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1 Florida Genetics 2013 Organizing Committee Chair: Michele Tennant Members: Charlie Baer, Chris Chase, Sixue Chen, Marty Cohn, John Davis, Julie Johnson, Thomas Yang Codified III Liaison : Connie Mulligan Poster Judges Akito Kawahara, Jim Maruniak, Lauren McIntyre, David Oppenheimer, Rolf Renne, Jianping Wang FG2013 Sponsors Platinum Level Department of Molecular Genetics and Microbiology; Department of Pathology, Immunology and Laboratory Medicine; Interdisciplinary Center for Biotechnology Research; University of Florida Genetics Institute Gold Level Center for Epigenetics; Center for Pharmacogenomics; Clinical and Translational Science Institute; College of Liberal Arts and Sci ences; Florida Agricultural Experiment Station Silver Level Emerging Pathogens Institute; Health Science Center Libraries Bronze Level Animal Molecular and Cellular Biology Graduate Program; Plant Molecular and Cellular Biology Program Special Thanks To Cornelia Frazier, Hope Parmeter, Matthew Daley Megan Kimmel Van Rysdam, Rolando Milian, Nikunj Syngal, Joseph Kelly, Connie Philebaum, Bret Boyd, Tamar Carter, Ana Carolina Costa S Dasharath Dixit, Lauren Douma, Justin Fear, Terry Felderhoff, Ruli Gao, Natya Hans, Lara Ianov, Heather Rose Kates, Lana McMillan, Katie O'Shaughnessy, Marcio Resende, Justyna Resztak, Yong Shen, Andrew Smith, Jared Stees Jessica Stone, Ming Tang, Solaleh Tusi, Dorianmarie Vargas, Yuanqing Yan Codified III Directors: Richard Heipp and Logan Marconi The exhibit in conjunction with the Florida Genetics 2013 symposium. The collaboration is now in its third year and the exhibit explores themes of genetics by UF SAAH students and alumni. Works of art from Codified Explore will be unveiled at 2:30, October 9 th during the FG2013 poster session and reception.
2 20 13 Florida Genetics Symposium Schedule Wednesday, October 9, 2013 12:00 p.m. 1:00 p.m.: Check-in and poster set-up at the CGRC posters no 158 1:00 p.m. 1:15 p.m., Opening Remarks: Patrick J. Concannon* Session I Translation of Discovery Chair: John M. Davis* *** 1:15 p.m. 2:00 p.m., Elaine A. Ostrander : Good dogs with bad genes: informing human health and biology by studying domestic dogs 2:00 p.m. 2:30 p.m., Gary F. Peter* : Conifer terpenes: manipulating an ancient plant defense pathway for production of renewable chemicals and biofuels 2:30 p.m. 4:30 p.m.: Poster Session I, Unveiling of Codified III, and Reception: Posters no. 158 **5:00 p.m. 6:00 p.m., William E. Evans: Genomic medicine: using genome variation to individualize cancer treatment (Introduction by Julie A. Johnson* ) *= UF Genetics Institute Faculty **All activities will be at the Cancer/Genetics Research Complex except the presentation by Dr. Evans, which will be at 5:00 p.m. Wednesday in the auditorium of the HPNP building on the Health Science Center campus. ***Drs. Ostrander and Huffaker were unable to participate due to the government shutdown. Please see the attached one-page sheet for the final slate of speakers. Thursday, October 10, 2013 8 :3 0 a.m. 9:00 a.m.: Checkin coffee, set up posters no. 59 -116 Session II Evolutionary Genetics Chair: Charlie Baer* 9:00 a.m. 9:45 a.m., Michael Lynch: 9:45 a.m. 10:15 a.m., Michael M. Miyamoto*: Scombroid fishes provide novel insights into the trait/rate associations of molecular evolution 10:15 a.m. 11:00 a.m., Gnter P. Wagner: Genetics and genomics of cell type evolution: the human decidual cell 11:00 a.m. 11:30 a.m., Stuart F. McDaniel*: "The genetics of sterility in populations of Ceratodon purpureus 11:30 a.m. 1:15 p.m.: Poster Session II and Lunch: Posters no. 59-116 Session III: Metabolomics Chair: Sixue Chen* 1:15 p.m. 2:00 p.m., Gerard Karsenty: The contribution of bone to whole organism physiology 2:00 p.m. 2:30 p.m., Arthur S. Edison: New approaches in metabolomics 2:30 p.m. 3: 15 p.m., Clint Chapple: Genetic analysis of phenylpropanoid metabolism in Arabidopsis *** 3:15 p.m. 4:00 p.m., Alisa Huffaker: Metabolism of the maize innate immune responses against insects and pathogens 4:00 p.m. 4:15 p.m.: Best Poster Awards
3 Presentation Abstracts (Listed in order of presentation, external speakers followed by internal speakers) Good dogs with bad genes: informing human health and biology by studying domestic dogs Ostrander EA Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD Domestic dogs represent closed breeding populations produced as a result of differential selection for traits associated with both behavior and appearance. This practice, coupled with small numbers of founders for many breeds has generated a population that is ideal for mapping genes underlying morphology, behavior, and disease susceptibility. Today we will update recent advances in the canine genome project and current approaches for finding genes controlling both simple and complex traits. We will discuss, specifically, genes controlling body morphology and disease susceptibility and consider ways in which the statistical methods developed for mapping morphologic traits can be used to find disease genes of interest for both human and canine health. Biography of Elaine A. Ostrander, PhD Dr. Elaine Ostrander is Chief of the Cancer Genetics Branch at the National Human Genome Research Institute (NHGRI) of NIH. She also heads the Section of Comparative Genetics. Dr. Ostrander received her PhD from the Oregon Health Sciences University (OHSU), and did her postdoctoral training at Harvard. She then went to UC Berkeley and the Lawrence Berkeley National Labs where, with collaborators, she began the canine genome project, initiating work on the canine map. She was at the Fred Hutchinson Cancer Research Center and University of Washington for 12 years, rising to the rank of Member in the Human Biology and Clinical Research Divisions, and Head of the Genetics Program where she led the canine genome project and studied the genetics of huma n breast and prostate cancer. She moved to NIH in 2004, where she took over as head of the Cancer Genetics Branch at NHGRI. Dr. including diseases such as cancer, and morphologic features such as body size, leg length and skull shape. Dr. Ostrander has published over 275 papers and articles, and is currently leading a team of postdocs and students on projects aimed at the mapping of several canine disease genes, finding genes regulating canine morphology, and understanding the population dynamics of modern domestic breeds. She has won multiple awards including the American Cancer Society Junior Faculty Award (1995), Burroughs Welcome Award for Functional Genomics (2001), OHSU Alumnus of the Year (2002), Asa Mays Excellence in Canine Health Research Award (2005), NIH Distinguished Investigator (2011), and the Genetics Society of America Medal (2013). Genomic medicine: using genome variation to individualize cancer tr eatment Evans WE Research Hospital, Memphis, TN The promise of genomic medicine has received great attention over the past decade, projecting how genomics will soon guide th e prevention, diagnosis, and treatment of many human diseases. However, this evolution has been slower than forecast, even where evidence is often strong (e.g., pharmacogenomics). Reasons include the requirement for institutional resources, the fractionated healthcare system in the US and inertia in overcoming barriers that are impeding health-care changes. This lecture will illustrate how inherited and acquired genome variation has been deployed to advance the treatment of childhood leukemia, a process that bega n 30 years ago and continues today. Examples of somatic and inherited genome variations that can influence the effects of cancer chemotherapy will be discussed, including SNPs, copy number alterations, focal deletions, and epigenetic changes that alter the sensitivity of cancer cells to specific anticancer agents (e.g., Holleman et al., 2004, N Engl J M ed 351(6):53342 ; Cheng et al. 2005, Nat Gen 37(8):87882 ; Diouf et al. 2011, Nat Med 17(10):1298-303). This presentation will also address ongoing strategies for identifying genome variations that alter drug response, including GWAS and whole genome sequencing (e.g. The St. Jude Washington University Pediatric Cancer Genome Project; Downing et al., 2012, Nat Genet 44(6):61922). Pharmacogenomic principles that apply to other human diseases will also be discussed.
4 Biography of William E. Evans, PharmD Dr. William E. Evans is Director and Chief Executive Officer of St. Jude Children's Research Hospital (SJCRH), and is a Professor of Pediatrics and Pharmacy at the University of Tennessee Colleges of Medicine and Pharmacy. For the past 36 years, his research at St. Jude has focused on the pharmacogenomics of anticancer agents in children, for which he has received three consecutive NIH MERIT Awards from the National Cancer Institute (1987-2015). The major disease focus of his pharmacogenomics research is acute lymphoblastic leukemia in children. He is a member of the Board of Trustees of Rhodes College and the Board of Memphis Tomorrow, and recently completed service on the Board of Scientific Counselors for the US National Cancer Institute of NIH. Dr. Evans has authored over 300 articles and numerous book chapters. He has received several national awards for his research, including the 2009 Pediatric Oncology Award from the American Society of Clinical Oncology (shared with Mary V. Relling of SJCRH), the 2009 Team Science Prize from the American Association for Cancer Research (shared with colleagues at St. Jude), the 2012 Remington Medal from the American Pharmacists Association and the 2013 Oscar B. Hunter Award from the American Society for Clinical Pharmacology and Therapeutics. He is recognized by the Institute for Scientific Information pharmacology, based on citations of his research publications. He was elected to the Institute of Medicine of the National Academy of Sciences in 2002. Dr. Evans has served as CEO of SJCRH since 2004, during which time St. Jude has been ranked annually in the top 10 best places to work in academia by The Scientist mag Parents Magazine (2009) and US News and World Report (2010), and listed by Fortune magazine among the top 100 companies to work for in the US (each year since 2011). St. Jude is the only NCI-designated comprehensive cancer center devoted solely to children. Mutation, drift, and the origin of subcellular features Lynch M Department of Biology, Indiana University, Bloomington, IN Achieving an understanding of the mechanisms of evolution requires information on the ways in which the population-genetic environment varies across phylogenetic lineages. The three major determinants of what selection can/cannot accomplish (mutation, recombination, and random genetic drift) have rates that vary by a few orders of magnitude across lineages in a particular manner. Thus, we should not be surprised that the paths of evolutionary change in different domains of life have been radically different. The drift-barrier hypothesis implies that natural selection pushes the refinement of molecular/cellular features to a level of perfection dictated by the power of drift. This fundamental constraint on the evolutionary process will be shown to apply to a diversity of previously unconnected observations: variation in error rates at the level of replication and transcription; the performance of metabolic enzymes; and the stability and multimeric structure of proteins. Biography of Michael Lynch, PhD Dr. nisms of evolution, with a recent concentration on issues at the genomic and cellular levels, and the development of methods for population-genomic analysis. The lab focuses on a number of model systems, most notably the microcrustacean Daphnia the ciliat e Paramecium and numerous other unicellular species. Currently a member of the Genome, Cell, and Developmental Biology faculty at Indiana University, he has previously held positions at the University of Illinois and the University of Oregon. Dr. Lynch is a member of the US National Academy of Sciences and of the American Academy of Arts and Sciences. He is current president of the Genetics Society of America, and past president of the Society for the Study of Evolution, the Society for Molecular Biology and Evolution, and the American Genetics Association. Genetics and genomics of cell type evolution: the human decidual cell Wagner GP Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT The overall goal of genetics and molecular evolution is to understand how organismal features and their variation within and between species are caused by differences in the genome. While there has been substantial progress in understanding the structure of the genome, the evolution of protein coding genes as well as cis -regulatory elements, and RNA, the explanation of genuinely
5 biological features is facing considerable challenges. Here I will argue that the evolution of novel cell types is the most likely place where major progress will be possible in the near future. Cell types are the basic building blocks of organismal complexity, at l east in the case of animals and plants. They are also the lowest level of complexity that presents many of the problems we face when we try to explain organismal diversity: the fact that cell types, unlike genes and proteins, are only indirectly transmitted from generation to generation or are direct translations of nucleotide sequences. Based on recent progress in cell biology I will propose a model of cell type identity that can be used as a guide to the study of cell type evolution. In this model cell type identity is caused by the activation of core-regulatory network which integrates external signals into a cell types specific response via the activation of effector genes. This model suggests that novel cell types arise through the modification of ancestral core regulatory networks. I will explore this idea by reporting on our current understanding of the evolution of a key cell type of placental mammals, the decidual cell. The decidual cell is part of the uterine lining and is critical for the establishment of pregnancy in all mammals that have invasive placentation, including humans. Our results suggest that there are at least two key events in the origin of the decidual cell: a modification of the core regulatory network by the recruitment of the Foxo1 and the origin of transcription factor cooperativity between Foxo1 and other the members of the core regulatory network. Biography of Gnter P. Wagner, PhD Dr Gnter P. Wagner is the Alison Richard Professor of Ecology and Evolutionary Biology at Yale University and a founding member of the Yale Systems Biology Institute, as well as Adjunct Professor of Obstetrics and Gynecology at Wayne State University. H e re ceived his initial training in biochemical engineering at the College for Chemical Industry in Vienna, Austria, and a PhD in Zoology from the University of Vienna. He did postdoctoral work at the Max-Planck-Institutes for Biophysical Chemistry in Gttingen and the Institute for Developmental Biology in Tbingen as well as the Department of Anatomy at the University of Gttingen, Germany. Dr current research on endometrial stromal cells was initiated as a model for the evolutionary origin of a major novelty in the phylogeny of mammals. Dr. Wagner has received the MacArthur Fellowship in 1992, was elected fellow of the American Association for the Advancement of Science, as well as the American Academy of Arts and Sciences and is a foreign associate member of the Austria n Academy of Sciences. The contribution of bone to whole organism physiology Karsenty G Department of Genetics and Development Columbia University Medical Center, New York, NY The mouse genetic revolution has shown repeatedly that most organs have more functions than expected. This has led to the realization that, in addition to a molecular and cellular approach, there is a need for a whole-organism study of physiology. The skeleton is an example of how a whole-organism approach to physiology can broaden the functions of a given organ, reveal connections of this organ with others such as the brain, pancreas and gut, and shed new light on the pathogenesis of degenerative diseases affecting multiple organs. Biography of Gerard Karsenty, MD, PhD Dr. Gerard Karsenty is the Paul A. Marks, MD, Professor, and chair of the Department of Genetics and Development at the Columbia University Medical Center. Dr. Karsenty received his MD and PhD from the University of Paris, France and completed his postdoctoral training at the University of Texas MD Anderson Cancer Center in 1990. His laboratory has studied every aspect of skeletal biology ranging from cell differentiation to function. His laboratory has studied all aspects of skeletal biology ranging fro m development to physiology. After having identified the master gene of bone formation and unravel ed the overall transcriptional mechanism leading to osteoblast differentiation his laboratory has focused its effort on skeletal physiology with the goal to link the skeleton to other organs. The overarching assumption of his work is that the appearance of bone during evolution has changed profoundly the physiology of animals because of the energetic cost that bone growth entails. His laboratory explored through genetic and molecular means every aspect of this hypothesis. This has resulted in the demonstration that bone via the hormone osteocalcin, regulates insulin secretion, energy metabolism, male fertility and cognitive functions. This has also led to the realization that osteocalcin is a molecular mediator of the maternal influence on brain development and function in the offspring.
6 Genetic analysis of phenylpropanoid metabolism in Arabidopsis Chapple C Department of Biochemistry Purdue University, West Lafayette, IN The products of the phenylpropanoid pathway, all of which are derived from phenylalanine, range from complex, insoluble polymers such as lignin and suberin, to soluble flavonoids and hydroxycinnamate esters, to volatile compounds used to attract pollinators. Among these diverse products, by far the most abundant in terms of carbon allocation is the complex heteropolymer lignin, which accounts for a significant portion of the dry weight of not only woody plants, but also of herbaceous plants like Arabidopsis. Indeed, because of its abundance in the biosphere, the lignin polymer is relevant in an ecological context because it provides a sink for over 4 X 10 11 kg of carbon annually. Lignin is found mainly in xylem, where it is important for resisting the negative pressure generated b y transpiration, and in fibers and sclerified parenchyma, where it provides the structural support required for plants to stand upright. In addition to its role in plant biology, another strong impetus behind the recent interest in lignin is that decreasing or altering lignin structure to provide increased cell wall digestibility could greatly increase the cost effectiveness of converting cell wall polysaccharides to ethanol or second-generation biofuels. Over the last ten to fifteen years, a number of experiments have greatly expanded our understanding of the phenylpropanoid biosynthetic pathway, such that now at least the catalog of enzymes and biochemical reactions in the pathway appears complete Particularly important in contributing to this knowledge has been the phenotypic analysis of plants in which phenylpropanoid metabolism is perturbed as a result of mutation or RNAi-mediated down-regulation of genes encoding pathway enzymes. Whereas the morphological impact of these particular manipulations of the pathway are relatively mild, defects at a number of other biosynthetic steps leads to a common suite of pleiotropic phenotypes such as dwarfing and sterility, the severity of which is dependent on the strength of the metabolic restriction. Although an understanding of the dwarfing and developmental arrest exhibited by many phenylpropanoid-deficient mutants is essential for determining the feasibility of future bioengineering approaches to decrease lignin, it is still unclear how these phenotypes arise. Consistent with the enormous commitment of energy and carbon it requires, the synthesis of lignin is under tight regulatory control. The picture emerging from recent work is that this control is effected by a complex, hierarchically organized network of transcription factors, many of them MYB and NAC domain-containing proteins, in which proteins near the top of the regulatory cascade act to regulate the expression of other transcription factors, while those located at the most downstream points in the network regulate the transcription of the phenylpropanoid biosynthetic enzymes directly. We previously identified several dominant mutants containing point mutations in the previously uncharacterized gene At2g48110 all of which contain lower levels of soluble phenylpropanoids and lignin, concomitant with a number of other phenotypes, including dwarfing and a pale seed coat commonly seen in plants in which phenylpropanoid pathway genes are mutated or down-regulated. As in several other mutants we have characterized, the decreased levels of the phenylpropanoid-derived UV protectant sinapoylmalate results in a reduced epidermal fluorescence ( ref ) phenotype, and we have named this mutant ref4 More recently we have obtained multiple lines of additional supporting evidence that the REF4 protein and its paralog, REF4-related 1 (RFR1), play a role in the suppression of phenylpropanoid biosynthesis in wild-type plants. REF4 and RFR1 have recently been shown to be components of Mediator, a large multi-protein complex that facilitates interactions between DNA-bound transcription factors and RNA polymerase II to activate or repress the expression of downstream genes. Mutants of Arabidopsis that lack REF4 and RFR1 hyperaccumulate phenylpropoanoids, are viable and show little in the way of developmental changes, making them a tractable system in which to examine the function of Mediator in plants and its role in phenylpropanoid homeostasis. Biography of Clint Chapple, PhD Dr. Clint Chapple is a Distinguished Professor in the Department of Biochemistry at Purdue University. He earned his BSc and MSc in botany and PhD in chemistry from the University of Guelph, in Guelph, Ontario Canada. He then conducted his post-doctoral work at the MSU-DOE Plant Research Laboratory in East Lansing, MI from 1990-1993. In 1993 he research focuses on the analysis and manipulation of the phenylpropanoid pathway in plants, with particular emphasis on Arabidopsis, using the tools of biochemistry, molecular biology and genetics.
7 Metabolism of the maize innate immune responses against insects and pathogens Huffaker A 1,2 Sims J 1 Christensen S 1 Kaplan F 3 Schmelz E 1 1 USDA-ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville FL 2 Plant Pathology Department, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL Like all plants, maize encounters attack by a diversity of pests and pathogens in the field and responds with a complex and multilayered defense response. The innate immune response in maize draws upon a full arsenal of defense chemicals. Benzoxazinoid hydroxamic acids were identified in the 1950s as phytoanticipin factors for resistance against both Fusarium rot and leaf feeding by Ostrinia nubilalis Recently the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-Glc (HDMBOA-Glc) was discovered to act as a phytoalexin, accumulating in response to both pathogen infection and herbivory. HDMBOA-Glc is highly reactive and seems to have multiple functions, capable of acting as an antimicrobial agent, a negative effector of pathogenicity, and a toxin to herbivores. Terpenoids are another well-characterized family of maize inducible defense chemicals, and have been associated primarily with indirect defenses against herbivores. Insect attack elicits production and emission of a suite of maize volatiles including terpenes that mediate tritrophic interactions by attracting parasitoids and predators that are the natural enemies of maize herbivores. Many of these volatile terpenes have antimicrobial activity and also contribute to pathogen resistance. We recently discovered two new families of non-volatile terpenoid phytoalexins that contribute to disease resistance in maize; the diterpenoid kauralexins and the sesquiterpenoid zealexins. Upon microbial attack, both families accumulate at the plant-pathogen interface and inhibit fungal growth. We are currently working to understand biosynthesis of these terpenoid phytoalexins as well as uncover the molecular mechanisms regulating production of maize secondary defense metabolites in response to pathogens and pests. By better understanding this concerted immune response, we hope to develop strategies for making maize plants more resistant to disease and herbivory. Biography of Alisa Huffaker, PhD Dr. Alisa Huffaker is a plant physiologist with the Chemistry Research Unit of the USDA-ARS Center for Medical, Agricultural and Veterinary Entomology (CMAVE) located on the University of Florida campus. She received her BS in Microbiology in 1999 and her PhD in Molecular Plant Sciences in 2005, both from Washington State University. Her graduate work was performed in the laboratory of Dr. Clarence A. Ryan at the Institute of Biological Chemistry and focused on isolation and characterization of a new plant peptide hormone, Arabidopsis Plant Elicitor Peptide 1 (AtPep1). She demonstrated that AtPep1 is a signal regulating innate immunity in Arabidopsis thaliana and that transgenic manipulation of AtPep1 gene expression can confer disease resistance. During three years of postother members of the AtPep gene family and worked to characterize specificity, breadth and overlap of responses regulated by each family member. Since joining CMAVE in late 2008, metabolism in response to biotic stress. She has discovered gene orthologs to AtPep1 in maize and over 50 plant species, and has demonstrated that these orthologs are regulators of anti-pathogen and herbivore defenses in diverse crop species. The Huffaker laboratory is working to understand the role of Peps in resistance to both pathogens and herbivores and to elucidate novel downstream signaling components. The laboratory is also focused on regulation and biosynthesis of maize defense metabolites. In conjunction with other scientists at CMAVE, two new families of maize terpenoid phytoalexins produced in response to fungal pathogens and stem-boring herbivores were recently identified, the zealexins and kauralexins. A current focus is delineation of zealexin and kauralexin biosynthesis and relevant regulatory mechanisms. Dr. Huffaker is presently active in mentoring research at the undergraduate level and has directed several undergraduate thesis projects of University of Florida students. Two have been awarded research fellowships; from the American Society of Plant Biology Summer Undergraduate Research Fellows program and the University of Florida Howard Hughes Medical Institute Science for Life undergraduate research program.
