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  • Undergraduate Poster Abstracts
  • Genetics

    FRI-637 POPULATION GENETICS DURING A MANGROVE RANGE EXPANSION

    • Jazmine Hernandez ;
    • Emily Dangremond ;
    • Jeremie Fant ;

    FRI-637

    POPULATION GENETICS DURING A MANGROVE RANGE EXPANSION

    Jazmine Hernandez1, Emily Dangremond2, Jeremie Fant1.

    1Chicago Botanic Garden, Glencoe, IL, 2Smithsonian Environmental Research Center, Smithsonian Institution, Edgewater, MD.

    Mangrove forests are diverse ecosystems that offer coastal protection and stability as well as shelter and food for both marine and terrestrial species. Mangrove trees grow in tropical coastal areas. The geographic range of the black mangrove Avicennia germinans spans from northern Florida to southern Brazil. Due to decreasing freezes in northern Florida, A. germinans is experiencing an expansion at its northern range limit in Florida. We hypothesized that A. germinans would have less genetic diversity at the northern range limit than more southern populations, and that seed dispersal would be higher than pollen dispersal due to the water-dispersed seeds. Leaf tissue was collected from 30 individuals at 4 different populations along the east coast of Florida. Nine neutral microsatellite markers were used to measure genetic diversity, gene flow, and inbreeding in nuclear DNA. Chloroplast DNA was sequenced from a subsample of individuals to measure haplotype diversity and seed-to-pollen flow ratios. We found no significant differences in genetic diversity (He) among populations, but the populations at the range edge were moderately isolated from populations further south in Florida (pairwise Fst = 0.124 - 0.158). Preliminary results show haplotype diversity to be lower in Florida (Hd = 0.70) than in Panama (Hd = 0.84). During its ongoing range expansion, A. germinans does not appear to be experiencing a loss of genetic diversity at its range edge.

    THU-636 THE GENETIC REGULATION OF ARSENITE OXIDASE

    • Benjamin Sanchez-Sedillo ;
    • Chad Saltikov ;

    THU-636

    THE GENETIC REGULATION OF ARSENITE OXIDASE

    Benjamin Sanchez-Sedillo1, Chad Saltikov2.

    1The University of New Mexico, Albuquerque, NM, 2University of California, Santa Cruz, Santa Cruz, CA.

    Microbes are known to contribute to biogeochemical cycling of arsenic, being able to interconvert arsenite and arsenate. While arsenate reduction is linked to high levels of arsenic in the environment, arsenite oxidizing microbes help to alleviate this problem. Although there are chemoautotrophs capable of aresnite oxidation, photosynthetic bacteria have recently been identified that can couple anoxygenic photosynthesis to arsenic oxidation. The 2 component regulatory system, arsenite oxidase, is a protein used by some of these bacteria in anaerobic conditions to oxidize arsenite, thereby detoxifying their environment while supplying necessary energy requirements. The regulation of this gene is directly involved in the arsenic biogeochemical cycle in which such bacteria play a role. Ectothiorhodospira sp. str. BSL- 9, was isolated from Big Soda Lake in Nevada, an alkaline, hyper-saline lake which is high in arsenic. The genes (arx) for photosynthetic arsenite oxidation have been identified in the recently completed genome. The metabolism of arsenite, though, has yet to be described on a genetic level. By comparing a gene induced by arsenite, such as arxA, to genes encoding normal cellular function, such as recA and gyrB, which are constantly being expressed, we can determine how these organisms respond to different environmental conditions. We hypothesize that by changing environmental conditions such as arsenite concentrations, light availability, and nutrient availability, we will see the expression of arxA being upregulated in the purple sulfur bacteria Ectothiorhodospira sp. str. BSL- 9. This study’s aim is to understand the environmental conditions which help promote arsenite oxidizing bacteria, thereby helping remediate toxic environments.

    THU-648 THE ROLE OF EXTRADENTICLE AND HOMOTHORAX IN SPECIFYING PARHYALE HAWAIENSIS APPENDAGE IDENTITY

    • Panda Kreidler ;
    • Erin Jarvis ;
    • Nipam Patel ;

    THU-648

    THE ROLE OF EXTRADENTICLE AND HOMOTHORAX IN SPECIFYING PARHYALE HAWAIENSIS APPENDAGE IDENTITY

    Panda Kreidler, Erin Jarvis, Nipam Patel.

    University of California, Berkeley, Berkeley, CA.

