Cell Biology & Molecular Genetics

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End date: 2009Subject: search.filters.subject.Biology, Genetics

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    Suppressors of etr1-2: I. etr1-11 is a loss-of-function mutation of the ETR1 ethylene receptor. II. REVERSION TO ETHYLENE-SENSITIVITY3 is a regulator of seedling growth.
    (2009) McClellan, Christopher Alan; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The plant hormone ethylene is an important regulator of plant growth and development, including senescence, abcission, fruit ripening, and responses to biotic and abiotic stresses. To find new members of the ethylene signaling pathway, a genetic screen for suppressors of the ethylene-insensitive mutant etr1-2 was performed. One mutant identified in this screen, etr1-11, is an intragenic mutation within ETR1. etr1-11 is a unique missense mutation that appears to eliminate ETR1-2 signaling. Mutant analysis further revealed that etr1-11 is a partial loss-of-function allele. The rte3 (reversion to ethylene sensitivity3) mutant was another mutant isolated in a genetic screen for suppressors of etr1-2. After testing other ethylene responses, such as leaf senescence, and performing epistasis analysis with other ethylene signaling mutants, it was determined that RTE3 is unlikely to play a direct role in the ethylene signaling pathway. Instead, RTE3 appears to be responsible for promoting hypocotyl elongation in etiolated seedlings in the ethylene triple response assay. The RTE3 gene was identified by positional cloning, and is predicted to encode a protein with an annotated SAC3/GANP domain. SAC3/GANP domains are present in proteins that participate in large multi-peptide complexes, such as the 26S proteasome regulatory subunit and the eIF3 translation initiation complex. Similarities in protein composition between these two complexes and the COP9 signalosome (CSN) suggest that a SAC3/GANP domain-containing protein may interact with members of the CSN. Interestingly, yeast two-hybrid analysis reveals that RTE3 interacts with EER5 and EIN2, proteins that have been shown to interact with members of the CSN. In addition, rte3-1 ein2-1 seedlings show a synthetic phenotype of delayed growth. Protein localization using a GFP tag reveals that RTE3 and EER5 both localize to the nucleus. These interactions suggest that RTE3, EER5, EIN2, and the CSN form a protein complex that regulates seedling growth.
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    EXPLORING THE ROLE OF NFκB HOMOLOGS IN AUTOPHAGIC CELL DEATH IN THE DROSOPHILA SALIVARY GLAND
    (2009) Ivory, Adrienne; Wu, Louisa P; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The innate immune response is an ancient, highly conserved means of defense against pathogens. An important mediator of innate immunity is the NF-κB (Nuclear Factor-Kappa B) family of transcription factors. Activation of immune-signaling pathways leads to the nuclear translocation of NFκB proteins which initiate the transcription of antimicrobial peptides (AMPs) that circulate and destroy microbes. In Drosophila, these AMPs are up-regulated during the destruction of larval salivary glands. Salivary gland cells are destroyed via autophagy during metamorphosis. This project sought to determine what, if any, role the NFκB transcription factors have in autophagic cell death. Using the Drosophila model, it was determined that a loss of AMP activity during metamorphosis results in a failure to completely degrade larval salivary glands, and this defect appears to be due to an inability to remove autophagic vacuoles. It is suggested that AMPs may serve to degrade the membranes of autophagic vacuoles.
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    Down Syndrome Cell Adhesion Molecule, Dscam Molecular Diversity Crucial for Survival in Drosophila Melanogaster
    (2008) Raghavan, Sangeetha; Pick, Dr. Leslie; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    There are 250,000 neurons and millions of synaptic connections in the fruit fly Drosophila Melanogaster. The molecular mechanism behind the precision and timing of these neural connections during development still eludes us. The Drosophila Down syndrome cell adhesion molecule or Dscam encodes 152,064 isoforms that are believed to be significant in regulating branching and targeting of neurites and, consequently in neuronal wiring and the viability of the organism. This study presents evidence that distinct set of Dscam isoform diversity is paramount to the survival of the organism. Single domain specific isoforms have been shown to rescue lethality caused by Dscam mutations up to the third instar larval stage (Wang, 2004). This study demonstrates that isoform specific single and multiple transgenes can rescue lethality caused by Dscam mutations up to the stage of adulthood with varying degrees of efficiency. The differences in rescuing abilities were found not only between isoforms belonging to different domains but also within the same domain. These individual differences reflect distinct functions for distinct isoforms in contributing to Dscam's overall function.