8 *= UF Genetics Institute Faculty Conifer terpenes: manipulating an ancient plant defense pathway for production of renewable chemicals and biofuels Peter GF 1,2, *, Westbrook J 1,2 Dervinis C 1,2 Davis JM 1,2, *, Kirby J 3 Rottmann W 4 Sykes R 5 Davis M 5 Wang Y 1 ,2 Keasling J 3 1 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Department of Bioengineering, University of California, Berkeley, CA 4 ArborGen, ArborGen, Ridgeville, SC 5 National Renewable Energy Laboratory, Golden, CO Terpenes are one of the most diverse sets of natural compounds with uses as industrial chemicals, biofuels, flavoring agents, fragrances and pharmaceuticals. Terpenes are important secondary metabolites involved in land plant defense to herbivores. Conifer trees synthesize and accumulate large amounts of monoterpenes and diterpenes in a specialized resin canal or duct system. In resin canals, terpenes are synthesized via a high flux plastidic pathway that is strongly conserved with bacteria. With the goal of increasing terpene synthesis and accumulation in the wood of conifers, we are 1) identifying regulators of resin canal differentiation and terpene biosynthesis, and 2) increasing carbon flux to terpenes by overexpressing rate limiting pathway enzymes and novel enzymes that shunt alternative precursors into the pathway. Scombroid fishes provide novel insights into the trait/rate associations of molecular evolution Qiu F 1 Kitchen A 2 Burleigh JG 1, *, Miyamoto MM 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Anthropology, University of Iowa, Iowa City, IA The study of which life history traits affect molecular evolutionary rates is often confounded by the covariance of these traits. Scombroid fishes are unusual in that their mass-specific metabolic rate is positively associated with body size. This study exploits this atypical pattern of trait variation to distinguish between mass-specific metabolic rate and body size as predictors of molecular ev olutionary rates. We inferred a phylogenetic tree for scombroids from a supermatrix of available molecular and morphological data. We then used new statistical approaches to assess the associations of body size and mass-specific metabolic rate with substitution rate in a phylogenetic context. As predicted by the body size hypothesis, there is a negative linear correlation between body size and substitution rate. However, we also find a negative bivariate association between mass-specific metabolic and substitution rates. The negative correlation between body size and substitution rate persists when controlling for metabolic rate. Conversely, no association remains between mass-specific metabolic and substitution rates when body size is held constant. These relationships are supported by analyses of the total molecular data, separate mitochondrial and nuclear DNA datasets, and individual genes, and they are robust to phylogenetic uncertainty. The molecular evolutionary rates of scombroids are affected b y body size and/or one or more of its covariates, and not by mass-specific metabolic rate. This study demonstrates that groups with novel patterns of trait variation can be particularly informative for identifying which life history traits are associated with rates of molecular evolution. The genetics of sterility in populations of Ceratodon purpureus McDaniel SF* Department of Biology, University of Florida, Gainesville, FL One of the central problems in modern genetics is identifying the genes that contribute to phenotypes of interest in natural populations. This effort may be complicated if the effects of allelic substitutions are population specific, have antagonistic effects on
9 other fitness-correlated traits, or depend upon environmental conditions. Here I document extensive genetic variation in meiotic spore viability, presumably an important component of fitness, in two populations of the moss Ceratodon purpureus I then evaluate the contribution of three allelic variants (a phytochrome presence-absence polymorphism, a putative chromosomal inversion, and the sex chromosomes) to variation in the outcome of meiosis. I report evidence that all loci are significantly associated with variation in spore viability, but that the strength and mechanism of this association varied among populations. In particular, pleiotropy and both population and sex-specific epistatic effects may be frequent. My lab is now undertaking genome-wide association studies of spore viability to interrogate the genetic architecture of the outcome of meiosis and evaluate the generality of these findings regarding the complexity of the genotype-phenotype map for sterility. New approaches in metabolomics Edison AS Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL Southeast Center for Integrated Metabolomics, University of Florida, Gainesville, FL The ultimate goal of global metabolomics is the universal detection, identification, and quantification of all the metabolites in a cohort. This is currently impossible. In this talk I will summarize recent developments and technology to improve the situation and show applications to metabolomics investigations of the nematode Caenorhabditis elegans
10 Poster Sessions *= UF Genetics Institute Faculty. Presenters are underlined. Poster Session I, Posters no. 1 58 Wednesday, October 9, 2:30 p.m. 4:30 p.m. 1. Demographic events shaping evolutionary history and genome-wide genetic diversity of Pinus taeda and Pinus elliottii Acosta JJ 1 Neves LG 2 Fahrenkrog A 2 Davis JM 1,2, *, Holliday JA 3 Kirst M 1,2, 1 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Department of Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and State University, Blacksburg, VA Clues of evolutionary history can be revealed by studying patterns of allelic variation, defined as DNA signatures. We applied DNA sequence-capture to analyze the genetic diversity and to understand demographic events that shaped the evolutionary history of the loblolly and slash pine genomes. Haploid DNA from megagametophytes was extracted from seeds of 24 trees of each species, collected from native populations in 11 states representing their natural range. For genotyping, libraries were prepared by sequence-capture of a small fraction (0.03%) of their genome representing 14,729 genes. Six multiplexed pools, of eight barcoded DNA samples each, were hybridized to complementary oligonucleotide probes. Captured molecules were sequenced using the Illumina HiSeq 2000 platform. Sequence reads were aligned using MOSAIK, and more than 65,000 SNPs were identified using FREEBAYES. No signs of population differentiation were found after running STRUCTURE. The proportion of similarity between the total number of genes captured on P. taeda and P. elliottii was high (0.989), which indicates that interspecific sequence capture in related species is very efficient. Genome-wide observed population genetic parameters were calculated using DNASAM and coalescent simulations were made assuming three demographic models (bottleneck, growth and neutral equilibrium). Using Approximate Bayesian Computation techniques, posterior probabilities for each demographic model given the observed population data were calculated. Our simulation results combined with observed population genetic parameters indicate that both species are undergoing population size expansion after a bottleneck event. 2. Microarray analysis of genes expressed in the developing urethral tube Armfield BA 1 Seifert A 1 Zheng Z 1 Merton EM 1 Lopez C 1 Baker HV 1 Cohn MJ 1-3 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL Identification of the molecular mechanisms of urethra development is vital for understanding congenital malformations of the lower urinary tract and for assembling the pathways that control external genital development. Hypospadias, for example, results from an incomplete closure of the urethral tube; however the etiology of this structural defect is unknown. Although a small number of genes have been implicated in external genital development, the regulatory networks that control urethral development are poorly understood. In order to discover new regulators of urethral tube formation, we performed a transcriptional profiling experiment aimed at identifying genes expressed specifically in the urethral epithelium. Transgenic mice expressing GFP and eYFP under the control of Shh fluoresce in the urethral plate epithelium. Genital tubercles were dissected at E12.5 and ShhGFP; eYFP cells were purified using FACS. cRNA from the urethral cells were hybridized using the Affymetrix Mouse Genome 430 2.0 GeneChip and data were analyzed with BRB ArrayTools. In situ hybridizations were conducted to validate the results. Eighty-four genes were identified that were expressed higher in the urethral plate epithelium relative to mesenchyme plus ectoderm In situ hybridization confirmed that the majority of genes identified as urethral markers are indeed expressed in urethral epithelial cells. Our data provide a tissue specific analysis of urethra development during a critical period of genitourinary morphogenesis. We identified numerous genes that were previously unknown to be expressed in the developing urethra. These results provide new molecular tools for investigating gene function during urethral development
11 3. Optimization of the capsid of recombinant adenoassociated virus 2 vectors: the final threshold? Aslanidi GV 1 Rivers AE 2 Ortiz L 1 Ling C 1 Van Vliet K 3 Govindasamy L 3 Agbandje-McKenna M 3, *, Srivastava A 1, 1 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL 2 Division of Hematology/Oncology, Department of Pediatrics, University of Illinois, Chicago, IL 3 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL Recombinant adeno-associated virus (AAV) vectors are currently in use in a number of Phase I/II clinical trials as delivery vehicles to target a variety of tissues to achieve sustained expression of therapeutic genes. We previously reported that substitution of several critical tyrosine (Y) and serine (S) and threonine (T) residues on the AAV2 capsid significantly increase the transduction efficiency of the wildtype (WT) AAV2 vectors by preventing capsid phosphorylation and subsequent proteasomal degradation. In the present studies, the most critical Y, S, and T mutations were combined on a single AAV2 capsid, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ~25-fold over the WT AAV2 vector in a murine hepatocyte in vitro and in vivo Next, we examined whether mutations on AAV2 capsid augmented the transduction efficiency by increasing nuclear transport of the quadruple-mutant vector. Indeed, only ~45% of the genomes from the WT AAV2 vectors were detected in the nuclear fraction 48 hrs post-infection and ~90% of the vector genomes from the quadruple-mutant vectors were detected in the nuclear fraction at the same timepoint. Although we identified a novel quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy the current studies raise the question of whether further optimization of AAV2 vectors by targeting surfaceexposed amino acid residues involved in capsid phosphorylation is feasible. The various combinations of surface residue modifications clearly showed that there is an optimal combination to achieve maximal augmentation. 4. Analysis of imprinted genes with developmental functions in the maize seed Bai F 1,2 Bagadion A 1,2 Chamala S 1,2 Davenport R 1,2 Tseung CW 1,2 Evans MM 3 Barbazuk WB 2,4, *, Settles AM 1,2, 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 4 Department of Biology, University of Florida, Gainesville, FL Genomic imprinting in plants is an epigenetic phenomenon by which a subset of genes is expressed in a parentof -origin dependent manner. Imprinted gene expression primarily occurs in the endosperm and is thought to influence seed size and embryo development. Although many maize imprinted genes have been identified through transcriptome analysis, imprinted genes with developmental functions in the maize seed have not been identified. We screened 178 rough endosperm ( rgh ) mutants for parentof -origin effects using reciprocal crosses to inbred parents. Six maternal rough endosperm ( mre ) and three paternal rough endosperm ( pre ) mutants were identified: mre1 mre*21, mre*40, mre*-217, mre*1014, and mre*-1147 as well as pre*58, pre*-144 and pre*-949. When inherited from the female parent, mre seeds show a rough, etched, or pitted endosperm surface as well as a reduced seed size and weight. The pre mutants show the converse inheritance pattern with pre pollen conferring a seed phenotype after fertilizing wildtype ovules. Preliminary characterization of the mre and pre isolates shows a range of seed defects with several mutants showing embryo defects in addition to the endosperm phenotype. Molecular mapping experiments have identified one locus, mre1 on chromosome 4, and current progress on mapping additional mre and pre mutants will be reported. 5. Assembly and validation of an evolutionary reference genome for flowering plants ( Amborella trichopoda ) Chamala S 1 Walts B 1 Chanderbali A 1 Albert V 2 Ayyampalayam S 3 Burnette J 4,5 dePamphilis C 6-8 Der J 6-8 Estill J 3 Lan T 2 Leebens-Mack J 3 Lee S 9 Ma H 6-8 Moore R 10 Palmer J 11 Ralph P 6-8 Rice D 11 Rounsley S 12,13 Schuster S 14 Tomsho L 14 Wessler S 4,5 Wing R 9,12,13 Yu Y 9 Jiao Y 6-8 Xiao N 10 Soltis DE 1,15, *, Soltis PS 15, *, Barbazuk WB 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo, NY 3 Department of Plant Biology, University of Georgia, Athens, GA 4 Department of Botany and Plant Sciences, University of California, Riverside, CA 5 Center for Plant Cell Biology, University of California, Riverside, CA 6 Intercollege Graduate Degree Program in Plant Biology, Pennsylvania State University, University Park, PA 7 Institute of Molecular Evolutionary Genetics, Pennsylvania State University, University Park, PA 8 Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 9 Arizona Genomics Institute, University of Arizona, Tucson, AZ 10 OpGen, Inc., Gaithersburg, MD 11 Department of Biology, Indiana University, Bloomington, IN 12 School of Plant Sciences, University of Arizona, Tucson, AZ 13 BIO5 Institute, University of Arizona, Tucson, AZ 14 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 15 Laboratory of Molecular Systematics and Evolutionary
12 Genetics, Florida Museum of Natural History, University of Florida, Gainesville, FL Amborella trichopoda as the sister to all other extant angiosperms, occupies a crucial phylogenetic position, and its genome sequence is therefore an important reference for comparative genomic studies across the angiosperms. A complete genome sequence of Amborella will help in understanding the evolution of key angiosperm traits and provide a baseline to examine genome organization throughout the angiosperms. We are using a whole-genome shotgun strategy to sequence the ~731-Mb Amborella genome. We assembled over 48 Gb of DNA sequence, comprising single-end (SE) 454-FLX, SE 454-FLX+, 11-kb paired-end (PE) 454-FLX, and 3-kb PE Illumina HiSeq, and 69,466 BAC-end reads. These sequences were extensively screened and filtered for organellar contaminants, short read lengths, poor quality, artificial duplicates, and chimeras. De novo sequence assembly was performed on these filtered sequence sets (~23 Gb) using Newbler. This resulted in 5,745 scaffolds totaling 706 Mb with a mean scaffold size of 123 kb and N50 size of 4.9 Mb covering over 96% of the Amborella genome. Significantly, about 90% of our assembled sequence resides within 155 scaffolds, each greater than 1.1 Mb in length. In the absence of genetic and physical maps for Amborella we employed a combination of chromosomal FISH analysis and optical mapping (OpGen) to evaluate the fidelity and the chromosomal positioning of our current scaffolds. Using this approach, our assembly continuity increased by 2X (N50 from 4.9 Mb to 9.3 Mb and N90 from 1.2 Mb to 2.9 Mb). We also evaluated the potential of this approach to serve as a surrogate to using long-range PE libraries to increase the contiguity of an assembly. 6. High resolution genotyping of Bacillus anthracis and spatiotemporal patterns of an anthrax epizootic in white-tailed deer, Odocoileus virginianus Mullins JC 1 Van Ert M 1,2 Hadfield T 2 Nikolich MP 3 Hugh-Jones ME 4 Blackburn JK 1,2 1 Spatial Epidemiology and Ecology Research Laboratory, Department of Geography, University of Florida, Gainesville FL 2 Emerging Pathogens Institute, University of Florida, Gainesville FL 3 Department of Emerging Bacterial Infections, Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 4 Department of Environmental Sciences, School of the Coast and Environment, Louisiana State University, Baton Rouge, LA Anthrax, caused by the bacterium Bacillus anthracis is a zoonotic disease enzootic in Texas. Despite a long history in human and veterinary medicine, little is understood about anthrax transmission during naturally occurring outbreaks. High resolution genotyping combined with individual level spatiotemporal analysis can provide insights into transmission pathways. Carcass locations from a large outbreak in white tailed deer, Odocoileus virginianus were analyzed for spatial clustering using K-function analysis and for space time clustering using the Mantel test and Jacquez-k. Directionality was assessed using trend surface analysis and the direction test and compared to results of high resolution genotyping. Spatial clustering results suggest that anthrax transmission events occur within limited spatial areas, with carcass locations occurring within home ranges of subsequent cases. The directionality of the outbreak corresponded to landscape features and there was agreement between the direction test and trend surface analysis. All isolates were identical on multiple locus variable number tandem repeat analysis. Four sub-genotypes were identified using single nucleotide repeat analysis. Two sub-genotypes were isolated from the carcass and soil at two locations. Areas of high transmission corresponded to higher genetic diversity. This study represents the first effort to combine genetics with individual level spatial analyses to understand anthrax outbreak dynamics 7. Elucidating activation pathways of the antioxidant response element using chemical and genomic approaches Bousquet MS, Liu Y, Luesch H Department of Medicinal Chemistry, University of Florida, Gainesville, FL The antioxidant response element (ARE) is a cis -acting enhancer sequence found upstream of many phase II detoxification enzymes, the major tools eukaryotes use to combat the oxidative damage associated with a wide variety of neurodegenerative diseases, aging, and cancer. While several activation methods have been characterized, the interplay between these pathways is poorly understood. We believe there are other major players which remain to be characterized, and that their discovery would lead to the identification of new drug targets with chemopreventative and chemotherapeutic potential. We use small natural product, cDNA, and siRNA library screens in an ARE-luciferase reporter assay to modify this pathway. Four analogues have been isolated from a marine subtropical alga with ARE-activity. Preliminary structure-activity relationship analysis has exposed the pharmacophore responsible for activating the ARE. Initial luciferase reporter data suggest that two analogues have higher potency and efficacy than the model compound, tert-butylhydroquinone. Additionally, genomic studies have revealed several genes whose proteins may be directly involved in ARE-activity. Preliminary transcriptional validation studies using RT-PCR r level appears to finely tune this highly regulated pathway. Full characterization of the small-molecule ARE-activators and genomic hits will aid in a global understanding of the eukaryotic antioxidant response, potentially revealing new drug targets for the prevention and treatment of cancer.
13 8. Intracellular symbionts of blood feeding lice converge on a common set of genes Boyd BM 1,2 Allen JM 3 Reed DL 2, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Mammalogy, Florida Museum of Natural History, University of Florida, Gainesville, FL 3 Illinois Natural History Survey, University of Illinois at UrbanaChampaign, Champaign, IL Bacteria in symbiosis with plants and animals have smaller genomes than do their free-living relatives. Their small genome size results from genome erosion during symbiosis. The beneficial endosymbiotic bacteria of insects are an extreme case, where erosion has lead to the smallest known bacterial genomes. These endosymbionts require a host for survival and are maternally inherited. The blood feeding lice of mammals possess such beneficial endosymbionts that synthesize vitamins closely related endosymbionts owing to a long-term pattern of coevolution. In contrast, different lineages of lice have very distantly related endosymbionts, each of which is more closely related to bacteria not associated with lice. Given this unique situation, we wanted to know if the genome content in these distantly related louse endosymbionts was similar or if each had a genome more similar to it closest living relative. Here we show that the endosymbionts of lice have converged on a similar set of genes and genome content. Lice present a unique opportunity to test questions about the evolution of insectbacteria symbiosis because unrelated bacteria have similar symbiotic roles in closely related species of lice that have nearly identical life history strategies. Using this powerful system we have shown that symbioses with lice can drive genome evolution to similar ends in unrelated bacteria. This suggests, at least for lice, that host association is more important than evolutionary history in determining genome content of endosymbiotic bacteria. 9. Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain Brant JO 1,2 Riva A 3, *, Resnick JL 3, *, Yang TP 1,2, 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Center for Epigenetics, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Reduced representation bisulfite sequencing (RRBS) was used to analyze high resolution DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the PraderWilli syndrome imprinting center (PWS-IC) on either the maternallyor paternally-inherited chromosome. The paternal PWS IC deletion resulted in hypermethylation of the differentially methylated CpG islands from paternally-expressed genes. The maternal deletion had little to no effect and showed methylation patterns similar to wild type animals. To analyze the effect of the PWS-IC deletion outside of the AS/PWS imprinted domain on DNA methylation, and to reduce the potential for false positives, only clusters of two or more CpG sites within 250 bases with coordinate changes in DNA methylation and with a read depth of least 30 were considered significant (q-value <0.01). Our data shows that there are CpG sites outside of the AS/PWS domain that showed changes in methylation levels in both the paternal and maternal PWS-IC deletions. For the pat PWS-IC deletion, we identified 52 such CpG sites associated with 17 loci/regions and for the mat PWSIC deletion we identified 87 CpG sites associated with 35 loci/regions. These results suggest that the PWS-IC may have effects on DNA methylation that extend beyond the imprinted AS/PWS domain. 10. Sustained AAV2/5-mediated overexpression of CRF in the central nucleus of the amygdala diminishes the dysphoric-like state associated with nicotine withdrawal in rats Qi X 1 Shan Z 2 Ji Y 1 Guerra V 1 Alexander JC 1 Ormerod BK 3, *, Bruijnzeel AW 1 1 Department of Psychiatry, University of Florida, Gainesville, FL 2 Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 3 J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL Smoking cessation leads to a dysphoric state and this increases the risk for relapse after a period of abstinence. Animal studies indicate that the dysphoric-like state associated with nicotine withdrawal is at least partly mediated by an increase in corticotropin-releasing factor (CRF) transmission in the central nucleus of the amygdala (CeA). Here we investigated whether a sustained overexpression of CRF in the CeA affects the dysphoric-like state associated with nicotine withdrawal in rats. To study brain reward function, rats were prepared with in tracranial self-stimulation (ICSS) electrodes in the medial forebrain bundle. An adeno-associated virus (AAV, pseudotype 2/5) was used to overexpress CRF or green fluorescent protein (GFP, control) in the CeA and osmotic minipumps were used to induce nicotine dependence. The AAV2/5-CRF vector induced a 40% increase in CRF protein and mRNA levels in the CeA. Administration of the nicotinic receptor antagonist mecamylamine (precipitated withdrawal) or nicotine pump removal (spontaneous withdrawal) led to elevations in ICSS thresholds. Elevations in ICSS thresholds are indicative of a dysphoric-like state. The overexpression of CRF did not affect baseline ICSS thresholds but diminished the elevations in ICSS thresholds associated with precipitated and spontaneous nicotine withdrawal. The RT-PCR analysis showed that the overexpression of CRF led to a decrease in CRF1 mRNA levels
14 and an increase in CRF2 mRNA levels in the CeA. In conclusion, the overexpression of CRF in the CeA diminishes the dysphoriclike state associated with nicotine withdrawal and this might be driven by changes in CRF1 and CRF2 receptor gene expression. 11. Spatial genetic structure, diversity, and effective size of invasive red lionfish, Pterois volitans in the Caribbean Butterfield JS 1,2 Hunter ME 2 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Southeast Ecological Science Center, US Geological Survey, Gainesville, FL The red lionfish ( Pterois volitans ) is an invasive predatory reef fish that is having significant adverse effects on coral reefs and other marine habitats. This non-indigenous invader has rapidly expanded its presence after introduction in the late 1980s along the eastern United States coast. Effects of this apex predator include substantially reducing native reef fish recruitment and outcompeting indigenous predatory fish. The result is a marked alteration in the biodiversity. Attributing to its success is the lack of predators, wide variety of prey, rapid reproduction and swift growth. It has established breeding populations in the Gulf of Mexico, Caribbean Sea, and southeastern United States. This study uses data from d-loop mitochondrial DNA sequences in untested regions, including northwestern and eastern Caribbean, and integrates it with current data. Vital to understanding current and future population boundaries and developing methods of control is the spatial genetic structure and effective population size. Spatial genetic structure gives insight into the introduction location or locations and subsequent dispersal, which likely relies heavily on ocean currents. Determining the effective population size provides understanding of how well this species can adapt to changing conditions, allowing assessment of the scope of the problem This information is essential to developing strategies for red lionfish population control and native species conservation. 12. Evolution of alternative splicing across legumes using Amborella as a reference Chamala S 1 Jackson SA 2 Schmutz J 3,4 Town CD 5 Barbazuk WB 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Crop and Soil Sciences, University of Georgi a, Athens, GA 3 Plant Genomics Program, Department of Energy Joint Genome Institute, Walnut Creek, CA 4 HudsonAlpha Institute for Biotechnology, Huntsville, AL 5 J. Craig Venter Institute, Rockville, MD Alternative splicing (AS) is the process by which a pre-mRNA can be processed into several alternate transcript isoforms. This is one of the mechanisms through which eukaryotes generate transcriptome and proteome diversity, as well as regulate protein abundance. There is increasing evidence suggesting a crucial role of AS in many essential plant functions such as photosynthesis, defense response, flowering, and cereal grain quality. Despite the important roles AS plays in angiosperms (flowering plants), the evolution and conservation of AS events across plant species is not well understood. We are interested in examining the evolutionary conservation and divergence of AS patterns in genes duplicated by independent polyploidy events during the evolutionary histories of the legumes Medicago truncatula Glycine max and Phaseolus vulgaris We will use Amborella trichopoda a basal angiosperm with no recent lineage-specific genome duplications, as an out-group for comparison. Our study first identifies AS events in the aforementioned species using public and private transcriptome data collections generated with multiple sequencing technologies. So far we successfully developed the software pipeline to identify AS events from transcriptome data collected with multiple sequencing technologies. Our current efforts focus on defining orthologous genes with OrthoMCL, and identifying AS events conserved between orthologous genes. This work will not only provide insights into the evolution of AS in legumes, but will lay the foundation for a comprehensive AS evolution study in angiosperms that will include multiple eudicot and monocot lineages. 13. Gene co-expression networks exhibit limited conservation across flowering plants Chanderbali AS 1,2 Soltis DE 1, *, Soltis PS 2, 1 Department of Biology, University of Florida, Gainesville, FL 2 Laboratory of Molecular Systematics and Evolutionary Genetics, Florida Museum of Natural History, University of Florida, Gainesville, FL Elucidating how developmental processes have been modified over time to create morphological diversity is the goal of regulatory genes are often conserved in protein function over vast expanses of evolutionary time, phenotypic diversity may have been shaped by the complex genetic networks they control. Global transcriptome profiling provides insights into these underlying gene expression networks and can therefor e be used to elucidate the evolutionary changes that shape phenotypic diversity. Here, we compare the transcriptional programs of plant tissues in species that are pivotal to understanding flowering plant evolution. Amborella trichopoda is sister to all other extant flowering plants, and is thus singularly important to elucidating the origin and evolution of angiosperms and their traits. Nuphar advena a water lily, represents the subsequent branch in flowering plant phylogeny, while Aristolochia fimbriata (pipe vine), Liriodendron tulipifera (tulip tree), and Persea americana (avocado) represent the most diverse extant basal angiosperm lineage, the magnoliids.
