Biochemistry Department Masters Theses Collection

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  • Publication
    Comparison of CcrM-dependent Methylation in Caulobacter Crescentus and Brucella Abortus through Nanopore Sequencing
    (2024-05) Campbell, Maxwell
    The gene ccrM, encoding a cell cycle-regulated methyltransferase, which is well conserved among Alphaproteobacteria and plays a crucial role in regulating gene expression in the alphaproteobacterium Caulobacter crescentus. However, it remains uncertain whether CcrM-dependent methylation plays a similar role in other members of the Alphaproteobacteria, such as Brucella abortus. To explore the both the conservation and regulation of CcrM-dependent methylation in Alphaproteobacteria, Nanopore sequencing in tandem with recent machine-learning based algorithms were utilized to explore the methylomes of both Caulobacter crescentus and Brucella abortusunder a variety of genetic conditions. In Caulobacter crescentus, the results for wild-type demonstrating a strong correlation between Nanopore-based detection and previously published results. With confirmation of Nanopore’s validity, the study then measures the impact of Lon-mediated CcrM degradation on the epigenome, confirming that the deletion of lon leads to widespread changes in methylation. Whereas the removal of alkB, a potential demethylase, did not yield significant changes in global methylation during normal growth. Applying a similar pipeline to Brucella abortus led to the first report of global DNA methylation dynamics in this species and revealed that CcrM-dependent methylation was conserved among the two Alphaproteobacteria. In contrast to Caulobacter, the removal of lon in Brucella exhibited both species and chromosome specific effects on the genome. Finally, the study examined a ∆mucR strain in Brucella, revealing that, akin to recent findings in Caulobacter, the transcription factor MucR plays a conserved role in protecting methylation sites from CcrM activity.
  • Publication
    Proteolytic Regulation of CtrA, the Master Regulator of Cell Cycle in Caulobacter crescentus
    (2012-09) Cantin, Amber M.
    Cell cycle progression in Caulobacter crescentus depends on the master regulator, CtrA. During the transition from swarmer to stalk cell (G1 to S phase), CtrA is degraded by the AAA+ protease ClpXP and levels rise again in the predivisional stage. The focus of this work is to explore how cyclic, regulated degradation is controlled. CtrA is known to bind to the origin of replication, thereby suppressing replication, so we first asked if DNA binding had an effect on CtrA stability. CtrA is readily degraded by ClpXP on its own, but when bound to DNA containing the proper binding sites, degradation is inhibited. Stabilization is dependent on DNA binding, as CtrA mutants deficient in DNA binding show the same degradation regardless of addition of DNA, as does CtrA in the presence of a mutant origin sequence lacking CtrA binding sites. Looking closely at CtrA degradation in the presence of auxiliary factors suggests that higher order complex formation may be a mechanism of protecting critical cell cycle regulators from premature proteolysis. In vivo study of over-expression of CtrA mutants revealed that accumulation of non-degradable CtrA, CtrA-DD, perturbs the cell cycle, leading to filamentation and a G2 arrest. Over-expression of the DNA-binding domain alone showed filamentation but no G2 arrest, suggesting that CtrA-DD is detrimental for reasons including, but likely not limited to, its ability to bind DNA. Exogenously expressing other domains of CtrA may further elucidate the mechanism of its regulated degradation in vivo.
  • Publication
    Probing the Peptidyl Transferase Center of Ribosomes Containing Mutant 23s rRNA with Photoreactive tRNA
    (2008-02) Caci, Nicole C
    There is strong crystallographic evidence that the 23S rRNA is the only catalytic entity in the peptidyl transferase center. Various mechanisms for the catalysis of peptidyl transfer have been proposed. Recently, attention has been given to the idea that the 23S rRNA simply acts to position the tRNA for spontaneous peptidyl transfer and that chemical catalysis may play only a secondary role. Conserved nucleotides U2585 and U2506 are thought to be involved in positioning the 3’ ends of A- and P-site substrates based on the crystallographic evidence, and because mutagenesis at these sites severely impairs peptide bond formation. In this study, pure populations of ribosomes with either U2585A or U2506G mutations in the 23S rRNA were analyzed to test the hypothesis that substitutions at nucleotides U2585 and U2506 in the peptidyl transferase center impair peptide bond formation by altering the position of the 3’ end of P-site tRNA relative to the 23S rRNA. Pure populations of mutant or wild-type ribosomes were obtained by an affinity tagging system and probed with 32P-labeled [2N3A76]tRNAPhe to determine how the 3’ end of tRNA interacts with the ribosomal proteins and 23S RNA at the peptidyl transferase center. Some of the data for the ribosomes with a G at position 2506 are consistent with a model suggested by Schmeing and coworkers in which nucleotide U2506 breaks from its original wobble base pair with nucleotide G2583 during A-site tRNA binding and swings towards the 3’ end of P-site tRNA, while nucleotide U2585 simultaneously moves away from the 3’ end of P-site tRNA.
