2006 Senior Thesis Abstracts
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Anthocyanins and Drought Responses in Delphinium nuttallii, D. leucophaeum, and Their Hybrids
Jillian H. Anderson
(Advisor: Karoly)
Flower color polymorphisms are traditionally considered the result of pollinator-mediated selection. However, recent studies indicate a relationship between flower color polymorphisms and plant performance during abiotic stress, suggesting that flower color polymorphisms are selected for by abiotic environmental selection. In particular, plants with anthocyanin based flower color are able to outperform plants with anthocyanin-lacking flower color in stressful environments. To test the hypothesis that anthocyanin pigment flower colored plants perform better than unpigmented flower colored plants in stressful environments, responses to drought and well-watered (wet) treatment were measured in 22 blue and 28 white flowered Delphinium. Physiological traits measured were carbon isotope ratio, variable chlorophyll fluorescence, and relative water content and morphological traits measured were anthocyanin reflectance index, flavonol reflectance index, and specific leaf weight. Both color morphs had a significantly higher water-use efficiency (as measured by the carbon isotope ratio) in the drought treatment, with a trend of the blue morphs performing better than the white morphs in both the drought and wet treatments. Drought treatment morphs also had significantly more foliar anthocyanins than wet treated morphs. However the trend differed from the water-use efficiency results in that blue morphs had less foliar anthocyanins than white morphs in the wet treatment. Drought treatment also resulted in significantly more foliar flavonol content (as measured by flavonol reflectance index) in both morphs, with the trend of blue morphs having more foliar flavonols than the white morphs in both treatments. These results can be interpreted as supporting the hypothesis that anthocyanins enhance drought tolerance through their ability to act as an osmoticum inside the leaf. This study was funded in part by an HHMI Undergraduate Research Program grant.
Understanding Virulence: in vitro Analysis of H-NS and Ler Mediated Regulation of
the LEE5 Regulatory Region in Enteropathogenic Escherichia coli
Anna Maria Carmona
(Advisor: Mellies)
Enteropathogenic Escherichia coli (EPEC) type III secretion system encoding genes are located within the locus of enterocyte effacement (LEE), a pathogenicity island that is controlled by the global transcriptional regulators histone-like nucleoid structuring (H-NS) protein and the LEE encoded regulator (Ler). Previous research has shown that transcription of the LEE5 operon, encoding the proteins necessary for the intimate attachment of the bacterium to the host epithelium, is repressed by H-NS at 25°C. Repression is achieved by H-NS binding to AT rich regions of DNA located upstream and downstream of the transcriptional start site, resulting in a predicted hairpin structure that traps RNA polymerase (RNAP) at the LEE5 promoter. Ler is predicted to activate transcription at 37°C by competitively binding to the H-NS upstream low-affinity binding region, effectively removing H-NS mediated repression. To further characterize these protein/DNA interactions, segments of the LEE5 regulatory region were subjected to increasing concentrations of purified H-NS and Ler proteins in an electrophoretic mobility shift assays (EMSAs) to determine the binding profile of each protein for DNA sequences located upstream and downstream of the transcriptional start site as well as the entire LEE5 regulatory region. H-NS binding profiles were consistent with previous H-NS DNase I footprinting assays that indicated the presence of an H-NS low-affinity biding site upstream of the LEE5 promoter (Kd = 540 nM) and a high-affinity binding site downstream of the promoter (Kd = 250 nM). The simultaneous and sequential competition assays support the hypothesis that H-NS mediated repression is lifted by Ler binding (Kd = 90 nM) to the Ler protected region of DNA overlapping the upstream H-NS low-affinity binding site. It has been proposed that RNAP/δ70 binding of LEE5 aids H-NS mediated repression by facilitating the formation of the hairpin structure, as observed for the rrnB P1 ribosomal promoter, which then traps the RNAP/70 holoenzyme at the transcriptional start site. Future research will involve additional EMSAs to further characterize the mechanism by which H-NS and Ler interact with RNAP/δ70 to regulate LEE5 transcription. The insight gained by these inquiries will reveal how H-NS and Ler interact to regulate virulence genes during EPEC infection, and help characterize their role as global regulators of gene expression. This study was funded in part by an HHMI Undergraduate Research Program grant.