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    Genetic regulation of autophagic cell death in Drosophila Melanogester
    (2008-11-20) Dutta, Sudeshna; Baehrecke, Eric H; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Apoptosis and autophagic cell death are the two most prominent morphological forms of programmed cell death that occur during animal development. While much is known about the mechanisms that regulate apoptosis, relatively little is known about autophagic cell death. The steroid hormone ecdysone coordinates multiple cellular processes during metamorphosis in Drosophila, including cell differentiation, morphogenesis and death. E93 is necessary and sufficient for larval tissue cell death during metamorphosis, including autophagic cell death of salivary glands. Here we characterize new mutant alleles of a dominant wing vein mutation Vein-off (Vno), and provide evidence that E93 and Vno are related. Our data also indicate that E93 functions in steroid regulation of both cell development and death during metamorphosis. E93 encodes a helix-turn-helix DNA binding motif and binds to specific regions of salivary gland polytene chromosomes. We have used genetic and genomic approaches to identify downstream targets of E93. We have identified numerous candidate E93 target genes using DNA microarrays, and have generated transgenic animals to identify downstream target genes of E93 by chromatin immune precipitation. We show that one putative E93 target gene, hippo (hpo), is required for salivary gland cell death. The Wts/Hpo tumor-suppressor pathway is a critical regulator of tissue growth in animals, but it is not clear how this signaling pathway controls cell growth. Our data indicate that salivary gland degradation requires genes in the Wts/Hpo pathway. Wts is required for cell growth arrest and autophagy in dying salivary glands, and regulates the degradation of this tissue in a PI3K-dependent manner.
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    Insights into the regulation of ethylene receptor signaling by RTE1
    (2008-10-10) rivarola, maximo lisandro; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ethylene is an important regulator of plant growth, development and responses to environmental stresses. The higher plant Arabidopsis thaliana perceives ethylene through five homologous receptors, which negatively regulate ethylene responses. The molecular mechanism by which these receptors signal to their next downstream component remains elusive. Genetic analyses have shown that the RTE1 locus is a positive regulator of ETR1. RTE1 encodes a novel protein of unknown molecular function, and is conserved in plants, animals and some protists. The goal of this research was to analyze the mechanisms involved in the regulation of ethylene receptor signaling by RTE1 and to enhance our understanding of the conserved cellular role of RTE1. Here we tested hypotheses for how RTE1 affects ETR1 and is specific to only ETR1, not the other ethylene receptor isoforms. We show that ETR1 and RTE1 gene expression patterns partially overlap and that the ETR1 receptor co-localizes with RTE1 within the cell. Moreover, RTE1 has no effect on ETR1 protein abundance or subcellular localization suggesting other mechanisms to regulate ETR1. We provide supporting evidence that RTE1 affects ETR1 signaling by restoring signaling of a non-functional ETR1 in an rte1 null through changes in ETR1 conformation(s). We next addressed the question of RTE1 specificity to ETR1. We discovered that ETR1 is surprisingly distinct from the other four ethylene receptor genes; in that RTE1-dependent mutations only confer insensitivity in ETR1 and not in the other ethylene receptors when the same mutations are introduced. In contrast, the RTE1-independent ETR1 insensitive mutations do give insensitivity in the closest receptor to ETR1, ERS1. Furthermore, we uncover that the ethylene binding domains are not completely interchangeable between ETR1 and ERS1. Our data point to a model in which RTE1 specifically promotes ETR1 signaling via conformational changes in a unique way that does not occur in other ethylene receptors. These findings highlight the importance and uniqueness of ETR1 signaling conformation(s) with respect to the other ethylene receptors, as well as advance our knowledge of RTE1 at the molecular and cellular level.
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    Role of ubiquitination in Caenorhabditis elegans development and transcription regulation during spermatogenesis
    (2008-08-12) Kulkarni, Madhura D; Mount, Stephen; Smith, Harold; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Regulation of gene function can be achieved through a variety of mechanisms. In this dissertation, I present the genetic and molecular characterization of two genes involved in two distinct mechanisms of control. Each gene was initially identified by its functional role in sperm development in the model organism Caenorhabditis elegans. The first gene, uba-1, is an essential enzyme involved in protein turnover through ubiquitin-mediated proteolysis. A temperature-sensitive allele, (uba-1)it129, was isolated in a classical genetic screen for mutations that cause sperm-specific sterility. The second gene, spe-44, encodes a putative transcription factor. Its identification by microarray screening for sperm-enriched genes led to the cytological analysis of the deletion allele spe-44(ok1400), by reverse genetics approach. it129 encodes a conditional allele of uba-1, the sole E1 ubiquitin-activating enzyme in C. elegans. E1 functions at the apex of the ubiquitin-mediated conjugation pathway, and its activity is necessary for all subsequent steps in the reaction. Ubiquitin is covalently conjugated to various target proteins. Poly-ubiquitination typically results in target protein degradation, which provides an essential mechanism for the dynamic control of protein levels. Homozygous mutants of uba-1(it129) manifest pleiotropic phenotypes, and include novel roles for ubiquitination in sperm fertility, control of body size, and sex-specific development. We propose a model whereby proteins normally targeted for proteasomal degradation instead persist in uba-1(it129ts) and impair critical cellular processes. The second gene, spe-44, was identified as a putative sperm gene regulator in C. elegans based on its up-regulated expression during spermatogenesis and its significant sequence homology to the DNA-binding SAND domain. Genetic analysis of a deletion allele of spe-44(1400) has revealed its functional role during sperm development. Cytological analysis of spe-44(ok1400) showed developmental arrest of spermatocytes prior to spermatid differentiation. spe-44 mRNA is expressed in a narrow spatial and temporal window, just prior to spermatocyte differentiation, consistent with its functional role during spermatogenesis. Future study will be directed to find putative targets of spe-44 and the mechanisms that regulate gene expression using microarray analysis and yeast-one hybrid screens. These studies will help to understand transcriptional regulatory aspects of spermatogenesis in C. elegans.
