Browsing by Subject "Transcriptome"

Now showing 1 - 5 of 5
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    Analysis of the Pumilio Repression Mechanism and its Impact on the Transcriptome
    (2021-05) Haugen, Rebecca
    The expression of genes in an organism is controlled by many methods. One of the most important means of regulation is post-transcriptional control of messenger RNA (mRNA). RNA binding proteins (RBPs) are crucial for this process. Here we describe the characterization of a functional domain within the Drosophila RBP Pumilio (Pum) and examine the effects of Pum on the transcriptome.Pum proteins are a highly conserved family of RBPs that control the expression of mRNA by binding to specific motifs in the 3’-UTR of transcripts. Through associations with other proteins, Pum proteins regulate the fate of an RNA, primarily causing decay of the target. Drosophila Pum contains autonomous domains capable of enacting decay independently from its RNA binding domain. The most conserved domain is repression domain 3 (RD3) which we characterize here. Using conservation analysis, we define conserved regions within RD3, and show their importance for the repressive activity of RD3. We identify two specific amino acids, F1033 and F1040, which are necessary for RD3’s function. Using yeast two-hybrid assays, we show that RD3 contacts several members of the CNOT deadenylase complex, the central scaffold unit Not1, and subunits Not2 and Not3. In Drosophila cells, we show that RD3 can interact with the CNOT complex. We further explore the endogenous targets of Drosophila Pum proteins by performing RNA sequencing experiments in which we deplete Pum and members of the CNOT complex. This is the first global analysis of the functional regulation of Pum targets in Drosophila. From this analysis we find that thousands of genes rely upon the regulatory framework provided by Pum. We demonstrate specific regulation of several targets of Pum using reporter assays and identify functional Pum binding sites within target 3’-UTRs. Finally, we show that regulation by Pum also affects the levels of expressed protein for two important targets, Pde11 and Raf. The work shown here enhances our understanding of Pum proteins for all organisms and the field of post-transcriptional control.
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    Expression profiling of the in vitro infection of cryptosporidium.
    (2011-10) Mauzy, Mary Jean
    Cryptosporidiosis a global health concern in large part because the causative organism, Cryptosporidium , is a ubiquitous water contaminant throughout the planet. Transcriptional analysis of the parasite has been limited to the pre-attachment stage, the sporozoite. This research describes the transcriptome of the attached C. parvum over a 72 hr in vitro infection utilizing real time PCR. These data provide direct insight into the parasite's biochemical requirements over its developmental life cycle. The parasite gene transcription is dependent upon the developmental stage present and stage specific gene predictions were deduced. Furthermore, a comparison of the gene transcription between C. parvum and C. hominis in duplicated infection protocols indicates that although these two species are almost identical at the nucleotide level, phosphorylation related genes are not expressed at concurrent time points. Additional promoter analysis indicates that genes with differing expression between these two species are more likely to contain a gap in the upstream sequence.