    Hox genes are a family of evolutionarily conserved genes that play a major role in animal body plan development and are largely responsible for determining segment identity along the anterior-posterior axis. They achieve this by producing proteins that bind to the regulatory regions of target genes responsible for the morphological structures in each segment, essentially acting as an on-off switch. A long-debated question is how Hox proteins are capable of binding to very specific regions of DNA, as they alone do not have such high binding specificity. Hox proteins often work in tandem with other DNA-binding proteins, such as those produced by the extradenticle (exd) and homothorax (hth) genes. These genes function codependently, and are referred to here as exd-hth. In Drosophila, exd-hth mutants display antenna-to-leg homeotic transformations, suggesting that exd-hth may cooperatively bind with certain Hox proteins to confer a given segmental identity. Exd-hth is also expressed proximally in many appendage types and mutations in these genes can result in loss of proximodistal specification. Exd-hth mRNA expression has been analyzed in the crustacean Parhyale hawaiensis, although a more thorough examination of their function is needed. This study explores the role of exd-hth in P. hawaiensis by determining expression via in situ hybridization and exploring function via CRISPR/Cas9-mediated knockout. We know exd-hth is expressed proximally in many Parhyale limbs, as seen in other arthropods. Based on this conserved expression, we predict that eliminating exd-hth function will result in altered proximodistal identity and homeotic transformation of Hox-exd-hth associated limbs.

    FRI-646 CHARACTERIZING THE PHENOTYPE OF TALEN ENGINEERED SLC26A4 MUTANT ZEBRAFISH

    • Ashley Scott ;
    • Alaa Koleilat ;
    • Lisa Schimmenti ;

    FRI-646

    CHARACTERIZING THE PHENOTYPE OF TALEN ENGINEERED SLC26A4 MUTANT ZEBRAFISH

    Ashley Scott1, Alaa Koleilat2, Lisa Schimmenti2.

    1University of Maryland Baltimore County, Baltimore, MD, 2University of Minnesota, Minneapolis, MN.

    Mutations in slc26a4 are associated with Pendred syndrome, a disorder characterized by bilateral sensorineural hearing loss and occasionally accompanied by thyroid goiter. Patients with Pendred syndrome may have an enlarged vestibular aqueduct, a malformation of the temporal bone. We have created mutations in slc26a4 via transcription-activator-like effector nucleases (TALEN) to mimic mutations found in humans and further characterize phenotypic changes using zebrafish. We hypothesize that the homozygous recessive mutant fish will respond less frequently, if at all, to sound stimuli and that the heterozygous mutants will not display an abnormal phenotype. In order to test for deafness, a hearing assay was performed in which fish were exposed to a 400 Hz sound. This frequency was chosen as it can specifically stimulate the inner ear and not the lateral line. Bright field imaging was conducted to look at differences in inner ear morphology. In order to quantitatively study the differences between mutant and wild-type ears, measurements of the otolith and outer-ear area were taken and standardized to trunk length. Preliminary results show that neither homozygous recessive mutants nor heterozygous mutants showed a change in hearing, indicating that this specific mutation is not deleterious enough to cause deafness. Furthermore, bright field imaging and measurements showed that heterozygous mutants and homozygous recessive mutants have significantly smaller otoliths and ears on average when compared to their wild-type counterparts. Based on this trend we can conclude that an abnormal protein Pendrin, is causing structural ear abnormalities in these mutant zebrafish.

    FRI-648 ROLE OF TEASHIRT AND TIPTOP IN THE REGULATION OF HOX GENES IN THE MARINE CRUSTACEAN PARHYALE HAWAIENSIS

    • Christian Garrido ;
    • Erin Jarvis ;
    • Heather Bruce ;
    • Nipam Patel ;

    FRI-648

    ROLE OF TEASHIRT AND TIPTOP IN THE REGULATION OF HOX GENES IN THE MARINE CRUSTACEAN PARHYALE HAWAIENSIS

    Christian Garrido1, Erin Jarvis2, Heather Bruce2, Nipam Patel2.

    1California State Polytechnic University, Pomona, Pomona, CA, 2University of California, Berkeley, Berkeley, CA.