  • Publication
    Novel Adaptor-Dependent Domains Promote Processive Degradation by ClpXP
    (2011-09) Rood, Keith L
    Protein degradation by ATP dependent proteases is a universally conserved process. Recognition of substrates by such proteases commonly occurs via direct interaction or with the aid of a regulatory adaptor protein. An example of this regulation is found in Caulobacter crescentus, where key regulatory proteins are proteolysed in a cell-cycle dependent fashion. Substrates include essential transcription factors, structural proteins, and second messenger metabolism components. In this study, we explore sequence and structural requirements for regulated adaptor mediated degradation of PdeA, an important regulator of cyclic-di-GMP levels. Robust degradation of PdeA is dependent on the response regulator CpdR in vivo and in vitro. Here, I structurally identify a novel PAS domain in PdeA that is necessary and sufficient for CpdR mediated PdeA degradation. The PAS domain was found to contain a unique dimerization element that is associated with PdeA function. I show specifically that PdeA engages ClpXP through C-terminal recognition motifs. Finally, we present evidence that PdeA contains cryptic ClpXP recognition sites that are revealed during partial processing. Due to these uncommon degradation characteristics of PdeA, unique proteolytic insights may be gained by investigating this model system.
  • Publication
    Pharmacological Chaperoning in Fabry Disease
    (2011-09) Rogich, Jerome
    Fabry Disease is an X-‐linked lysosomal storage disorder characterized by a variety of symptoms including hypohydrosis, seizures, cardiac abnormalities, skin lesions, and chronic pain. These symptoms stem from a lack of functional endogenous α-‐ Galactosidase A (α-GAL), which leads to an accrual of its natural substrate. The severity of the disease symptoms can be directly correlated with the amount of residual enzyme activity. It has been shown that an imino sugar, 1-deoxygalactonojirimycin (DGJ), can increase enzymatic activity and clear excess substrate. This pH-‐dependent chaperoning phenomenon is believed to arise from the presence of aspartic acid 170 in the active site. This key residue may become protonated at lower pH, preventing a buried salt bridge from being formed. We mutated this residue to an alanine, abolishing activity, and making traditional assays impractical. We have measured the KD of chaperone for this modified active site through crystallography. Previous crystallographic studies on this enzyme have also shown a preliminary second binding site on the surface of α-Galactosidase that prefers the β-Galactose anomer. When β-Galactose binds it buries a greater surface area than when α‐Galactose binds to the active site. Binding of this site by a small molecule should stabilize the native state of the enzyme, but would be sterically occluded from inhibiting active site. We have probed this second site by soaking crystals of α‐Galactosidase with a small library of compounds.