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    Surviving Ionizing Radiation: General Stress Response and Mechanisms for the Prevention and Repair of DNA Damage in Halobacterium sp. str. NRC-1
    (2007-12-18) Kish, Adrienne; DiRuggiero, Jocelyne; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The effects of ionizing radiation on the extremely halophilic Archaeon Halobacterium sp. str. NRC-1 can be divided into three central themes: protection from oxidative damages, response to ionizing radiation, and repair of DNA double strand breaks (DSBs). Intracellular salts used to maintain osmotic balance in the hypersaline conditions Halobacterium cells require are shown in this study to provide in vivo protection from oxidative damages through the scavenging of hydroxyl radicals produced from the radiolysis of water by gamma radiation. These results highlight both the importance of the intracellular environment in determining radiation resistance and the multiplicity of pathways resulting in radiation resistance that can be utilized by various microbes resulting from their adaptations to common environmental stresses such as desiccation. The global stress response to gamma radiation was measured using both genomic and proteomic methods. The resulting systems view reveals cooperation amongst several cellular processes including DNA repair, increased protein turnover, apparent shifts in metabolism to favor nucleotide biosynthesis and an overall effort to repair oxidative damage. Further, we demonstrate the importance of time dimension while correlating mRNA and protein levels and suggest that steady state comparisons may be misleading while assessing dynamics of genetic information processing across transcription and translation. The repair of DNA DSBs incurred after exposure to gamma radiation was examined in greater detail. The in vivo role of the Mre11/Rad50 complex was determined in an archaeal model system to determine if these proteins performed the same role in homologous recombination repair as their eukaryotic homologs. Deletion of mre11 was found to reduce the rate of DSB repair, but not the overall survival of the cells. Taken together, the data presented here provide a halophilic model for radiation resistance that shares some common elements with other radiation resistant organisms such as Deinococcus radiodurans while presenting alternative mechanisms specific to extreme halophiles.
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    An Internal tRNA-Like Structure Regulates the Life Cycle of a Plus-Sense RNA Virus
    (2007-12-12) McCormack, III, John Crisler; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Turnip crinkle virus (TCV) is a 4054 b plus-sense RNA virus that belongs to the genus Carmovirus in the Family Tombusviridae. The 3' terminal 200 b of TCV are predicted to fold into 5 hairpins labeled in the 3' to 5' direction as the promoter (Pr), hairpin 5 (H5), hairpin 4b (H4b), hairpin 4a (H4a), and hairpin 4 (H4), using 3' UTR phylogenetic comparisons with other carmoviruses and the RNA structural prediction program, mfold. H5 was found to be a highly-conserved structure containing a large symmetrical loop (LSL) that formed a tertiary interaction between the 3' side of the LSL and the 3' terminal nucleotides using compensatory mutational analysis in vivo. In plants, LSL mutations resulted in a mutation frequency that was increased by as much as 12-fold without inducing error catastrophe. The original mutations frequently reverted and led to second site alterations biased for uridylate to cytidylate and adenylate to guanylate changes. These results suggest that H5 may function as a chaperone to properly fold the RdRp. The TCV 5' UTR, which binds 40S ribosomal subunits, contains two short segments exhibiting IRES activity that function synergistically with the 3' terminal region to enhance cap-independent translation in vivo. In the TCV 3' UTR, H4a, H4b, H5, and flanking sequences, form an internal tRNA-like structure (iTLS) that binds 60S ribosomal subunits and the P-site of salt washed 80S ribosomes. The iTLS may therefore mediate assembly of 80S ribosomes, which are then transported to the 5' end for translation of virally-encoded proteins. Phylogenetic comparisons of carmovirus 3' UTRs revealed that Cardamine chlorotic fleck virus (CCFV) and Japanese iris necrotic ring virus (JINRV) are capable of forming the 5 elemental features comprising the iTLS. Ribosome binding and plant cell culture assays showed that only the CCFV iTLS bound 80S ribosomes and could functionally replace the TCV iTLS. These results suggest that closely-related members of the same viral genus may utilize different strategies for cap-independent translation.