    Hox genes are characterized by their role in anterior-posterior body plan formation, specifying appendage development and identity. Arthropods are useful models for studying segment morphology because of the obvious changes displayed when particular mutations have occurred. Several Hox genes have been identified as having a significant role in the production of various appendages (e.g., legs, claws, and feeding appendages) in the crustacean species Parhyale hawaiensis. However, little is known about the different regulatory factors that drive Hox genes. Teashirt (tsh) is a non-Hox gene, which contains 3 zinc-finger motifs and is known to distinguish homeotic function in trunk segment diversity in Drosophila. Tiptop (tio) is considered a paralogue of tsh, sharing an acidic N-terminal domain and additional zinc-finger motif. While other evidence has suggested teashirt family genes are not conserved within insects, we seek to determine if tsh and tio may have coregulatory function in Parhyale. We refer to tsh and tio as tiotsh, a possible single teashirt family related homolog found in Parhyale. By using the emerging genomic editing technology CRISPR/Cas9, tiotsh can be knocked out in Parhyale by specifically targeting the sequence, cutting it, and rendering the gene no longer functional. Hox transcription following tiotsh knockout can be visualized using in situ hybridization. The loss-of-function phenotype can be visualized using light microscopy methods. We present our preliminary data on identifying tiotsh zinc-finger domains in the Parhyale transcriptome. Understanding the role of tiotsh as a possible regulatory Hox cofactor in Parhyale will ultimately allow insight into body plan evolution.

    THU-646 MATRIX STIFFENING INDUCES ENDOTHELIAL DYSFUNCTION IN PULMONARY ARTERIAL HYPERTENSION

    • Thaius Boyd ;
    • Paul Dieffenbach ;
    • Laura Fredenburgh ;
    • Anna Coronata ;

    THU-646

    MATRIX STIFFENING INDUCES ENDOTHELIAL DYSFUNCTION IN PULMONARY ARTERIAL HYPERTENSION

    Thaius Boyd1, Paul Dieffenbach2, Laura Fredenburgh2, Anna Coronata2. Christina Mallarino-Haeger2.

    1Haskell Indian Nations University, Lawrence, KS, 2Brigham and Women's Hospital, Boston, MA.

    Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive breathlessness, worsening hypoxemia, and right-heart failure. Pathologically, PAH leads to vascular remodeling of pulmonary arteries. Our lab has shown vessel stiffening occurs early in PAH and worsens as the diseases progresses. To understand the effect of stiffness on vascular cell phenotypes, we examined pulmonary artery endothelial cells (PAECs) in vitro on discrete matrix stiffnesses recapitulating normal and remodeled vessels. We subsequently disrupted mechanical signaling by siRNA knockdown of YAP and TAZ, key regulators of mechanotransduction, and tested for alterations in remodeling phenotypes. By preventing remodeling through disruption of mechanical signaling, we hope to identify new therapeutic targets in PAH. Human primary PAECs were plated onto discrete-stiffness polyacrylamide gels and assessed for cell proliferation, cell toxicity, gene expression, and cytoskeletal organization. SiRNA transfection was then used to knockdown YAP and TAZ, after which cells were assessed for YAP cellular localization, cell proliferation, cell toxicity, and gene expression. Results show stiff matrix conditions result in increased cell proliferation and YAP/TAZ activity. In contrast, YAP/TAZ knockdown shows reduced cell proliferation, increased cell death, and increased expression of COX-2, an important inhibitor of vascular remodeling. We conclude that matrix stiffness mimicking pathologically remodeled vessels leads to increased cellular proliferation and YAP/TAZ mechanical signaling in PAECs. Reducing YAP and TAZ gene expression reverses these phenotypic changes in stiff matrix and induces genes associated with normal vascular homeostasis. These results suggest that the YAP/TAZ pathway may be an interesting target for future PAH therapy.

    FRI-636 FUNCTIONAL ANALYSIS OF GENE THERAPY FOR MUSCULAR DYSTROPHY

    • Lara Roach ;
    • Christophe Pichavant ;
    • Michele Calos ;

    FRI-636

    FUNCTIONAL ANALYSIS OF GENE THERAPY FOR MUSCULAR DYSTROPHY

    Lara Roach1, Christophe Pichavant2, Michele Calos2.

    1University of California, Los Angeles, Los Angeles, CA, 2Stanford University, Stanford, CA.