  • Publication
    Probing the Activation Mechanism of Transcription-Coupled Repair Factor Mfd
    (2010-09) Hsieh, Chih-heng
    Cells dedicate tremendous amounts of energy to express essential genes for survival. During transcription, RNA polymerase (RNAP) actively scans the template strand of DNA, stalling when it meets DNA damages. Stalled RNAP prevents repair by the nucleotide excision repair pathway (NER); a sub-pathway of NER named transcription-coupled repair (TCR) resolve this problem by removing RNAP and recruiting repair proteins. In Escherichia coli, a TCR protein named “Mutation Frequency Decline” (Mfd) couples removal of RNAP through its motor activity with recruitment of the NER repair proteins. Mfd can be divided into two functional halves; the N-terminal region (MfdN, domains 1-3) is essential for NER protein recruitment, and the C-terminal region (MfdC, domains 4-7) is responsible for RNAP-interaction and motor activity. Data suggest Mfd undergoes large conformational movement to activate RNAP removal and repair protein recruitment. To study the activation mechanism of Mfd, we created several full-length “hyperactive” mutants by perturbing interactions between MfdN and MfdC. In all mutants, residue 79 in domain 1 is changed from aspartic acid to arginine (D79R), disrupting a key salt bridge interaction with arginine 804 in domain 6. The linker connecting MfdN and MfdC was made cleavable to allow separation of MfdN and MfdC, which enable us to study activities in equal molar concentration. We have studied the effect of the D79R mutation in vivo (cytotoxicity and UV sensitivity) and in vitro (enzyme activity and thermal stability), and demonstrate that this single residues change render the enzyme “hyperactive”. This confirms our model of activation: activation of Mfd results from breaking communication between MfdN and MfdC
  • Publication
    Dopamine Controls Locomotion by Modulating the Activity of the Cholinergic Motor Neurons in C. elegans
    (2009-05) Allen, Andrew T
    Dopamine is an important neurotransmitter in the brain, where it plays a regulatory role in the coordination of movement and cognition by acting through two classes of G protein-coupled receptors to modulate synaptic activity. In addition, it has been shown these two receptor classes can exhibit synergistic or antagonistic effects on neurotransmission. However, while the pharmacology of the mammalian dopamine receptors have been characterized in some detail, less is known about the molecular pathways that act downstream of the receptors. As in mammals, the soil nematode Caenorhabditis elegans uses two classes of dopamine receptors to control neural activity and thus can serve as a genetic tool to identify the molecular mechanisms through which dopamine receptors exert their effects on neurotransmission. To identify novel components of mammalian dopamine signaling pathways, we conducted a genetic screen for C. elegans mutants defective in exogenous dopamine response. We screened 31,000 mutagenized haploid genomes and recovered seven mutants. Five of these mutants were in previously-identified dopamine signaling genes, including those encoding the Ga proteins GOA-1 (ortholog of human Gao) and EGL-30 (ortholog of human Gaq), the diacylglycerol kinase DGK-1 (ortholog of human DGK0), and the dopamine receptor DOP-3 (ortholog of human D2-like receptor). In addition to these known components, we identified mutations in the glutamate-gated cation channel subunit GLR-1 (ortholog of human AMPA receptor subunits) and the class A acetycholinesterase ACE-1 (ortholog of human acetylcholinesterase). Behavioral analysis of these mutants demonstrates that dopamine signaling controls acetylcholine release by modulating the excitability of the cholinergic motor neurons in C. elegans through two antagonistic dopamine receptor signaling pathways, and that this antagonism occurs within a single cell. In addition, a mutation in the putative Rab GTPase activating protein TBC-4 was identified, which may suggest a role for this Rab GAP in synaptic vesicle trafficking. Subsequent behavioral and genetic analyses of mutants in synaptic vesicular trafficking components implicate RAB-3-mediated vesicular trafficking in DOP-3 receptor signaling. These results together suggest a possible mechanism for the regulation of dopamine receptor signaling by vesicular trafficking components in the cholinergic motor neurons of C. elegans.
  • Publication
    Probing for Conformational Changes in the Repair Enzyme Mfd Using Mutant Protein Constructs
    (2008-05) Hunnewell, Mary E.
    DNA repair is essential for survival, as damage to the genome can interrupt the precarious balance of cell functions, causing further mutations and possibly leading to cancer. The bacterial transcription repair coupling factor, Mfd, is capable of recognizing a stalled RNA polymerase at a site of DNA damage. The Mfd works both to remove the RNA polymerase through its motor function (utilizing the energy of ATP to translocate along DNA), and to recruit the DNA repair complex UvrA/B/C. To study conformational changes in the protein, we are creating multiple mutants of the full length Mfd protein. My approach is to use a cleavable mutant of full-length Mfd as a template for further mutations. This will allow us to probe for conformational changes by changing interactions at the interface of the two halves of Mfd, and then using the ability to cut with TEV protease as a sensor to identify and characterize the open state of the protein. By introducing this TEV protease cut site at residue 450 in the protein linker region between the N (amino-) and C (carboxy-) terminal domains, we can then assess the conformational changes Mfd must undergo to obtain activity. We can study the effect of further mutations on the full length and cut versions of the protein. Another approach attempted in this study involves using cysteine modification of the full length Mfd protein as a sensor for these conformational changes. Mfd acts as a model system for studying the DNA repair mechanisms found in humans, and the elucidation of functional and conformational changes in Mfd contributes to studying disease phenotypes resulting from aberrant transcription coupled repair.