15 We also include a cycad, Zamia vazquezii to facilitate comparisons between angiosperms and gymnosperms. Our analyses extend from these phylogenetically pivotal non-model species to the eudicot genetic model Arabidopsis thaliana to provide a systems-level approach for evaluating the dynamics of gene expression changes accompanying angiosperm evolution. 14. A pharmacogenomic genome-wide association study (GWAS) for new onset diabetes (NOD) in the INternational VErapamil SR-Trandolapril STudy (INVEST) Chang SW 1,2 McDonough CW 1,2 Gong Y 1,2, *, Pepine CJ 3 Johnson JA 13, *, Cooper-DeHoff RM 1-3 1 Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 2 Center for Pharmacogenomics, University of Florida, Gainesville, FL 3 Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville, FL Background: We conducted a GWAS to identify genetic determinants contributing to the inter-individual variability of antihypertensive drug-related NOD in hypertensive patients with coronary artery disease from INVEST. Methods: NOD cases (n=334) and matched controls (n=806) were genotyped on the OmniExpressExome chip. Patients were randomized to either a Verapamil SR-based strategy (CCB) or an atenolol-based strategy (BB) both with hydrochlorothiazide and trandolapril as add-on therapy. NOD was defined as any new occurrence of self/physician reported diabetes mellitus (DM) or use of DM medication during follow-up (avg 2.7 yrs). Drug-gene interactions were analyzed by race/ethnicity (white and Hispanic) separately, using logistic regression, controlling for age, sex, principal components for ancestry, and treatment strategy. Between race meta-analysis was performed. Results: SLC9A4 was the strongest signal from meta-analysis (interaction p = 9.18 x 10 -7 ). A allele carriers had increased risk for NOD when treated with BB (OR=2.66 (1.64-4.33), p<0.001), but decreased risk when on CCB (OR=0.56 (0.39-0.80), p=0.002). Consistent direction was observed in whites (BB: OR=2.87 (1.53-5.37), CCB: OR=0.48 (0.28-0.82), interaction p=4.02 x 10 -5 ) and Hispanics (BB: OR=2.38 (1.10-5.15), CCB: OR=0.64 (0.40-1.03), interaction p=5.29 x 10 -3 ). Conclusion: The chromosome 2q12 locus, consisting of SLC9A4 and the interleukin 1 receptor family cluster, may be important for antihypertensive related NOD. This region is enriched with inflammatory diseases GWAS signals, suggesting inflammation may play a role in antihypertensive drug-related NOD. 15. Redox regulation of a stress hormone inducible serine/threonine protein kinase in Brassica napus Zhu M 1 Ji W 1,2 Zhang T 1 Silva-Sanchez C 3 Song W-Y 4,5, *, Harmon A 1,5, *, Chen S 1,3,5, 1 Department of Biology, University of Florida, Gainesville, FL 2 Plant Bioengineering Laboratory, College of Life Science, Northeast Agricultural University, Harbin, China 3 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 4 Plant Pathology Department, University of Florida, Gainesville, FL 5 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Protein kinase mediated phosphorylation events have been recognized as pivotal regulatory processes in stomatal function under abiotic and biotic stress conditions. A serine/threonine protein kinase belonging to the SnRK2b subfamily was identified to be ABA-responsive in B. napus guard cells. Recombinant BnSnRK2b exhibited kinase activity in a manganese-dependent manner and preferentially phosphorylated myelin basic protein -casein. Phosphorylation sites of the kinase were mapped by mass spectrometry. Interestingly, the autophosphorylation activity was inhibited by treatment of oxidants, including hydrogen peroxide, oxidized glutathione and nitric oxide donor S-nitrosoglutathione. Isotope tagging analysis revealed that specific cysteines are responsive to certain treatment and contribute to the redox responsiveness. Additionally, the recovery of the inhibited kinase activity by reductant such as dithiothreitol or thioredoxin isoforms indicated the autophosphorylation activity is reversibly regulated by redox of the microenvironment. Futhermore, the ability to phosphorylate a B. napus slow anion channel protein is also reversibly redox regulated. The BnSnRK2b is preferentially expressed in guard cells and the activity is activated by ABA and MeJA treatment. All these data demonstrate a novel redox regulatory mechanism underlying hormone response in stomatal guard cells. 16. Aberrantly activated AREG EGFR signaling is required for the growth and survival of CRTC1 MAML2 fusion-positive mucoepidermoid carcinoma cells Chen Z 1,2 Chen J 3 Gu Y 1,2 Hu C 1,2 Li J-L 4 Lin S 1,2 Shen H 5 Cao C 2,6 Gao R 2,6 ,7 Li J 8 Ha PK 9 Kaye FJ 2,6, *, Griffin JD 2 Wu L 1,2, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 University of Florida Shands Cancer Center, Gainesville, FL 3 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 4 Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL
16 5 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China 6 Department of Medicine, University of Florida, Gainesville, FL 7 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 8 Teaching Hospital of Fujian Medical University, Fujian Province Tumor Hospital, Fuzhou, Fujian, China 9 Department of Otolaryngology Head and Neck Surgery, Johns Hopkins University, Baltimore, MD Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1 MAML2 fusion oncogene, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a frequent genetic alteration found in >50% of mucoepidermoid carcinomas (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1 MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1 MAML2 fusion gene and its downstream AREG EGFR signaling in human MEC cancer cell growth and survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1 MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and in vivo The CRTC1 MAML2 oncoprotein induced the upregulation of the epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) by co-activating the transcription factor CREB, and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1 MA ML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG EGFR signaling is required for CRTC1 MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapie s will benefit patients with advanced, unresectable CRTC1 MAML2-positive MEC. 17. -associated herpesvirus (KSHV) latencyassociated genes down-regulate TGF-beta signaling by inducing cellular miRNAs Choi HS 1 Jain V 1 Whitby D 2 Renne R 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Viral Oncology Section, AIDS and Cancer Virus Program, SAICFrederick, NCI-Frederick, Frederick, MD Aberrant miRNA expression contributes to the development of many types of cancer. KSHV is a DNA tumor virus that causes of latent genes are expressed. We found that KSHV infection regulates host gene expression, and hypothesized that latent genes also modulate the expression of host miRNAs. Arraybased miRNA profiling revealed that all six miRNAs of the oncogeneic miR-17/92 cluster are up-regulated in KSHV infected endothelial cells. Among the latent genes, vFLIP and vCyclin were shown to activate the miR-17/92 promoter. MiR17/92 was previously shown to target TGF-beta signaling. We demonstrate that vFLIP and vCyclin induce the expression of the miR-17/92 to strongly inhibit TGF-beta by down-regulating SMAD2. Moreover, TGF-beta activity and SMAD2 expressio n were fully restored when miR-17/92 antagomirs (inhibitors) were transfected in cells expressing either vFLIP or vCyclin. In addition, we utilized viral genetics and produced vFLIP or vCyclin knock-out viruses and infected endothelial cells. While single-knock out mutants showed a marked reduction of expression of SMAD2, TIVE cells infected by a double-knock out mutant virus fully restored SMAD2 expression, compared to non-infected TIVE cell. In summary, these data demonstrate that the KSHV latent genes, vFLIP and vCyclin induce the oncogenic miR-17/92 cluster in endothelial cells to inhibit TGFbeta, a pathway often targeted early during tumorigenesis. 18. Gene signaling pathways in liver of largemouth bass by dietary exposure to EE2 Colli-Dula RC, Kroll KJ, Barber DS, Denslow ND* Department of Physiological Sciences, University of Florida, Gainesville, FL Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 17 alpha-ethinylestradiol (EE2), used for birth control in humans, is a potent estrogen that is found in wastewater and has the ability to interfere with the endocrine system in fish. We used a transcriptomics approach (microarray analysis and gene set enrichment analysis (GSEA) using Pathway Studio V9) to investigate pathway signatures in the livers of female largemouth bass (LMB) caused by dietary exposure to 0.07 mg EE2/kg feed at 1% of their body weight over two months. Our data showed that a large group of genes were differentially regulated and these are involved in key signaling pathways including protein-kinase cascades (MAPK), phosphoinositol 3kinase, as well as in DNA-damage, DNA methylation, tumorigenesis and cancer. GSEA revealed that some cell process pathways including tight junction assembly and RNA ge ne silencing were significantly changed. In addition several genes normally attributed to transactivation through soluble estrogen receptors were also differentially expressed (e.g., associated with vitellogenesis and female gonad development). Our results suggest that exposure to EE2 via the diet activates both genomic and nongenomic pathways, some of which are implicated in tumorigenesis and cancer in female LMB. These data provide new insights into the mechanism of action of EE2 and its health implications for fish.
17 19. Studying the Topless co-repressor complex Collins J 1 Gurley WB 1,2, *, Chen S 1,3,4, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 3 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 4 Department of Biology, University of Florida, Gainesville, FL Repression of gene expression is critical for proper development and response to environmental stimuli. Down regulation of specific target gene is typically accomplished via the recruitment of chromatin remodeling factors which condense and ultimately shut-down the expression of specific regions in the genome. In plants, the transcriptional corepressor known as Topless interacts with a variety of transcription factors in a wide range of pathways. However, the mechanism by which Topless achieves repression remains poorly understood. Utilizing an affinity-tagged protein purification approach, we hope to identify novel proteins in the Topless complex via mass spectrometry. In addition, truncated versions of the Topless protein are being studied to elucidate the functional roles of the intrapeptide domains of Topless. Current studies involve assaying transgenic plants for protein expression. 20. A novel regulator of actin depolymerizing factor is localized to the ER Cuddy KK 1 Grey PH 1 Oppenheimer DG 1,2, 1 Department of Biology, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Actin filament turnover is required for many actin-dependent cellular processes including cell motility and membrane trafficking. Members of the actin depolymerizing factor/cofilin (ADF) family of actin binding proteins are essential for regulating actin dynamics. We identified a new ADF regulator in Arabidopsis named IRREGULAR TRICHOME BRANCH 3 (ITB3). Our in vitro analysis of ITB3 function showed that it inhibits actin binding to ADF. Interestingly, half of ITB3 family proteins possess a putative signal sequence. Because signal sequences direct proteins for secretion, we must reconcile the fact that ADF, which interacts with ITB3, is not secreted. We thus hypothesize that the putative signal sequences are novel Nterminal transmembrane anchors. To test this hypothesis, we conducted in vitro transcription/translation of ITB3L-10 in the presence of canine pancreatic microsomal membranes to test for co-translational membrane insertion. Results show that the N-terminus of ITB3L10-YFP does not behave as a canonical signal sequence. We also constructed YFP fusions to the putative signal sequence containing protein, ITB3L-10, and transiently transformed Arabidopsis epidermal cells using particle bombardment. Results show that the ITB3L10-YFP anchor co-localizes in vivo with an endoplasmic reticulu m marker. ITB3L10 -YFP also localizes to ER patches and punctate structures that we hypothesize to be ER exit sites (ERES). Experiments to confirm ERES localization are underway. 21. Florida manatee (Trichechus manatus latirostris ) Davis MC 1,2 Tringali MD 3 Bonde RK 2 Hunter ME 2 1 Aquatic Animal Health Program, University of Florida, Gainesville, FL 2 Southeast Ecological Science Center, US Geological Survey, Gainesville, F L 3 Fish and Wildlife Research Institute Florida Fish and Wildlife Conservation Commission, St. Petersburg, FL By performing the first pedigree analysis of Florida manatee cow-calf pairs, we will deepen our understanding of manatee relatedness and the consequences of inbreeding. Pedigree analysis is the study of multigenerational populations that are either known or can be deduced through behavioral observation studies and genetic parentage assessment. Parentage and sibling relationships can be inferred using molecular tools combined with relevant statistical analyses. They can also reveal estimates of relatedness, which is necessary in quantifying inbreeding levels. Little is known about the level of genetic relatedness in Florida manatees; reproduction takes place in mating herds, involving one female and multiple males attempting copulation. An extended cowcalf dependency period has been observed, but very little information is known about the paternity of calves since visual identity of successful males is typically not possible. Captive pedigrees and simulation data were used to determine the accuracy of these molecular tools and individual genetic identification was established for all specimens to confirm relationships. A suite of 31 microsatellite markers and multiple pa rentage assessment programs were used to delineate relatedness. Using these molecular tools, 160 cow-calf pairs were analyzed to genetically verify maternity and 152 sibling pairs were examined to determine sibling relationships. Preliminary results indicate that parent-offspring relationships and overall relatedness can be identified with high accuracy, while exact sibling and extended relationships are more difficult to determine.
18 22. Phylogeny of Caprimulgiformes and Apodiformes based upon complete mitochondrial genome sequences Dayton JB 1 White ND 2,3 Faircloth BC 4 Glenn TC 5 McCormack JE 6,7 Brumfield RT 8,9 Crawford NG 10 Braun MJ 2,3 Braun EL 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, DC 3 Behavior, Ecology, Evolution, and Systematics Program, University of Maryland, College Park, MD 4 Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 5 Department of Environmental Health Science, University of Georgia, Athens, GA 6 Moore Laboratory of Zoology, Occidental College, Los Angeles, CA 7 Department of Biology, Occidental College, Los Angeles, CA 8 Louisiana State University Museum of Natural Science, Baton Rouge, LA 9 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 10 Center for Comparative Genomics, California Academy of Sciences, San Francisco, CA Traditional Caprimulgiformes is an avian order of birds comprising the families Aegothelidae (owlet-nightjars), Caprimulgidae (nightjars), Podargidae (frogmouths), Nyctibiidae (potoos), and Steatornithidae (oilbirds). The order Apodiformes (swifts and hummingbirds) is closely related to Caprimulgiformes and likely nests within them as the sister of Aegothelidae. Caprimulgiformes are nocturnal or crepuscular but Apodiformes are diurnal, like most other birds. There are several hypotheses as to how this could occur. There could be a single transition from diurnal to nocturnal at the base of Caprimulgiformes followed by a transition back to diurnal at the base of Apodiformes. Alternatively all developed nocturnality independently in four separate transitions. Another hypothesis is that Aegothelidae is not sister to Apodiformes and instead belongs with the rest of Caprimulgiformes so that they form two separate groups and nocturnality only arose once, although this is least supported by existing data. We used whole mitochondrial genomes to explore the evolutionary relationships among the different families. Using maximum likelihood bootstrap analysis we generated a tree using 34 species of Caprimulgiformes and Gallus to root the tree. The results of our tree show strong support for the families themselves but limited for relationships between the families, with the exception of the relationship between Aegothelidae and Apodiformes. 23. Spreading of Sinorhizobium meliloti on semisolid surfaces: a quorumDilanji GE 1 Teplitski M 2, *, Hagen SJ 1, 1 Department of Physics, University of Florida, Gainesville, FL 2 Soil and Water Science Department, University of Florida, Gainesville, FL When growing on soft agar, Sinorhizobium meliloti exhibits an unusual spreading behavior that does not require generating propulsive forces, unlike other well studied forms of bacterial surface motility such as swarming and twitching. The S. meliloti colony secretes a galactoglucan exopolysaccharide (EPS-II) to generate a spreading mucoid layer, which carries within it the actively dividing bacteria. In the process complex patterns of cells and aggregates are deposited on the agar surface. We studied the dynamics of the surface spreading and pattern formation by collecting time-lapse images of spreading colonies of wild-type and mutant strains of S. meliloti Our data show that the spreading is primarily driven by two entropic effects associated with the high concentration of EPS-II: the osmotic extraction of water from agar and the molecular crowding mechanism known as the depletion attraction. EPS-II causes an osmotic pressure that draws water out of the agar and spreads the mucoid layer across the surface. Meanwhile the suspended bacteria are swept to the boundary of the mucoid by the depletion attraction. The swimming and aggregation of daughter cells shed by the cells at the boundary give rise to the complex patterns seen in the growing colonies. Finally, the osmolyte EPS-II and consequently its entropic effects are tightly regulated by th e S. meliloti quorum sensing system. A quorum sensing receptor, ExpR, regulates the operon for EPSII synthesis as well as some of the motility genes in S. meliloti Therefore, S. meliloti a quorum sensing activated, of translocation. 24. Uncovering the roles of the E. coli clamp loaders in DNA replication and repair Douma LG 1,2 Bloom LB 1, 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL During DNA replication in all organisms, ring-shaped sliding clamps bind DNA polymerases to keep the enzyme on the template strand used to produce the nascent strand. Without the sliding clamp, the DNA polymerase would lose contact with the DNA and replication would not be completed. The sliding clamp is loaded onto the DNA by the clamp loader, an AAA+ ATPase. In E. coli two known clamp loaders exist, tau complex and gamma complex. The same gene, dnaX, encodes both the
19 gamma and tau subunits of the clamp loaders. Tau is the full gene product while gamma is a C-terminally truncated product produced by a ribosomal frameshift. Tau complex is required at the replication fork to organize protein-protein interactions, but currently, the function of gamma complex in vivo is unknown. Here we show that gamma complex has a role in maintaining genome stability. We found that E. coli cells with a knockout of gamma are sensitive to UV irradiation, MMS and HU treatment, which is a novel phenotype. Although a function for the fulllength gene product and the presence of the truncated gene product have been known for more than 20 years, a function for the shorter gamma subunit has remained elusive and our results are the first clues as to what gamma may be doing. Determining the role of gamma complex in vivo would provide insight to the roles of other alternative clamp loaders, like those found in eukaryotic cells, and into repair pathway mechanisms. 25. Cumulative mutational effects on susceptibility of C. elegans to the bacterial pathogen Pseudomonas aeruginosa : a large mutational target but a miniscule average effect Etienne V 1 Andersen EC 2 Baer CF 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Molecular Biosciences, Northwestern University, Evanston, IL Understanding the relationship between an individual's genotype and its susceptibility to pathogens is an important goal of medical genetics and evolutionary biology. Of fundamental importance is the nature of genetic variation in susceptibility to pathogens introduced into a population of (potential) hosts by mutation. Characterizing the "mutational variance" (VM) provides a first step toward understanding the evolutionary factors underlying genetic variation within and between populations: all else equal, the genetic variation present in a population is expected to be proportional to the input of genetic variation by mutation. Here we quantify the cumulative effects of spontaneous mutations on susceptibility to the pathogenic bacterium Pseudomonas aerugino sa in a population of the nematode Caenorhabditis elegans Perhaps surprisingly, spontaneous mutations do not affect the expected time to death ("LT50") of a population of worms exposed to P. aeruginosa However, VM for susceptibility to P. aeruginosa is on the order of VM for many life history traits in the same population, which implies that the trait "pathogen susceptibility" provides a large mutational target. The previous finding of high genetic variance for the same trait among natural isolates of C. elegans is straightforwardly explained by the high VM and weak purifying selection on deleterious mutations; maintenance of genetic variation for pathogen susceptibility by balancing selection need not be invoked. 26. Exome resequencing and population structure analysis in Populus deltoides Fahrenkrog AM 1 Neves LG 1 Acosta JJ 2 Barbazuk B 1,3, *, Kirst M 1,2, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL Poplars are models for the study of the biology and genetics of wood formation and biomass growth in woody, perennial plants. Poplars also have the potential to be used as feedstock for biofuel production, which, together with their high nucleotide diversity and low linkage disequilibrium (LD), makes them especially suited to conduct association genetics studies to identify genes that regulate bioenergy traits. Genome-wide association studies require genotyping the samples with a high number of markers. Additionally, determining the population structure is crucial to avoid spurious associations between the genotype and the trait of interest. In this study we are genotyping Populus deltoides (eastern cottonwood) by exome capture and resequencing of 18,153 genes in an association population composed of 579 unrelated individuals, collected in 13 States of the southeastern US. A total of 1,103,014 SNPs was identified in the population using this high-throughput genotyping method. A random subset of 10,000 SNPs was selected to determine the genetic structure of the population using the software STRUCTURE, where the presence of one to te n subpopulations (K) was tested. The true number of methodology proposed by Evanno et al. (2005, Mol Ecol 14(8):2611-20). These preliminary results are going to be confirmed after sequencing part of the population again to increase depth of coverage and generate a higher confidence SNP set. Those SNPs will be used in the future for genome-wide association and population genetics studies. 27. Proteomic and genomic analysis of transcription factor TFII -I reveals insight into the stress response Fan AX 1-3 Papadopoulos GL 4 Hossain MA 1-3 Lin I-J 1-3 Hu J 5 Kilberg M 13, Renne R 5, *, Strouboulis J 4 Bungert J 13, 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Center for Epigenetics, University of Florida, Gainesville, FL 3 Powell Gene Therapy Center, University of Florida, Gainesville, FL 4 Division of Molecular Oncology, Biomedical Sciences Research 5 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL
20 Transcription factor TFII-I was originally identified as a protein that binds to the initiator and recruits the RNA polymerase II transcription complex in the absence of a TATA box. Subsequent studies have shown that TFII-I functions as a transcription modulator that exerts both positive and negative effects. TFII-I is a relatively large protein that contains DNAbinding and multiple repeated protein/protein interaction domains. Using biotinylation tagging technology we identified TFII-I associated proteins and determined sites of chromatin interactions for TFII-I in the human erythroleukemia cell line K562. The data show that TFII-I interacts with TBP associated factor (TAF) 15, E components of SWI/SNF chromatin remodeling complexes including Brg1, SMARCC1, and SMARCC2. The genome wide chromatin binding analysis revealed three different patterns of TFII-I occupancy. TFII-I binds upstream of the transcription start site of expressed genes, including genes encoding GATA-1, DNMT1, and MLL2. The second pattern is characteristic of nonexpressed genes where TFII-I binds both upstream and downstream of the transcription start site. Finally, TFII-I binds immediately downstream of paused Pol II at inducible and stress responsive genes. Induction of expression of one of these genes, ATF3, leads to increased association of Pol II and Elongin A with the coding region. The data suggest that TFII-I is involved in the conversion of stalled to elongation productive transcription complexes at stress response genes. 28. In utero caffeine exposure alters the transcriptome in embryonic heart Fang X, Poulsen R, Rivkees SA, Wendler CC Child Health Research Institute, Department of Pediatrics, University of Florida, Gainesville, FL Introduction: Our previous research demonstrated that in utero caffeine exposure at embryonic day (E) 8.5 increases body weight, alters cardiac morphology and function, and changes DNA methylation patterns and cardiac gene expression in adult male mice. Hypothesis: We hypothesize that the changes in DNA methylation patterns observed with in utero caffeine exposure leads to long lasting effects on gene expression and cardiac function in adulthood. Method: Pregnant CD-1 mice were injected with 20 mg/kg of caffeine daily from E6.5-9.5. On E10.5, total RNA was isolated from embryonic ventricles and used for transcriptomic RNA sequencing with Illumina HiSeq2000. Results: Differential expression (DE) analysis revealed that 59 genes were significantly up-regulated and 65 genes were downregulated by prenatal caffeine treatment (fold change >1.5 or <1.5; p-value with FDR<0.05). These DE genes include DNA methylation enzymes, cardiac structural genes, and transcription factors. Many of these changes correlate with the changes in adult DNA methylation that were identified in our previous studies. Further analysis discovered that 181 genes exhibited abnormal alternative splicing at the exon level after in utero caffeine treatment. Pathway analysis by MetaCore and DAVID revealed that many pathways related to cardiovascular development and diseases were significantly enriched. Conclusion: In utero caffeine exposure caused gene expression changes in embryonic ventricles and these changes may lead to long-term effects on cardiac morphology and function. 29. Goodness of fit: structural equation modeling methods to reconcile gene regulatory networks Fear JM 1,2 Campo D 3 Nuzhdin SV 3 McIntyre LM 2, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Section of Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA Perturbations to gene regulatory networks (GRNs) can affect important complex traits. Understanding what genes are involved and how these genes interact will provide insights which can be used. Traditionally, GRNs have been constructed from molecular studies by observing phenotypic outcomes after grossly perturbing a regulatory network by over-expressing or knocking down/out specific gene(s). These studies have also focused on specific developmental stages or tissue/cell types. However, in natural populations, variations in gene regulatory networks are the result of segregating alleles in regulatory regions. We hypothesize that natural variants can be used to reconstruct GRNs. We are interested in whether GRNs are static or change by cell type or in response to environmental stimuli. In order to compare the topology of GRNs a formal test for the goodness of fit is needed. Here we show, using structural equations modeling, how several goodness of fit statistics can be applied to GRN to identify differences in network topology. We demonstrate the utility of our approach using RNA-Seq data from a D. melanogaster heterozygous panel of 53 lines. Focusing on two well characterized pathways (sex determination, InR/TOR), we test fit of existing molecular models in virgin and mated female head tissue. We identified statistically significant differences in transcriptional regulation between these two experimental conditions. We also identified
21 30. Identification of anthracnose resistance loci in Sorghum bicolor Felderhoff T 1 Saballos A 2 Vermerris W 3, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Agronomy Department, University of Florida, Gainesville, FL 3 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Colletotrichum sublineolum is an aggressive fungal pathogen that causes anthracnose in sorghum. The symptoms of anthracnose are leaf blight and stem rot. Sorghum bicolor the fifth most grown cereal crop in the world, can be highly susceptible to the disease, most notably in environments that favor spread and infection of the fungus. Yield losses of up to 70% have been reported in susceptible germplasm. The sweet sorghum acreage in the southeastern U.S. is increasing steadily, spurred by growing interest in biofuel production and established sugarcane industry. Resistance to anthracnose is, humid environment. We generated a biparental mapping population of 125 F4:5 sorghum lines to identify anthracnose resistance genes in the highly resistant cultivar Bk7. Two field trials have been performed in Florida and the population has been mapped using genotyping by sequencing. Preliminary QTL have been identified using QTL Cartographer. These data will be used to ultimately identify the molecular basis of disease resistance and to introgress this trait in susceptible germplasm with otherwise attractive agronomic properties. Supported by the Southeastern Sun Grant Center and USDA-NIFA Award No. 2010-38502-21854 and USDA-BRDI Award No. 2011-1000630358. 31. Global gene and microRNA array studies to define the molecular etiology of salivary gland tumors Gao R 1,2 Zhang M 1 Cao C 1 Lopez MC 3 Baker HV 3, *, Renne R 3, *, Zajac-Kaye M 1,4 Kaye F 1, 1 Department of Medicine, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 4 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL Salivary gland tumors (SGT) comprise a heterogeneously group of tumors. The most common subtypes are classified as pleomorphic adenomas (PA), mucoepidermoid carcinomas (MEC), adenoid cystic cancer (ACC), and adenocarcinoma (ADC) not otherwise specified. Interestingly, PA, MEC, and ACC are now known to arise due to somatic chromosomal translocations. For example, PA usually arises from a HMGA2-NFIB translocation, MEC from a fusion between CRTC1-MAML2, and ACC from the recently published MYB-NFIB rearrangement. Our laboratory previously cloned and characterized the functional properties of the CRTC1MAML2 fusion oncogene. We have now focused on studying the genetics and biology of the MYB-NFIB oncogene in ACC, which is the most lethal subtype of SGT with no known systemic treatment. To study the mRNA and miRNA expression patterns and define the functional properties of MYB-NFIB fusion oncogene to develop new therapeutic targets, we have collected 74 samples of malignant SGTs from ACC, MEC, and ADC with matched normal samples. We focused on ACC tumor biology and have discovered a highly specific pattern of mRNA and miRNA gene expression in ACC that confirms the importance of deregulated MYB gene expression. We have also identified 20 MYB regulated genes that may serve as biomarkers for prognosis and targets for therapy in patients with ACC. In summary, we have demonstrated that ACC is a unique genetic, biological, and clinical entity from other subtypes of salivary gland tumors. 32. The N-terminus of the maize ROUGH ENDOSPERM3 splicing factor is necessary for the proper splicing of many U12-type introns Gault CM 1 Mei W 2 Xiong Y 3 Felderhoff T 4 Barbazuk WB 1,2, *, Settles AM 1,5, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 4 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 5 Horticultural Sciences Department, University of Florida, Gainesville, FL The splicing of introns from mRNA transcripts is catalyzed by two different spliceosomes in eukaryotic cells. The major spliceosome removes U2-type introns, which are the most common type of intron. In contrast, the minor spliceosome removes the rarer U12-type introns. The U2AF 35 -related protein is a core splicing factor that is necessary for the removal of both types of introns in human cells, but the splicing factor plays different roles in each spliceosome. The domains of the U2AF 35 related protein that are necessary for the function of each spliceosome remain unknown. Here we show that an insertion/deletion mutation in the N-terminus of the ROUGH ENDOSPERM3 splicing factor, a maize U2AF 35 -related protein ortholog, selectively impairs the splicing of U12-type introns in the maize rough endosperm3 ( rgh3 ) mutant. Out of all U12-type introns within genes that are expressed at an early seedling stage, 43% exhibit splicing defects in rgh 3 mutants. These U12type intron splicing defects include intron retention and cryptic splice site activation, and they occur in genes related to cell cycle and cytoskeletal organization. Due to the reduced
22 function of the RGH3 protein, the endosperm cells of rgh3 mutants fail to differentiate into specialized cell types, and rgh3 seedlings die shortly after germination, as shown in a previous study. Our findings give insight into the molecular function of the RGH3 splicing factor as well as its critical importance in maize development. 33. Targeted deep sequencing in multiple-affected sibships as a tool to identify rare variants contributing to risk of type 1 diabetes Ge Y 1 Onengut-Gumuscu S 2,3 Quinlan AR 3 Mackey AJ 3 Wright JA 1 Buckner JH 4 Rich SS 2,3 Concannon PJ 1, 1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2 Department of Public Health Sciences, University of Virginia, Charlottesville, VA 3 Center for Public Health Genomics, University of Virginia, Charlottesville, VA 4 Translational Research Program, Benaroya Research Institute at Virginia Mason, Seattle, WA Although over 40 risk loci have been identified for type 1 diabetes (T1D), the causative genes largely remain unknown. Here, we report a family-based approach to identifying key T1D genes by exploring rare deleterious variants in multiple-affected T1D families. Based on the hypothesis that rare susceptibility variants were enriched in T1D causative genes, we searched, by targeted deep sequencing, for such variants in 301 known protein-coding genes located in 49 T1D risk loci (excluding the human leukocyte antigen region). We sequenced 70 T1D patients of European ancestry whose families had three or more affected children, and found genes harboring two or three splice regions, including ICAM3 RAVER1 PTPN22 KCNH7 BACH2 BAZ2A, ALDH2, SULT1A2, SIRPB1 and NF2 PTPN22 was the only gene in the T1D risk region on chromosome 1p13.2 that had any rare deleterious variant (two novel frameshift deletions), indicating PTPN22 as the causative gene in this region. Furthermore, the rare allele of rs56048322 (c.2250G>C) in PTPN22 was independently associated with increased risk in T1D-affected sibpair/trio families, and led to elevated levels of two alternative PTPN22 transcripts and an additional isoform of LYP (the protein encoded by PTPN22 ), which binds to CSK as the full-length LYP does. These findings show that our strategies were effective in identifying rare risk variants in PTPN22 and can be used as a tool to identify causative genes for complex diseases. 34. AAV8 is preferential candidate for neonatal gene transfer in Sanfilippo syndrome B model Gilkes JA Bloom MD, Heldermon CD* Department of Medicine, University of Florida, Gainesville, FL Sanfilippo syndrome type B, referred to as mucopolysaccharidosis IIIB (MPS IIIB), is an autosomal recessive lysosomal storage disease caused by defective production of the e -N-acetylglucosaminidase (NAGLU) and is characterized by intracellular retention and accumulation of heparan sulfate. It is defined by severe and complex central nervous system (CNS) pathology leading to profound mental degeneration and ultimately premature death. Treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry to the CNS. Here, we assess the relative effectiveness of several adeno-associated virus vectors (AAV) for therapeutic utility in gene therapy based treatment of this disease. Using the MPS IIIB mouse model, we perform a comprehensive comparative study of transduction and distribution patterns of AAV-GFP serotypes -5, -8, -9 and rh10 administered by two different routes-intracranial six site (IC6) and thalamic. The serotype exhibiting the best treatment potential is defined by broadest distribution and transduction profiles. The cortex, hippocampus, thalamus and cerebellum were assessed in each animal. Of the two methods compared, IC6 administration of AAV resulted in the broadest distribution pattern regardless of serotype, while AAV8 transduced the largest number of cells and cell types compared to other serotypes. Interestingly, differential distribution and transduction profiles were observed in wild-type compared to MPS IIIB animals. Given its superior distribution and tropism capacity, this data suggests that AAV8 represents the best therapeutic gene transfer vector for the treatment of MPS IIIB via the IC6 method. 35. Genome-wide association analysis (GWAS) of blood pressure response to atenolol: results from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) study Gong Y 1, *, McDonough CW 1 Wang Z 2 Langaee TY 1, *, Beitelshees AL 3 Chapman AB 4 Gums JG 1, 5 Bailey KR 6 Boerwinkle E 2 Turner ST 6 Cooper-DeHoff RM 1,5 Johnson JA 1,5, 1 Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville FL 2 IMM Center for Human Genetics, University of Texas at Houston, Houston, TX 3 Division of Endocrinology, Diabetes and Nutrition, University of Maryland, Baltimore, MD 4 Renal Division, Emory University, Atlanta, GA 5 Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville FL 6 Division of Nephrology and Hypertension, Mayo Clinic,
23 Rochester, MN Background: blockers (which lower plasma renin activity, PRA) usually respond poorly to thiazide diuretics (which elevate PRA). We hypothesized that polymorphisms (SNPs) associated with good response to atenolol would also be associated with PRA lowering and worse response to thiazide diuretics. Methods: PEAR evaluated blood pressure (BP) response in uncomplicated hypertensive patients randomized to either atenolol or hydrochlorothiazide (HCTZ) monotherapy then the combination. GWAS analysis of BP response after atenolol monotherapy was performed in 233 white patients using linear regression adjusting for baseline BP, age, gender and ancestry. SNPs with p<10 -5 for BP response were evaluated for association with PRA change with same direction, and then for BP response to HCTZ in the opposite direction in 228 independent whites. Results: The A allele of rs12346562 near PTPRD was associated with better diastolic BP (DBP) response to atenolol (p=3.2*10 -6 -2.4 mmHg), with same direction of association in change in -0.10 ng/ml/h) and a directionally opposite association with DBP response to HCTZ (p=0.001 mmHg). Similar associations were observed for systolic BP. Conclusion: We identified a novel locus associated with BP response to atenolol with concurrent PRA change and opposite association with response to HCTZ. 36. Strigolactones in kernel size, ear architecture, and maize domestication Guan JC, Koch KE*, Suzuki M, Klee HJ*, McCarty DR* Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Ear-architecture and kernel size are critical components of maize domestication that led to improved grain yield. Teosinte glume architecture 1 ( Tga1 ) was previously identified as a major domestication gene controlling exposure of kernels on the ear surface. Here, we show that strigolactone (SL) signaling also regulates ear architecture. Ears of the SL-biosynthetic mutant, zmccd8 have elongated outer glumes and rachis internodes, a phenotype reminiscent of tga1 Confirmation that these phenotypes are specifically mediated by SL was obtained by analysis of a wild-type revertant derived from the zmccd8::Ds mutant, and by application of a synthetic SL, GR24, to zmccd8 mutants using a steady-state, string-feeding method. In addition, kernel weight of the mutant was reduced by 22%. The small-grain phenotype was also observed in four, SL-mutants of rice, indicating a conserved role for SLs in grain development. In contrast to the zmccd8 mutant, tga1 did not affect seed size. In the tga1 zmccd8 double mutant, the ear architecture phenotype was additive; whereas seed size was determined by zmccd8 alone. Expression of Tga1 paralog, Neighbor of Tga1 ( Not1 ), showed small, but significant increases at both mRNA and protein levels in zmccd8 singleand tga1 zmccd8 double mutants, implying that SL signaling acts partly by downregulating Not1 during ear development. In teosinte, levels of ZmCCD8 mRNA were 43-fold lower than in maize, suggesting a reduced potential for SL biosynthesis. Hence, an enhanced expression of ZmCCD8 may have played a key role in maize domestication by increasing seed size and down-regulating Not1 37. Impact of CYP2D6 polymorphisms on the clinical response and tolerability of immediate release metoprolol Hamadeh IS, Langaee TY*, Gong Y*, Burkley BM, Johnson JA* Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL Purpose: Metoprolol is a cornerstone therapy for the treatment of several cardiovascular diseases. In clinical practice, it is administered as a racemic mixture of Rand Senantiomers which undergo extensive metabolism by the polymorphic enzyme, CYP2D6. The primary purpose of this study was to determine whether CYP2D6 polymorphisms influence the clinical efficacy and tolerability of metoprolol. Methods: Following antihypertensive washout, participants took 50 mg twice daily with titration to 100 mg twice daily, guided by clinical response. CYP2D6 genotyping and copy number assessment were performed by pyrosequencing. Patients were assigned genotype-derived metabolism phenotypes of poor (PM), intermediate (IM), extensive (EM), or ultrarapid (UM) metabolizers, and appropriate parametric analyses were conducted. Adverse events (fatigue, tiredness, dizziness, depression, wheezing and bradycardia) were monitored to assess metoprolol tolerability. Results: Among 218 patients, there were 11 (5%) PMs, 17 (8%) IMs, 181 (83%) EMs, and 9 (4%) UMs. Heart rate (HR) response to metoprolol differed significantly by CYP2D6 phenotype (pvalue < 0.0001), but systolic and diastolic blood pressure did not. Adverse event rates were comparable, 14% in PMs and IMs versus 10% in EMs and UMs (p=0.5). Conclusion: Other than a significant difference in HR response, CYP2D6 polymorphism was not a determinant of the variability in response & tolerability to metoprolol therapy.
