Cell Biology & Molecular Genetics Theses and Dissertations
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- ItemGenome-wide identification and analysis of imprinted genes in strawberry seed development(2022) Joldersma, Dirk; Liu, Zhongchi Anne; Taneyhill, Lisa Anne; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The activation of zygotic gene expression is of fundamental importance to reproductive biology, but its regulation remains poorly understood. Within Angiosperm plants, fertilization occurs simultaneously in two locations, the embryo and its genetic twin, the endosperm, a nutritive tissue that is a defining feature of Angiosperm reproduction. Auxin hormone synthesized in the endosperm is essential to seed and fruit development. In the diploid strawberry Fragaria vesca, that auxin synthesis is regulated by FveAGL62, which is expressed specifically after fertilization in endosperm. How fertilization activates FveAGL62 expression in the endosperm, however, is presently unknown. I investigated the hypothesis that epigenetically regulated maternally- and paternally expressed genes (MEGs and PEGs, and together, “imprinted genes”) regulate the expression of FveAGL62. I hybridized two F. vesca accessions, isolated the endosperm from the F1 seeds, and sequenced the transcriptome of the F1 endosperm—a result facilitated by strawberry’s uniquely accessible seed. To identify imprinted genes within the endosperm, I assembled and annotated the genome of the maternal parent, F. vesca accession “Yellow Wonder” (FvYW5AF7), a model for the commercial strawberry. The paternal parent genome was obtained from a collaborator. 809 PEGs and 825 MEGs were identified from RNA sequencing reads that align uniquely to the maternal or paternal genome. MEGs are enriched in genes catabolizing auxin and hence limit seed growth, while PEGs are enriched in genes involved in histone modification, thereby promoting cell differentiation and seed growth. The distinct roles of MEGs and PEGs supports and can be explained by parental conflict and kinship theories, which predict a maternal genome tends to restrict progeny consumption of maternal resources, while a paternal genome will encourage such consumption. In contrast to findings in other species, I find that the endosperm-specific auxin biosynthetic gene FveYUC10 is maternally expressed, but while its imprinting status has changed, it may still function as a fertilization sensor. The maternally expressed gene FveMYB98 contains a binding domain that targets motifs present in FveAGL62’s promoter and its homolog binds AtAGL62 promoter in Arabidopsis. With collaborators, I showed that overexpression and CRISPR knockout of FveMYB98 changes seed size. Transient expression, yeast one hybrid and quantitative PCR analyses suggest that FveMYB98 represses FveYUC10 expression directly and FveAGL62 expression indirectly. These results suggest that FveMYB98 expression is a vehicle for maternal regulation of the level of auxin in the endosperm and thereby endosperm proliferation and seed size. My dissertation research has produced a new genome assembly of a model strawberry, a transcriptome of strawberry endosperm, and identified imprinted genes at genomic scale. I find FveMYB98 regulates seed size—a function echoed broadly within MEG and PEG classes—providing supporting evidence for the parental conflict theory within the developing progeny. These results improve our understanding of zygotic expression in developing seeds, addressing a fundamental scientific gap and, more tangibly, may enable future production of fertilization-independent seeds and seedless fruits.
- ItemIDENTIFICATION AND ANALYSIS OF GENETIC VARIANTS PERMITTING DISSEMINATED COCCIDIOIDOMYCOSIS(2022) Hsu, Amy Pepper; Mosser, David M; Holland, Steven M; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the Southwestern United States and Mexico. While the majority of those infected have minor symptoms or remain asymptomatic, illness requiring medical attention occurs in approximately 30%, with <1% developing extrapulmonary dissemination. To address why some individuals allow dissemination, we performed whole-exome sequencing on an exploratory cohort of 67 DCM patients. Using standard genetic analysis for identification of novel or rare Mendelian mutations only two patients were identified, both with STAT3 premature termination codons causing haploinsufficiency. Since Coccidioides are geographically isolated, I explored the possibility that dissemination could be a combination of more common genetic variants plus exposure. Defects in sensing and response to -glucan, the major component of Coccidioides cell wall, were seen in 34/67 (50.7%) cases. Damaging variants in CLEC7A, encoding DECTIN-1, (n=14) and PLCG2 (n=11) were associated with impaired production of -glucan-stimulated TNF from peripheral blood mononuclear cells compared to healthy controls (P<0.005). Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were over-represented in DCM (P=0.0206, P=0.015, respectively) including CLEC7A Y238* (P=0.0105) and PLCG2 R268W (P=0.0025). A validation cohort of 111 DCM patients confirmed over-representation of the specific variants, PLCG2 R268W (P=0.0276), CLEC7A I223S (P=0.044), and CLEC7A Y238* (P=0.0656). Lastly, I identified a novel pathway of pulmonary-epithelial fungal recognition by DECTIN-1 leading to activation of the NADPH oxidase complex, DUOX1/DUOXA1. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived H2O2 in transfected cells. Heterozygous DUOX1 or DUOXA1 variants which impaired H2O2 production were overrepresented in discovery and validation cohorts. Together these studies highlight the importance of fungal recognition and response for control of infections. Patients with DCM have impaired -glucan sensing or response affecting TNF and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.