  • Publication
    Investigation of the Effect of Dimerization on Human α-Galactosidase Activity
    (2014-02) Dooley, Scott R
    Fabry disease is an X-linked lysosomal storage disease that results from a deficiency in the enzyme α-galactosidase (α-GAL). α-GAL hydrolyzes α-galactosides, and patients with Fabry disease suffer from an accumulation of these undegraded substrates. Human α-GAL naturally occurs as a homodimer, as determined through SEC and crystallographic analysis. This means its quaternary structure consists of two identical α-GAL subunits that are associated together into a single unit. Other species, such as rice, produce a monomeric form of α-GAL, consisting of only a single subunit. If α-GAL is functional as both a homodimer and monomer, then how does homodimerization affect the activity of human α-GAL? This can be answered through two model systems. First, a monomeric form of human α-GAL can be produced, testing the activity of human α-GAL in a monomeric state. A variant of α-GAL was engineered (called α-GALF273G/W277G) that appeared promising. Secondly, another system can be produced capable of stabilizing one active site of the dimer and testing the other active site for activity. Another lysosomal enzyme, α-N-acetylgalactosaminidase (α-NAGAL), shares 46% amino acid sequence identity and share 11 of 13 active site residues. Previously, an α-GAL variant (called α-GALE203S/L206A) was produced, that maintained the antigenicity of α-GAL, but had acquired the enzymatic specificity of α-N-acetylgalactosaminidase (α-NAGAL). A heterodimeric form of α-GAL can be produced combining one subunit of α-GAL with the engineered variant. The engineered site can be stabilized, while the wild-type site can be tested for activity. SEC analysis suggests α-GALF273G/W277G is a monomer, and its kinetic properties are reported. Evidence shows monomeric α-GAL could be useful as an improved enzyme replacement therapy. Western blotting and activity assays suggest the presence of the α-GAL/ α-GALE203S/L206A heterodimer.
  • Publication
    Assessing Stress Tolerance of Organelle Small Heat Shock Protein Mutants in Arabidopsis Thaliana
    (2020-09) Patel, Parth
    Molecular chaperones are proteins found in virtually every organism and are essential to cell survival. When plants are heat stressed, they upregulate and downregulate multiple genes, many of which are associated with the heat shock response. Small heat shock proteins (sHSPs) are one class of molecular chaperones that are upregulated during heat shock. They are proposed to act as the first line of defense by binding to heat sensitive proteins and preventing their irreversible aggregation. However, many details of sHSP function remain to be discovered and exactly what proteins they protect is unresolved. In addition to cytosolic sHSPs found in other organisms, plants also produce sHSPs that are targeted to organelles. In this study, I focus on the mitochondria and chloroplast localizing sHSPs: HSP23.5-MTI/CP, HSP23.6-MTI/CP, HSP25.3-CP, and HSP26.5-MTII in Arabidopsis thaliana. The heat tolerance of knockout mutants of these different organelle-localized sHSPs, including single, double, triple, and quadruple knockouts was assessed through various stress assays. A hypocotyl elongation assay indicated a mild heat sensitive phenotype for many of the sHSP knockout mutants and plants lacking all four sHSPs showed the greatest reduction in hypocotyl elongation following heat stress. In an vi assay with light grown seedlings, I observed plants that lacked the chloroplast-localizing HSP25.3-CP were sensitive to acute heat stress. In stress assays involving arsenic, plants that did not express mitochondrial sHSPs were the most sensitive to excess arsenic. Interestingly, plants lacking the four sHSPs were more resistant to salt and cadmium stress. The phenotypes of these sHSPs will bring us closer to defining their mechanism of action during heat or heavy metal stress and the mutants will provide a platform for further studies of sHSP structure and function.