    Duchenne muscular dystrophy (DMD) is the most severe muscular dystrophy, characterized by the absence of the protein dystrophin. DMD causes muscle degeneration and loss of ambulation, resulting in death from respiratory or cardiac failure. Currently, there is no effective long-term treatment for DMD, although many pharmaceutical, cell, and gene therapy methods have been proposed. The current approaches have been plagued by poor delivery, immunogenicity, and/or lack of effectiveness. To address these limitations, we developed a novel non-viral gene therapy technique involving vascular fluid-push delivery of plasmid DNA and genomic integration mediated by phiC31 integrase. To test the functional efficacy of the proposed therapy, a treadmill test protocol was developed. Preliminary data indicated a significant decrease in average time to exhaustion in dystrophin-deficient mice, compared to wild-type controls. These results validated the treadmill protocol that will be used to functionally analyze the effectiveness of our non-viral gene therapy technique. We also examined the times to exhaustion on the treadmill of mouse models for two other forms of muscular dystrophy. Using the fluid-push vascular delivery procedure, we injected plasmid DNA coding dystrophin in DMD model mice, and treated animals will be analyzed on the treadmill. This gene therapy treatment offers a potential alternative to viral or cell therapies for muscular dystrophy and may significantly impact the lives of muscular dystrophy patients.

    FRI-645 THE INVOLVEMENT OF THE MYOGENIC GENE NAUTILUS IN MUSCLE DEVELOPMENT

    • Jonathon Cordova ;
    • Richard Cripps ;

    FRI-645

    THE INVOLVEMENT OF THE MYOGENIC GENE NAUTILUS IN MUSCLE DEVELOPMENT

    Jonathon Cordova, Richard Cripps.

    The University of New Mexico, Albuquerque, NM.

    In vertebrates, MyoD belongs to a family of myogenic regulatory factors (MRFs) that are essential for muscle differentiation. In Drosophila, the only MyoD homolog, nautilus, plays a role in the differentiation of progenitor cells into mature muscle fibers. However, there is controversy over the requirement for MyoD in this process. To help resolve this controversy, we investigated the requirement and sufficiency of nautilus to promote muscle development. Individuals homozygous for nautilus mutations were examined at stage 16 of embryonic development so that the nautilus mutant phenotype could be characterized. These mutant embryos exhibited slight to severe alterations in a subset of muscles, indicating that a deficiency of nautilus leads to poor muscle development. To visualize the importance of nautilus for myogenic conversion, we transfected a line of Drosophila cells with cDNA expressing nautilus, daughterless, and Mef2, which allowed us to observe myogenic conversion of the cells. Our objective was 2-fold: to determine if nautilus is able to initiate myogenic conversion in tissue culture by itself or when coexpressed with other basic-helix-loop-helix (bHLH) or MADS-domain box transcription factors and to better understand how these transcription factors interact with one another to help decipher the overall mechanism of muscle differentiation in Drosophila. In preliminary data, we found that the expression of nautilus, daughterless, and Mef2 was sufficient to induce myogenesis. This supports a basic understanding of how orthologous genes for muscle development function in various organisms.

    FRI-647 APOE, IRON AND AMYLOID-B INTERACTIONS WITH BRAIN MICROGLIA

    • Anna Roland ;
    • Ryan McCarthy ;
    • Jack Sanford ;
    • Marianne Wessling-Resnick ;

    FRI-647

    APOE, IRON AND AMYLOID-B INTERACTIONS WITH BRAIN MICROGLIA

    Anna Roland1, Ryan McCarthy2, Jack Sanford2, Marianne Wessling-Resnick2.

    1Trinity Washington University, Washington, DC, 2Harvard T. H. Chan School of Public Health, Boston, MA.

    Alzheimer’s disease is the most common form of dementia, affecting the lives of 5 million Americans. This neurodegenerative disease is associated with the presence of amyloid plaques in the brain. Amyloid-β (Aβ) is normally cleared by microglia cells, which become activated and phagocytose Aβ oligomers. When activated, microglia produce nitric oxide, cytokines, and other pro-inflammatory mediators. Chronic microglial activation promotes neuronal cell damage and death by these mediators. Under these conditions, a decrease in Aβ receptors also occurs and plaque formation is enhanced. A role for both iron and apolipoprotein E (ApoE) in Aβ interactions with microglia has been proposed, and these interactions may help to limit their activation and alter Aβ clearance. While iron binds to Aβ, its possible interactions with ApoE remain to be characterized. To study ApoE iron binding and Aβ-induced microglia activation, this project had 3 goals: express and purify recombinant ApoE, use isothermal calorimetry (ITC) to examine iron binding to ApoE, and examine the effects of Aβ, iron, and ApoE on M1 activation of immortalized microglia (IMG) cells using the Griess assay to detect NO production. Our results showed that ApoE was successfully inserted into a bacterial plasmid and that iron has an affinity to ApoE, causing an exothermic reaction. Further, we concluded that Aβ induces a pro-inflammatory response that is exacerbated by iron, but that an ApoE iron complex quenches this pro-inflammatory response.