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    Hepatotoxic and Immunomodulatory Transcriptome Responses to Aflatoxin B1 in the Turkey (Meleagris gallopavo)
    (2015-05) Monson, Melissa
    Hepatoxicity and immunotoxicity from dietary exposure to aflatoxin B1 (AFB1) adversely affect poultry health and production. Domestic turkeys (Meleagris gallopavo) are especially sensitive to AFB1 since they have a deficiency in glutathione-mediated detoxification of the reactive AFB1 intermediate. Changes in gene expression can be used to characterize the molecular mechanisms of toxicity; transcriptome analysis allows investigation of differential expression at the genome-wide level. In this research, Illumina RNA-sequencing (RNA-seq) was used to examine transcriptome responses to AFB1 exposure in the turkey. As the liver is the primary site of AFB1 activation and toxicity, the effects of dietary AFB1 on the domestic turkey liver transcriptome was first investigated by sequencing 4 pooled libraries representing 3 individuals for each of 4 treatment groups. As detailed in Chapter 2, predicted transcripts were de novo assembled and differential expression analysis identified significant effects on transcripts from genes involved in apoptosis, cell cycle regulation and lipid metabolism (like E3 ubiquitin-protein ligase Mdm2 and lipoprotein lipase). In Chapter 3, RNA-seq and de novo transcriptome assembly were performed on 3 individual spleen samples per treatment group (n = 12) collected from the same AFB1 challenge trial. Significant down-regulation of antimicrobial genes (like beta-defensin 1) and up-regulation of cytotoxic and antigen presentation genes (such as granzyme A) were observed after AFB1 treatment. Another aspect of these studies was to evaluate the ability of a Lactobacillus-based dietary probiotic to reduce AFB1-effects in the liver and spleen. Addition of probiotics during AFB1 exposure modulated expression in both tissues. Many AFB1-induced expression changes were not mitigated in liver, and although probiotics had some amelioratory effects in the spleen, they were also broadly suppressive of immune genes. Multiple genes impacted in the spleen transcriptome belonged to the Major Histocompatibility Complex (MHC), a region of the genome with genes essential to immune functions. The functions and expression patterns of many of the genes located in the turkey MHC have not been characterized. A single-gene investigation (Chapter 4) characterized expression patterns of 29 MHC genes in the domestic turkey and provided first evidence for expression of B-butyrophilin 2 in muscle tissue. Understanding these expression profiles will help determine MHC gene functions and provide background for expression changes from immunological challenges like AFB1. Unlike the domestic turkey, Eastern wild turkeys (M. g. silvestris) are more resistant to aflatoxicosis due at least in part to their ability to detoxify AFB1. In Chapter 5, an in ovo exposure model was utilized to directly compare the effects of AFB1 exposure in domestic and wild turkey embryos. Embryonic exposure has applicability to poultry production since AFB1 can be maternally transferred into eggs. RNA-seq datasets from embryonic liver tissue in domestic (n = 24) and wild (n =15) turkeys were mapped to a MAKER turkey gene set. Differential expression and pathway analysis identified conserved effects on cell cycle regulators (like E3 ubiquitin-protein ligase Mdm2) and variable effects in genes encoding detoxifying and anti-oxidant enzymes (like glutathione S-transferases) in domestic and wild turkeys. Overall, transcriptome analysis identified hepatic and splenic responses to AFB1, evaluated the use of probiotics, directly compared domestic and wild turkeys, and provided gene targets for future investigation of the molecular mechanisms of aflatoxicosis.
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    Salmonella’s Desiccation Survival and Thermal Tolerance: Genetic, Physiological, and Metabolic Factors
    (2017-07) Maserati, Alice
    Salmonella can survive for long periods under extreme desiccation and low water activity conditions (aw < 0.6) while becoming tolerant to heat. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular processes involved in this adaptation mechanism and its potential for cross-protection. This dissertation consists of three distinct studies focused on elucidating this mechanism. The objective of the first study was to identify the genes involved in Salmonella’s resistance to desiccation. A global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low aw (aw 0.11) and cells equilibrated to high aw (aw 1.0) determined that 719 genes (16% of the total number of genes in the genome) were differentially expressed between the two conditions. The genes that were up-regulated at aw 0.11 (290) were mostly involved in metabolic pathways, DNA replication/repair, regulation of transcription and translation, and virulence. Based on the transcriptomic analysis, we created deletion mutants for two virulence genes, sseD and sopD, and tested their ability to survive desiccation and low aw on glass beads. The two mutants exhibited significant cell viability reductions after desiccation compared to the wild-type and additional decrease after