  • Publication
    Predicting Successful Chaperoning of Fabry Disease Mutants via Computation
    (2019-09) Patel, Priyank
    Fabry disease is an inherited X-linked recessive disorder caused by mutations in the galactosidase alpha (GLA) gene, leading to deficiencies in α-galactosidase A (α-GAL) enzyme production. α-GAL, a lysosomal glycosidase, catalyzes the removal of a terminal α-galactose; however, loss of α-GAL activity leads to accumulation of globotriaosylceramide (an endogenous substrate) and the eventual onset of the disease. Approved treatments for Fabry disease include enzyme replacement therapy and pharmacological chaperone therapy. In the latter treatment, 1-deoxygalactonojirimycin (DGJ), a pharmacological chaperone, is administered to Fabry disease patients, leading to increased enzymatic activity. The DGJ iminosugar acts as a competitive inhibitor of α-GAL, and upon addition at sub-inhibitory concentrations, the α-GAL activity in the cell increases. At pH 7.5, the DGJ binds and stabilizes both wild type and mutant α-GAL and can thus drive the folding of the α-GAL protein (Guce 2011). DGJ has been clinically approved to treat a subset of the more than 900 known mutations in the GLA gene. These approvals come from the chaperone activity data published by Amicus Therapeutics (Benjamin 2017). However, these assays cost money, time, and effort to perform, and novel mutations are discovered annually. Using molecular dynamics energy calculations in the Schrödinger software package, we developed a model to predict successful chaperoning of the mutants. Overall, the results are directly applicable to Fabry disease, but could also be applied to the much larger family of protein folding diseases, including Alzheimer's, Parkinson's and Huntington's diseases.
  • Publication
    Analyzing the Biochemical and Functional Interactions of the RALF1-FERONIA-LLG1 (a peptide ligand-receptor kinase-GPI-anchored protein complex) Signaling Pathways in Arabidopsis thaliana
    (2019-05) Jordan, Samuel
    Signal transduction pathways play a critical role in plant growth and reproduction by perceiving extracellular signals, leading to a cellular response. FERONIA (FER) is a transmembrane receptor kinase found on the plasma membrane in the model plant Arabidopsis thaliana and plays critical roles in growth, development, and fertilization. FER works upstream of master molecular switch RAC/ROP GTPase to regulate signaling into the cytoplasm. LORELEI-Like Glycosylphosphatidylinositol (GPI)-Anchored Protein 1(LLG1) is a GPI-anchored protein and co-receptor of FER on the plasma membrane. LLG1 is responsible for chaperoning FER from the endoplasmic reticulum (ER) to its functional location on the plasma membrane. Rapid Alkalinization Factor 1 (RALF1) is a small, secreted growth-regulatory peptide that interacts with FER, regulating signaling activity. This interaction, among other, regulates the activity of a downstream plasma membrane proton ATPase (AHA2) which impacts cell growth. Additionally, published pulldown data indicates LLG1, FER, and RALF1 complex together. My data suggests that LLG1, in addition to localizing and chaperoning FER, binds directly to RALF1. My results show that this RALF1-LLG1 interaction is required for proper RALF1 mediated signaling through FER. Data also indicates that FER and LLG1 regulate RALF1 location on the plasma membrane. Additionally, RALF1 binds the MALA domain of FER. Another aspect of my thesis focuses on LURE1. LURE1 is a secreted cysteine-rich, defensin like protein which guides incoming pollen tubes to the ovule in a process called pollen tube guidance. LURE1 guides pollen tubes by binding with pollen-specific receptor kinase 6 (PRK6), located on the plasma membrane of the incoming pollen tubes, to facilitate proper fertilization. My data also shows that the ovule derived signaling molecule nitric oxide (NO), also regulated by FER, negatively impacts the property of LURE1, causing it to fall out of solution and aggregate. Furthermore, the negative impact of NO on LURE1 disrupts the binding affinity of LURE1 to PRK6. Together with data from my lab showing pollen tube arrival at the ovule triggers NO production in a FER dependent manner, my findings provide a biochemical explanation for why pollen tubes do not target fertilized ovules.