24 38. The impact of ozone on growth and development in Zea mays Hans N 1,2 Yendrek C 3 Ainsworth L 3 Brown P 3 Leakey ADB 3 Dalsing B 3 Rios L 3 Sorgini C 3 Barrios-Perez E 3 Erice G 3 Shim S 3 Leisner C 3 McIntyre LM 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 USDA-ARS Global Change and Photosynthetic Research Unit, Urbana, IL Within the next 40 years, global demand for cereal production will increase by 60% as the population rises from 7.0 billion today to 8.7 11.3 billion in 2050. Meeting this increased demand must be achieved not only against a background of damaging effects of global atmospheric change. Approximately 200 diverse maize inbred lines were screened for O3 tolerance at the FACE facility in Champaign, IL. Replicated plots of each of the 200 lines were grown at ambient [O3] (~40 ppb) and elevated [O3] (100 ppb). Each FACE ring had 52 genotype plots, and 12 B73 checks. We measured height and leaf number at three times during the growing season, leaf reflectance, anthesis, silking, and sterility (male and female). We modeled the effect of ozone on each genotype and identified lines with a differential response to ozone. 39. A direct organogenesis system for Agrobacterium mediated transformation of peanut ( Arachis hypogaea L.) Hsieh YF 1 Jain M 2 Wang J 1,2, *, Gallo M 1-3 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Agronomy Department, University of Florida, Gainesville, FL 3 Molecular Biosciences and Bioengineering Department, Leaf spot caused by Cercospora spp has been one of the major disease threaten the peanut production across the world. The transgenic approach is an effective alternative way to decrease peanut yield loss caused by leaf spot disease. Published peanut direct regeneration protocols for transformation are highly genotype-dependent. The repetitive somatic embryogenesis system is suitable only for biolistic gene delivery and requires prolonged in vitro sub-culturing. We have optimized a facile and rapid method to obtain direct shoot organogenesis from cotyledonary node (CN) explants excised from peanut seedlings germinated on cytokinin-supplemented basal salt medium. To maintain the photosynthesis performance and decrease the yield loss results from leaf spot disease, the optimized regeneration system combined with Agrobacterium -mediated transformation systems was applied to transfer a construct PSag12::IPT into a leaf spot susceptible peanut cultivar, New Mexican Valencia. The regenerated putative Psag12::IPT transgenic plantlets were subjected to genomic PCR validation. The results showed that 15 out of 53 T0 putative transgenic plantlets were positive yielding a total transformation rate of 6.5%. Further confirmation and characterization will be conducted on the regenerated plants. The results of this study implied that our regeneration and transformation systems can be successfully used in peanut germplasms improvement. 40. Modulation of tumorigenic potential of osteosarcoma cells through re-patterning of the epigenome Hyddmark EMV, Levings PP, Guijamo MV, Nasri E, Zarezadeh A, White ME, Ghivizzani SC*, Gibbs CP Department of Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL Osteosarcoma (OS) is the most frequent primary malignant bone tumor in children and adolescents, with a high propensity for lung metastases. Despite currently used aggressive treatment, 40% of the pat Our lab has previously shown that OS tumors are composed of two phenotypically distinct cell populations exhibiting differences in global gene expression, suggesting an underlying epigenetic mechanism to the observed intratumoral heterogeneity. Therefore, we hypothesized that treatment of OS cells with the histone deacetylase inhibitor TSA and the DNA methyltransferase inhibitor 5-aza would destabilize the epigenome of the cells and potentiate their re-programming response in vivo TSA treatment caused G1/S arrest of the cells, whereas 5-aza did not induce any changes on cell cycle distribution. When these cells were injected into mice, they formed tumors with vastly different phenotypes. Control tumors were morphologically heterogeneous, highly organized and had well defined vasculature. On the contrary, tumors developed from cells that had received either treatment were highly pleomorphic, poorly structured, and hemorrhagic. Furthermore, tumors arising from TSA treated cells showed a significant increase in metastatic dissemination. Therefore, comparing the gene expression between these different tumors and relating it to the histological phenotype could provide new information on how epigenetics contribute to OS biology and malignancy.
25 41. Gene therapy with the caspase activation and recruitment domain (CARD) ameliorates the inflammatory response in the endotoxin induced uveitis (EIU) mouse model Ildefonso CJ 1 Jaime H 2 Li Q 3 Lewin AS 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Department of Ophthalmology, University of Florida, Gainesville, FL Uveitis is an inflammatory response within different tissues of the eye. The current treatment involves prolonged use of steroids which is associated with several side effects such as increased risk of glaucoma. Our goal is to test anti-inflammatory genes delivered by an adeno-associated virus (AAV) vector as potential treatments for uveitis. We developed a secretable and cell penetrating form of the CARD domain from the ASC gene that binds and inhibits the inflammasome mediated activation of caspase-1. The secretion and cell penetration characteristics of our construct were validated in vitro by measuring its effects on inflammasome signaling. This construct was then packaged in AAV viral particles for further tests in the EIU mouse model. Mice were injected in the vitreous compartment with either control or CARD-expressing vector. The gene expression was determined one month after injection by fluorescence fundoscopy. Mice were then injected in both eyes with E. coli lipopolysaccharide. The eyes of these mice were harvested 24 hrs. later and fixed for histological analysis. To quantify the number of infiltrative cells, the eyes were embedded in paraffin and sectioned through the cornea-optic nerve axis for staining with hematoxylin and eosin. Eyes injected with the secretable and cell penetrating CARD AAV vector had a significantly lower number of infiltrating cells. These results suggest that the use of anti-inflammatory genes such as the CARD could be used to treat inflammatory diseases such as uveitis. This gene therapy could provide therapeutic benefits with a single injection while decreasing side effects of steroids. 42. Functional roles of LAFL and VAL transcription factors regulating the transition from seed formation to seedling development Jia H, Suzuki M, McCarty D* Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL The transition between seed and seedling is coordinately regulated by a family of B3 transcription factors (TFs). The embryo maturation program is positively regulated by AFL B3 TFs (ABI3, FUS3 and LEC2) and two HAP3 family TFs (LEC1 and L1L) that comprise the LAFL network, whereas VAL B3 factors (VAL1, VAL2 and VAL3) repress the LAFL network during germination. The normally seed specific LAFL network is derepressed in val1 val2 mutant seedlings. A key hypothesis is that the B3 DNA binding domains of VAL and AFL proteins both recognize Sph/RY elements in LAFL genes. To dissect the nonredundant roles of individual LAFL and VAL TFs in regulating the seed to seedling phase transition, the ability of LAFL mutants to suppress val1 val2 seedling phenotype was analyzed. Our results indicate distinct roles of individual LAFL genes in maintaining activation of the LAFL network in val1 val2 seedlings. To probe the functions of B3 domains in AFL and VAL proteins in planta a series of chimera proteins in which the B3 domain of maize ABI3 ortholog VP1 was replaced with B3 domains of ABI3, FUS3, LEC2 and VAL1, were over-expressed in abi3-6 mutant Arabidopsis. VP1/FUS3 and VP1/LEC2 chimeras induced ectopic embryonic phenotypes in vegetative tissues. In the VP1/ABI3 VP1/FUS3 and VP1/LEC2 transgenic lines activation of the downstream CRC gene was strongly correlated with transgene expression level, but only weakly so in VP1/VAL1 lines. ABA signaling enhanced activation of CRC in plants that had relatively low transgene expression. The results have delineated functional differences among the B3 domains of the AFL proteins as well as between the B3 domains of the AFL and VAL proteins. 43. Preliminary molecular phylogeny of the endangered Hawaiian leaf mining moth genus Philodoria Johns CA 1,2 Rubinoff D 3 Kawahara AY 1,2,4, 1 Department of Entomology and Nematology, University of Florida, Gainesville, FL 2 McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, Gainesville, FL 3 Department of Plant and Environmental Protection Sciences, 4 Department of Biology, University of Florida, Gainesville, FL The leaf mining moth genus Philodoria includes 30 poorly studied species that are endemic to the Hawaiian Islands. Most species are monophagous, feeding internally within the leaf of a single plant species, but the genus is known to feed on as many as 12 d host plants are threatened or endangered (IUCN 2012), making these moths particularly vulnerable. A comprehensive systematic treatment has not been conducted in over thirty years, and the systematics, phylogenetics, and conservation status of many of these moths remains largely unknown. In this poster, we present preliminary data from recent systematic and phylogenetic work on Philodoria
26 44. Root zone chemical ecology: new techniques for below ground sampling and mass spectrometric analyses of volatile semiochemicals Alborn HT 1 Kaplan F 2,3 1 USDA-ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville FL 2 Kaplan Schiller Research LLC, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL In nature volatile organic compounds (VOCs) produced by plant leaves, flowers, fruit and roots are an important part of multitrophic interactions. While above ground VOCs are well studied, research below ground lags because of the complexity of the system. In addition to plant roots, VOCs can be released by soil microorganisms, insects and nematodes into a virtually static airspace within the soil where they disperse solely by diffusion. VOCs that are released at a constant low rate as well as a short time burst will accumulate similarly in the soil surrounding the root. Since these factors have made it difficult to differentiate root released VOCs from soil background, root volatiles have typically been sampled by uprooting and moving a plant to an artificial environment. Then root related VOCs have been sampled by simple solvent extraction or by drawing most of the air surrounding the exposed roots through an adsorption filter that traps VOCs. These methods destroy, or stress the plants, and produce volatile profiles that are hard to relate to the dynamics of a natural situation. To solve this problem we designed non-invasive probes that allow sampling of VOCs directly from intact plants in soil. We also improved thermal desorption GC/MS analyses for sensitivity and reduced degradation which allowed us to sample small air volumes (short sampling times) leading to minimal effect to the rhizosphere VOC dynamics. The new method, a non-invasive probe coupled with more sensitive GC/MS technique, makes it possible to continuously monitor root VOCs below ground and to differentiate constitutively released and stress induced VOCs. Our method allows us to study in vivo below ground plant interaction with the environment. 45. Genetic analyses on hybrid populations of two largemouth bass subspecies based on SNPs analysis Kitagawa T 1, 2 Okuda K 2 Takata K 3 Hargrove J 1 Austin J 1, 1 Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 2 Faculty of Agriculture, Kinki University, Nara, Japan 3 Faculty of Science, Shinshu University Matsumoto, Nagano, Japan The largemouth bass Micropterus salmoides one of the most intensively managed sport fish species in the United States, has been introduced into freshwater habitats around the world. In Japan, the northern subspecies ( M. s. salmoides ) was first introduced in 1925. This subspecies subsequently expanded its distribution from a single lake to the whole of Japan. In 1988, the Florida subspecies ( M. s. floridanus ) was also introduced to central Japan, where it interbreeds with the northern subspecies. Since the 2000s, the Florida subspecies or hybrid progeny have been detected in other lakes in central Japan. Secondary introductions of the Florida subspecies into water bodies already inhabited by populations of the northern subspecies may result in fish with altered ecological traits. Our research aims to quantify the genetic impacts of secondary introductions of Florida subspecies on the genetic architecture of the previously established northern subspecies. The population genetic structure of both subspecies in sympatry were analyzed using seven new sub-species diagnostic SNP markers. Analyses on populations from three lakes revealed that both subspecies had mated randomly with each other and that for most gene frequencies of the Florida subspecies alleles had been constantly increasing since their introduction These results suggest that some positive selective advantages may exist for ecological traits of the Florida subspecies. 46. Fungal communities in ambrosia beetles: high-throughput community sequencing reveals promiscuity between beetle farmers and fungal crops Kostovcik M 1-3 Stelinski LL 4 Hulcr J 1 1 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2 Institute of Microbiology, Czech Academy of Sciences, Prague, CZ 3 Faculty of Science, Charles University, Prague, CZ 4 Department of Entomology and Nematology, Citrus Research and Education Center, University of Florida, Lake Alfred, FL Agriculture has evolved in at least four groups of organisms: in humans, fungus-growing ants, termites and wood-boring ambrosia beetles. The beetle-fungus relationship is thought to involve significant mutual specificity. However, despite the economic importance of the symbiosis, very little is known about these relationships, and mutual specificity is currently an assumption based on limited culturing studies. To quantify the specificity between beetle farmers and their fungal crops we employed culture-independent community survey by highthroughput sequencing. We analyzed 95 beetles from three species from across the eastern US, and focused on fungi in mycangia beetle organs for fungus transmission. The diversity of fungi in our sample beetles is an order of magnitude richer than previously reported. Yet the most consistent members of the fungal community are still those known as the primary nutritional symbionts. Symbiont community composition is specific to beetle species or clade, not to locality. Fungal diversity differs significantly between beetle species with different mycangia: fungal community in beetles with mesonotal mycangium ( Xylosandrus crassiusculus ) is heavily dominated by a single symbiont, while communities in beetles
27 with mandibular mycangia ( Xyleborus affinis and X. ferrugineus ) resemble loose assemblages. Low-abundance and low specificity community members appear to be sampled by the beetle from the environment, as they often include common soil saprophytes, plant pathogens, or plant endophytes. Overall it is evident that the beetle-fungal symbiosis is more dynamic and the community structure depends on the beetle vector clade and the mycangium type. 47. Improving heat tolerance in rice through early heat shock treatment Krishnamurthy A, Kilasi N, Rathinasabapathi B* Horticultural Sciences Department, University of Florida, Gainesville, FL Increases in average global temperature and extreme temperature ranges are frequently recorded in the 20th century. In this scenario, developing innovative techniques to improve high temperature tolerance in food crops is important. Rice is a major food temperature stress is one of the common problems in rice growing countries in the world. Increased temperatures during reproductive growth stages make rice highly susceptible to heat. Few hours of heat stress during flowering is known to reduce the yield severely. Seed hot water treatment is a practice followed in rice production for protection against seed borne pathogens. There are few studies on acquired thermotolerance in rice where a mild heat treatment induces heat tolerance in plants. However, the effect of early heat shock on improving tolerance to heat stress after long recovery periods is not studied. We demonstrated that an early heat shock treatment of Oryza sativa cv. Nipponbare seeds at 47C for 5 min improved heat tolerance of seedlings for high temperature stress imposed after 10 days. A 24h heat stress at 45C on 10 day old seedlings reduced the shoot dry weight by 56% while the pre-heat shocked plants had only 24% reduction. Early heat shock also improved the survival rate of seedlings to 78% compared to 27% in control. Biochemical and gene expression analyses to understand the effect of early heat shock on seedling heat tolerance are in progress. 48. Transcriptomic analysis of maize embryo size mutants Kudo T 1 McCarty DR 1,2, *, Suzuki M 1,2 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Horticultural Sciences Department, University of Florida, Gainesville, FL The maize seed is comprised of embryo and endosperm. Embryo size is an important agricultural trait correlated with seed oil content (Moose et al., 2004, Trends Plant Sci 9(7):35864). In a screen of the UniformMu transposon population, we identified several mutants that alter embryo size. The small embryo 3 (sem3) gene encodes a putative transcription factor, whereas big embryo 2 (be2) and big embryo 4 (be4) encode an inositol trisphosphate kinase and a putative acyl-CoA decarboxylase, respectively. To obtain clues toward understanding the physiological function of these genes in embryo development, global gene expression was analyzed in mutant embryos using RNA-seq. Significant changes in transcript levels of cell-cycle related genes were detected in sem3 and be2 embryos. In addition, consistent with the metabolic function of be2, RNA-seq data revealed altered expression of key inositol-phosphate metabolic genes in be2 embryos. The implication that sem3 and be2 have opposite effects on cell-cycle regulation was supported by profiling of nuclear ploidy obtained by flow cytometric analysis. Cells in sem3 and be2 embryos show relative depletion and enrichment of 4C nuclei, respectively. On the other hand, cell-cycle related gene transcripts and ploidy were not changed in be4 embryos, while several genes implicated in carbon and amino acid metabolism were affected. Together, these results implicate multiple mechanisms in regulation of embryo size in maize. 49. Gene therapy via magnetic particles for cystic fibrosis Dobson J 1,2 Batich CD 1 Kwan MWC 1 1 Department of Materials Science and Engineering, University of Florida, Gainesville, FL 2 J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL Cystic fibrosis (CF) is an autosomal recessive disorder that affects about 1 out of 4000 individuals each year. The main cause of morbidity from this disease is the repeated bacterial The gene responsible for the mutation had been identified over 20 years ago but a cure for this disease has yet to be developed. This study focuses on non-viral gene therapy and in particular, the gene delivery agent. Non-viral gene therapy is appealing due to the limited immune response elicited and the ease of repeated administration. In addition to the reticuloendothelial system, gene delivery must also overcome other barriers such as the thicker sputum, beating cilia, cellular membrane, cellular lysosomes and nuclear membrane. The work presented here would involve the use of magnetic particles to overcome some of these barriers. Oscillating magnetic fields can provide the transverse force necessary to penetrate the thick sputum typically found in CF patients and also improve entry past the cellular membrane. Additionally, local heating by an alternating magnetic field was examined for improvement in sputum viscosity and particle diffusivity in sputum.