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    Mechanisms Utilized by Neisseria gonorrhoeae to Persist in the Human Cervical Epithelium: Signaling, Invasion, Intracellular Survival, and Escape
    (2007-11-07) Bish, Samuel Earl; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The obligate human pathogen Neisseria gonorrhoeae (gonococci) causes the sexually transmitted disease gonorrhea. Interactions between gonococci and human cervical epithelial cells that promote bacterial invasion and post-invasion survival were examined. The data showed that gonococci activated MAPK signaling in cervical epithelial cells prior to invasion. Gonococci triggered prolonged ERK phosphorylation (possibly through EGFR signaling), which could facilitate gonococcal invasion. Furthermore, gonococci expressing lacto-N-neotetraose LOS decreased JNK activation and elicited lower IL-8 production from host cells compared to variants expressing lactosyl LOS. I propose that gonococci can manipulate MAPK signaling to facilitate invasion into cervical epithelial cells and to evade the innate immune response. To investigate gonococcal invasion, I expressed β-lactamase on the outer membrane of gonococcal strain FA1090 to generate the reporter strain FA1090 psi(bla-iga'). Utilizing a β-lactamase reporter assay, I demonstrated that FA1090 psi(bla-iga') only invaded a subpopulation of cervical epithelial cells. Nonviable FA1090 psi(bla-iga') did not invade host cells and failed to recruit F-actin to sites of adherence. Viable gonococci required pili expression but not Opa expression to invade susceptible host cells. These data suggest that viable gonococci elicit invasion-promoting interactions with host cells not triggered by nonviable gonococci. Finally, the data demonstrated that gonococci realize different intracellular fates after entry into cervical epithelial cells. I developed a tannic acid survival assay to show that intracellular gonococci survived within host cells. Intracellular survival did not require catalase or the NfsB nitrogen reductase. A subpopulation of gonococci could escape from cells back into the extracellular milieu. Gonococci utilized an exocytosis pathway to escape after invasion that was blocked by treating cells with tannic acid, but not cytoskeletal inhibitors. I propose a working scenario whereby gonococci undergo cycles of invasion into and escape from a susceptible subpopulation of cervical epithelial cells. Gonococci that survive within host cells may represent bacteria that progress into deeper host tissues to disseminate to secondary sites. Escaping gonococci could invade into other host cells and are well-positioned for transmission to another host. Gonococci interact with the cervical epithelium through invasion, intracellular survival, and escape to establish bacterial persistence in the female host.
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    Characterization of Ribosomal Protein L2 and Analysis of Missense Codon Discrimination in the Yeast Saccharomyces cerevisiae
    (2007-07-27) Russ, Johnathan Robert; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Translational fidelity is of vital importance to the ribosome as it strikes an optimal balance between speed and accuracy. The study of factors contributing to the structure and function of the ribosome by assaying effects on fidelity yields important insights into the mechanism of the ribosome. Ribosomal protein L2 is vital to the structure and function of the ribosome with importance in RNA interaction and positioning, ribosomal subunit binding, and peptidyl transfer. Sixteen unique alleles of the RPL2A gene were studied for their effects on translational fidelity and general ribosome function. Mutations at V48D and L125Q of the highly structured globular domain proved generally deleterious to growth, but resistant to anisomycin, and markedly increased -1 programmed ribosomal frameshifting. H215Y, a mutation at the site of interaction with H93 of the peptidyl transferase center (PTC), was both sparsomycin and anisomycin resistant, indicating a functional impact on the PTC structure. Mutations at F185L and W195C border conserved rRNA binding regions and proved hyperaccurate in misincorporation studies. Understanding of fidelity and proper tRNA selection in translation requires analysis of how the ribosome discriminates between cognate, near- cognate, and non-cognate tRNAs. Measuring misreading of several codon substitutions in the context of paromomycin as well as mutations and deletions of contributing elongation factors and ribosome structural elements allowed further understanding of interactions governing tRNA selection and tRNA misincorporation events. A mutant of eEF1Balpha, the nucleotide exchange factor for eEF1A, promoted a general increase in fidelity. Interestingly, a mutated ribosomal protein L5, though distant from the decoding center, also stimulated hyperfidelity in both near- and non-cognate cases. Distinction between near- and non-cognate tRNA interactions on the ribosome was observed in tests using paromomycin, and in studying mutations of eEF1Bgamma with both demonstrating increased misreading of near-cognate codons but hyperaccurate decoding of non-cognate codons.