    THU-637 RAI1 MUTANT MICE EXHIBIT SMITH-MAGENIS SYNDROME PHENOTYPES

    • Nalini Rao ;
    • Katherina Walz ;

    THU-637

    RAI1 MUTANT MICE EXHIBIT SMITH-MAGENIS SYNDROME PHENOTYPES

    Nalini Rao1, Katherina Walz2,3.

    1University of Miami, Coral Gables, FL, 2The Dr. John T. Macdonald Foundation, University of Miami, Miller School of Medicine, Miami, FL, 3John P. Hussman Institute for Human Genomics, University of Miami, Miller School of Medicine, Miami, FL.

    Mutations on chromosome 17 result in Smith-Magenis syndrome (SMS), a neurodevelopmental disorder. About 1 in 25,000 people worldwide suffer from SMS as a result of mutations within 17p11.2, however, because of phenotypic similarity to autism, the true prevalence may be closer to 1 in 15,000. Smith-Magenis syndrome is characterized by intellectual disability, delayed motor and speech functions, craniofacial abnormalities, sleep disturbances, and social behavior abnormalities. The retinoic acid induced 1(RAI1) gene has been associated with clinical manifestations of SMS. Thirty percent of SMS patients have mutations within the RAI1 gene. We hypothesize that a single gene mutation in RAI1 causes behavioral characteristics of SMS to manifest in mice. Social behavior of normal and RAI1 mutant mice were assessed using 5 behavioral assays looking at levels of social interest, obsessive compulsive tendencies, dominance in social situations, and social interaction. Our results suggest that RAI1 mutant mice were significantly different from normal mice, especially in social behavior, showing significant lacks in social interest and social stimuli, obsessive compulsive tendencies, and submissive behavior. These differences among the social tendencies and behaviors of the RAI1 mutant mice and wild-type mice are important because it shows that a mutation in RAI1 gene is sufficient for abnormal social behavior, but not other SMS symptoms. These results may implicate other gene mutations as the basis of the remaining SMS phenotypes.

    THU-645 INVESTIGATION OF THE MOLECULAR MECHANISMS THAT MEDIATE NEURAL CIRCUIT FORMATION

    • Vivianna Mas ;
    • Aruna Varshney ;
    • Raakhee Shankar ;
    • Katie Watters ;

    THU-645

    INVESTIGATION OF THE MOLECULAR MECHANISMS THAT MEDIATE NEURAL CIRCUIT FORMATION

    Vivianna Mas, Aruna Varshney, Raakhee Shankar, Katie Watters.

    San Jose State University, San Jose, CA.

    Our nervous system is a network of neuronal circuits that mediate perception, behavior, and thought. To form circuits, neurons must identify their correct synaptic partners among the many neurites in a target region and assemble synapses. Elucidating the molecular mechanisms neurons employ to identify the correct partners and build synapses may aid in understanding neurological disorders such as autism. To study synaptic partner recognition, we use a transgenic trans-synaptic marker called Neuroligin 1-mediated GFP reconstitution across synaptic partners (NLG-1 GRASP), which fluorescently labels synapses between specific neurons in live animals. Using this marker, our group previously found that the UNC-6/Netrin ligand and its receptor UNC-40/DCC regulate synaptic partner recognition between PHB sensory neurons and AVA interneurons in C. elegans. We hypothesize that additional molecules act downstream of these recognition molecules to target synaptic components. To identify these molecules, our group has tested a panel of synaptogenesis mutants. We found that the presynaptic organizer unc-69/SCOCO is required for normal synapse formation between PHB and AVA neurons. Our genetic analysis also indicates that unc-69/SCOCO acts with unc-6/Netrin and unc-40/DCC in PHB-to-AVA synapse formation. In unc-69 loss-of-function mutants, the region of contact between PHB sensory neurons and AVA interneurons is also reduced; however, work with other mutants suggests that the remaining contact is sufficient for normal synaptogenesis. In conclusion, we have discovered a gene that acts in the unc-6/Netrin synapse formation pathway. Our ultimate goal is to elucidate the entire pathway from recognition of synaptic partners to synaptic assembly and maintenance.

    THU-647 EVALUATING A DROSOPHILA MODEL SYSTEM TO DETERMINE PROTEIN INTERACTIONS WITH WERNER EXONUCLEASE

    • Imad Eddine Rebiai ;
    • Angel Aguilar ;
    • Elyse Bolterstein ;

    THU-647

    EVALUATING A DROSOPHILA MODEL SYSTEM TO DETERMINE PROTEIN INTERACTIONS WITH WERNER EXONUCLEASE

    Imad Eddine Rebiai, Angel Aguilar, Elyse Bolterstein.