28 50. An extensive evaluation of healthy immunoglobulin repertoires using 454-pyrosequencing Lange H 1 Yin L 1 Yao J 2 Chang K 1 Zhang X 1 Rodriguez C 3 Sriaroon P 4 Sleasman J 4 Goodenow M 1, 1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2 Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 3 Division of Infectious Disease and International Medicine, Department of Medicine, University of South Florida, St. Petersburg, FL 4 Division of Allergy, Immunology and Rheumatology, Department of Pediatrics, University of South Florida, St. Petersburg, FL Deep sequencing has revolutionized the capacity to evaluate the depth and breadth of the Ig repertoire along the B cell developmental pathway, and can be used to pinpoint defects of primary or acquired B-cell associated diseases. In this study healthy IgM and IgG repertoires were studied by 454pyrosequencing to establish the healthy controls for diseased repertoires. Amplicon libraries of Ig heavy chain [IGH] variable region of IgM or IgG was generated from mRNA from four healthy individuals by a RT-PCR followed by a nested PCR. For each library 3,500-10,500 quality sequences from deep pyrosequencing were obtained. A novel IgSEQ software developed by us for automated analysis of IGH pyrosequences through IMGT/V-QUEST and IMGT/junction analysis was applied to obtain information about somatic hypermutation [SHM], use of IGHV, IGHD and IGHJ alleles and IGHD reading frame, length in complementarity determining regions [CDR], and junctional modifications. IgM repertoire is significantly different from IgG repertoire in healthy young adults. A higher percentage of sequences with SHM was observed in IgG compared to IgM. IgG sequences contain significantly more non-silent mutations in CDR1, CDR2 and FR3 regions than IgM sequences. When comparing sequences with SHM, IgM sequences showed greater fraction of sequences with nucleotide [N] insertion in VD-J junctions. Frequency distribution of CDR3 length was Gaussian in IgM repertoire but relatively variable among individuals in IgG. IgM repertoire is significantly different from IgG repertoire in healthy young adults. IgG Ab repertoire comprises more diversity than IgM mostly due to acquisition of greater extent of SHM. 51. Excess androgen prevents vaginal migration Larkins CE 1 Cohn MJ 1-3, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL Congenital absence of the vagina (vaginal atresia) is found in approximately 1 in 4000 female births and is commonly associated with congenital adrenal hyperplasia (CAH), a condition in which the adrenal glands produce excess androgen beginning at embryonic stages. Prenatal androgen exposure in females often leads to urethral/vaginal fistula with absence of a vaginal opening, but precisely how androgens prevent proper vaginal development is not clear. The vagina forms from the Mullerian ducts, which connect to the urogenital sinus-derived sinovaginal bulbs just posterior to the bladder. The sinovaginal bulbs and Mullerian ducts then move caudally along the urogenital sinus until they reach the base of the external genitalia, where the developing vagina separates from the urethra. The mechanism that controls displacement of the vagina from the rostral to the caudal end of the urethra is unknown; however, the observation that CAH results in urethral/vaginal fistula led us to hypothesize that androgen inhibits caudal movement of the sinovaginal bulb along the urethra. To test this hypothesis, we treated embryos with testosterone at early stages of vaginal migration. We found that androgen exposure during this time caused lengthening of the urethra, and the vagina failed to migrate caudally resulting in urethral/vaginal fistula. We are currently determining the targets of androgen that result in a failure to reposition the sinovaginal bulb to the caudal urethra. 52. Loss of limbs by regulatory evolution: conservation of the limb-specific enhancer of Sonic hedgehog ( Shh ) gene in the snake Python regius Leal F 1 Cohn MJ 13, 1 Department of Biology, University of Florida, Gainesville, FL 2 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Limb loss has occurred many times during vertebrate evolution, however the underlying molecular mechanisms are largely unknown. The Shh gene mediates the activity of a key signaling region of the limb buds, the zone of polarizing activity (ZPA), which controls digit development along the anteroposterior (thumb-pinky) axis. Shh expression is regulated at the ZPA by a limb specific enhancer called ZRS. Previous studies have failed to identify a ZRS in limbless reptiles, linking loss of this enhancer to the evolution of limblessness. We isolated the ZRS from two basal snakes, the blind snake Leptotyphlops sp. and the ball python Python regius which lack forelimbs but conserve rudimentary hindlimbs. We found that in python snakes, there is transient Shh expression in the ZPA in very early hindlimb buds, which shuts down prematurely. This result suggests the ZRS in pythons is still functional, although Shh is not maintained. We also found that Fgf8 which mediates activity of a second signaling region in the limb, the apical ectodermal ridge (AER) is expressed robustly at early stages but also
29 disappears prematurely. Taking together these results suggest a premature break of the ZPA-AER feedback loop leads to the premature arrest of python hindlimb bud development. To determine whether misregulation of Shh is due to disruption of upstream regulators we found that the expression of genes like Gli3 Hoxd13 Hoxa13 and Wnt7a is conserved and cannot account for the premature shutdown of Shh expression in the python ZPA. Finally, to test whether the temporal changes in Shh regulation are due to evolution of the ZRS, we are making transgenic mice carrying the Python ZRS sequence 53. Mbnl compound and conditional mouse knockout models for studies on RNA processing and RNA-mediated disease mechanisms Lee KY 1,2 Li M 1 Manchanda M 1 Batra R 1 Charizanis K 1 Mohan A 1 Finn D 1 Hong H 1 Swanson MS 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Neurology, Chang Gung Memorial Hospital, Keelung, Taiwan Myotonic dystrophy (DM) is an RNA-mediated disease caused by the expression of C(C)UG expansion RNAs that alter the activities of several factors including the muscleblind-like (MBNL) family of RNA-binding proteins. MBNL proteins, which are sequestered by C(C)UGexp RNAs, have been implicated in the regulation of alternative splicing and mRNA localization. Studies on Mbnl constitutive knockout (KO) mice have revealed that Mbnl1 plays a major role in skeletal and heart muscle splicing regulation while Mbnl2 performs an analogous function in the brain. Because Mbnl1 and Mbnl2 KOs fail to recapitulate the full spectrum of DM skeletal muscle and heart symptoms, we generated Mbnl1; Mbnl2 compound KOs. While Mbnl1-/; Mbnl2-/were embryonic lethal, Mbnl1-/-; Mbnl2+/mice, which expressed no Mbnl1 and reduced levels of Mbnl2, were viable and developed cardinal DM disease manifestations in skeletal and cardiac muscles. Mbnl2 protein was upregulated in both Mbnl1-/and Mbnl1-/-; Mbnl2+/KOs where Mbnl2 relocalized into the nucleus and targeted Mbnl1-regulated genes by binding to YGCY motifs. To overcome the embryonic lethality of Mbnl1; Mbnl2 constitutive DKOs, a skeletal muscle specific knockout for Mbnl was generated using Myo-Cre transgenic mice. Surprisingly, Mbnl1-/-, Mbnl2cond/cond; Myo-Cre+/(Myo DKO) mice exhibited severe muscular atrophy and profound splicing misregulation, recapitulating advanced-stage features of adult DM1. These findings support the MBNL compound lossof -function model for DM and provide novel Mbnl compound knockout cell and animal models for investigating RNA processing and localization pathways. 54. Imprint setting transcripts at the Angelman/Prader-Willi locus Lewis M 1 Kramer J 2 Williams RS 2 Resnick J 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two genetic imprinting disorders that arise from disruption of either paternal or maternal gene expression at chromosome 15q11-q13. Allele specific gene expression in this region is regulated by a bipartite imprinting center (IC) composed of the PWS -IC and AS-IC. The PWS-IC is a positive acting element that promotes gene expression on the paternal allele, while the ASIC functions in the oocyte to epigenetically inactivate the PWSIC on the future maternal allele. Thus the AS-IC sets the imprint by establishing a maternal specific mark at the PWS-IC. The PWS -IC and AS-IC have been identified by shared deletion overlaps in PWS and AS individuals. The PWS-IC spans SNRPN exon 1. The AS-IC is 35 kb upstream of SNRPN and encodes two of its alternative upstream exons. Our lab recently demonstrated that AS-IC imprint-setting activity consists of ensuring transcription across the PWS-IC in mouse oocytes. However, transcripts transiting the PWS-IC in human oocytes have not been identified. To explore mechanisms of the human AS-IC to establish the maternal imprint, we used 5' rapid amplification of cDNA ends on human oocytes to map transcripts transiting the PWS-IC. This study has revealed novel transcripts that initiate within or nearby the AS-IC. These oocyte transcription patterns indicate that the human AS-IC acts to redirect SNRPN transcription so that all transcripts transit the PWS-IC for imprint establishment. 55. Proteomics and mass spectrometry applications i n biological and medical research Li J, Chow M, Zheng R, Koh J, Silva-Sanchez C, Chen S* Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Proteomics and mass spectrometry have provided unprecedented tools for fast, accurate, high throughput biomolecular separation and characterization, which are indispensable towards understanding biological and medical systems. Studying at the protein level allows researchers to investigate how proteins, their dynamics, modifications and interactions affect cellular processes and how cellular processes and environment affect proteins. The mission of our facility is to provide excellent service and training in proteomics and mass spectrometry. Here we present our capabilities in proteomics, highlighting differential proteomics, which include a gel-based 2D-DIGE (two dimensional difference gel electrophoresis) and
30 gel-free iTRAQ (isobaric tags for relative and absolute quantitation). 56. An oxalate decarboxylase gene functions in the early infection processes of Sclerotinia sclerotiorum Liang XF, Rollins J* Plant Pathology Department, University of Florida, Gainesville, FL Sclerotinia sclerotiorum is a devastating necrotrophic fungal plant pathogen and its pathogenesis requires the accumulation of oxalic acid. The S. sclerotiorum genome contains two putative oxalate decarboxylase genes, Ss odc1 and Ss odc2 which we hypothesize are involved in the fine-tune regulation of oxalate homeostasis, pathogenesis and morphogenesis. We used northern blot hybridization to determine their expression patterns across major development stages and in vegetative hyphae exposed to pH and exogenous oxalate treatments. The Ss odc1 transcript was detected across all sampled life stages and showed no inductive accumulation in response to low pH (3.0) or exogenous oxalate (up to 40 mM at pH 4.8). In contrast, Ss odc2 transcript was specifically detected during compound appressoria differentiation but not in hyphae in liquid culture. Ss -odc1 knock out mutant showed wild type-like growth, morphogenesis, and virulence while Ss -odc2 knock out mutants showed less efficient compound appressorium differentiation, a threeto four-fold increase in oxalate accumulation during compound appressorium differentiation, and significantly reduced virulence on bean petioles, soybean leaves, soybean petioles and celery stems. Wounding host tissues prior to inoculation rescued all the virulence defects. We hypothesize that oxalate decarboxylase activity functions in creating a beneficial microenvironment for compound appressorium differentiation and function during the early infection processes of S. sclerotiorum. 57. A novel role of AAV terminal repeat as an RNA element in polyA-deleted mRNA translation Wang Y 1-4 Wang L 1,3,4 Lu Y 5 Van Vliet K 6 Kim J-H 3 Aslanidi GV 3,4 Agbandje-McKenna M 6, *, Ling C 1,2 Srivastava A 3,4,7,8, *, Ling C 3,4 1 Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China 2 Shanghai University of Traditional Chinese Medicine, Shanghai, China 3 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL 4 Powell Gene Therapy Center, University of Florida, Gainesville, FL 5 Department of Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL 6 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 7 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 8 University of Florida Shands Cancer Center, Gainesville, FL The recombinant AAV2 (rAAV2) genome is flanked by two 145 nucleotides cis -DNA sequences, termed inverted terminal repeats (ITRs). It is now well accepted that the ITRs at both ends of the rAAV2 genome are the only cis -DNA elements necessary for viral genome replication, encapsidation, integration into as well as rescue from host chromosomal DNA. Although it is generally believed that the ITRs are not transcribed, we hypothesized that the right ITR in the rAAV genome might be transcribed if the viral genome lacked the polyA signal. To this end, we generated two rAAV2 vectors in which the polyA sequence was deleted. The polyA-deleted vector genome could be successfully packaged, and following transduction of cultured cells, expressed robust amounts of polyA-less mRNA transcripts, which contained the entire right ITR sequences. Surprisingly, the polyA-deleted vectors mediated detectable levels of transgene expression that was at ~5% compared with those containing an authentic polyA sequence. Remarkably, in the presence of AAV2 Rep proteins and adenoviral proteins, the extent of transgene expression from the polyA-deleted vectors was indistinguishable from that from vectors containing an ends of the polyA-less mRNA transcripts contain the entire viral ITR, which interacts with the AAV large Rep proteins to mediate efficient transport to the cytoplasm, resulting in robust transgene expression from polyA-less mRNA transcripts. These studies provide new insights into the role of ITRs in the life cycle of AAV2. 58. Poster withdrawn. Poster Session II, Posters no. 59 116 Thursday, October 10, 11:30 a.m. 1:15 p.m. 59. Investigating a replicative mechanism for the establishment and maintenance of recombinant adenoassociated viral vector genomes in vitro and in vivo Lyles JK 1 Penaud-Budloo M 2 Moullier P 1-3 Snyder RO 1,2,4, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 INSERM UMR1089, Nantes Cedex, France 3 GENETHON, Evry, France 4 Department of Pediatrics, University of Florida, Gainesville, FL Over the past few decades, adeno-associated virus (AAV) has been developed as a recombinant vector for gene transfer. Recombinant AAV (rAAV) vectors consist of a transgene cassette flanked by the viral inverted terminal repeats encapsidated in a non-enveloped icosahedral capsid. rAAV
31 vectors have been used successfully in multiple clinical trials, but there are still aspects of the biology of the vector that remain unknown. Upon infection with rAAV, the vector is trafficked through the cell and delivered to the nucleus, where the vector genome persists. It is known that these vector genomes can persist in vivo and maintain stable transgene expression for at least 10 years in large animal models. It was previously thought that integration of the vector genome into the host cell chromosome was responsible for this long-term persistence. However, it has now been demonstrated in muscle and other tissues that rAAV vector genomes persist in the host cell nucleus as chromatinized episomal circular monomers and concatemers, and these forms are believed to be responsible for the observed long-term expression of the transgene. While there is sufficient evidence for the presence of these episomal circular forms, it is unknown how they are established and maintained over time. To elucidate this aspect of the vector life cycle, we are investigating the role of DNA replication in the establishment and maintenance of episomal circular rAAV monomers and concatemers in vitro and in vivo. 60. The maize ( Zea mays ) mutant narrow leaf rough endosperm2 (nlr2) impacts cell division planes and maps to chromosome 8 Malidelis IA 1 Gustin JL 1 Jackson S 1,2 Settles AM 1, 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Florida Agricultural and Mechanical University, Tallahassee, FL Maize grain is used extensively for food, feed, and increasingly for fuel. Identifying and characterizing maize mutants that have altered or defective grain can uncover the genetic components involved in the production of this important commodity. We have isolated a mutant, from an Activator/Dissociation transposon tagging population that we named narrow leaf rough endosperm2 (nlr2). Mature nlr2 kernels have a pitted and etched endosperm surface with a viable embryo. Mutant nlr2 seedlings develop into stunted plants with narrow leaves. Preliminary characterization of seedling leaf epidermal cell files shows incomplete and disorganized cell division planes. This suggests that the seed and plant phenotypes are caused by improper cell division possibly due to defects in cell plate formation or insertion. An F2 mapping population was used to map the nlr2 locus to the long arm of chromosome 8 using highthroughput single nucleotide polymorphism (SNP) genotyping. This position was confirmed and refined using traditional SSR markers. The nlr2 physical interval has been narrowed to 0.57 Mb, which contains 20 predicted gene models. Additional marker development will fine-map this interval to identify candidate genes for nlr2. 61. Enabling identification and selection of gummosis resistance QTLs in peach Mancero-Castillo D, Chaparro JX* Horticultural Sciences Department, University of Florida, Gainesville, FL Peach ( Prunus persica (L) Batsch) is an important fruit tree crop in the United States with a farm gate value of 631 million dollars in 2012 (USDA). A significant threat to the peach industry is peach tree fungal gummosis caused by fungal pathogens in the Botryosphaeriaceae family. Botryosphaeria dothidea B. rhodina and B. obtusa have been identified and associated with peach gummosis. Disease symptoms include exudates on the stems and branches and peach trees with severe infections ultimately die. Highly susceptible peach cultivars present significant decreases in fruit growth and fruit yield reductions of up to 40%. Currently, there are no effective and suitable cultural or chemical controls for peach gummosis in the southeastern US. Thus, scientists and growers are concerned with the serious detrimental effects on tree longevity and fruit production. Preliminary research indicates that almond may provide a new source of genes for resistance. Segregating interspecific backcross populations have been generated and planted at Gainesville, FL. Clonal replicates in Byron, GA under a trellis designed to enhance infection. All trees will be evaluated according to a rating system based on the severity of gumming. The populations have been genotyped for SSR markers sourced from the Prunus reference genome map. QTL analysis will be performed on the segregating populations. The aim of the present study is to identify the number and genomic location of loci associated with peach gummosis resistance. Additionally, molecular markers closely related to gummosis resistance will be developed to facilitate marker assisted selection. 62. A pharmacogenomic genome-wide association study for adverse cardiovascular (CV) outcomes in the INternational VErapamil SR-Trandolapril STudy (INVEST) McDonough CW 1,2 Gong Y 1,2, *, Langaee TY 1,2, *, Pepine CJ 2 Cooper-DeHoff RM 1-3 Johnson JA 13, 1 Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 2 Center for Pharmacogenomics, University of Florida, Gainesville, FL 3 Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville, FL We sought to identify novel pharmacogenomic markers for antihypertensive drug-associated long term CV outcomes in study participants with hypertension and coronary artery disease (CAD) A matched case-control cohort of 998 INVEST participants was
32 genotyped on the Illumina OmniExpressExome chip. During INVEST, participants were randomized to a verapamil SR-based calcium channel blocker strategy (CCB) or an atenololblocker strategy (BB). Trandolapril and hydrochlorothiazide were available as add-on drugs. Cases experienced the primary outcome (PO) during INVEST: all-cause death, nonfatal myocardial infarction (MI), or nonfatal stroke. The primary analysis was a SNP x treatment interaction analysis, using logistic regression, and adjusting for age, sex, ancestry, history of MI, heart failure, and diabetes. Results from each race group were combined using meta-analysis. The G allele of rs1800792 in the fibrinogen gene cluster was associated with lower risk of the PO in whites on the CCB strategy (OR=0.44 (0.29-0.68)) but higher risk in whites on the BB strategy (1.64 (1.04-2.60)) (interaction p=5.22x10 -5 ). This replicated in Hispanics (CCB: 0.79 (0.42-1.49), BB: 3.36 (1.328.55), one-sided interaction p=0.033). The meta-analysis interaction p=1.31x10 -5 Other top regions were RAB27A /PIGB at 15q21.1 (white/Hispanic meta interaction p=4.80x10 -6 ), and SIK3 /ZNF259 at 11q23.3 (white/Hispanic/black meta interaction p=1.29x10 -5 ). In conclusion, the fibrinogen gene cluster, and other regions, are associated with treatment related CV outcomes in INVEST. If validated, these SNPs could be incorporated into individualized clinical management of hypertensive patients with CAD. 63. ITRAQ analysis of SAMP2 conjugates in the haloarchaeon Haloferax volcanii McMillan LJ 1 Miranda HV 2 Nembhard N 2 Maupin-Furlow JA 2, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL The proteasome degrades misfolded or damaged proteins and hydrolyzes proteins responsible for cell signaling. The 20S core particle of the proteasome is conserved through the three type subunits. Small archaeal modifier proteins, or SAMPs, are structurally similar to ubiquitin and presumed to function similarly in archaea to the ubiquitin dependent degradation pathway in eukaryotes. The model haloarchaeon Haloferax volcanii will be used in this study to improve understanding of SAMPs and their association with proteasomes. Hfx. volcanii grown in the presence of commercial proteasome inhibitor bortezomib increases SAMP2 conjugates, suggesting SAMP2 targets proteins to the proteasome. DMSO, an alternate electron acceptor, also increases conjugates when present in the growth media. Here we show DMSO increases and bortezomib inhibits activity of purified 20S core particles in vitro To identify the accumulated SAMP2 conjugates after inhibiting the proteasome of Hfx. volcanii a FLAG-SAMP2 integrant strain will be constructed and grown in the presence of DMSO and/or bortezomib and conjugates will be analyzed via iTRAQ or other quantitative proteomic method. 64. Using next gen sequence data to understand the evolution of gallopheasants (Galliformes) Meiklejohn KA 1 Braun EL 1, *, Glenn TC 2,3 Faircloth BC 4 Kimball RT 1, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Environmental Health Science, University of Georgia, Athens, GA 3 Georgia Genomics Facility, University of Georgia, Athens, GA 4 Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA Approximately 60% of Galliformes belong to the Phasianidae, which includes but is not limited to groups such as the pheasants, tragopans, junglefowl and gallopheasants. A rapid radiation among the six gallopheasant genera ( Catreus, Chrysolophus, Crossoptilon, Lophura, Phasianus, and Syrmaticus ) has made relationships among them difficult to resolve, even in studies utilizing a number of intron and mitochondrial loci. Considering this, our study was aimed at evaluating the utility of next generation sequence data, in particular ultraconserved elements (UCEs) and flanking DNA, for inferring relationships within this group. A complete matrix of ~620,000 sites from 1,479 UCE loci, 15 nuclear introns and three mitochondrial loci was established for 18 species, re presenting all gallopheasant genera and basal phasianid lineages. Bootstrapped maximum likelihood analyses were performed on a range of datasets, in order to evaluate the phylogenetic signal of the data both separately and when combined. As expected, some conflict was noted for relationships among the gallopheasants, mainly for the placement of Chrysolophus and Phasianus. The UCE loci do appear to be reliable markers however, as they have low misleading signal and did not give high bootstrap support for all nodes. Our results indicate that UCE loci provided informative phylogenetic signal for the gallopheasant radiation, and therefore could prove useful for resolving relationships in other problematic groups. 65. The role of cilia in external genital development Moore GL 1 Larkins CE 1 Cohn MJ 13, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL Congenital defects in external genitalia, such as micropenis, have been associated with ciliopathies, diseases caused by mutations in cilia proteins. It remains unknown how penile anomalies occur in the presence of poorly functioning cilia. Cilia are thin microtubule-based structures located on the apical cell membrane and act as cellular antennae that bind ligands required
33 for signal transduction and transport. Among these proteins is Arl13b, a small GTPase that is required for localizing Shh signaling components to the cilium. A null mutation for Arl13b in mice, the hennin mutation (hnn), causes shortened cilia and expansion of low levels of Shh signaling activity in the neural tube. Shh signaling has proven to be necessary for the growth and development of the genital tubercle, the embryonic precursor to the penis and clitoris. Because hnn mutants have defective Shh activity in the neural tube, we predict that these mutants will also have defects in external genital development. Here we show that hnn mutants display smaller genital tubercles, consistent with a defect in Shh signaling. To determine if the phenotype we see is due to reduced and/or expanded levels of Shh signaling, similar to what was found in the neural tube of hnn mutants, we are examining expression patterns of Shh target genes in the genital tubercle. This study will define the role of cilia in the regulation of Shh signaling during development of the external genitalia, and will add to our understanding of the complex phenotypes found in ciliopathies. 66. Differential miRNA expression in osteosarcoma tumorinitiating cells and their reverted progeny Nasri E, Levings PP, Guijarro MV, Zarezadeh A, Hyddmark EM, White ME, Ghivizzani SC*, Gibbs CP Department of Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Previously we isolated tumorigenic and non-tumorigenic cells within the same tumor based on their ability to activate an exogenous Oct-4/GFP human promoter. GFP+ cells probed to be at least 100-fold more tumorigenic than GFPcells. Gene expression profile revealed G2/M checkpoint override in GFP+ cells and stress induced reversion/differentiation more in GFPcells. In this study we investigate heterogeneity at miRNA level in osteosarcoma. A total of 24 miRNAs in OS521 and 16 miRNAs in OS156 were found downregulated in the GFPrelative to the GFP+ cells. miR-15b, 18a, 20a, 93, 106a were differentially downregulated in GFPcells in both OS cell lines and play a crucial role in controlling the G2-M checkpoint (ATM, CHEK1, WEE1). MiR-221, 155, 93 have been shown to regulate G1/S checkpoint by targeting TP53, CDKN2A and CDKN2B. Four miRNAs (miR-206,134,202, 153) in OS156 and one miRNA in OS521 (miR-153) were found upregulated in GFPvs. GFP+ cells. MiR-153 was differentially upregulated in GFPcells in both cell lines, and it has been shown to act as a tumor suppressor by specifically targeting important growth signaling mediators. Our miRNA expression profiles show clear differences among tumor initiating and non-tumor initiating cells, supporting differential gene expression analysis data. This is the first study to examine the expression of miRNAs in osteosarcoma tumor initiating cells and following their reversion in vivo Our results suggest that miRNA expression may be a key regulatory mechanism behind intratumoral heterogeneity. 67 Adeno associated virus mediated somatostatin gene therapy reduces the severity of seizures in an animal model of epilepsy Natarajan G 1,2 McElroy JA 1,3 ,4 Zafar R 1,3-6 King MA 7,8, *, Carney PR 16, 1 B. J. and Eve Wilder Center for Excellence in Epilepsy Research, University of Florida, Gainesville, FL 2 J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 3 Department of Pediatrics, University of Florida, Gainesville, FL 4 Department of Neurology, University of Florida, Gainesville, FL 5 Department of Neuroscience, University of Florida, Gainesville, FL 6 Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, FL 7 Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 8 Malcom Randall VA Medical Center, NF/SGVHS, Gainesville, FL We tested the putative anticonvulsant role of the neuropeptide somatostatin (SST) overexpression in the rat amygdala electrical kindling model of temporal lobe epilepsy (TLE). We used the adeno associated virus (AAV) serotype 5 to introduce the rat preprosomatostatin gene into the bilateral hippocampi of rats were fully kindled to five consecutive grade five seizures. After waiting for a period of three weeks to allow for high gene expression, fully kindled rats were administered test stimulations at periodic intervals. Results demonstrated that all rats that showed optimal expression of the virus in the hippocampus after histological verification (n=7) showed a reduction in seizure grade. Forty-three percent of these rats (three out of seven rats) were also seizure free poststimulation. We also observed that the extent of seizure grade reduction was correlated to the final observable levels of expression of the virus vector in the hippocampus. In summary, our results demonstrate that the neuropeptide somatostatin exhibits anticonvulsant properties in TLE. It also raises the most important question in current gene therapy trials regarding the relationship between therapeutic efficacy, optimum expression levels of gene expression and target specificity. The findings of our study could be a significant development to further understand and study the clinical relevance of SST gene therapy in human TLE and to optimize gene expression.