exposure to aw 0.11 for 7 days. Under scanning electron microscopy, the mutants displayed a different cell morphology and extracellular matrix production when compared to the wild-type under the same conditions. The findings of this study suggested that sopD and sseD are required for Salmonella’s survival during desiccation. The objective of the second study was to determine the effect of food and inert matrices, nutrient availability, and growth conditions on desiccation survival and thermal tolerance of S. enterica serovar Typhimurium. Salmonella was grown in LBglc and M9 media, in the presence or absence of EDTA and dipyridyl. Cultures were inoculated on toasted oat cereal (TOC) or glass beads, dried, and equilibrated for a week at aw 0.11 and 1.0, before being thermally treated at 75, 85, 90, and 95oC. For all growth conditions and temperatures tested, cells exposed to aw 0.11 had inactivation rates (δ-values) at least 10-fold longer than cells equilibrated at aw 1.0. Our results showed that growth in the presence of EDTA or Dipyridyl did not have any effect on Salmonella’s thermal tolerance at either aw on TOC. In control conditions, recovery after drying and thermal tolerance was higher on TOC than on glass beads, suggesting that the food matrix was protective for desiccation and thermal treatment. Growth in M9 resulted in lower survival to drying and exposure to low aw on glass beads, compared to LBglc. On the contrary, thermal tolerance increased in cells grown in M9 compared to LBglc at both aw. Cells grown in LBglc and M9 displayed differences in the production of extracellular matrix, in particular during equilibration to aw 0.11 and after thermal treatment at both aw. Additionally, when Salmonella was grown on glass beads in LBglc as biofilm, the thermal tolerance was greater than free cells dried on beads. Our observations suggest that the presence of nutrients during growth and before exposure to desiccation and thermal treatment influenced Salmonella’s ability to survive desiccation and develop thermal tolerance. The objective of the third study was to identify proteins involved in Salmonella’s resistance to desiccation and thermal treatment using iTRAQ. Proteins were extracted from S. enterica servorar Typhimurium cells dried, equilibrated at high aw (1.0) and low aw (0.11), and thermally treated at 75°C. Our analysis determined that 734 proteins were differentially expressed among samples, and of these 175 proteins were the most significant in determining differences in the proteomic profiles among treatments. Based on their proteomic expression profiles, the samples were clustered in two main groups by PCA analysis, “dry” samples and “wet” samples, while we did not observe significant differences between the thermally treated samples and the non-heated samples, at both aw. Protein profiles indicated shifts in cell metabolism in both samples, as well as a strict regulation of DNA repair, replication, transcription, and translation. “Dry” samples had higher levels of 50S and 30S ribosomal proteins, indicating that ribosomal proteins might be important for extra-ribosomal regulation of cellular response even when the synthesis of proteins is slowed down. Stress response proteins were more frequently present in “wet” samples compared to “dry” samples, including SspA, GorA, and Dps, suggesting that “wet” cells were activating stress systems in response to rehydration. In conclusion, our study indicated that pre-adaptation to dry conditions was linked to increased thermal tolerance, while reversion from a dry state into a wet state implied a significant change in protein expression that is linked with reduced thermal tolerance.
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    Transcriptome analysis of wheat and barley near-isogenic line pairs carrying contrasting alleles for different Fusarium head blight resistance QTLs.
    (2009-06) Jia, Haiyan
    Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is a major disease problem on wheat and barley around the world. Grain yield and quality are reduced due to infection and the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). Previously, quantitative trait loci (QTL) conferring FHB resistance and reduced DON accumulation have been identified on wheat and barley chromosomes. A major FHB resistance QTL (Fhb1) was identified on wheat chromosome 3BS. Two major QTLs associated with reduced FHB severity have been detected on barley chromosome 2H Bin 8 and 2H Bin 10, which are associated with heading date and spike type, respectively. A QTL associated with reduced deoxynivalenol (DON) accumulation was identified on chromosome 3H Bin 6. Near-isogenic line (NIL) pairs carrying the resistant and susceptible allele for a wheat QTL and three barley QTL were used to examine DON concentration, fungal biomass and transcript accumulation during F. graminearum infection. Based on the disease phenotypes, transcript accumulation data, and comparative analysis of the wheat and barley host response to F. graminearum infection, we developed an integrated model for the wheat and barley-F. graminearum interactions. In addition, gene transcripts that are differentially expressed in the NIL pairs during infection provide a set of genes that may play a role in FHB resistance.