    Northeastern Illinois University, Chicago, IL.

    Every day, our DNA is subjected to damage which, if not repaired properly, can lead to mutations that cause aging and cancer. In humans, deficiencies in the DNA repair proteins Werner (WRN) and Bloom (Blm) are associated with Werner syndrome and Bloom syndrome, which are diseases characterized by accelerated aging and high susceptibility to cancer, respectively. Previously, we have shown that flies mutant in Blm and the Drosophila homolog of WRN, WRNexo, have hatching defects and are sensitive to the fork-stalling drug hydroxyurea, suggesting that these proteins interact when responding to stress during DNA replication. Currently, we are investigating a possible physical interaction between WRNexo and Blm. We designed a transgenic WRNexo mutant in which WRNexo is fused to green florescent protein (GFP) and have confirmed by western blot that the WRNexo-GFP fusion protein is expressed. Next, we will determine if the WRNexo-GFP fusion protein is functional by testing hatching frequency and hydroxyurea sensitivity. We predict that the WRNexo-GFP fusion protein will be functional in these flies, and they will not exhibit defects associated with WRNexo deletion mutants. We will then use antibodies specific to GFP in a co-immunoprecipitation assay to pull down the WRNexo-GFP fusion protein along with any other proteins that are attached to it. Finally, we will use mass spectrometry analysis of the protein complex to identify bound proteins. We predict that Blm will be bound to WRNexo. Demonstrating the physical interactions of these proteins will yield further insights into the molecular bases of aging and cancer predisposition.

    THU-639 IDENTIFICATION AND CHARACTERIZATION OF THE NOVEL TUMOR SUPPRESSOR GENE GONAD-14 IN C.BRIGGSAE MULTIVULVA MUTANTS

    • Jennifer Patritti Cram ;
    • Helen Chamberlin ;

    THU-639

    IDENTIFICATION AND CHARACTERIZATION OF THE NOVEL TUMOR SUPPRESSOR GENE GONAD-14 IN C.BRIGGSAE MULTIVULVA MUTANTS

    Jennifer Patritti Cram, Helen Chamberlin.

    The Ohio State University, Columbus, OH.

    Cells make decisions to divide based on the activity of intracellular proteins and extracellular signals. Tumor suppressor genes normally prevent inappropriate cell division, and loss-of-function mutations in these genes are associated with cancer. This project uses the nematodes C. elegans and C. briggsae as systems to identify tumor suppressor genes. Our research aims to characterize the signaling features of C. briggsae tumor-suppressor-like mutant gon-14. We have shown that these mutants exhibit increased cell division during development of the egg-laying structures. This is a system in which signals from non-epithelial cells, including LIN-3/EGF and the LIN-12/Notch pathway promote vulva fate. Mutants that exhibit the multivulva phenotype are important because they exhibit inappropriate division of distal vulva precursor cells and therefore identify tumor suppressor genes. Our lab has identified new C. briggsae mutants that exhibit the Muv phenotype and, in preliminary results, we have found that one of these genes is Cbr-gon-14. In C. elegans, gon-14 encodes a nuclear protein that influences gonad development, but has only a modest impact on vulva development. Thus, we have identified a novel tumor suppressor gene that influences cell division differently in the distinct genetic backgrounds. To better understand this gene, we will evaluate the expression of LAG-2, a ligand of the LIN-12/Notch pathway. We will also test whether the excess cell division is dependent on LIN-12/Notch. Finally, we will assess the time and location that gon-14 is required by determining the temperature-sensitive period for the gon-14 gene in C. briggsae.

    FRI-644 MAXIMIZING DNA RETENTION IN NEXT GEN LIBRARY PREP

    • Courtney Knauer ;
    • Christopher Bird ;

    FRI-644

    MAXIMIZING DNA RETENTION IN NEXT GEN LIBRARY PREP

    Courtney Knauer, Christopher Bird.

    Texas A&M University-Corpus Christi, Corpus Christi, TX.