34 68. Sexually dimorphic fin development: implications for the evolution of intercourse O'Shaughnessy K 1 ,2 Dahn R 3 Cohn MJ 2,4,5, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 7322 Country Wood Lane, Madison WI 53719 4 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 5 Department of Biology, University of Florida, Gainesville, FL The origin of internal fertilization is an important evolutionary event. The first vertebrate copulatory organs are paired structures called claspers, which are extensions of the posterior pelvic fins in ancient fishes. Claspers are found in the fossil record extending back to arthrodires, jawed fishes that predate the origin of sharks by 25 million years. Today only male cartilaginous fishes (sharks, skates, and rays) develop claspers, and the morphology is remarkably similar to those found in the most basal jawed vertebrates. Using the little skate ( Leucoraja erinacea ) as a model system, this study seeks to determine the molecular mechanisms involved in formation of this important evolutionary novelty. Analysis of embryonic skeletogenesis demonstrates that the male pelvic fin undergoes a prolonged period of outgrowth; this suggests that the fin development circuit may remain active in male fins, and is responsible for clasper formation. In support, analysis of fin patterning genes ( Hoxd12, Hoxd13, Shh and Fgfs ) by in situ hybridization demonstrates sustained gene expression in male fins when compared to stage-matched females. To identify an upstream factor that could be regulating this sexually dimorphic gene expression, we next investigated whether hormone receptors were present in the fins of L. erinacea Interestingly levels of androgen receptor (AR) transcript and protein were higher in male pelvic fins, suggesting that androgen signaling may regulate clasper development. These results suggest that the earliest male copulatory organs may have evolved by a hormonally regulated modification of the fin development program. 69. The coding potential of Pseudomonas aeruginosa : conservation and expression analyses of newly predicted genes Oden S 1 Picca A 1 Zhang Y 1 Jin S 1, *, Tornaletti S 2 Brocchieri L 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL In frame analysis of high GC content sequences, contrasts in GC usage among codon positions, plotted as S-profiles allow visual detection of coding regions. Previously we introduced N-PACT (NProfile Analysis Computational Tool), which implements two procedures for detecting potential coding regions, quantifying the principle of frame analysis and extending it to sequences of any nucleotide composition. One approach considers the local compositional biases of the sequence to score the composition of codon positions (Method 1). A second approach is based on the general expectation that coding sequences induce a compositional periodicity three in sequence content resulting in significant non-randomness in nucleotide distributions (Method 2). We used N-PACT to perform ab initio gene prediction in nine strains of Pseudomonas aeruginosa The N-PACT predictions were compared with the published gene annotations for each strain to discover their level of concordance and to uncover new genes that are not included in the published annotations. RNA-seq and ribosome footprinting were employed to validate certain highconfidence gene predictions in the strain PAO1. 70. Arabidopsis polygalacturonase1 beta subunit related mutant alters cell wall composition affecting plant growth Park J 1 Kang B-H 13, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 3 Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL The plant cell wall is composed of multiple polysaccharides, structural proteins, and enzymes. The polysaccharides are mostly cellulose and hemicellulose embedded in gel-like pectin polysaccharides. The cell wall undergoes differentiation including cell wall remodeling and repairing throughout the plant life cycle. During the modifications, numerous polysaccharide modifying enzymes and structural proteins are involved. Polygalacturonase1 (PG1) is a pectin-digesting enzyme composed of two subunits, catalytic PG2 and non-catalytic PG1beta with BURP domain. BURP domain proteins are reported that they are expressed in response to stress or interact with other cell wall proteins that promote cell elongation. The Arabidopsis polygalacturonase1 beta subunit (AtPG1b) is homologous to the tomato PG1b subunit. GUS genes under the control of AtPG1b promoter are detected mostly in cells that undergo elongation or cell wall remodeling. When GFP protein is fused to AtPG1b under its native promoter, the fusion protein was seen in the leaf trichomes cell wall where rapid cell expansion occurs with exclusive cell wall characteristics. Overexpressed AtPG1b transgenic plants showed enhanced growth rate while loss of function mutant of atpg1b delayed in growth rate. Moreover, immunogold labeling with antibodies against several pectic polysaccharides suggested alteration of cell wall composition in the gain and loss of function mutants. Coexpression analysis suggests that functions of AtPG1b are linked to those of cell wall expanding proteins. Taken together, with possibility of having binding
35 partners, AtPG1b is involved in cell expansion probably by interacting with cell wall extensibility. 71. Phylogenomics of basidiomycete fungi Pendleton A 1 Davis JM 1,2, *, Burleigh JG 1,3, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 3 Department of Biology, University of Florida, Gainesville, FL The availability of whole genome sequence data provides unprecedented opportunities to resolve the most perplexing relationships within the Tree of Life and infer the major processes of genome evolution. The goal of this study is to construct a well-supported phylogenetic tree of major lineages of basidiomycete fungi from whole genome data and use this tree to identify gene groups that may be involved in establishing pathogenic or symbiotic life histories. We assembled predicted proteomes of 15 Basidiomycetes and one ascomycete (as outgroup) from public sequence databases. The species were chosen to represent pathogenic, free-living, and symbiotic taxa. We used OrthoMCL to categorize 200,313 proteins from all 16 genomes into 19,489 gene families, which range in size from two to 334 proteins and together comprise 75.6% of input proteins. We first estimated the maximum likelihood phylogenic tree from 2404 single-copy genes that had sequences from at least eight of the 16 species. In spite of an alignment of over 2.1 million characters in length, the position of Mixia osmundae was unresolved. This is due to conflicting phylogenetic signal among genes; nearly equal numbers of genes support the placement of the pathogen Mixia with the yeast clade or with a clade of other pathogens. Patterns of duplications and losses and deep coalescence events in 4582 single and multi-copy gene trees were also evaluated. We similarly found mixed support among multi-gene families for the position of Mixia Thus, rather than resolving the phylogeny, the genomic data reveals phylogenetic ambiguity in Basidiomycota, likely reflecting a complex evolutionary history. 72. Comparing PCR conditions that detect rAAV harboring human erythropoietin cDNA: applications for gene doping Perez IC 1 Ni W 1 Le Guiner C 2 Moser D 3 Simon P 3 Moullier P 1,2 Snyder RO 1,2,4, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 3 Department of Sports Medicine, Johannes GutenbergUniversity, Mainz, Germany 4 Center of Excellence for Regenerative Health Biotechnology, University of Florida, Alachua, FL The development of molecular assays that are capable of detecting gene doping in sports can serve as a deterrent and can identify athletes who may be illicitly using gene transfer for performance enhancement (gene doping). In this study, assay parameters (specificity, LOD, and LOQ) for three different PCR assays were evaluated for the detection of a rAAV vector harboring a promoterless human EPO (phEPO) complementary DNA (cDNA) cassette. Following assay validation these assays were used to detect phEPO on samples of known copy numbers of phEPO plasmid spiked into 500ng nave human genomic DNA (hgDNA). Data shows that the Taqman real-time PCR assay which involves the amplification of a 114 bp product size has a LOD of 10/80 copies of circular plasmid in the absence/presence of nave hgDNA. The LOQ for this assay is 40/80 copies of circular plasmid in the absence/presence of nave hgDNA. The LNA real-time PCR assay designed to amplify a product size of 293 bp has an LOD of 5/160 copies of linearized plasmid in the absence/presence of nave hgDNA. The LOD of the nested PCR assay is five copies of circular plasmid in the absence/presence of nave hgDNA. The processing of samples with known copy numbers of phEPO plasmid showed that two out of the three assays were able to reliably detect and/or quantitate samples. Future work involves the detection of rAAV-phEPO in WBCs of injected macaques mixed with DNA from pooled human WBCs at different dilutions. 73. NOD2 signaling protects against ischemia/reperfusioninduced intestinal injury Perez-Chanona E 1-4 Muhlba er M 2-4 Jobin C 1-4 1 Department of Medicine, University of Florida, Gainesville, FL 2 Department of Medicine, University of North Carolina, Chapel Hill, NC 3 Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 4 Department of Pharmacology, University of North Carolina, Chapel Hill, NC NOD2 is an intracellular pattern recognition receptor that induces an autophagic response upon detection of muramyl dipeptide (MDP); a component of microbial cell walls. The contribution of NOD2 signaling towards hypoxia-mediated autophagy and its impact on epithelial wound-healing response has not been addressed. Here, we showed that the microbiota protects against ischemia/reperfusion (I/R)-induced injury as germ-free (GF) wildtype (WT) mice showed worse injury scores than conventionally-derived (CVN-D) mice (10.71.5 vs 4.11.6, p<0.05). Importantly, microbial-driven protection against injury is abrogated in CVND Nod2 -/mice and showed comparable injury as GF Nod2 -/mice (9.3 2.3 vs 9.22.3, n.s. Also, WT mice raised in specific pathogen-free (SPF) conditions fare better against I/R-induced injury than SPF Nod2 -/mice (4.50.3 vs. 8.61.4, p<0.05). SPF WT mice administered MDP (i.p., 10mg/kg), were protected against I/R-induced injury compared
36 to mice administered the control peptide (6.60.9 vs 3.30.8, p<0.05); an effect lost in Nod2 -/mice. In vitro studies showed that MDP signaling, coupled to hypoxic stress, induces a synergistic elevation in autophagic flux, comparable to the rapamycin control. Rapamycin protected Nod2 -/mice against I/R-induced injury compared to the controls (5.81.2 vs 9.01.2, p<0.05). These findings demonstrate that NOD2 protects against I/R and promotes wound healing, likely through the induction of the autophagy response. 74 A multi-locus phylogeny of Tetraoninae, the grouse and ptarmigan Persons NW, Kimball RT*, Braun EL*, Meiklejohn KA Department of Biology, University of Florida, Gainesville, FL The Tetraoninae, now a recognized subfamily of the large avian family Phasianidae (Order: Galliformes), includes all grouse and ptarmigan species. These ground-nesting pheasants are found in a wide variety of northern habitats ranging from U.S. sagebrush to high-latitude Siberian tundra. Several species are studied ecologically as game birds significant to small North American and Eurasian economies, while other research has focused on their exhibition of sexual dimorphism, ornamental traits, and mating habits. In addition, conservation efforts have been important during the last half of the 20th century, as >50% of phasianid species have faced an increasing danger of extinction. During this time the fine generic and species boundaries within Tetraoninae have been distinctly reshaped. Despite this research effort, the results of existing molecular phylogenetic studies lack confidence regarding many of these evolutionary relationships. Addressing this need, we analyzed all 19 species of grouse (and eight outgroup species) to produce a better supported species-level phylogeny. Nuclear regions are used to provide support for ancestral nodes while analysis of rapidly evolving mitochondrial DNA provides resolution for inter-species relationships. Considering this, we integrated data from four nuclear loci from published studies with data we collected for six additional nuclear regions and the mitochondrion, in attempts to provide better support for these relationships. 75 Investigation of the interaction between the Fanconi anemia E3 ligase and the FANCD2-FANCI heterodimer Polito D 1 Cukras S 1 Wang X 2 Spence P 1 Moreau L 3 AD 2 Kee Y 1 1 Department of Cell Biology, Microbiology and Molecular Biology University of South Florida, Tampa, FL 2 Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA 3 Cytogenetics Core L ab oratory, Dana-Farber Cancer Institute, Boston, MA Fanconi anemia (FA) is a cancer predisposition syndrome characterized by chromosomal abnormalities and cellular hypersensitivity to DNA crosslinking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 15 FA proteins. A central player of the pathway is a multisubunit E3 ubiquitin ligase complex, or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. Here we analyze a series of mutations of FANCE and report that a phenylalanine located at the highly conserved extreme C-terminus, referred to as F522, is a critical residue for mediating the FANCD2/FANCI monoubiquitination and conferring cellular resistance to mitomycin C. Consistent with previous structural analysis of FANCE, we further show that a mutation in the F522 residue abolishes physical interaction with FANCD2, but preserves the integrity of the FA core complex. Intriguingly, ectopic expression of the FANCE Cterminus fragment alone in FA-normal cells disrupts DNA repair, consolidating the importance of the FANCD2-FANCE interaction in the DNA crosslink repair. 76. Quality assessment in RNA-seq Polvadore T 1,2 Fear JM 2,3 Morse AM 2 Hans N 2,3 New FN 2 Bloom DC 2, *, Nuzhdin SV 4 Concannon PJ 5, *, McIntyre LM 2, 1 Advanced Concentration in Genetics, Interdisciplinary Program in Biomedical Sciences, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 4 Section of Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 5 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL RNA-seq is the use of high throughput sequencing to study transcription. While the data produced by RNA-seq are brimming with potential for discovery, there are technical issues that need to be addressed in every RNA-seq experiment. Unfortunately, not all RNA-seq data are of the same quality, and there are inherent sources of error that can accompany even the most diligently executed experiments. There are a myriad of potential technical as well as biological sources of variation that should be considered. Metrics that can be used on datasets that are too large to look at by eye have been developed, yet a standardized quality control (QC) pipeline that can be quickly and easily applied to RNA-seq datasets has yet to be developed. Such evaluation is critical, as valuable time and resources can be devoted not only to analysis but also to downstream follow-up experiments that are expensive. We have developed a QC pipeline that enables an intuitive grasp of
37 the quality of each sample in an RNA-seq experiment. We show the results on several datasets. 77. Measuring mitochondrial DNA diversity and demographic patterns of tribal and caste populations from the northeast Indian state of Assam Rej PH 1 Deka R 2 Norton HL 3 1 Department of Anthropology, University of Florida, Gainesville, FL 2 Department of Environmental Health, University of Cincinnati, Cincinnati, OH 3 Department of Anthropology, University of Cincinnati, Cincinnati, OH In an effort to place the northeast Indian state of Assam in the overall scheme of Indian mitochondrial (mtDNA) diversity, we used sequence information from the hypervariable region of the mtDNA molecule (HVR-1 and HVR-2), along with select diagnostic mutations from the mtDNA coding region. Focusing on both the tribal (Ahom, Sonowal Kachari, and Rabha) and caste (Assamese) populations of Assam, we have made a number of inferences on the population history and cultural practices of the populations in our study, finding that the people of Assam predominantly express autochthonous Indian haplogroups (M, M2, M3, M4, M5, M6, R5), but also express a higher degree of East Asian markers (A, F, D, M7, M8, M9, and B) due to their close proximity to south China and Southeast Asia. A general lack of northern Chinese, Tai, and IndoEuropean ancestral markers, however, led us to conclude that many of the population migrations to Assam were male specific; the ethnically Tai Ahom in particular reflect next to no Southeast Asian specific haplotypes, indicative of their 13th century male specific migration. In general, we found that haplogroup affiliation was more closely associated with geographical location rather than ethnolinguistic heritage. This study also identified two novel haplogroups: one subhaplogroup was found within macrohaplogroup M, the other in haplogroup R. Both novel haplogroups were discovered in the Rabha, an ethnically Tibeto-Burmese tribe hailing from Lower Assam. 78. Use of genomic selection to predict the performance of yetto be -made crosses Resende MFR 1,2 Munoz PR 3 Kirst M 1, 1 School of Forest Resources and Conservation, University of Flo rida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 Agronomy Department, University of Florida, Gainesville, FL The main goal/challenge of a researcher in a breeding program is to select the best individuals for a trait of interest and define which of these will be crossed in order to optimize the progeny. Genomic selection is a molecular breeding tool proposed to predict the individual performance based on DNA markers. However, given the costly effort to produce crosses, the use of genomic selection for efficient selection of crosses is possibly even more critical in a breeding program. To predict the outcome of yetto be -made crosses, we adapted genomic selection models that considered additive and non-additive effects. A loblolly pine population with 926 trees was used to predict, in silico all possible pairwise combinations. The resultant families were ranked according to their genetic value for total height. In addition, the molecular markers were used to simulate 50,000 meioses and predict the distribution of the future progeny around its mean. The results indicate that the dominance deviation is not homogeneous across all the crosses because different markers contribute differently to the dominance effect. The predicted crosses using genomic selection shows different patterns of variation compared to the phenotype estimates (rank correlation of 0.82). Nonetheless, the use of genomic information allows the prediction of the variance around the mean progeny value. In conclusion, the use of DNA markers in plant breeding should be exploited beyond the selection of individuals and extended to identify superior crosses. This approach also breaks a paradigm because it allows the use of non-additive variance in conventional sexuallypropagated breeding. 79. Reverse genetics analysis of the OPPP in maize seed development Ribeiro C, Spielbauer G, Boehlein SD, Tseung CW, Shaw JR, Hannah LC*, Settles AM* Horticultural Sciences Department, University of Florida, Gainesville, FL Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Central carbon metabolism provides precursors for the synthesis of storage molecules in maize seeds, such as starch, oils and protein. Within central carbon metabolism, the pentose phosphate pathway (PPP) is a major source of reducing power through the production of NADPH. The PPP also produces metabolic intermediates for the synthesis of nucleotides, amino acids, and fatty acids. Recent data from our lab showed that the chloroplast-localized PPP enzyme, PGD3, is required for starch accumulation. PGD3 appears to be heat labile, suggesting that the protein can be engineered to improve storage molecule accumulation. We are using a reverse genetics approach to test if additional PPP enzymes are required for maize endosperm development. We have identified 25 loci in the B73 genome that encode the eight enzymes required for the complete PPP cycle. Nineteen of these genes have been confirmed and annotated by cloning full-length cDNAs. Using UniformMu and MuIllumina transposon flanking sequence tags (FSTs), we obtained at least one transposon insertion for 20 PPP loci. We are currently
38 screening for homozygous lines for each locus and are generating double, triple, and higher order mutants to reduce or eliminate individual PPP enzymes in the plastid, cytosol, or peroxisome. Subcellular localization predictions are being confirmed or tested using transient expression of GFP fusions with the full length ORFs from the cloned cDNAs. The subcellular localization experiments will be used to guide double and higher order mutant crosses. We are also developing enzyme assays for all the OPPP enzymes to test the knockdown and knockout effects on the enzyme activity at the mutants obtained. 80. HC1 regulates growth and hydraulic conductivity in Poplar Ribeiro CL 1 Milles B 2 Dervinis C 1,2 Martin T 2 Peter G 1,2, *, Kirst M 1,2, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 School of Forest Resources and Conservation, University of Florida, Gainesville, FL Hydraulic conductivity (HC) is hypothesized to be a critical factor for rapid growth and biomass productivity in forest trees. Trees with low HC are expected to experience more extreme withintree water potential gradients and more frequent stomatal closure, reducing carbon gain and growth. We previously quantified genetic and phenotypic variation for stem hydraulic properties and growth in a pseudo-backcross population of Populus Quantitative trait loci (QTL) analysis identified a major locus that regulates vessel diameter, HC and growth. A genetical genomic approach integrating differentiating xylem gene expression and trait QTL data identified a previously uncharacterized gene referred hereafter as Hydraulic Conductivity 1 (HC1) as a potential regulator of the traits. Downregulation of HC1 expression in the hybrid genotype 717 was lethal. However, 717 transgenic lines up-regulating HC1 accumulate over 50% more biomass than wild-type. Analysis of the stem properties of these plants indicate they have vessels with larger diameter, count per area and longer vessel elements than those observed in the wild-type. The measured sapwood and leaf specific conductivity is significantly higher in the transgenic. However, no significant difference in the vulnerability to cavitation was observed indicating that other factors, such as pit membrane morphology, may contribute more to droughtinduced cavitation. 81. Gene therapy with self-complementary recombinant adeno-associated virus in a model of retinal degeneration based on mutant rhodopsin Rossmiller B 1 Mao H 1 Hauswirth WW 1,2, *, Lewin AS 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Department of Ophthalmology, University of Florida, Gainesville, FL Retinitis pigmentosa is the leading cause of hereditary blindness with 30-40% of cases attributable to autosomal dominant retinitis pigmentosa (ADRP). ADRP arises from mutations in at least 24 genes with 30% arising in the rhodopsin gene (RHO). Given the large heterogeneity of mutations in RHO leading to ADRP, we propose knocking down of endogenous RHO and changes that preserve the amino acid sequence but decrease the efficiency of knock-down. Here we report the use of a scAAV serotype 8 (Y733F) to express a hardened mouse rhodopsin and either miRNA or ribozyme under the control of the mouse opsin promoter. Each miRNA and ribozyme were first tested against the wild-type and hardened target using a 24 and 48 hour knock-down utilizing a dual luciferase plasmid. Following screening the I307N mouse model was used. I307N Rho mice were created through the use of N-ethyl-Nnitrosourea (INU) and will be used to serve as a model of ADRP. The I307N mouse model exhibits very slow degeneration under ambient light but is reduced in visual response to light by 50% in one week post exposure to a 100W light for five minutes in a mirror lined box. Viral injections will be given in the right eye at p30 with the left eye served as control. One month post injection photoreceptor preservation will be assessed following light damage through electroretinography (ERG) and optical coherence tomography (OCT). 82. Alternative targets of Chlamydia trachomatis small n on coding RNA IhtA Runac J 1 Tattersall JS 2 Grieshaber SS 2 Grieshaber NA 2 1 Advanced Concentration in Biochemistry and Molecular Biology, Interdisciplinary Program in Biomedical Sciences, University of Florida, Gainesville, FL 2 Department of Oral Biology, University of Florida, Gainesville, FL The obligate intracellular bacterium Chlamydia trachomatis is responsible for more than 3% of worldwide blindness and a common cause of pelvic inflammatory disease. C. trachomatis progresses through a biphasic development cycle alternating between a metabolically active phase and an inactive, infectious phase. The small non-coding RNA IhtA regulates expression of a key gene critical to controlling this development cycle. To investigate alternate targets of the IhtA, we have developed a surrogate system using CheZ deficient E. coli transformed with cheZ fused to potential target sequences of IhtA. Detecting the impact on chemotaxis of E. coli by expression of IhtA in these transformed bacteria will allow us to identify alternate targets of IhtA. CTL0322 has been identified as a potential target of the sRNA IhtA based on conservation of key nucleotides involved in the interaction between hctA and IhtA, and our surrogate system demonstrated that expression of the IhtA sRNA does repress expression of CTL0322. Bio-layer interferometry (BLI) was used to verify in vitro interaction between the CTL0322 transcript and IhtA. We will use the surrogate system to
39 investigate other potential IhtA targets and identify sequences within each target responsible for interaction with IhtA. Preliminary BLI data has detected interaction between purified CTL0322 and chlamydial DNA. DNase-seq will be used to identify regions of the chlamydial genome that may be under regulatory control of CTL0322. 83. ATP2B1 locus is associated with resistant hypertension (RHTN) in the International VErapamil SR-Trandolapril STudyGENEtic Substudy (INVEST-GENES) Fontana V 1 McDonough CW 2,3 Gong Y 2,3, *, El Rouby NM 2,3 S ACC 2-4 Pepine CJ 5 Cooper-DeHoff RM 2,3,5 Johnson JA 2,3,5, 1 Department of Pharmacology, University of Campinas, Campinas, SP, Brazil 2 Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 3 Center for Pharmacogenomics, University of Florida, Gainesville, FL 4 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 5 Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville, FL RHTN is a clinical condition defined as lack of blood pressure (BP) control despite use of three antihypertensive drugs or any BP level requiring four or more antihypertensive drugs. Increased risk for cardiovascular outcomes has been associated with RHTN compared to controlled hypertension. The aim of this study was to identify SNPs associated with RHTN in INVESTGENES. We used a gene-centric array containing ~50,000 SNPs to identify polymorphisms associated with RHTN in white and Hispanic hypertensive patients with coronary artery disease (CAD) in INVEST-GENES. Logistic regression analysis was performed in white (n=1023) and Hispanic (n=940) race/ethnic groups, adjusting for age, gender, treatment assignment, body mass index, principal components for ancestry, and history of diabetes, heart failure, myocardial infarction, stroke, left ventricular hypertrophy and peripheral vascular disease. In a meta-analysis, both race groups were combined using inverse variance-weighted methods. The top gene was followed up through expression analysis in RNA isolated from whole blood. The A allele of rs12817819 in the ATP2B1 locus was associated with RHTN in both whites (p=3.68 x 10 -4 OR=1.67 [1.26-2.22]) and Hispanics (p=4.72 x 10 -4 OR=1.77 [1.28-2.43]) and the meta-analysis result reached chip-wide significance (p=6.02 x 10 -7 OR=1.71 [1.39-2.12]). Expression studies revealed that the rs12817819 A allele was associated with significantly lower ATP2B1 expression compared to the G allele. ATP2B1 genetic variation and associated alteration in expression may contribute to causality of RHTN in CAD patients. Additional functional studies and replication of these findings are needed. 84. Novel roles for hSETD1A histone methyltransferase in regulation of canonical WNT-signaling pathway, cellular proliferation, and invasion Salz TH 1 Li G 2 Pampo CA 3 Kaye FJ 4, *, Siemann DW 3 Qiu Y 5, *, Zhou L 2, *, and Huang S 1, 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 3 Department of Radiation Oncology, University of Florida, Gainesville, FL 4 Department of Medicine, University of Florida, Gainesville, FL 5 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL Genomic patterns of H3K4 methylation are altered in transformed cells and during epithelial-mesenchymal transition. The reason for such changes is not clear. H3K4 methylation is catalyzed by the COMPASS family of histone methyltransferases. While hSETD1A and hSETD1B regulate promoter-H3K4me3 and significantly affect global gene expression, MLLs are mainly responsible for enhancerH3K4me1/2 and demonstrate only minor effect on global gene expression. The goal of this study was to elucidate the role of human hSETD1A in colorectal and breast cancers. Here, we report for the first time that hSETD1A is upregulated in colorectal and breast cancer specimens. Depletion of hSETD1A led to a dramatic decrease in global H3K4me3 and to retardation in cellular proliferation, invasion, and migration in vi tro and in vivo Genome wide microarray analysis in HCT116 cells depleted of hSETD1A led to a significant decrease in the expression of Wnt-signaling target genes. We further found that -catenin and that these interactions facilitat -catenin responsive promoters, enrichment of H3K4me3, TAF3 occupancy, and activation of Wnt targ Finally, this work provides new biologic and mechanistic insights into the role of hSETD1A in cancer and provides a potential candidate target for cancer therapeutics. 85. Development of a summer Ruby grapefruit via accidental cybridization Satpute AD 1 Grosser JW 1 Chen CX 1 Gmitter FG 1 Ling P 1 Grosser MR 2 Chase CD 3, 1 Citrus Research and Education Center, University of Florida, Lake Alfred, FL 2 Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 3 Horticultural Sciences Department, University of Florida, Gainesville, FL