  • Publication
    FERONIA-RELATED RECEPTOR KINASE 7 AND FERONIA AND THEIR ROLE IN RECEIVING AND TRANSDUCING SIGNALS
    (2018-09) Vyshedsky, David
    Receptor kinases (RKs) are transmembrane proteins that have been shown to regulate an array of important processes in A. thaliana, including polar cell growth, plant reproduction, and many other plant growth processes. In this thesis, I examine RECEPTOR KINASE 7 (RK7) and FERONIA (FER), two closely related transmembrane RKs, and their effects on plant reproduction. The RK7 gene when knocked out (rk7) in conjunction with FER resulted in delayed plant growth, decreased seed yield, and a lower percentage of the seeds germinating as compared to the single FER knockout. Transgenic plants with GUS reporter driven by RK7 promoter and RK7 promoter expressed GFP-tagged RK7 (RK7-GFP) were generated to study, respectively, the expression property of the RK7 gene and characterize the location of the RK7 protein. RK7 expression increased in the papillary cells as a direct result of pollination. Transgenic plants with RK7-GFP showed that RK7 protein localizes to the plasma membrane of stigma cells and pollination induces prominent internalization of this protein. RK7 is also expressed during seedling growth. rk7 mutant seedlings had a much weaker physiological response to brassinosteroids than wild type plants, implicating an involvement of RK7 in brassinosteroid signaling. Taken together this data point to the importance of RK7 in plant growth and reproduction through its ability to receive and transduce signals.
  • Publication
    In Vitro S-Glutathionylation of S-Nitrosoglutathione Reductase from Arabidopsis Thaliana and Phenotype Determination of Sensitive to Formaldehyde 1 Knockout Strains of Saccharomyces Cerevisiae
    (2018-02) Truebridge, Ian
    Cells are constantly exposed to different stresses – one being redox stress, which is induced by metal, reactive oxygen species and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR) helps modulate redox stress by two different mechanisms – either by reducing S-nitrosoglutathione (GSNO) to oxidized glutathione (GSSG) or by oxidizing hydroxymethyl glutathione (HMGSH), a biproduct of glutathione and formaldehyde, to formic acid. GSNO has the potential to posttranslational modify proteins in two different manners, either by S-nitrosation or by S-glutathionylation. Interestingly, GSNOR can be modified by its substrate GSNO, either by S-nitrosation, which has previously been reported, or, as discussed in this thesis, by S-glutathionylation. As S-glutathionylation has been reported to occur through intermediate species, the S-glutathionylation of GSNOR appears to occur though the S-nitrosated intermediate, instead of the most common route of an oxidation pathway. It is hypothesized that the S-glutathionylation, and the overall presence of glutathione, can act as a buffer to regulate the amount of nitrosation that GSNOR experiences, and thus the enzymatic activity. It is has reported that the S-nitrosation occurs on three different non-structural, non-catalytic, solvent-accessible cysteine residues. Experimentation was conducted using Saccharomyces cerevisiae as a model organism to determine how those three cysteine residues of the GSNOR homolog Sensitive to Formaldehyde 1 (SFA1) participate in the indirect detoxification of formaldehyde, through the hydroxymethyl glutathione pathway. It has been determined that cysteine 370 is not as important as previously thought, but the other one or two cysteines (either cysteine 10 or 271) do indeed play a role in the detoxification, but further analysis needs to be conducted.
  • Publication
    THE EVOLUTION OF THERMOTOLERANCE A CHARACTERIZATION OF A DIRECTIONALLY EVOLVED CYANOBACTERIUM
    (2015-09) Bopp, nathen Emil
    Chaperone proteins are essential components in the maintenance and turnover of the proteome. Many chaperones play integral functions in the folding and unfolding of cellular substrates under many conditions, including heat stress. Most chaperones can be characterized into two categories; the typical ATP dependent chaperones and the ATP independent chaperones. One ATP independent chaperone class it the Small Heat Shock Proteins (sHSPs), which as molecular life vests and are thought to protect misfolding proteins from irreversible aggregation. One such organism, the cyanobacterium Synechocystis sp. PCC 6803, is an excellent model for the study and understanding of these proteins and their functions in vivo. The genome of Synechocystis encodes only one sHSP, Hsp16.6, and it has be shown to be essential for acquired thermotolerance. Two mutant derivatives of Hsp16.6 with single amino acid substitutions in the N-terminal arm (L9P and E25K) have loss-of-function phenotypes similar to knock out strains, but each has very different biochemical properties. The mutant L9P has an inability to interact with putative substrates during heat stress in vivo, while the mutant E25K appears unable to release substrates. Using a directed evolution approach, suppressors have been isolated that recover the lost thermotolerance of their respective parent strains, either L9P (16 suppressors) or E25K (10 suppressors). Illumina sequencing and comparative genomics have been used to identify alterations in the genomes of the suppressor strains in order to define genetic circuits involved in thermotolerance.