    One challenge in next generation sequencing is minimizing the loss of DNA during library preparation. If these losses can be minimized, then very small quantities of DNA can be used. Paramagnetic beads are almost universally employed in library preparations in order to purify reactions and size-select DNA fragments because they are inexpensive and effective. However, paramagnetic beads have also been connected to the loss of DNA. In order to maximize the retention of DNA, we identified the factors within a paramagnetic bead clean-up that could contribute to DNA losses. A fully factorial design was employed, and the following factors were manipulated: DNA concentration, NaCl PEG concentration, bead brand, elution buffer, elution volume, and number of elutions. Gel electrophoresis was used to quantify the concentration and size range of DNA recovered from the beads (retained), supernatant (loss), and reaction tube (loss). The factors most strongly tied to the loss of DNA were the concentration of DNA, elution number, and elution volume. These results indicate that the number of paramagnetic beads in a reaction relative to the DNA concentration is the single greatest source of DNA loss in library preparation. The number of beads in a clean-up reaction is not considered in any protocol that we know of, and the concentrations of DNA (3, 30 ng/uL) are well within ranges typically used. By using appropriate ratios of the number of beads to the concentration of DNA and multiple elutions, it is possible to substantially increase the retention of DNA in paramagnetic bead clean-ups.

    FRI-639 GENETIC CHARACTERIZATION OF INTEGRONS FROM ENVIRONMENTAL AEROMONAS BACTERIA

    • Josselyn Pena ;
    • Luis Mota-Bravo ;
    • Andrey Tatarenkov ;

    FRI-639

    GENETIC CHARACTERIZATION OF INTEGRONS FROM ENVIRONMENTAL AEROMONAS BACTERIA

    Josselyn Pena, Luis Mota-Bravo, Andrey Tatarenkov.

    University of California, Irvine, Irvine, CA.

    Antibiotic resistance is a growing concern for public health. While the immediate concern is the resistance found in clinical settings, it is also important to study the spread of these genes in the environment. Integrons are genetic units able to capture and incorporate antibiotic resistant genes known as gene cassettes through site-specific recombination. The objective of this study was to characterize and describe the integrons found in environmental bacteria belonging to the genus Aeromonas. Water samples were taken from streams and beaches in Southern California. Bacterial DNA was isolated and PCR amplification was conducted with primers described in the literature and designed by our laboratory to screen the isolates for the presence of integrons. PCR products were sequenced and then analyzed for gene composition. Eight out of 19 (42%) total Aeromonas isolates contained integrons, which ranged from 1,833 to 3,574 base pairs in length. The species containing integrons were A. culicicola and A. caviae. All integrons discovered belong to class 1. The most common gene cassettes contained dfrA12 and aadA2 genes, which code for trimethoprim and aminoglycoside resistance, respectively. Other gene cassettes included dfrA17,aadB, and dfrA15. This is the first report of the gene dfrA15 in Aeromonas suggesting that environmental Aeromonas are acquiring new resistance genes, perhaps due to physical proximity of diverse bacteria in natural habitats. The presence of integrons suggests that the environment is a formidable source for the spread of antibiotic resistance genes through horizontal gene transfer.

    THU-644 DEFINING THE ROLE OF THE NOVEL PROTEIN CG1674 IN ADULT MUSCLE DEVELOPMENT

    • Marilyn Cisneros ;
    • Richard Cripps ;

    THU-644

    DEFINING THE ROLE OF THE NOVEL PROTEIN CG1674 IN ADULT MUSCLE DEVELOPMENT

    Marilyn Cisneros, Richard Cripps.

    The University of New Mexico, Albuquerque, NM.

    In Drosophila, the study of muscle formation and structure can provide a better understanding of the factors that trigger muscle disease in humans. Our current goal is to determine if the protein CG1674 is a component of the sarcomere and if it is required for muscle structure and function. CG1674 was discovered through proteome sequencing of dissected flight muscles. This sequencing data suggests that this protein is a functional component of the flight muscles of Drosophila. We are currently generating a CG1674-GFP fusion protein to determine the localization of our protein within the muscle cell. In parallel, we are determining the requirement for CG1674 in normal muscle formation by expressing in the flight muscles an RNAi targeting the CG1674 transcript. When crossing UAS-CG1674 RNAi with the drivers Mef2Gal4 and 1151Gal4, the flies are flightless. Immunohistochemical staining of the muscles reveals defects in myofibril formation and structure indicating that the presence of the CG1674 protein is required for sarcomere assembly. In addition to creating our CG1674–GFP fusion construct, we are working on creating a knockout using the CRISPR method. The results will indicate the impact this protein has on flight muscles when it is absent. These studies identify a potentially novel muscle protein that is required for normal muscle assembly and function.

