated over time from three individuals with cystic fibrosis, I identified 24 genes showing significant expression changes consistently across all strains, as evidence of parallel evolution of common traits that were beneficial in establishing chronic infection. Also, by analyzing proteome and transcriptome of two reference Pseudomonas aeruginosa strains, PAO1 and PA14, under growth condition mimicking in vivo nutrition environment in cystic fibrosis patients, I revealed that protein abundances are less correlated than mRNA abundance between them, and many proteins known as virulence factors showed different abundances only in protein level, demonstrating that post-transcriptional regulation is important to understand pathogenesis of Pseudomonas aeruginosa. To boost sensitivity both in identification and quantification in shotgun proteomics, I also created a novel integrative database search algorithm, and released freely available software package termed in MSblender. These results would be valuable information for the research community to understand the regulatory mechanism of gene expression both in transcription and translation, especially related to infectious diseases caused by pathogenic bacteria. Also, I present an integrative analysis method would be generally beneficial to extract more information from conventionally used shotgun proteomics experiments.