Identification of Chemotherapeutic Agents Against Enterotoxigenic Escherichia coli Using the Model Organism Caenorhabditis elegans
Dorothea Hope Coffer
(Advisor: Mellies)
Enterotoxigenic Escherichia coli (ETEC) is responsible for pediatric and traveler's diarrhea in developing countries and, like many other bacterial strains, is rapidly becoming resistant to commonly prescribed antibiotics. Alternate therapies must be developed to combat these multi-resistant strains in an effort to prepare for what many scientists believe to be the end of the antibiotic age. An effective screening method to test the efficacy of various chemotherapeutic agents in vivo is needed to expedite this process. The soil dwelling nematode Caenorhabditis elegans can be used as a small animal model to study bacterial pathogenicity as well as the effect of antimicrobial compounds on human pathogens in vivo. This study demonstrated that ETEC killed C. elegans faster than a non-pathogenic lab strain, establishing that ETEC acts as a pathogen in both human and nematode hosts. We developed a screen to assess the antimicrobial ability of thymol and carvacrol, potentially therapeutic molecules found in many plant essential oils, to inhibit ETEC colonization of C. elegans. Fluorescent microscopy of nematodes fed on bacteria expressing green fluorescent protein (GFP) prior to treatment with potential antimicrobial compounds illustrated that thymol greatly decreased the number of bacteria recoverable from the nematode gut. Our findings support the use of the nematode model to screen for potential therapeutics against ETEC infection and demonstrate the powerful antimicrobial potential of plant-derived compounds such as thymol. This study was funded in part by an HHMI Undergraduate Research Program grant.
What Does This Have to do with Goats: Identifying Host Genes Involved in Stop Codon Readthrough in Barley Yellow Dwarf Virus
Dorien Skye Coray
(Advisor: Russell)
Stop codon readthrough is a non-canonical translation event that occurs when the ribosome does not terminate protein synthesis at the stop codon of a gene and continues on to a second stop codon. Barley yellow dwarf virus is an RNA virus with programmed readthrough of the coat protein stop codon (about 1% in plants) to produce a protein essential for transmission between plants via aphid transmission vectors. Here, yeast was used as a model organism to find host genes affecting readthrough in a yeast overexpression library. Four yeast overexpression library plasmids, UK1F1(3), UK1F11(2), UK1C(12)1, and UK2C(2), previously shown to increase RT of the Barley yellow dwarf virus stop codon with the lacZ single reporter system were tested with a more quantitative lac-luc double reporter system. The rate of RT was 0.67% (160% relative to the control) with the UK1F1(3) library plasmid, 0.52% (120%) with the UK1F11(2) library plasmid, 0.36% (84%) with UK1C(12)1, and 0.19% (44%) with UK2C(2). From this, the UK1F1(3) library plasmid gave the greatest increase in readthrough over the controls. Two individual genes on the UK1F1(3) plasmid, YAR066W and YAR062W were subcloned and tested individually for readthrough using the double reporter system. YAR066W, a protein of unknown function thought to be involved with nuclear export of RNA and tRNA, and trehalose synthesis, gave 5.0% RT, 4500% relative to the control (100%). YAR062W, a hypothetical protein/pseudogene possibly involved in flocculation gave 2.3% readthrough, 2300% relative to the control. Both genes were identified as potential increasers of stop-codon readthrough in Barley yellow dwarf virus. This study was funded in part by an HHMI Undergraduate Research Program grant.