    FRI-638 SET-4 PROMOTES DAUER ARREST IN C. ELEGANS INSULIN SIGNALING MUTANTS

    • Jacqueline Graniel ;
    • Colin Delaney ;
    • Patrick Hu ;

    FRI-638

    SET-4 PROMOTES DAUER ARREST IN C. ELEGANS INSULIN SIGNALING MUTANTS

    Jacqueline Graniel, Colin Delaney, Patrick Hu.

    University of Michigan, Ann Arbor, MI.

    Insulin-like growth factor signaling (IIS) is a conserved pathway that regulates development, metabolism, and longevity in metazoans. Caenorhabditis elegans experience an alternative developmental stage called dauer that allows them to endure harsh environments. Mutations that reduce IIS activity cause animals to enter dauer constitutively in a manner requiring the transcription factor DAF-16/FOXO. DAF-16/FOXO function is antagonized by DAF-2/IIS signaling through AKT-mediated phosphorylation, leading to its nuclear export. When AKT activity is reduced, DAF-16/FOXO translocates to the nucleus, where it is inhibited by EAK-7/TLDC1, a conserved protein of unknown function. While eak-7 and akt-1 single mutants do not have strong dauer-constitutive phenotypes, eak-7;akt-1 double mutants always arrest as dauers. Previously, we conducted a forward genetic screen to discover novel regulators of DAF-16/FOXO by mutagenizing eak-7;akt-1 animals, looking for rare progeny that did not arrest as dauers. Mapping and whole genome sequencing revealed that one mutant strain harbored a mutation in set-4, which encodes a conserved histone H4 lysine 20 methyltransferase. We hypothesize that SET-4 promotes dauer specifically through the IIS pathway. To test how SET-4 promotes dauer arrest, we created set-4 compound mutants with mutants from known dauer regulatory pathways. Here we show that SET-4 promotes dauer arrest though the IIS pathway but not through the TGF-β and steroid hormone pathways. We also show that integrating an extra copy of wild-type set-4 rescues the dauer phenotype in set-4 null animals. Future work will study the effect of SET-4 over-expression on lifespan as well as deduce mechanistically how SET-4 promotes DAF-16/FOXO activity.

    THU-638 DETERMINING THE OUTCOME OF OVEREXPRESSING SUCROSE PHOSPHATE SYNTHASE AND GLUTAMINE SYNTHETASE, KEY ENZYMES IN C AND N METABOLISM, AT THE PHYSIOLOGICAL, BIOCHEMICAL AND MOLECULAR LEVELS IN TRANSGENIC ALFALFA

    • Karen Acosta ;
    • Amanda Peel ;
    • Jose Ortega-Carranza ;
    • Champa Gopalan ;

    THU-638

    DETERMINING THE OUTCOME OF OVEREXPRESSING SUCROSE PHOSPHATE SYNTHASE AND GLUTAMINE SYNTHETASE, KEY ENZYMES IN C AND N METABOLISM, AT THE PHYSIOLOGICAL, BIOCHEMICAL AND MOLECULAR LEVELS IN TRANSGENIC ALFALFA

    Karen Acosta, Amanda Peel, Jose Ortega-Carranza, Champa Gopalan.

    New Mexico State University, Las Cruces, NM.

    Plant growth and performance is largely determined by the ability of a plant to use carbon (C) and nitrogen (N). The C and N metabolic pathways closely interact with each other. The goal of this project is to determine the consequences of manipulating individual pathways by transgenic methods in alfalfa plants. We have selected sucrose phosphate synthase (SPS) and glutamine synthetase (GS) as the 2 key enzymes in C and N metabolism, respectively, as targets for manipulation. While SPS catalyzes the synthesis of sucrose, GS produces glutamine from glutamate and ammonia. Alfalfa, like all leguminous plants, is capable of fixing atmospheric N2 by forming a symbiotic relationship with N2-fixing bacteria (Rhizobia). A result of this association is the formation of an organ, the root nodule, where the bacteria reside and fixes N2, and the ammonia produced is assimilated by GS. The assimilated ammonia, in the form of glutamine, is exported and used to make all N containing macromolecules. Photosynthate, in the form of sucrose, is exported to the nodule and used for both N2-fixation and ammonia assimilation. We have produced 2 sets of alfalfa transformants, 1 containing the SPS gene and the other a GS gene, both driven by a constitutive promoter. The 2 sets of transformants have tested positive for the functionality of the transgenes. We will present the data on growth, photosynthetic rates, chlorophyll content, nodule number, and function. Furthermore, we will analyze the expression of other key genes in C and N metabolism in leaves and nodules.