40 The harvest season for grapefruit in Florida generally ends in May. After this time, fruits begin to lose acidity and juice content, and seeds germinate within the fruit, making the fruit unmarketable. To potentially generate seedless triploid citrus, we fused diploid protoplasts isolated from an embryogenic suspension culture of Dancy tangerine ( Citrus reticulata Blanco) with haploid tetrad-derived protoplasts isolated from anthers of Ruby grapefruit ( Citrus paradisi Macf.). Only one triploid plant was recovered, but several diploid plants with typical grapefruit morphology were regenerated. All of these trees maintained excellent fruit quality throughout the summer. This fruit remained firm with exceptional sweetness and good flavor into August, with no seed germination. Genetic analysis of the test grapefruit trees revealed that they were all cybrids. The nuclear composition of the test grapefruit trees was determined by eight SSR markers, with all alleles equivalent to those of commercial Ruby grapefruit. The mitochondrial genomes in the test grapefruit trees were determined by intron marker analysis to have originated from the embryogenic Dancy suspension culture, whereas the plastid genomes of individual trees originated from either fusion partner. We hypothesize that the haploid protoplast preparations contained contaminant diploid protoplasts, which fused with the Dancy suspension culture protoplasts, resulting in cybrid cells following the loss of the Dancy nucleus. These novel nuclear-organelle genome combinations associated with desirable fruit traits promise new market opportunities for Florida citrus. 86. Cross-talk between viral microRNAs (miRNAs) and host long nonassociated herpesvirus (KSHV) infected B-cell tumors Sethuraman S 1,2 Gay LA 1,3 Haecker I 1 Renne R 1, 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2 Advanced Concentration in Genetics, Interdisciplinary Program in Biomedical Sciences, University of Florida, Gainesville, FL 3 Advanced Concentration in Immunology and Microbiology Interdisciplinary Program in Biomedical Sciences, University of Florida, Gainesville, FL LncRNAs regulate cellular gene expression by a variety of mechanisms. Recently, it was shown that viruses (like HCV) can target host cell lncRNAs that function in host innate immunity. KSHV is a dsDNA virus that causes three different human miRNAs, which are highly expressed in latently infected tumor cells. Analysis of the KSHV miRNA targetome by HITS-CLIP assays revealed an enrichment of 230 host lncRNAs apart from mRNA targets (Haecker et al., 2013, PLoS Pathog 9(3)). Based on these observations and recent bioinformatics evidence for lncRNA-miRNA interactions, we hypothesize that KSHV miRNAs regulate host cellular gene expression by targeting lncRNAs. Since LncRNAs aid regulation at a transcriptional level in the nucleus and post-transcri ptionally in the cytoplasm, as an initial step, we determined the subcellular location of lncRNAs that were HITS-CLIP enriched in PEL cells. We found both nuclear and cytoplasmic lncRNAs. Further, subcellular fractions were screened for viral miRNAs and interestingly we detected mature KSHV miRNAs in the nucleus. We are currently investigating the pull-down of RISC complexes from total cell extracts for enrichment of both cytoplasmic and nuclear lncRNAs. Together, these observations strongly suggest that KSHV viral miRNAs interact with host lncRNAs, some of which might function in the nucleus as transcriptional regulators. Additionally, targeting of host lncRNAs by KSHV miRNAs could affect mRNA maturation, alternative splicing, and RNA turnover. 87. VKORC1 Asp36Tyr: a coding missense variant that shows more promise in individualizing warfarin therapy in northeastern African populations Shahin MHA 1,2 Cavallari LH 3 Perera MA 4 Khalifa SI 5 Dvorak A 6 Langaee T 1,2, *, Patel S 3 Perry K 4 McLeod HL 6 Johnson JA 1,2, 1 Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 2 Center for Pharmacogenomics, University of Florida, Gainesville, FL 3 Department of Pharmacy Practice, University of Illinois at Chicago, Chicago, IL 4 Department of Medicine, University of Chicago, Chicago, IL 5 Pharmaceutical Sciences Section, College of Pharmacy, Qatar University, Doha, Qatar 6 Institute for Pharmacogenomics and Individualized Therapy, University of North Carolina, Chapel Hill, NC The VKORC1 Asp36Tyr single nucleotide polymorphism (SNP) is considered one of the most promising predictors of high warfarin dose, but data on its population prevalence is incomplete. We determined the frequency of this SNP in participants from seven countries on four continents and investigated its effect on warfarin dose requirement. 1000 samples were analyzed to define the population prevalence of this SNP. Those samples included individuals from Egypt, Ghana, Sudan, Kenya, Saudi Arabia, Peru and African Americans from the United States. 206 Egyptian samples were then used to investigate the effect of this SNP on warfarin dose requirements. This SNP was highest among Kenyans and Sudanese, with a minor allele frequency (MAF) of 6% followed by Saudi Arabians and Egyptians with a MAF of 3% and 2.5%, respectively. It was absent in West Africans, based on our data from Ghana, and a large cohort of African Americans. Egyptian carriers of the VKORC1 Tyr36 showed higher warfarin dose requirement (57.129.4 mg/week) than those with the Asp36Asp genotype (35.816.6 mg/week; P<0.0001). In linear regression analysis, this SNP had the greatest effect size among the genetic factors (16.6 mg/week increase in dose per allele), and improved the warfarin dose variability explained in Eg yptians (model R 2 from 31% to 36.5%). The warfarin resistant
41 VKORC1 Asp36Tyr appears to be confined to north-eastern Africa and nearby Middle-Eastern populations, but in those populations where it is present, it has a significant influence on warfarin dose requirement and the percent of warfarin dose variability that can be explained. 88. LifNet: one mathematical model to optimize networks based on phenomics and molecular omics Shen Y Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL During the post-genomic era, one of the key issues is how to orchestrate various omics to reveal more details about the biological processes. We are struggling with obscurity of the graphic representation of molecular networks: crowdedness one mathematical algorithm, called LifNet, has been developed in order to optimize networks for more clearness and dynamics. The model took into consideration network-related phenotypes and molecular components in it. The phenotypes were quantified from phenomics and the weight of molecular components were defined by omic data. Both of them were impact to the network. We used aging network in C. elegans as an example to test efficiency of the model. We collected phenotypic data from literatures of aging study during past two and gene expression values from GEO microarray database (entry number: GDS583). Here, we show the optimized network highlighted functional components controlling longevity and it also demonstrated more biological dynamics during whole lifespan. 89. Genetic analysis of root growth and architectural traits associated with domestication of common bean ( Phaseolus vulgaris L.) Singh J 1 Gezan S 3, *, Vallejos CE 1,2, 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL Selection for the most desirable alleles causes the phenotypic alterations between wild and domesticated populations of various crop plants. Genetic analysis of various domesticationassociated traits has detected major genes that regulate those traits. However, previous analyses of domestication have mainly focused on the above-ground plant organs, while root traits have been largely neglected because of underground nature of this organ. This problem was further complicated by time consuming and destructive methods of phenotyping root traits by digging and washing roots from the soil. However, it is very likely that changes in root growth and architecture might have happened due to selection for above-ground plant parts as these organs need to operate in functional equilibrium with each other. Thus, we hypothesized that domestication of the common bean brought about changes in root traits controlled by two types of genes, one that controls root growth only, while the other regulates resource allocation to maintain a functional balance between root and shoot. We have performed a QTL analysis using a recombinant inbred population derived from a cross between a landrace (G19833) and a wild accession (G23419), both from the Andean gene pool. A SNP-based linkage map was constructed using the genotypingbysequencing approach. Time series root images were collected using a novel 2-dimensional phenotyping platform. Like previous domestication studies, QTLs for different traits were clustered on various genomic regions. 90. Estimating genetic diversity, migration, and population structure across a biogeographic barrier in Mexican free-tailed bats Speer KA, Reed DL* Mammalogy, Florida Museum of Natural History, University of Florida, Gainesville, FL Within the West Indies, the Bahamas provide a unique system where dispersal can be evaluated in a relatively controlled setting when compared to mainland communities. Previous research indicates that the oceanic channel separating the Great and Lesser Bahama Banks acts as a barrier to gene flow between bat populations. This break is interesting because it is unclear whether characteristics specific to the oceanic channel or habitat and resource differences between the Great and Lesser Bahama Banks are most important in deterring dispersal. To identify factors influencing gene flow and test the effects of this barrier on an excellent disperser, we examined the population structure of Tadarida brasiliensis across banks of islands. We collected tissue from a total of 160 individuals from localities on Grand Bahama and Abaco of the Lesser Bahama Banks, and Eleuthera and Long Island of the Great Bahama Bank. We amplified nine variable microsatellite loci, which were analyzed using STRUCTURE to test for population cohesion and MIGRATE to estimate movement between populations. Preliminary mitochondrial sequence data shows T. brasiliensis populations are distinct across the Great and Lesser Bahama banks. By characterizing dispersal within islands and between islands of the same bank, we can potentially identify attributes important in maintaining structure across these banks. These attributes may provide novel information for elucidating key mechanisms underlying biogeographic processes (e.g. colonization, speciation, extinction).
42 91. Using genotypeby -sequencing to uncover the population history of fungus farming ambrosia beetles Storer C 1 Hulcr J 1,2 1 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 2 Department of Entomology and Nematology, University of Florida, Gainesville, FL Many insects display haplo-diploidy as their mode of reproduction, producing haploid males from the unfertilized eggs of diploid females. Several groups of bark and ambrosia beetles (Coleoptera: Curculionidae: Scolytinae, Platypodinae) combine haplo-diploidy with complete inbreeding: a female produces many diploid daughters and one haploid son, who mates with his sisters. This allows the beetles to colonize new regions with a single individual, which has led to the global distribution of many of these species. Despite their ecological and genetic uniqueness and their increasing economic importance as exotic pests, little is known about the population history and dynamics of ambrosia beetles. This is in part due to the lack of genomic tools for studying these small and common species. Here we combine high-throughput genomic marker discovery and genotyping using restriction site associated DNA (RAD) sequencing to examine the population history of one of beetle Xylosandrus crassiusculus Specifically, we test whether a population that colonized the USA is a homogeneous clone from a single female, several clones, or a heterogeneous outcrossing population. We used two restriction enzymes to reduce the size of the X. crassiusculus genome into fragments that are the same size and that are shared among individuals for Illumina sequencing. Using RAD-Seq we recovered over 200 southeastern USA. Currently, population structure is being as sessed using summary statistics, generated through the STACKS pipeline, and the program STRUCTURE. 92. Correlating neuropathogenesis with brain infection dynamics in the SIVmac251 infected CD8-depleted rhesus macaque model using Bayesian phylogeography Strickland SL 1 Lamers SL 2 Veras NM 1 Prosperi MC 1 Burdo TH 3 Nolan DJ 1 Goodenow MM 1, *, Suchard MA 4-6 Williams K 3 Salemi MM 1, 1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 2 BioInfoExperts, LLC, BioInfoExperts, Thiobodaux, LA 3 Biology Department, Boston College, Chestnut Hill, MA 4 Department of Biomathematics, University of California, Los Angeles, CA 5 Department of Biostatistics, University of California, Los Angeles, CA 6 Department of Human Genetics, University of California, Los Angeles, CA The infection of CD8-depleted rhesus macaques with the SIVmac251 viral swarm is a rapid disease model of neuroAIDS. We hypothesized that early infection of the brain from specific tissue reservoirs can lead to the emergence of neurovirulent strains and the onset of neuropathogenesis. Approximately 1200 SIV gp120 sequences collected longitudinally from multiple sites and post mortem brain tissues from four infected primates were evaluated. Bayesian phylogeographic analysis was performed to deduce the mode of transmission, the tempo of the appearance, and the spatial distribution of the virus in the brain. Phylogeographic analysis showed multiple brain seeding events occurred throughout the infection, with the initial event occurring as early as seven days post infection (dpi), and the median of the transitions taking place between 52-68 dpi. Two primates showed a peak in the effective population size (Ne) at about 50 dpi; whereas one primate showed two peaks at 50 and 85 dpi and the last primate had three peaks occurring around 20, 50, and 85 dpi. Viral strains from multiple tissues were the precursors for the brain transitions throughout the infection. Interestingly, viral migrations from bone marrow occurred at approximately the same time as the observed peaks in viral Ne in the animals that developed SIV-associated encephalitis. The data indicates the majority of the migration events to the brain occurred several weeks post infection, near the rise in the Ne. Flow from the bone marrow showed a possible correlation with the increase in Ne and neuropathogenesis, which may account for ranges in neuropathology and provide a novel therapeutic site for treatment. 93. Defining a retinal phenotype in Usher IIIA mouse models Stupay RM 1 Smith WC 2 Deng W 2 Zhu P 2 Hauswirth WW 2,3, *, Dinculescu A 2 1 Advanced Concentration in Genetics, Interdisciplinary Program in Biomedical Sciences, University of Florida, Gainesville, FL 2 Department of Ophthalmology, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Usher syndrome type III (USH3A) is an autosomal recessive disorder that presents with progressive deafness as well as retinal degeneration in humans. This phenotype is a result of mutations in the Clarin-1 (CLN1) gene which encodes a tetraspanin protein of unknown function. As of yet, there is no discernable retinal phenotype in the current USH3A mouse models, however the mice present with early hearing loss and vestibular dysfunction. In the present study we attempt to reveal a retinal phenotype in the existing mouse models through the comparison of Arrestin-1 translocation upon exposure to light. CLN1 wild-type (WT) and mutant (MT) mice
43 were dark-adapted overnight and exposed to light for 1 hour at 1000 lux intensity. Both WT and MT photoreceptors display Arrestin-1 migration upon exposure to light; however, MT photoreceptors appear to have a delay in Arrestin-1 migration out of the OPL compared to the WT controls. Upon quantification of Arrestin-1 staining, the signal intensity from the OPL was significantly greater for CLN1 MT photoreceptors compared to WT, indicating that this delay in Arrestin1 migration may be indicative of a potential retinal phenotype. These results document a retinal phenotype for Ush3A mice in regards to the Arrestin translocation in response to light. This phenotype can be further used to test potential retinal gene therapies for USH3A, including AAV-mediated delivery of the missing wild-type CLN1 cDNA to the knock-out and N48K knockin retinas. 94. Characterization of the mechanisms by which SKN-1/Nrf influence oxidative stress resistance and longevity Tang L, Choe KP* Department of Biology, University of Florida, Gainesville, FL The SKN-1/Nrf family of inducible transcription factors are principle regulators of oxidative stress responses and are emerging as important determinants of longevity. We identified a protein named WDR-23 as a repressor of SKN-1 in the nematode Caenorhabditis elegans Loss of wdr23 causes accumulation of SKN-1, induction of detoxification genes, resistance to oxidants and electrophiles, increased longevity, and delayed age-related degeneration. Loss of wdr23 also slows growth and reduces brood size suggesting that SKN1 activation has important consequences. However, little is known about the mechanisms by which SKN-1 influences diverse processes. Epistasis analysis suggests that wdr-23/skn-1 acts independently of insulin/IGF-1 signaling pathway and mitochondria pathways to influence lifespan. Genes that are involved in glutathione metabolism are up-regulated by SKN1 in wdr23 mutants and they have a large influence on oxidative stress survival, suggesting that SKN-1 promotes oxidative stress resistance by increasing glutathione metabolism. RNA interference screening of SKN-1 target genes suggests that SKN1 may slow aging by activating genes that were previously reported to be required for promoting longevity and aging. It also suggests novel roles for SKN-1 in regulating protein translation and the extracellular matrix to promote stress resistance. Taken together, this study will begin to address the gap in our understanding regarding the mechanisms by which SKN -1 influence oxidative stress resistance and longevity. 95. The MBT domain protein SFMBT1 is an integral chromatin reader component of the LSD1 demethylase complex for epithelialto -mesenchymal transition Tang M 1,2 ,5 Shen H 3,4 Lin T 1,2 ,5 Pinard MA 1 Jin Y 1, 5 Cai Q 1, 5 McKenna R 1 Wu L 3,5, *, Lu J 1, 5, 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 4 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China 5 University of Florida Shands Cancer Center, Gainesville, FL Histone modifications critically impact chromatin structure and gene transcription. Chromatin readers recognize such marks and recruit specific chromatin-modifying effectors for subsequent epigenetic redrafting. The Snail family transcription factors are central drivers of epithelialto -mesenchymal transition (EMT), a developmental program believed to contribute to cancer progression. Snai1 represses epithelial gene expression by directly recruiting the LSD1 (KDM1A) demethylase complex to its target genes to remove dimethylation of histone H3 lysine 4 (H3K4me2), a mark associated with active transcription. But the LSD1 complex seems to lack a subunit to recognize modified histones. Here, we identified SFMBT1, a malignant brain tumor (MBT) domaincontaining protein, as a new component associated with the Snai1-LSD1 complex. MBT domains are histone readers with a preference for monoand di-methyl lysines. We found that SFMBT1 selectively binds diand unexpectedly, tri-methyl H3K4, both of which are enriched at active promoters. Depletion of SFMBT1 impairs LSD1 recruitment, Snai1/LSD1-mediated H3K4 induced EMT and cell invasion. LSD1 is overexpressed in many cancers, high levels of LSD1 are correlated with cancer and tumor relapse during therapy. Because SFMBT1 functions as an indispensable methyl H3K4 reader component of the LSD1 complex in interpreting epigenetic modifications, antagonist of SFMBT1 reader function may offer unique opportunity to interfere with EMT and cancer progression. 96 The genetic program for cartilage development evolved in the common ancestor of Bilateria Tarazona OA 1 Slota L 1 Cohn MJ 13, 1 Department of Biology, University of Florida, Gainesville, FL 2 Howard Hughes Medical Institute, University of Florida, Gainesville, FL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Cartilage has been proposed to be a defining character of vertebrates, however this tissue type has evolve d independently in a number of invertebrate lineages. In vertebrates, Collagen2A1 is the major structural protein in the cartilage extracellular matrix, and the Collagen2A1 gene is directly regulated by Sox9, Sox5 and Sox6 transcription factors. Wnt\beta-catenin and Sonic hedgehog signaling pathways play
44 important roles in chondrogenesis as well, by either activating or repressing sox mediated transcription of cartilage genes. To test the hypothesis that invertebrates and vertebrates use a common genetic program to build cartilage, we studied chondrogenesis in the cephalopod Sepia sp and the horseshoe crab Limulus polyphemus two distantly related invertebrates. We cloned the invertebrate orthologs of the ancestral (preduplication) Collagen2A1, Sox9, Sox5/6 and Hedgehog genes and examined their expression during embryonic development by in situ hybridization. We found that Clade A collagen and Sox genes are expressed in pre-chondrogenic cells of both species, and the expression of Wnt and Hedgehog signaling proteins in adjacent tissues suggests a possible regulatory role. The expression data presented here demonstrate that invertebrate cartilage is collagen-based and chondrogenesis is probably regulated by SoxE and SoxD genes. Therefore, invertebrate cartilages share homologous molecular building blocks, which c cartilage by a deeply conserved genetic program for chondrogenesis for Bilateria. 97. Lignin nanotubes as vehicles for gene delivery into human cells Ten E 1 Ling C 2 Wang Y 3-6 Srivastava A 2,5,6, *, Dempere LA 7,8 Vermerris W 1, 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Department of Pediatrics, University of Florida, Gainesville, FL 3 Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China 4 Shanghai University of Traditional Chinese Medicine, Shanghai, China 5 Powell Gene Therapy Center, University of Florida, Gainesville, FL 6 University of Florida Shands Cancer Center, Gainesville, FL 7 Department of Materials Science and Engineering, University of Florida, Gainesville, FL 8 Major Analytical Instrumentation Center, University of Florida, Gainesville, FL Lignin is a phenolic cell wall polymer that remains as a waste product after lignocellulosic biomass is converted to fermentable sugars that can be used for the production of fuels and chemicals. Development of high-value co-products from this waste stream is expected to make the production of fuels and chemicals from biomass economically more viable. We have developed lignin-based nanotubes synthesized in an alumina membrane template. Specifically, isolated lignin was linked to the inner walls of activated alumina membranes and then layers of dehydrogenation polymer were added onto the base layer and crosslinked via a peroxidase-catalyzed reaction. By using phenolic monomers displaying different reactivities, we were able to change the thickness of the polymer layer, resulting in the synthesis of nanotubes with a wall thickness of approximately 45 nm or nanowires with a diameter of 200 nm. SEM and nanoindentation studies indicated that the nanotubes exhibit different morphology and mechanical properties depending on the biomass species and lignin isolation procedure. Lignin nanotubes (LNTs) exhibited low cytotoxicity. LNTs enable the uptake of DNA, as shown by the expression of green fluorescent protein (GFP) following transfection with plasmid DNA. Confocal microscopy imaging revealed that all LNTs enter HeLa cells, and that LNTs made from NaOH-ligni n accumulate inside the cell nucleus. We further show that DNA can adsorb to LNTs, enabling the transfection of HeLa cells. The ease with which lignin nanotubes can be functionalized makes them attractive candidates for the delivery of DNA in gene therapy, as well as for the smart delivery of pharmacological agents. 98. Synthesis of threonylcarbamoyladenosine in Saccharomyces cerevisiae mitochondrial tRNAs Thiaville PC 1-3 Perrochia L 2 El Yacoubi B 3 Hecker A 2 Forterre P 2 Namy O 2 Basta T 2 de Crcy-Lagard V 3, 1 Graduate Program in Genetics and Genomics, University of Florida, Gainesville, FL 2 Institut de Gntique et Microbiologie, Universit Paris-Sud, Orsay, France 3 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Threonylcarbamoyladenosine (t 6 A) is a universal modification located in the anticodon stem loop of tRNAs, and its synthesis pathway was recently elucidated. Two of the protein families involved in t 6 A synthesis, TsaC/Sua5 and TsaD/Kae1, are found in all kingdoms of life. In addition, Bacteria generally require the TsaB and TsaE families while Eukarya and Archaea need the KEOPS complex. t 6 A is also present in mitochondrial tRNAs and in most organisms its synthesis occurs in this compartment. Qri7, a nuclear encoded homolog of the bacterial TsaD, localizes to the mitochondria and when targeted to the cytoplasm can complement kae1 S. cerevisiae TsaB and TsaE are specifically bacterial and the KEOPS complex proteins along with Sua5 are not known to localize to the mitochondria. Sua5 and Qri7 have recently been shown to form t 6 A in vitro (Wan et al., 2013, Nucleic Acids Res 41(12):6332-46), and here we demonstrate that in yeast, mitochondrial t 6 A synthesis is minimal, requiring only two components. Indeed, Sua5 and Qri7 are necessary and sufficient to synthesize t 6 A both in vitro and in vivo in a heterologous system using E. coli Like Trm5 involved in the formation of the m 1 G37 modification in tRNA, Sua5 localizes both to the cytoplasm and to the mitochondria through alternative AUG start sites. This minimal t 6 A pathway could be reminiscent of the ancestral system, which raises the question of what is the role of the added components in extant organisms.
45 99 Evolution of recombination rate in angiosperms Tiley GT, Burleigh JG* Department of Biology, University of Florida, Gainesville, FL The genomic recombination rate varies both among and within species, with fine scale variation at the population level. Although recombination rate may be highly variable among closely related species or populations, the evolution of recombination rate is not well understood on a macroevolutionary scale, and little is known about the patterns of variation in recombination rate among plant lineages. The evolution of global recombination rate in angiosperms, as well as its relationship with genome size and affects on genome evolution, has been disputed over the years. A better understanding of the evolution of recombination rate and genome size may elucidate some emergent properties about genome evolution. Specifically, are there detectable HillRobertson effects at the macroevolutionary scale? Also, since the rate of recombination should affect the efficacy of selection, are there concerted patterns of molecular evolution that vary with recombination rate? Here we investigate the mode and tempo of the evolution of global meiotic recombination rate from 30 angiosperm species, and we examine how it varies with genome size. We also estimate the proportions of sites under different selective regimes from gene clusters constructed from the 30 genomes and evaluate a possible link between rates of evolution change and recombination rate across the genome. 100. Testing the function of a new regulator of actin depolymerizing factor Turcotte M 1,2 Grey PH 1 Emmanuel M 1 Cuddy K 1 Oppenheimer DG 1,3, 1 Department of Biology, University of Florida, Gainesville, FL 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 3 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL The activity of the actin cytoskeleton is central to a wide variety of cellular processes such as cell motility, membrane trafficking, and intracellular transport. These processes are regulated by a variety of proteins and factors that facilitate the polymerization and depolymerization of actin subunits into filaments or monomers. The actin depolymerizing factor/cofilin (ADF) family of proteins plays a key role in regulating the depolymerization of actin filaments. Our lab recently discovered a novel protein that inhibits ADF function in Arabidopsis we named this protein ITB3. There are 21 additional related proteins within this family, which we named ITB3-like (ITB3L) proteins. This study focuses on whether a distantly related member of this family, ITB3L-6, is also a regulator of ADF. To do this, we created a bacterial protein expression vector that contains a yellow fluorescent protein tag and affinity purification tags to optimize expression, isolation, and purification of ITB3L-6. Results show successful cloning and expression of ITB3L-6, as evidenced by sequencing data, visualization of the co-expression of YFP, and confirmation by protein gel electrophoresis. Purified ITB3L6 was then analyzed using in vitro actin polymerization assays followed by fluorescent microscopy and actin filament length measurement. Our results show that polymerized actin filaments are longer in the presence of ITB3L-6 and ADF than in the presence ADF alone. These results suggest that all members of the ITB3 family are negative regulators of ADF. Further in vitro analyses of ITB3L-6 and other ITB3L proteins will continue to shed light on this new family of ADF regulators. 101. Enhancing magnetic nanoparticle based DNA tr ansfection: intracellular-active cassette features Vernon MM 1 Dean DA 2 Dobson J 1,3,4 1 J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 2 School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, NY 3 Department of Materials Science and Engineering, University of Florida, Gainesville, FL 4 Institute for Cell Engineering and Regenerative Medicine, University of Florida, Gainesville, FL Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique we have developed that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability (McBain et al., 2008, Nanotechnology 19(40):405102). Improvements to this technique have occurred mostly with particle functionalization and transfection parameter optimization, which cannot assist the nuclear translocation of delivered plasmid DNA and influence subsequent gene expression. In this study, features incorporated into the plasmid DNA sequence such as a cellappropriate promoter, Kozak sequence and a DTS sequence demonstrated the most significant improvements in observed transfection efficiency. Utilization of a cell-appropriate promoter and Kozak sequence increased transfection efficiency by ~400% in HeLa cells. Incorporation of a DTS sequence enhanced the proportion of transfected differentiated SH-SY5Y cells by ~450%. Incorporation of cell-appropriate features in the delivered plasmid DNA improves transfection efficiency in the tested cell types.