Gene Expression of Structural Genes in the Anthocyanin Biosynthesis Pathway of Delphinium nuttallii and Delphinium leucophaeum
Brian Gareth Howell
(Advisors: Karoly)
Two species of Northwestern Larkspur, Delphinium leucophaeum and Delphinium nuttallii are primarily distinguished from each other by a pigment polymorphism that causes the D. nuttallii to have blue-purple flowers and D. leucophaeum to have predominantly white flowers. The anthocyanin pathway responsible for the production of the blue-purple pigmentation of D. nuttallii has been characterized in a number of other species and thus provides a useful framework to study the evolution of this pigment polymorphism. Unlike similar polymorphisms in other species, in these two the white phenotype is dominant to the more pigmented purple type, indicating a potentially interesting genetic mechanism rather than a simple loss of function mutation. Using genetic sequence information from studies with related species our lab has been able to develop Delphinium specific primers for key biosynthetic enzymes to use real-time PCR to assess the RNA expression of this pathway's structural genes (CHS, F3H, F3'5'H, DFR and ANS) in various stages of bud development and between species. In a survey of six plants (3 D. nuttallii and 3 D. leucophaeum) taken from southwestern Washington and northwestern Oregon, significant expression differences were detected between genes, but not between species or stages of bud development. These results imply that differential regulation of these genes between species or over the course of bud development is not occurring at the level of transcript synthesis. Potential alternative regulatory mechanisms for these genes may include post-transcriptional mRNA regulation or loss of function at the translational or enzymatic levels. This study was funded in part by an HHMI Undergraduate Research Program grant.
Morphogenetic Movements during Inversion of the Spider Embryo
Colin Stephen Magowan
(Advisor: Black)
Inversion is the blastokinetic movement of the germ band of spider embryos. During these acrobatic movements of cells, the overall curvature of the germ band changes from a C-shape to an S-shape intermediate, which then snaps back, resulting in an inversion of the original C-shape. Through the 150 years that spider development has been studied, this remarkable process has remained poorly understood at the cellular level. Therefore, I examined the cellular changes that occur in the embryo before and during inversion. The cells of the ventral sulcus and dorsal extra-embryonic ectoderm were found to be especially interesting as changes correlated with the changes in the curvature of the germ band. Leading up to inversion, the columnar ventral sulcus cells actively increase their area and become squamous. In addition, these cells were found to have a highly stereotypic orientation of mitosis. The consequence of both changes was to increase the area of the ventral sulcus. The dorsal extra-embryonic cells were found to undergo spatial re-arrangments that coincided with the active migration of germ band cells. These observations are discussed in the context of the evolutionary origin of inversion in the lineage leading to the spiders. This study was funded in part by an HHMI Undergraduate Research Program grant.
Finding Neverland: An Examination of Female Hormone Replacement Therapy
Marc William Nolan
(Advisor: McClellan)
Aging is a fact of life and the pursuit of therapies that slow or even prevent symptoms of aging are very attractive. The sex steroid estrogen is a vital protector of bone strength, heart function, and even cognition in women of reproductive age. Menopause signifies the cessation of ovarian function and results in a precipitous decline in circulating estrogens. Hormone replacement therapy (HRT) was introduced to prevent the symptoms of aging women and until recently was accepted as relatively safe. When a large-scale clinical study, the Women's Health Initiative (WHI), reported increased risks for diseases rather than protection clinicians recommended against HRT. This thesis reinterprets the findings of clinical trials in light of recent molecular mechanisms of estrogen action and HRT alternatives. The results prove that much of the angst over potential harmful effects of HRT is unfounded and linked to ancillary disease conditions. Strategies for replacing estrogens with synthetic or plant-derived compounds are discussed. This study was funded in part by an HHMI Undergraduate Research Program grant.
The Role of Indoleamine 2, 3-dioxygenase (IDO) in the Immune Response in Xenopus laevis
Diana Christine Parrish
(Advisor: Ruben)
Mammalian pregnancy and amphibian metamorphosis are immunologically similar in that both are situations in which cells expressing foreign antigens, which would normally evoke an immune response and be removed, are allowed to survive and proliferate in the host. Indoleamine 2,3-dioxygenase (IDO) has been found to be important for successful pregnancy; its inhibition results in spontaneous abortion soon after implantation in mice. Here, we explore whether IDO may be present in Xenopus laevis and may playing a role in amphibian metamorphosis. To determine the presence or absence of IDO in Xenopus, we tested the effects of the IDO inhibitor 1-methyl-L-trypyophan (MLT) on cell proliferation, apoptosis, helper function, and suppressor function in Xenopus cells, as well as the effects of increased tryptophan catabolites on Xenopus lymphocytes. We found that IDO inhibition with MLT results in significantly less lymphocyte apoptosis and significantly less suppression of specific immune responses. These results suggest that IDO is present and active in Xenopus, and that Xenopus could potentially be used as a model in which to further study IDO. This study was funded in part by an HHMI Undergraduate Research Program grant.
