Biology

2004 Senior Thesis Abstracts

(CLASS OF 2004: If your thesis abstract is not currently included on this page and you would like it to be, please follow this link.)

Gene Overexpression and Barley Yellow Dwarf Virus-Programmed Stop Codon Readthrough in Yeast

Anees Muhammed Ahmed

(Advisor: Russell)

ABSTRACT

Viruses have developed various mechanisms that play a crucial role in the viral-host relationship. One such mechanism utilized by both plant and animal viruses is programmed stop-codon readthrough, which occurs when ribosomes are occasionally directed by the surrounding sequence of a stop codon to continue the process of in-frame translation while bypassing the translational termination signal. While this mechanism has been studied in the Tobacco mosaic virus (TMV), a single-stranded, positive-sense, RNA plant virus, another virus that has exhibited this phenomenon is the Barley yellow dwarf virus (BYDV), which has the same genomic character as TMV. As viruses are entirely dependent upon host proteins for gene expression and replication, the overall goal of this thesis was to assist in the effort of identifying host genes that are involved in stop-codon readthrough for BYDV by using yeast as a model host. Specifically, this study aimed to examine whether the overexpression of STU2, a gene encoding a microtubule-binding protein, resulted in increased readthrough efficiency, which has been exhibited in the TMV sequence context. As an initial approach to characterizing this relationship, a lacZ reporter plasmid was constructed that contains the BYDV stop-codon context and fused to the 5' end of lacZ, so that the beta-galactosidase activity was a measure of the rate of stop-codon readthrough. A control construct that would allow for 100 % readthrough or a mutant construct had not yet been produced at the time of this study. When STU2 was overexpressed in yeast, the data were unclear as to whether this increased dosage had any effect upon readthrough. There was a slight indication that STU2 may be a down regulator of readthrough, but more research with additional numbers of repeated, consistent experiments as well as additional techniques such as a double reporter system that can control for variables such as mRNA stability, will provide a much clearer answer to the question posed here. With this future research, another important step towards characterizing the relationship between viruses such as BYDV and their hosts can be made.

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Isolation of Non-H-NS Negative Regulators of Virulence Genes of Enteropathogenic Escherichia coli

Alexander G. Beebe

(Advisor: Mellies)

ABSTRACT

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea among infants in developing countries. The genome of EPEC contains a 35.6-kb pathogenicity island known as the Locus of Enterocyte Effacement (LEE), which confers the attaching and effacing phenotype. The LEE genes encode a type-III secretion system and associated molecules, and are expressed from five known operons: LEE1, LEE2, LEE3, LEE4, and LEE5. The regulation of these operons, and the environmental signals they respond to are incompletely understood. Studies on LEE operons show that most are repressed by H-NS. Ler displaces H-NS at LEE2, LEE3 and LEE5. In addition, repression of LEE5 is mediated by a non-HNS regulator. This additional negative regulator is known to be disrupted by osmolarity and DNA damage. While H-NS primarily represses at 26°C , the additional negative regulator functioning at LEE5 apparently represses at host body temperature, 37°C. Of particular interest is the LEE4 operon, which encodes the injectosome of the type-III secretion system and an initial attachment factor. As the LEE operons are likely regulated in a similar manner, additional negative regulators may function at LEE4 as well. In order to study the regulation of the LEE4 operon, I used a LEE4 promoter-lacZ fusion strain and attempted to identify a second negative regulator by disrupting its function via transposon mutagenesis. I found four of mutants that appeared to exhibit greater b-galactosidase activity in the presence of X-gal. These were tested and none appeared to be due to a disruption in hns. These mutations were associated with sensitivity to MacConkey media, indicating that the regulator had pleiotropic effects. Though the identity of the negative regulator remains unknown, it appears that LEE4--like the other LEE operons--is responsive to non-H-NS regulatory proteins at 37°C.

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Isolation of the 5' end of Xenopus tropicalis TRF1 mRNA

Royd Litanu Carlson

(Advisor: Shampay)