- ItemA MULTI-OMICS APPROACH TO CHARACTERIZING THREE HEALTH RELEVANT FUNCTIONS OF THE HUMAN GUT MICROBIOME(2022) Braccia, Domenick James; Hall, Brantley; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The human gut is home to trillions of microorganisms that routinely interact with their human host in both beneficial and detrimental ways. The advent of next-generation sequencing and high-throughput “omics” technologies has created new opportunities to examine the role that the human gut microbiome plays on human health, especially in regard to gastrointestinal diseases such as Inflammatory Bowel Disease and colorectal cancer. In my dissertation, I utilize genomic, transcriptomic, metabolomic, and protein sequence datasets to characterize three health-relevant functions of the human gut microbiome. First, I performed a multi-omic, bioinformatic analysis to identify the bacterial enzyme, bilirubin reductase. While bilirubin reduction to urobilinogen and stercobilinogen is a well-known function of the human gut microbiome, the enzyme(s) responsible for the conversion of bilirubin to non-toxic reduced products have yet to be fully characterized. In this chapter, I review how I leveraged publicly available metabolomic, metagenomic, and metatranscriptomic data to explore over 2 million putative reductase genes and identify a candidate operon encoding bilirubin reductase. Second, I examined sources of microbial hydrogen sulfide (H2S) production by bacteria of the human gut microbiome. H2S is a sulfuric gas produced by various bacterial phyla of the human gut microbiome and is implicated in the etiology of gastrointestinal diseases such as Inflammatory Bowel Disease and colorectal cancer. In this chapter, I show via bioinformatic analysis that the capacity to produce H2S via cysteine degradation is ubiquitous in the human gut. Third, I explored bacterial prodrug activation required for the activation of immune system modulators such as sulfasalazine. After curating amino acid sequences of known azoreducing genes and performing a protein sequence search across the Unified Human Gastrointestinal Genomes (UHGG) collection containing 4,644 genomes, I identified putative azoreducing and non-azoreducing bacterial strains to be experimentally validated. Together, these results highlight a successful mult-omic approach to characterizing three diverse but health-relevant functions of the human gut microbiome.
- ItemINVESTIGATING MOLECULAR MECHANISMS SPECIFYING DIVERSE ROSACEAE FRUIT TYPES THROUGH COMPARATIVE TRANSCRIPTOMIC ANALYSIS(2022) Li, Muzi; Liu, Zhongchi; Mount, Stephen; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rosaceae is a plant family with over 3,000 species including a number of economically important fruit-bearing species. Although plants in Rosaceae family have similar basic flower structure, their fruit flesh comes from distinct floral tissues. In drupe fruit, such as peach and plum, the ovary wall becomes enlarged and fleshy. In pome fruit, such as apple and pear, the fruit fleshy is mainly derived from the hypanthium that encases the ovary. In drupetum fruit, such as raspberry, numerous unfused ovaries each grow into a fleshy drupelet. In achenetum fruit, such as strawberry, the numerous unfused ovaries eventually dry up, but the receptacle, the stem tip that supports these ovaries, instead develops into the fruit flesh. By investigating and comparing the transcriptomes from these four Rosaceae fruits, peach (Prunus persica), apple (Malus x domestica), strawberry (Fragaria vesca), and raspberry (Rubus idaeus), at the earliest stages of fruit development, we gain important insights into the genetic mechanisms underlying fleshy fruit diversity. The expression of B class MADS-box genes, PISTILLATA, APETALA3 and TM6, shows negative correlation with the ability to form fleshy fruit tissues. Based on RNA transcript and phylogenetic analysis, FBP9, a MADS-box gene related to the E class, appears to be necessary but insufficient for flesh formation. In addition to the regulatory roles MADS-box genes play in fruit identity specification, extensive lignification of the strawberry ovary wall may contribute to the inability of strawberry ovary to become fleshy. Finally, a database (ROsaceae Fruit Transcriptome database, ROFT) is established for researchers to query for orthologous genes and their expression patterns during fruit development in the four species as well as to query for the tissue-specific and tissue- and stage-specific genes. Together, these findings provide the framework for functional investigations of fruit type specification and insights into the evolution of diverse fruit types in the Rosaceae family. The knowledge gained will advance our understanding in the evolution of fleshy fruits, a defining feature of angiosperm, and enable the creation of new fruit types for consumers.
- ItemA SYNTHETIC TMRNA PLATFORM FOR ELUCIDATION OF BACTERIAL PROTEOME REMODELING UNDER STRESS(2022) Turner, Randi; McIver, Kevin; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Translational reprogramming is a key component of the bacterial stress response and is a function of mRNA stability, protein turnover and proteolysis. Total proteome measurements give a view of the stable proteome but can fail to capture dynamic changes under stress, including incomplete polypeptides that result from cleaved mRNAs or stalled translation events. Bacteria employ a nearly ubiquitous native ribosome rescue system, transfer-messenger RNA (tmRNA), that rapidly resolves stalled translational complexes and tags the incomplete polypeptides for degradation. Characterization of these tmRNA-tagged polypeptides could reveal previously unknown aspects of the bacterial stress response. To address this information gap, we have developed a synthetic tmRNA platform that reprograms the native system to allow for co-translational labeling of the incomplete polypeptides in live bacteria. A short tag reading frame (TRF) encoded on native tmRNA facilitates the addition of a natural peptidyl degradation tag to the polypeptides, and therefore offers an attractive modular domain to introduce synthetic peptide tag sequences and study the “degradome”. To study translational remodeling under stress, we modified the native tmRNA with an 6x-HIS isolation tag with the specific purpose of stabilizing, isolating, and characterizing the degradome in Escherichia coli. Using our inducible system, we have successfully isolated 6xHis-tagged proteins, verified dynamic controlled tagging, assessed broad-spectrum tag introduction with mass spectrometry. Our results capture known tmRNA substrates and excitingly show that tagged protein profiles are markedly different under stress. We investigated the shifting degradome in cells experiencing translational stress associated with serine starvation induced by serine hydroxamate. In cells lacking RelE, the mRNA interferease toxin that cleaves mRNA in the ribosome A site, we find a dramatic shift away from catalytic protein degradation and distinct, disparate enrichment of ribosomal proteins in the degradome under stress. These latter results suggest a new specific role for RelE in regulating ribosome protein abundance under translational stress conditions