Molecular Concerted Evolution of the 5.8 S rDNA Multigene Family in Delphinium
Leila Elizabeth Rieder
(Advisor: Karoly)
Concerted evolution is the process by which multigene families, for example rRNA genes, evolve as a unit. Concerted evolution is thought to proceed through two mechanisms collectively termed molecular drive: biased gene conversion and unequal crossing over. The former produces a directed change in favor of one allele at the expense of the other while keeping gene copy number constant. The later produces both a change in gene copy number and with it allele ratio. A single nucleotide polymorphism in the 5.8 S rDNA of two species of Delphinium provides a unique study system for concerted evolution. Both the evolution of individual genomes over time as well as the inheritance of rDNA within 3-generation crosses were studied. The techniques used in this experimentÑPCR-RFLP with polyacrylamide gels to study gene conversion and quantitative PCR to study unequal crossing overÑhave not been used before to study the effects of concerted evolution.
Changes in allele ratios, a measure of gene conversion, were assayed by PCR of 5.8 S rDNA, digestion with HaeIII, and polyacrylamide gel electrophoresis. Relative intensities of bands representing the two alleles resulting from the single nucleotide polymorphism were scored and converted to allele copy numbers.
New and old DNA was used to address gene conversion effects within 21 individuals. The mean allele ratios across new DNA was higher than the mean of the old DNA ratios with a 5% bias in the direction expected for biased gene conversion. Across generations the average F2 (N = 38) ratio was more positive than the F1 ratio in all families, again supporting a role for biased gene conversion.
Change in gene copy number was used to investigate the process of unequal crossing over. Copy numbers were determined for 3 generations of individuals through quantitative PCR analysis of genomic DNA. Chalcone synthase, a gene thought to occur in a single copy in the genome, was used for comparison. A change in 5.8 S rDNA copy number was observed across generations, suggesting that unequal crossing over may be an important mechanism of concerted evolution in Delphinium.
It is also possible that both mechanisms, acting in opposition or in concert, are actively driving concerted evolution. If the 75 allele confers some phenotypic benefit it is possible that selection, in addition to the mechanisms of concerted evolution, acts to increase the frequency of the 75 allele within populations. However, even absent the effects of selection, gene conversion and unequal crossing over may have profound impacts on rDNA evolution. This study was funded in part by an HHMI Undergraduate Research Program grant.
Effects of the Overexpressed Host Genes STU2 and YHR213W-A on Barley Yellow Dwarf Virus Stop Codon Readthrough in Saccharomyces cerevisiae
Lindsay Kathryn Swanson
(Advisor: Russell)
In the positive-sense, single-stranded RNA virus Barley yellow dwarf virus (BYDV) translation of ORF3 continues past the usual stop codon into ORF5 to make an extended version of its coat protein that is required for the infectivity of the virus. The model organism Saccharomyces cerevisiae was used in this study to identify and characterize host genes that influence the unconventional translational recoding event of programmed stop codon readthrough of BYDV's ORF3 stop codon. In this study, a dual luciferase reporter plasmid containing a Renilla luciferase gene directly upstream from the viral readthrough sequence proximal to the stop codon, a firefly luciferase gene located immediately downstream of the sequence, and the more distal BYDV readthrough sequence element located further downstream was used. Readthrough rates were quantified by dividing the ratio of enzyme activities of the experimental plasmid by the ratio of activities from a control plasmid that contained a sense codon in place of the viral stop codon. This study sought to confirm that the host gene STU2 increases BYDV stop codon readthrough rates when the gene is overexpressed in yeast. STU2, which encodes a microtubule-binding protein, had been previously shown to increase Tobacco mosaic virus (TMV) stop codon readthrough rates when overexpressed and assayed in yeast. Preliminary studies with BYDV had also indicated that STU2 could increase readthrough rates of its leaky stop signal. This thesis presents data from dual luciferase assays that show a five-fold increase in BYDV readthrough rates when STU2 is overexpressed compared to the control, suggesting STU2 plays a general role in affecting termination efficiency. Yeast cells with overexpressed STU2 were analyzed for a secondary phenotype associated with a translation mutation: sensitivity to osmotic pressure caused by ethylene glycol. No apparent difference in sensitivity to ethylene glycol was observed between yeast with and without overexpressed STU2. In a search for other yeast genes performed previous to this thesis, a dual lacZ-luciferase reporter plasmid identified increased readthrough rates caused by an overexpressed yeast genomic library plasmid. The gene YHR213W-A was subcloned from the library plasmid to test for its effects on BYDV readthrough rates. It was hypothesized that YHR213W-A would cause an increase of BYDV's ORF3 stop codon readthrough when overexpressed, however these results were not obtained. Lastly, the hypothesis that two genes independently responsible for causing increased readthrough rates would cause an increase of BYDV readthrough when simultaneously overexpressed in yeast could not be tested due to time constraints. This study was funded in part by an HHMI Undergraduate Research Program grant.