ABSTRACT

The mRNA sequence for TTAGGG-repeat binding protein 1, TRF1, has been obtained in human, mouse, and chicken, as well as Xenopus laevis. Here, by PCR amplification from cDNA, TRF1 mRNA transcription has been shown to occur in ovary tissue of Xenopus tropicalis. Additionally, the 5' end of Xenopus tropicalis TRF1 mRNA has been isolated using the Rapid Amplification of cDNA Ends (RACE) procedure, and analyzed. The Sanger Institute maintains a Xenopus tropicalis cDNA library, but it was not certain whether the true 5' end of the Xenopus tropicalis TRF1 mRNA indicates the presence of a start codon, and thus suggests that the entire 5' coding region of the mRNA has been isolated. Additionally, information about the acidic domain structure in Xenopus tropicalis was obtained. The TRF1 mRNA in both Xenopus species is curious because it codes for a shortened acidic domain as compared to human and mouse TRF1. In humans, the acidic domain binds to the protein tankyrase 1, which negatively regulates TRF1. This raises the questions about whether the same mechanism occurs in Xenopus, where the acidic domain is truncated. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Vesicle recycling at the axon terminals in the neuroendocrine cells of Aplysia californica

Thea Simone Doidge

(Advisor: Arch)

ABSTRACT

There has been considerable debate in recent years as to whether or not exocytosis of secretory vesicles can occur without full fusion of the vesicular membrane with the plasmalemma. Especially controversial is the suggestion that such a mechanism would be advantageous for cells that secrete large dense-core vesicles containing only peptides. Until now, no evidence has been presented to indicate that such fast exo/endocytosis events occur in peptide-only vesicles. Here a new method for detecting exocytosis was employed to test for faster modes of exo/endocytosis in the neuroendocrine bag cells of Aplysia californica. Cells were incubated in medium containing tritium-labeled neurotransmitter (3H-dopamine). After the cells had loaded their secretory vesicles with 3H-dopamine, exocytosis was stimulated with insulin. One treatment group was exposed during the loading period to bafilomycin, an inhibitor of the proton pump V-ATPase. This protein has been implicated in the process of vesicle acidification and in the refilling of vesicles with neurotransmitter. Bafilomycin had no effect on vesicle loading or secretion. We concluded that vesicles load with 3H-dopamine through endocytosis, rather than by the action of V-ATPase. The results of this study strongly imply that recycling of peptide-only vesicles can occur near the release site. If this implication is born out, this study will have provided the first evidence of rapid exo/endocytosis in peptide-only vesicles. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Survey of Glutathione S-Transferase Expression in Soybean (Glycine max) Root Nodules

Lisa Marie Fuller

(Advisor: Dalton)

ABSTRACT

Nitrogen fixation is a fundamental requirement for the persistence of life on Earth. The majority of the accessible nitrogen supply is provided by symbiotic associations between prokaryotic rhizobia, capable of fixing atmospheric nitrogen into biologically active forms, and leguminous plants that house the symbiont in nodules and provide resources that fuel nitrogen fixation. This projects sought to explore the gene expression of glutathione S-transferase (GST) in soybean (Glycine max) root nodules, a hitherto unexplored aspect of root nodule physiology. Twenty-five GST genes have been identified in soybean (McGonigle et al., 2000), although the tissue specific pattern of GST expression has not been ascertained. GSTs are a large and diverse enzyme superfamily, present in almost all aerobic life. These proteins are functionally defined as catalyzing the conjugation of the tripeptide glutathione to electrophilic centers of foreign and endogenous toxins. The connection between GST and root nodule functionality was presumed to involve regulation and neutralization of reactive oxygen species (ROS) that are universally cytotoxic, but also major determinants of the efficiency of nitrogen fixation in root nodules. Previous research in this lab has confirmed that GSTs are in soybean root nodules (Coan, 2003).To identify the particular GST genes that were expressed in soybean root nodules, a nodule-specific cDNA library was constructed and screened via PCR, using GST-gene specific primers. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Peptidergic Vesicle Structure

Michael Hoppa

(Advisor: Arch)

ABSTRACT

Neurons primarily communicate between themselves and with other cells by releasing different types of chemicals, collectively known as neurotransmitters. Therefore a key feature of neural communication is the release of chemicals, which is achieved by a vesicle fusing with the presynaptic membrane of the neuron. Vesicles are subcellular organelles that act as storage containers for multimolecular packets of neurotransmitter. Recent studies from synaptic vesicles have pointed to the existence of an intravesicular gel-matrix as a means to prevent collapse of the vesicle. Immunological data have implicated involvement of the vesicle protein SV2 in this structure. The current study was undertaken to determine whether this protein might also play a similar role in peptidergic vesicles. A vesicle fraction was immuno-isolated from the bag cells of Aplysia californica. Western blots revealed the presence of SV2 from these isolated fractions. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Colonization of Caenorhabditis elegans by Enteropathogenic Escherichia coli

Andrew Steffes Korson

(Advisor: Mellies)