46 102. The impact of spatial variation in ozone on growth and development in Zea mays Wang C 1 Ainsworth L 2 McIntyre LM 3, *, Young LJ 1 Brown P 4 Leakey ADB 5 Dalsing B 4 Rios L 5 Sorgini C 4 Barrios-Perez E 4 Erice G 4 Shim S 4 Leisher C 4 1 Department of Statistics, University of Florida, Gainesville, FL 2 USDA-ARS Global Change and Photosynthetic Research Unit, Urbana, IL 3 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 4 Department of Crop Sciences, University of Illinois, UrbanaChampaign, IL 5 Department of Plant Biology, University of Illinois, UrbanaChampaign, IL Approximately 200 diverse maize inbred lines were screened for O3 tolerance at the FACE facility in Champaign, IL. Replicated plots of each of the 200 lines were grown at ambient [O3] (~40 ppb) and elevated [O3] (100 ppb). Each FACE ring had 52 genotype plots, and 12 B73 checks. The 200 lines were divided into four sets, where each set was replicated independently in two control and two ozone rings. The 200 lines included the nested association mapping (NAM) founder lines, which represent most of the current genetic variation in modern maize germplasm. Quantity and duration of O3 release was controlled by a proportional integral derivative algorithm for computer feedback to maintain a constant concentration of elevated [O3] (100 ppb). Centrally located sensors measured ambient background O3 concentration at the site, and sensors in the center of each ring [O3] measured wind speed and wind direction. The success of the ozone treatment, and the impact of the ozone distribution were examined spatially. The B73 checks were analyzed according to spatial models and the impact of ozone treatments over space was assessed. 103. S male-sterile maize a novel tool for the forward genetics of plant mitochondrial function Wang Y 1 Williams-Carrier R 2 Chamusco K 1 Zhao L 3,4 Hannah LC 1, *, Gabay-Laughnan S 5 Barkan A 2 Chase C 1, 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 2 Institute of Molecular Biology, University of Oregon, Eugene, OR 3 USDA-Animal and Plant Health Inspection Service, New Brunswick, NJ 4 Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 5 Department of Plant Biology, University of Illinois, Urbana, IL Interaction of the plant mitochondrial and nuclear genetic systems is exemplified by mitochondrial-encoded cytoplasmic male sterility (CMS) under the control of nuclear restorerof fertility (restorer) genes. Many restorer genes encode pentatricopeptide repeat (PPR) proteins that arose through adaptive evolution for the silencing of specific mitochondrial CMS gene targets. The CMS-S system of maize is characterized by a pollen collapse phenotype and by a novel paradigm for fertility restoration. Numerous restorer mutations rescue CMSS pollen but condition homozygous-lethal seed phenotypes. Seven independent, restorerof -fertility, seed-lethal (rfl) mutants recovered from Mutator (Mu) transposon-active, CMSS lines were not allelic based on the seed-lethal phenotype. Profiling pollen mitochondrial proteins and transcripts from normal-cytoplasm and restored CMS-S plants associated several non-allelic restorers with post-transcriptional failure to accumulate mitochondrial-encoded respiratory subunits. Illumina sequencing of Mu-flanking regions identified Mu insertions in nuclear genes encoding mitochondrial ribosomal proteins and mitochondrial protein complex assembly factors as candidate rfl mutations. The rfl mutations therefore rescue CMS-S pollen by disrupting central features of mitochondrial gene expression that are expendable in pollen but essential for seed development. CMS-S fertility restoration affords a novel genetic approach to investigate the nuclear regulation of mitochondrial biogenesis and function in plants. 104. Generation of a polyadenylation signal deleted and infectious wild-type adeno-associated virus serotype 2 Wang Y 1-4 Wang L 1,3,4 Lu Y 5 Aslanidi GV 3,4 Srivastava A 3,4,6,7, *, Ling C 1,2 Ling C 3,4 1 Department of Traditional Chinese Medicine, Second Military Medical University, Shanghai, China 2 Shanghai University of Traditional Chinese Medicine, Shanghai, China 3 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL 4 Powell Gene Therapy Center, University of Florida, Gainesville, FL 5 Department of Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL 6 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 7 University of Florida Shands Cancer Center, Gainesville, FL The single-stranded nature of the wild-type (WT) AAV2 genome, which renders it transcriptionally inactive, raises the intriguing possibility that AAV2 utilizes this feature to minimize expression of the viral genes during a natural infection. In our recent studies with recombinant AAV2 (rAAV2) vectors, in which the polyA sequence upstream from the right ITR in the viral genome was deleted, we observed detectable levels of transgene expression, which could be dramatically improved in the presence of AAV2 Rep proteins and adenoviral proteins. Therefore, we reasoned that WT AAV2 might also utilize the absence of a polyA signal as yet another mechanism to limit viral gene expression. To this end, we generated a recombinant
47 plasmid containing the WT AAV2 genome in which the polyA sequence was deleted. Following transfection of this plasmid in HEK293 cells, and in the presence of adenovirus co-infection, the polyA-deleted AAV2 genomes underwent successful rescue, replication, gene expression, encapsidation, and progeny virion production, which were indistinguishable from the WT AAV 2 genomes containing an authentic polyA sequence. The progeny virions were biologically active in secondary infections in the presence of co-infection with adenovirus. These data demonstrate that a conventional polyA signal is dispensable for WT AAV2, and can be substituted by the viral ITR to mediate efficient viral gene expression. Although it remains to be seen whether polyA-deleted AAVs exist in nature, our studies generated the first AAV genome that can maintain its life cycle without polyadenylation. 105. Differential accumulation of carotenoids and expression of carotenogenic genes in sweet orange fruits Wei X 1,2 Chen C 1,3 Yu Q 1 Gady A 1 Yu Y 1 Liang G 2 Gmitter FG 1 1 Citrus Research and Education Center, University of Florida, Lake Alfred, FL 2 College of Horticulture and Landscape, Southwest University, Chongqing, China 3 USDA-ARS Southeastern Fruit and Tree Nut Research Laboratory, Byron, GA Carotenoid composition and accumulation levels during citrus fruit maturation were compared in Valencia orange (Val) and Rohde Red Valencia orange (RRV). Samples were collected from the two varieties in July, September, November, January and February. Twenty three gene family members associated with carotenoid biosynthesis were analyzed by qRT-PCR. In RRV flavedo at the green stages, there was uncoordinated carotenoid accumulation and gene expression, which might be related to the expression of certain gene(s) in the MEP pathway. At the orange stages, more violaxanthin accumulated in Val flavedo than in RRV, while the expression levels of PSY, CCS and LCYB-Contig21 were rapidly increasing in RRV. These results suggest differential regulation of carotenoid biosynthesis pathways in flavedo of the two varieties. In juice sacs during the green stages, the low carotenoid content in both varieties coincided with low expression of LCYE-Contig03 and LCYE-Contig24, which suggested LCYE was the limiting step of carotenoid accumulation in early fruit maturation. During the orange stages, Val mainly accumulated violaxanthin, but RRV -cryptoxanthin and violaxanthin, which corresponded to differences in juice color. The upstream genes (PDS-Contig17, all ZDS family members, LCYB-Contig19) and the downstream gene (ZEP) were more highly expressed in RRV juice sacs than in Val, while the two PSY and four CHYB members were expressed at similar levels, which might be cryptoxanthin in RRV juice sacs; and the high expression of ZEP increased the accumulation of violaxanthin in juice sacs of RRV relative to Val. 106. Understanding differences in olfaction acuity through investigation of olfactory receptor gene repertoires in two genera of New World vultures (family Cathartidae) Wright JE, Braun EL*, Burleigh JG*, Kimball RT* Department of Biology, University of Florida, Gainesville, FL Olfactory receptors (ORs) are the largest multigene family in mammals. Among animals that have been examined, OR numbers vary widely, ranging from ~100-2000. The number of ORs in a acuity and percent of pseudogenized ORs are found to be high in mammals that have shifted to reliance on visual systems. Olfaction has been poorly studied in most bird groups. Chicken ( Gallus gallus ), which does not demonstrate reliance on olfaction, has ~500 OR genes. Black ( Coragyps atratus ) and turkey ( Cathartes aura ) vultures occupy the same habitat, yet demonstrate differing reliance on discrete sensory systems for foraging. Black vultures appear to be dependent on vision to locate food, while turkey vultures rely on olfaction, capable of foraging using smell alone. Turkey and black vultures present an opportunity to study the underlying genetic basis for reliance on olfaction in two related avian species. Additionally, investigation of the olfactory receptor repertoires in these species will contribute to our knowledge of what evolutionary changes are necessary in order for animals to shift reliance on sensory systems. We found that turkey vultures have three times as many OR genes compared to the chicken and our outgroup, the red-tailed hawk ( Buteo jamaicensis ), which has numbers similar to the chicken. Surprisingly, black vultures have an intermediate number of OR genes between turkey vultures and red-tailed hawks. Our results also indicate that a large amount of OR gene duplication occurred after the divergence of black and turkey vultures. 107. Adaptive divergence of recently duplicated genes in the maize Meg family Xiong Y 1 Mei W 2 Mukherjee K 1 Hassanein H 1 Barbazuk WB 2, *, Kolaczkowski B 1, *, Kang B-H 1,3, 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Department of Biology, University of Florida, Gainesville, FL 3 Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Gene duplications play a crucial role in the evolution of new gene function. Gene duplications can be broadly divided as large-scale (whole genome) and small-scale duplication events. One third of genes in the maize genome are estimated to be tandem duplicates. A number of evolutionary models have
48 been proposed to interpret the diversification of gene functions accompanying duplication but it is not easy to explain a specific duplication event with a specific model. This is because information for each duplicate such as its biological function and polymorphism in the population is not available or multiple models could be assigned to a particular gene family, Here we present results from phylogenetic and expression analyses of tandem duplicated family of the Meg genes. The founding member of the family, Maternally expressed gene1 ( Meg1 ) encodes a small cysteine-rich peptide that is required for the normal development of the maize basal endosperm transfer cells. Twelve more paralogs originated from Meg1 mostly by unequal recombination constitute a tandem duplicate gene family. Copy number variations of the Meg gene family suggest that the duplicated genes were fixed quickly after their appearance in the population. Phylogenetic analysis revealed a strong signature of positive selection in the youngest copies, suggesting a functional diversification after gene duplications. The burst of amplification in the Meg family fits the adaptive radiation model of gene duplications, which involves accelerated gene duplication rate, heterogeneous levels of gene expression, directional positive selection on amino acids, and higher ratio of pseudogenes and genes in the process of nonfunctionalization. 108. Embryo lethal plastid translation mutants and their genetic suppressors in maize Yang J 1 Suzuki M 1,2 McCarty DR 1,2, 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Horticultural Sciences Department, University of Florida, Gainesville, FL In plants, mutations that disrupt plastid translation typically cause embryo lethal (emb) phenotypes. In maize seed, such mutations severely perturb embryo development, but have little impact on endosperm development, indicating that plastid translation is essential for embryo development but not for endosperm formation. We have observed that in certain genetic backgrounds, some embryo lethal mutants are suppressed, conditioning viable embryos that germinate to produce albino seedlings. For example, when crossed to the B73 inbred, a mutant in plastidial Ribosomal Binding Protein A gene that has an emb phenotype in the W22 inbred background, segregates with an albino seedling phenotype in the F2. These findings led us to hypothesize that genetic background dependent suppression of emb mutants is specifically associated with plastid translation defects in maize. To test our hypothesis, we isolated additional mutants in plastid translation with emb phenotype from UniformMu W22 inbred transposon population. We have crossed these mutants with B73 inbred and are generating F2s to determine whether these mutants are also suppressed to produce albino seedlings. In addition, mapping of the suppressors is under way to determine the number of genes involved in suppression of embryo lethality in the W22/B73 hybrid. Our genetic and molecular study of these suppressors will provide insight into the specific role of plastid in embryogenesis. 109. Developing a reference genomic map for sugarcane resistance gene mapping Yang X 1 Comstock J 2 McCord PH 2 Wang J 1, 1 Agronomy Department, University of Florida, Gainesville, FL 2 US DA -ARS, Sugarcane Field Station, Canal Point, FL Genetic maps with positions of markers and known genes based on recombination frequency are an important tool for gene mapping and cloning, marker-trait linkage analysis, marker assisted selection, and other studies. Though efforts have been made towards sugarcane map construction for over a decade, the existing genetic maps are still far from saturated due to the features of large genome size, polyploidy, and heterozygosity of sugarcane. The objective of this research is to establish a high density genomic map for sugarcane resistance gene mapping, which can be utilized for efficiently mapping disease resistance and other traits for a marker-assisted selection approach in sugarcane breeding program. In this study, the complete sorghum genome was used as a reference to align available sugarcane SSR primers and plant disease resistance genes (R genes) for the reference genomic map construction. In total, 6,149 unique SSR primers were identified from a literature search, which were successfully amplified in sugarcane. In addition, 104,910 R genes were downloaded from the Plant R Gene Data Base, including 112 manually curated R genes. Of the 6,149 pairs of SSR primer sequences, 1,151 were aligned to the sorghum reference genome with less than three base pairs mismatch. Of the 104,910 R genes, 35,926 were mapped to the sorghum genome and matched to 1,890 sorghum gene models. This reference genomic map of ten chromosomes each has an average of 115 SSR markers and 189 R genes. With this map, SSR markers and R gene derived markers can be used to screen any population segregating for disease resistance, to quickly locate corresponding gene loci, and more markers in the targeted region can be designed for fine mapping the candidate genes. 110. BSMA: a bioinformatics pipeline for genome-wide bisulfite sequencing methylation analysis Yao J 1 Yu F 1 Farmerie W 1, *, Mou Z 2, 1 Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL Bisulfite conversion of genomic DNA combined with nextgeneration sequencing (BS-seq) is the gold standard of DNA
49 methylation analysis. Bisulfite treatment introduces specific changes in the DNA sequence. After sequencing, unmethylated cytosine (C) reads as T, whereas methylated C remains as C. We developed bioinformatics software (BSMA) that identifies methylation status of individual cytosines in the model plant Arabidopsis thaliana The workflow of this pipeline is as follows: 1) Alignment: reads were aligned with Novoalign against in silico bisulfite converted sense and antisense references of the Arabidopsis genome; 2) Filtering: reads with >10% mismatches, PCR duplicates, and non-converted reads are removed; and 3) Calling methylcytosines: a binomial probability distribution was applied to call a methylated cytosine with 95% confidence based on the coverage, total reads, bisulfite conversion rate and random mutation rate. This pipeline identified 3,910,680 and 6,109,887 methylcytosines with greater than 10x coverage accounting for 9.08% and 14.18% of the total cytosines in the wild-type Col-0 and the elp2 (Elongator subunit2) mutant, respectively. In Col-0, the average methylation levels for the sequence contexts CG, CHG, and CHH are 70.16%, 32.98% and 14.65%, respectively. The majority (72.8%) of the methylcytosines in all sequence contexts were shared in both Col-0 and elp2, and the elp2 mutation increased the total number of methylcytosines, but decreased average DNA methylation levels. We scanned chromosome 1 and identified a large group of genes in which DNA methylation levels varied dramatically between Col-0 and elp2. Our results demonstrate that our genome-wide bisulfite sequencing pipeline is a powerful tool for DNA methylation analysis. 111. Systems analysis of transcriptomic and metabolic reprograming of Arabidopsis in response to perturbation of jasmonate signaling Yoo M-J 1 Koh J 2 Yan H 1,3 Liu L 1,3 Chen Y 1 Acikgoz D 1 Wang Q 3 Chen S 1,2,4, 1 Department of Biology, University of Florida, Gainesville, FL 2 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 3 Department of Horticulture, Zhejiang University, Hangzhou, China 4 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL JASMONATE ZIM-DOMAIN (JAZ) proteins have recently been characterized as repressors in jasmonate (JA) signaling. In Arabidopsis thaliana 12 JAZ protein encoding genes were identified, but only a few were studied in detail. In this study, we focused on characterizing the molecular networks involving JAZ2 and JAZ7. We have isolated homozygous T-DNA mutants of JAZ2 and JAZ7, and observed their growth phenotypes in the presence or absence of methyl jasmonate (MeJA). To understand the phenotypes and elucidate the regulatory functions of JAZ2 and JAZ7, shoot and root tissues from wild type (WT), jaz2 and jaz7 were harvested for RNA sequencing and metabolomics experiments. Approximately 5.5 million RNA sequencing reads from shoots and 9.3 million reads from roots mapped onto 35,386 transcripts were evaluated for differential gene expression in the three genotypes after MeJA treatment. Besides, a total of 316 metabolites were identified, among which 16 exhibited unique changes after MeJA treatment. Transcript and metabolite patterns in jaz7 were remarkably different from those in WT and jaz2 Distinct changes of genes and metabolites in JA biosynthesis, primary and secondary metabolism, and oxidative stress were observed among the three genotypes, and new findings on pathway/network reprograming were discussed. 112. Proteomics and metabolomics analyses provide insight into flavor volatile production and regulation in tangerine fruit Yu Q 1 Plotto A 2 Baldwin EA 2 Bai J 2 Gmitter FG 1 1 Citrus Research and Education Center, University of Florida, Lake Alfred, FL 2 USDA-ARS Horticultural Research Laboratory, Fort Pierce, FL The biochemistry and biosynthesis of plant volatiles is complex, and involves many biochemical pathways and genes. Although many of the volatile constituents of flavors and aromas have been identified, the molecular mechanism and regulation of volatile production are still poorly understood. In order to screen tentative proteins involved in specific metabolic network related to aroma production, a comparative proteomic approach of the isobaric tags for relative and absolute quantification (iTRAQ) technique was used to identify differentially expressed proteins in juice sac. Results showed that the Temple and Murcott have two contract volatil e profiles, especially in chemical family of sesquiterpene and ester. We identified 798 unique proteins in Temple and Murcott. Of these identified proteins, 93 were differentially expressed in juice sac in three time points of ripening stages (fold change >1.5, P<0.05). Function analysis of the differential expressed proteins between two varieties revealed that a set of candidates was associated with volatile biosynthesis and regulation. An enzyme encoded sesquiterpene synthaseencoding gene ( Cstps1 ) was over expressed 25 times higher in Temple than Murcott. The enzyme is to convert farnesyl diphosphate (FPP) to valencene, a single sesquiterpene. No valencene was detected in Murcott during fruit development. Transcript was found over 217 and 2720 times significantly higher in Temple than Murcott on Dec 22, 2008 and March 11, 2009, respectively. The result suggests that up-regulated valencene synthesis and down-regulated isopentenyl pyrophosphate isomerase in terpene biosynthesis pathway might play important role of difference in composition of terpenes and carotenoids of the two varieties. In addition, other differentially expressed proteins and their potential roles were also discussed in the present study.
50 113. Proteasome inhibitors induce differentiation, cell cycle arrest and apoptosis in osteosarcoma Zarezadeh A, Levings P, Guijarro MV, Nasri E, Hyddmark EM, White ME, Ghivizzani SC*, Gibbs CP Department of Orthopaedics and Rehabilitation, University of Florida, Gainesville, FL Osteosarcoma (OS) is the most common primary bone tumor of childhood and adolescence. Despite numerous treatment approaches, OS remains difficult to treat and often reoccurs after primary treatment. This may lie in heterogeneity of the cells in response to commonly used treatments like doxorubicin. Our group works on intratumoral heterogeneity and previously we could fractionate two cell populations based on ability of the cells to activate exogenous human Oct4/GFP reporter. In OS156 cell line, we found up regulation of proteasomal degradation pathway genes in GFP positive, tumor initiating cells, suggesting this pathway might be a good candidate to target for treatment. Our data show that tumor cells are heterogenic in terms of response to treatment and some cells are almost three times more resistant to doxorubicin. Although we have a variation in bortezomib sensitivity among cell populations, bortezomib shows a range of favorable dose dependent anti-cancer effects on osteosarcoma including differentiation, cell cycle arrest at G2/M and apoptosis. Also the doxorubicin resistant population is the most sensitive one for bortezomib. Bortezomib can inhibit cell migration as a surrogate measure of metastasis in compared to doxorubicin. Interestingly, the bortezomib IC50 in osteosarcoma cell lines is similar to the IC50 previously demonstrated for multiple myeloma and lymphoma cell lines, suggesting that bortezomib can be potentially used in osteosarcoma in the same doses as lymphoma and multiple myeloma. All these data show that PIs are good candidates for treatment of osteosarcoma but further in vivo assessment is necessary to validate these findings. 114. The maize defective kernel5 (dek5) locus encodes a plant specific protein required for starch accumulation in seeds Zhang J 1 Wu S 2 ; McCarty, DR. 1,2 ; Settles AM 1,2 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 2 Horticultural Sciences Department, University of Florida, Gainesville, FL defective kernel 5(dek5) is one of the classic dek mutants in maize, but little is known about the biochemical and molecular function of the dek5 gene. The dek5 mutant has a shrunken seed phenotype with a severely reduced starchy endosperm. However, dek5 mutants can produce a normal embryo that germinates poorly. The dek5 seed phenotype is similar to starch biosynthesis mutants such as brittle1 and brittle2, and we hypothesize that dek5 affects endosperm starch biosynthesis in the amyloplast, a non-green form of the chloroplast in plant. The dek5 seedling leaves are pale green and occasionally develop white stripes. The pale green seedling phenotype of dek5 suggests the gene is also required for chloroplast function. Genetic mapping of dek5 with ~700 mutant kernels from Mo17 X dek5-25 F2 population narrowed the locus to a 460kb interval on chromosome 3. By co-localizing the genetic map position with transposon flanking sequence tags (FSTs) from the dek5 mutant, we identified a candidate insertion at the first exon of a gene of ~30kb with unknown function. Mu insertion cosegregation analysis and complementation tests with additional alleles identified from UniformMu reverse genetics resources confirmed the identity of the dek5 gene. 115. Oxidation and phosphorylation of MAP kinase 4 causes protein aggregation Zhang T 1 Zhu M 1, Song W 2,3, *, Harmon AC 1,3, *, Chen S 1,3,4, 1 Department of Biology, University of Florida, Gainesville, FL 2 Plant Pathology Department, University of Florida, Gainesville, FL 3 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 4 Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling events in response to environmental cues during plant growth and development. MPK4 is a stressresponsive protein kinase. It is activated by phytohormone abscisic acid (ABA) and hydrogen peroxide. However, little i s known about how redox contributes to the regulation of kinase activity. Our data show that the in vitro kinase activity of MPK4 is not affected by the redox state. In absence of a reducing reagent, phosphorylation of MPK4 caused protein aggregation. The aggregated MPK4 retained kinase activity and could be reversed to the monomeric form by the reducing reagent. To understand the protein conformational change caused by phosphorylation, the phosphorylation sites were mapped. Two amino acids within the activation loop were phosphorylated. In addition, protein aggregation was abolished in a Cys232A mutant. In summary, our data suggest the role of cysteines in protein conformation change during phosphorylation under different redox states, and aggregation may constitute a mechanism for kinase feedback regulation.
51 116. Methylated celastrol extracted from traditional Chinese medicine herbs enhances rAAV-mediated transgene expression both in vitro and in vivo Wang Y 1-4 Wang L 1,3,4 Zhang Y 1-4 Aslanidi GV 3,4 Srivastava A 36, *, Ling C 1,2 Ling C 3,4 1 Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China 2 Shanghai University of Traditional Chinese Medicine, Shanghai, China 3 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL 4 Powell Gene Therapy Center, University of Florida, Gainesville, FL 5 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 6 University of Florida Shands Cancer Center, Gainesville, FL Accumulating data have suggested that the bioactive monomeric compounds extracted from traditional Chinese medicine (TCM) herbs can be utilized together with viral vectorbased gene therapy. For instance, celastrol, a chemical monomer, isolated from Tripterygium wilfordii has the ability to significantly enhance rAAV1 serotype vector-mediated transgene expression in adipose tissues of mice through inhibition of the cellular proteasome activity. However, in our pilot studies, we observed that celastrol only modestly facilitated rAAV2 serotype vector-mediated transgene expression. Further investigations on the structure of celastrol revealed that a methylated isomer of celastrol, named pristimerin, also exists, which is isolated from Celastus hypoleucus Since pristimerin is also used widely in TCM to treat inflammation and detumescence and it is known to potently inhibit the cellular proteasome activity, but at a lower IC50 compared with Celastrol, we systemically evaluated the ability of these two bioactive compounds to enhance rAAV2 serotype vector-mediated transgene expression in both human cell lines in vitro and in murine hepatocytes in vivo In the present studies, we observed that treatment with pristimerin, at a lower concentration than celastrol, significantly enhances rAAV2 vector-mediated transgene expression both in cultured cells in vitro and in murine hepatocytes in vivo The underlying molecular mechanisms include both inhibition of ubiquitination of the capsid proteins and enhanced nuclear translocation of the vector genomes. These studies suggest the potential beneficial use of pristimerin in future liver-targeted gene therapy with rAAV2 vectors.
52 Best Poster Awards 2013 Best Student Poster Brian Rossmiller Poster 81. Gene therapy with self-complementary recombinant adeno-associated virus in a model of retinal degeneration based on mutant rhodopsin Rossmiller B, Mao H, Hauswirth WW, Lewin AS Honorable Mention Student Juan J. Acosta Poster 1. Demographic events shaping evolutionary history and genome-wide genetic diversity of Pinus taeda and Pinus elliottii Acosta JJ, Neves LG, Fahrenkrog A, Davis JM, Holliday JA, Kirst M Xiaofei Liang Poster 56. An oxalate decarboxylase gene functions in the early infection processes of Sclerotinia sclerotiorum Liang XF, Rollins J Best Post-doctoral Associate Poster Andre S. Chanderbali Poster 13. Gene co-expression networks exhibit limited conservation across flowering plants Chanderbali AS, Soltis DE, Soltis PS