Bisphenol A Enhances ER Recruitment to Estrogen-Responsive Genes in MCF-7 Breast Cancer Cells
Susanna Holley Wegner
(Advisor: McClellan)
Bisphenol A (BPA) is a synthetic estrogen mimic with detrimental health effects in humans and other vertebrates following very low levels of exposure. Though BPA can be rapidly metabolized, the Center for Disease Control found detectable levels of BPA in the urine of 95% of the Americans tested. Based upon in vivo experiments, BPA is believed to exert physiological effects through interactions with the estrogen receptor (ER). However, no direct evidence proves that BPA influences endogenous ER activity at relevant genes in the cell. This thesis reports in vivo association of ER with estrogen target genes following exposure to BPA. In a chromatin immunoprecipitation assay analyzed with quantitative real time PCR, nanomolar concentrations of BPA dramatically enhanced ER recruitment to estrogen-responsive genes in a dose-dependent and gene-specific manner. The effect of BPA relative to estrogen proved to be variable among assays, but BPA consistently had a dramatic effect relative to controls. This study was funded in part by an HHMI Undergraduate Research Program grant.
One Plant, One Pollinator: Morphology Makes a Marriage
Jessica Youngberg
(Advisor: Karoly)
Nectar spurs are a recognizable but little understood floral trait that has been thought to lead to the ability of flowering plant taxa to exploit a range of niches in pollination. Spurs often hold nectar far from the flower opening and allow a flower to adapt to the precise anatomy of a pollinator, resulting in enhanced pollen transfer and even reproductive isolation. This exploitation of niches is associated with diversification providing a rapid increase in species number over a very short period of time. This explosion of species is called adaptive radiation.
This thesis attempts to define a nectar spur and then assess the variability and prevalence of the trait on the scale of flowering plant orders worldwide. The resulting phylogeny shows that nectar spurs have multiple origins all over the angiosperm phylogeny, even multiple origins within the same order or even family, indicating that there are many possible structural and developmental routes to spur evolution. The definition of a spur is likely a reliable one, and the information gathered on spur ratios and distribution suggests that a more detailed study of the world's flora is in order.
In order to assess the role of nectar spurs in pollinator specialization, I performed a quantitative analysis of observational data to compare pollination diversity and specialization across three floral morphologies (nectar spurs, tubular flowers and those without an isolated nectary), based on two different definitions of pollinator specialization. In a large scale assessment of an Illinois community (N=271), flowers with nectar spurs specialize just as frequently as flowers with tubes (36-72% versus 44-79%), and unrestricted flowers had the lowest specialization frequencies (28-49%). A smaller assessment of a community in Colorado (N=16) does not show as significant results, but the pattern of nectar spurs correlated with increased specialization remains. Because increased specialization leads to greater reproductive isolation and speciation, we can identify nectar spurs as being a trait that can unlock a previously unavailable niche and thus contribute to adaptive radiation.