ABSTRACT

Enteropathogenic Escherichia coli (EPEC) is a prominent cause of diarrheal illness, particularly in developing countries. EPEC causes disease by adhering to host cells and disrupting host cell function. Expression of the bundle-forming pilus (BFP) is known to be important for EPEC pathogenesis in humans. Excluding the human, a model organism for the study of EPEC pathogenesis has not been identified. The nematode, Caenorhabditis elegans, has arisen as a model organism for the study of bacterial pathogenesis in a wide variety of mammalian pathogens. Bacterial accumulation in the nematode intestinal tract is known to be dependent on the expression of virulence factors involved in mammalian pathogenesis. In the present study, C. elegans was examined as an animal model for the study of EPEC pathogenesis. Bacterial accumulation of EPEC in C. elegans was evaluated by standard plate count following the homogenization of nematodes exposed to EPEC. EPEC strains were found to accumulate in the nematode in larger amounts than a non-adherent lab strain. Through the use of strains expressing green fluorescent protein (GFP), EPEC was shown to accumulate in the alimentary tract of C. elegans. Surprisingly, the BFP was not found to be involved in the accumulation of EPEC in the nematode, and potential explanations of this result are discussed. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Deducing the JNK MAPK Pathway Using RNAi in Drosophila Cells Exposed to Oxidative Stress

Molly Marie Ross O'Quinn

(Advisor: McClellan)

ABSTRACT

Organisms encounter stimuli in the environment that are detrimental to their survival, causing stress. Work in flies and mammals suggests that organisms respond to stress by activating the c-Jun N-terminal kinase pathway (JNK), a highly conserved stress pathway. To discover how oxidative stressors are linked to the steps in the JNK pathway, RNA interference (RNAi) was used to eliminate upstream activators of JNK and cells were then treated with the oxidative agent phorone. Surprisingly, we discovered that oxidative stress caused by phorone activated JNK through the combined action of several upstream kinases. Unexpectedly, phorone deactivated the other stress-activated kinase p38 MAPK. In contrast, cells treated with ethanol, also believed to cause oxidative stress, were activated by only one upstream kinase. Although phorone is believed to deplete glutathione, replenishing glutathione did not override the phorone effect. These data imply that oxidative stress acts via different MAP kinase pathways.

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Investigating Polyploidy and Genetic Variation in Garlic Mustard (Alliaria petiolata, Brassicaceae), an Invasive Species

Micha Louise Rahder

(Advisor: Karoly)

ABSTRACT

Invasive species are a growing environmental and biological concern on a global scale. Ecological studies have revealed a great deal about the processes by which new species come to inhabit and dominate local ecosystems. Recently, population and evolutionary genetic frameworks have brought a new perspective to this research. In this study I have used several genetic techniques to investigate the mechanisms by which Alliaria petiolata (Brassicaceae) is successfully invading North American forest ecosystems. A. petiolata is a particularly aggressive invader across much of the United States and Southern Canada, and can invade relatively undisturbed habitats and seriously disrupt local ecosystems and biodiversity. I studied thirty individuals from each of four Portland, OR populations of Alliaria petiolata using allozymes, inter-simple sequence repeat (ISSR) markers, and direct observation of chromosomes in order to determine the mode of polyploidy and levels of genetic variation in this species. A. petiolata is a known polyploid, but whether it is an allo- or autopolyploid has never been investigated. I attempted to answer this question by direct observation of chromosome behavior during meiosis, but was unable to achieve success. By analyzing patterns of gene expression in allozymes I have determined that A. petiolata is most likely an allopolyploid, and that it displays gene duplication and fixed heterozygosity at two to four of six surveyed loci. Very little variation was found within or between populations in allozyme expression, with only one of eight loci showing a polymorphism. I also surveyed genetic diversity using ISSRs and the Shannon-Weaver index of diversity, and found an average diversity of H0=0.646. This level of diversity is much higher than that indicated by the allozyme data, but lower than ISSR diversity reported in other North American populations of garlic mustard. The drop in variation from other North American populations may be due to a bottleneck event associated with the range expansion of A. petiolata into Oregon. Allopolyploidy has been posed as a possible mechanism for maintaining genetic diversity in invasive species undergoing severe population bottlenecks, and this may be key to understanding the invasive success and vigor of A. petiolata. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Early Spider Development and the Role of Extracellular Matrix in Major Cell Movements

Mercedes Bridie Ramirez

(Advisor: Black)