  • Publication
    RNAi Mediated Silencing of Cell Wall Invertase Inhibitors to Increase Sucrose Allocation to Sink Tissues in Transgenic Camelina Sativa Engineered with a Carbon Concentrating Mechanism
    (2015-05) Zuber, Joshua
    Plant invertases are a class of proteins that have enzymatic function in cleaving sucrose to fructose and glucose. Cell wall invertase, located on the exterior of the cell wall of plant cells, plays a key role in the unloading of sucrose from the apoplast to the sink tissues. Cell wall invertase interacts with an inhibitor, cell wall invertase inhibitor, post-transcriptionally to regulate its activity. The inhibitor is constitutively expressed in pollen development, early developing seeds, and senescing leaves: indicative of sucrose allocation being a limiting factor at these stages of development. We introduced algal bicarbonate transporters LCIA/CCP1 to Camelina sativa for the purpose of increasing photosynthetic capacity. The bicarbonate transporters concentrate CO2 at RuBisCO by pumping CO2 in the form of bicarbonate through the membrane, then converting it back to CO2 at RuBisCO, increasing CO2 concentration. Results from these plants have shown an increase in seed number, but not seed mass, along with a faster rate of maturity and senescence. This is indicative of acclimation to high CO2 conditions, resulting from insertion of the bicarbonate transporters. RNA sequencing was performed and a putative invertase inhibitor was recognized as being expressed in the transgenic C. sativa but not in the wild type. Our strategy is to knock out two invertase inhibitors using induced RNA silencing, dramatically altering sucrose allocation into developing seeds and resulting in an increase in seed biomass. It is the aim of this research to increase the biomass of C. sativa seeds in order to increase its effectiveness as an agent to create sustainable biofuels.
  • Publication
    Investigation of SHOT1-binding ATPases in Arabidopsis thaliana
    (2020-09) Zelman, Sam
    Mitochondria play critical roles not only in primary metabolism as a central organelle for ATP generation, but also in responding to abiotic stresses. We identified a mutation in the MTERF18 (Mitochondrial Transcription Termination factor)/SHOT1 (Suppressor of hot1-4 1) gene in Arabidopsis thaliana that enables plants to better tolerate heat and oxidative stresses, presumably due to reduced oxidative damage, but the exact molecular mechanism of the heat tolerance is unknown. In order to reveal the stress tolerance mechanisms of mterf18/shot1 mutations, it is critical to understand the molecular defects of the mutant and to identify the molecular targets of the MTERF18/SHOT1 protein. MTERF18/SHOT1, a mitochondrial matrix protein, was found to bind to membrane-spanning mitochondrial AAA+ proteins homologous to ATAD3a of humans and other multicellular eukaryotes. A. thaliana has four ATAD3a homologues in two clades, and plants require one gene from each clade for viability. Previous studies of the topology and ATPase activity of ATAD3a suggest a role in endoplasmic reticulum (ER)-mitochondria contact sites. These sites are poorly defined in plants, and their relationship to heat stress tolerance is intriguing. To better understand ATAD3 function I expressed and purified the soluble, matrix-located, catalytic C-terminal ATPase domain of these proteins in order to assay their ATPase activity and oligomerization states. Transgenic plants with fluorescently labelled ER and mitochondria have been generated to observe effects of the MTERF18/SHOT1 mutation on ER-mitochondria dynamics. These studies of the four ATAD3 proteins will provide insights into ER-mitochondrial contact sites in plants, and into their link to MTERF18/SHOT1 and heat stress tolerance. I also provide a review of our current knowledge of ER-mitochondria contact site protein components in plants with reference to these proteins in A. thaliana.