ABSTRACT

The early development of spiders, as with virtually all metazoans, involves the internalization of cells at a single point to form the internal organs and organize the development of the body. This process is called gastrulation, and in spider embryos is usually thought to take place by involution of cells. The exact location of involution, however, is less well agreed upon. Using time-lapse videography, I have identified a potential site of internalization, the primitive groove in {\it Zygiella x-notata} and established its position in relation to a later structure, the cumulus. By sectioning and staining embryos at the cumulus stage, I found evidence of ingression of the top layer of cumulus cells. Measurements of width: height ratios of presumptive ingressing and non-ingressing cells supported my visual observations. Moreover, investigation into the nature of the extracellular matrix of the embryo revealed that it contains a protein recognized by an antibody against chick chondroitin sulfate proteoglycan (CSP). Most of the spider protein is localized to the apical surfaces of top layer cells, and there appears to be a decrease in the amount of protein at the cumulus stage. Since in other systems CSP inhibits cell adhesion, it is possible that the decrease in the putative spider CSP might faciliatate ingression, in addition to involution, at the cumulus. Finally I developed a staining technique that visualizes the cytoplasm of vitellophages. Vitellophages are cells involved in yolk utilization in oviparous invertebrates, but little else is known about their function or formation. My preliminary data show that the relative proportions of vitellophages near the surface and deep within the yolk mass changed during development. More deep than surface cells were found at the blastoderm stage, while the opposite was true for the germ disc and cumulus stages.

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Host Factors Involved in Stop Codon Readthrough on Barley Yellow Dwarf Virus RNAs in the Model Host Saccharomyces cerevisiae

Jonathan Charles Rupp, BMB

(Advisor: Russell)

ABSTRACT

Barley yellow dwarf virus (BYDV) is a positive-sense plant pathogenic RNA virus. BYDV (PAV serotype) uses the non-standard translational mechanism, stop codon readthrough, for the translation of an extended version of its coat protein, which is required for the infectivity of the virus. This investigation sought to characterize host factors involved in this programmed readthrough, using the model host Saccharomyces cerevisiae. lacZ-luc double reporter plasmids were constructed to quantify the rate of readthrough in S. cerevisiae. The plasmids contain (reading 5' to 3') the E. coli lacZ gene, a 144 bp section of the BYDV chromosome containing the ORF3 stop codon and readthrough conferring sequence, and the fire y luc gene. Read-through rate was calculated by dividing the ratio of activities from the experimental plasmid by the ratio of activities from a control plasmid containing a sense codon in the place of the leaky stop codon. Plasmids containing the BYDV sequence in the reverse orientation were constructed as a negative control. A readthrough rate of 0.6% was observed for wild-type S. cerevisiae, which is similar to values observed from other reporter systems in S. cerevisiae, as well as values reported for BYDV in its natural hosts. These results indicate that the reporter system is effective for the BYDV-S. cerevisiae experimental system. The double reporter system has advantages over single reporter systems used in previous studies, as it controls for varying mRNA levels. Further work, quantifying rates of readthrough in strains of S. cerevisiae containing mutated or overexpressed genes, has potential to reveal information about the mutated or overexpressed genes' involvement in readthrough.

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Response of the Soybean (Glycine max) Root Nodule Antioxidant System to Exogenous Hydrogen Peroxide Application

Laura Clare Shaffer

(Advisor: Dalton)

ABSTRACT

Reactive oxygen species (ROS) are a common danger to all aerobic life. ROS cause damage and destruction of essential biomolecules by rapidly reacting with and oxidizing them. Carrying out the processes of photosynthesis, respiration and nitrogen fixation make plants particularly prone to ROS formation. To cope with this problem, they have evolved an impressive array of antioxidant defenses including both proteins and small molecules. In the nodule, the site of nitrogen fixation, conditions are particularly conducive to ROS formation. Symbiotic bacteria convert N2 to NH3 using the nitrogenase enzyme, which requires both a microaerobic environment and high rates of respiration to sustain it. A high concentration of catalytic iron in the nodule also contributes to ROS formation. For these reasons, antioxidants are required in high concentrations in the nodule. This study sought first to identify an effective means of causing oxidative stress in nodules, and, second, to identify the contribution of phenolic antioxidants to antioxidant defense in the nodule. Results show that hydrogen peroxide treatment is an effective means of oxidative stress induction in nodules as measured by acetylene reduction activity. Treatment of nodules with hydrogen peroxide elicited a change in ascorbate peroxidase activity, a key antioxidant enzyme. The activity of phenylalanine ammonia lyase, an enzyme involved in phenolic compound synthesis, was also measured in response to hydrogen peroxide treatment, but no activity was found. Results from this study do not provide any evidence of increased phenolic production in response to oxidative stress. The contribution of phenolic compounds to antioxidant defense in nodules could not be measured.

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