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1 Florida Genetics 2006 Organizing Committee Chair: Indra Vasil Members: Nicholas Muzyczka, Diana Nolte, John Pastor, Sanjay Ranka, A. Mark Settles, Pamela Soltis, Michele Tennant FG2006 Sponsors University of Florida Genetics Institute, Evelyn F. & William L. McKnight Brain Institute, Graduate Program in Plant Molecular and Cellular Biology, College of Engineering, UF Health Science Center Libraries Special Thanks To David Brumbaugh, Dwight Bennett, Mickey Cuthbertson, Ned Davis, Martine Horrell University of Florida Genetics Institute Director: Kenneth Berns Associate Directors: Henry Baker, Donald McCarty, Connie Mulligan, Indra Vasil Executive Board: William Allen, Henry Bake r, Kenneth Berns, Su-Shing Chen, Robert Ferl, Terry Flotte, Janie Fouke, William Hauswirt h, Julie Johnson, Donald McCarty, Michael Miyamoto, Connie Mulligan, Nicholas Muzyczka, Winfred Phillips, Pam Soltis, Douglas Soltis, Michele Tennant Indra Vasil, Thomas Yang UFGI Strategic Plan The discovery of the three-dimensional double helix architecture of DNA in 1953 was not only a defining moment for biology, but arguably one of the most significant scientific discoveries of all time. It fundamentally and permanently changed the course of biology and genetics. The unraveling of DNAs structure, combined with its elegant mechanism for self-replication and the existence of a universal genetic code for all livin g beings, have together provided the basis for the understanding of fundamental cellular proc esses, mutation and genetic repair, genetic variation, the origin of life and evolution of species, and the structure/function/regulation of genes. The double helix is also proving to be of immense significance to advances in agriculture, medicine and such other diverse fields as an thropology, criminology, computer science, engineering, immunology, nanotechnology, etc. It was the study of DNA that led to the development of tools that brought about the biotechnology revolution, the cloning of genes, and the sequencing of entire genomes. Yet, most knowledgeable people agree that what has been achieved in DNA science thus far is only the beginning. Bigger and better applications, which will impact directly on the quality of human life and su stainability of life on earth, are yet to come. In order to attain these objectives, the digital natu re of DNA and its complementarity are beginning to be exploited for the development of biology as an information-based science. Indeed, a paradigm shift is already taking place in our vie w of biology, in which the natural, physical, engineering and environmental sciences are beco ming unified into a grand alliance for systems biology. Indeed, biology in the 21st century will be surely dominated by this expanded vision. The Genetics Institute is committed to fostering excellence in teaching and research, and in promoting cross-campus interdisciplinary interactio ns and collaborations. In the pursuit of these objectives, it offers a graduate program in ge netics, and has identified the following four key areas for teaching, research and development: Bioinformatics, Comparative Genomics, Population and Statistical Genetics, and Epigenetics.
2 FLORIDA GENETICS 2006 Schedule Wednesday, November 1, 2006 1:00-6:15 p.m.: Check-in Grand Ballroom Regist ration Desk, 2nd floor, J. Wayne Reitz Union 2:30-2:45 p.m.: Opening Remarks Indra K. Vasil, Ph.D., and Kenneth I. Berns, M.D., Ph.D. Session I Chair: Kenneth I. Berns, M.D., Ph.D. 2:45-3:30 p.m.: Thomas J. Kelly, M.D., Ph.D. Director, Sloan-Kettering Institute DNA replication and cell cycle checkpoints 3:30-4:15 p.m.: Jeffrey D. Palmer, Ph.D. Distinguished Professor, Department of Biology, Indiana University Plant mitochondrial genomes: unexpecte d bounties of lateral gene transfer 4:15-6:15 pm: Poster Session and Reception Thursday, November 2, 2006 8:00 a.m.-3:00 p.m.: Check-in Grand Ballroom Registration Desk, 2nd floor, Reitz Union 8:00-8:30 a.m.: Continental Breakfast Session II Chair: Nick Muzyczka, Ph.D. 8:30-9:15 a.m.: Steven A. Goldman, M.D., Ph.D. Professor, Departments of Neurology, Neurosurgery, and Pediatrics, and Chief of the Division of Cell and Gene Ther apy, University of Rochester Medical Center Isolation, induction and use of adult neural progenitor cells 9:15-9:45 a.m.: Dennis A. Steindler, Ph.D. Executive Director, Evelyn F. and William L. McKnight Brain Institute and Professor, Department of Neuroscience, University of Florida Embryonic, adult and cancer stem cells in vitro and in vivo 9:45 a.m.-10:15 p.m.: John M. Davis, Ph.D. Professor, School of Forest Resources and Conservation, University of Florida Genetic analysis of adaptive traits in forest trees 10:15-10:45 a.m.: Coffee Break
3 10:45-11:30 a.m.: Michael S. Waterman, Ph.D. Professor, Departments of Biological Sciences, Mathematics, & Computer Science, University of Southern California Whole genome optical mapping 11:30 a.m.-12:00 p.m.: Julie A. Johnson, Pharm.D. Professor and Chair, Department of Pharmacy Practice, and Director, Center for Pharmacogenomics, University of Florida Pharmacogenomics 12:00-2:00 p.m.: Poster Session and Lunch Session III Chair: Pamela Soltis, Ph.D. 2:00-2:45 p.m.: Gerald F. Joyce, M.D., Ph.D. Professor, Departments of Chemistry & Molecular Biology, The Scripps Research Institute and Investigator, The Skaggs Institute for Chemical Biology Genetics in vitro: synthesizing the RNA world 2:45-3:15 p.m.: Douglas E. Soltis, Ph.D. Professor, Department of Botany, University of Florida Darwins abominable mystery: developm ental genetic/genomic insights into angiosperm evolution 3:15-3:45 p.m.: Sean M. Sullivan, Ph.D. Associate Professor, Department of Pharmaceutics, University of Florida Development of tumor vasculature targeted nanoplexes for treatment of malignant brain cancer
4 Presentation Abstracts DNA replication and cell cycle checkpoints Kelly TJ Sloan-Kettering Institute, New York, NY Cell cycle checkpoints are complex signal transducti on pathways that protect the integrity of the genome by regulating cell cycle progression when the duplication or segregation of the chromosomes is perturbed or the chromosomal DNA is damaged by endogenous or exogenous factors. Checkpoints function to preserve cell viability and to prevent gene tic alterations that can lead to cancer. We have studied two cell cycle checkpoints in the model organism S. pombe: (1) the replication checkpoint which stabilizes the replisome and delays mitosis when DNA synthesis is inhibited by nucleotide starvation or other factors and (2) the DNA damage checkpoint that blocks cell cycle progression when the genome is damaged by ul traviolet (UV) irradiation. The re plication checkpoint is activated at blocked replication forks by the protein kinase Rad3 (ATR in humans) acting through a mediator protein, Mrc1 (claspin in humans), which appe ars to be a component of the replisome. Rad3 phosphorylates Mrc1 to generate docking sites for a second protei n kinase Cds1 (Chk2 in humans). Cds1 is then activated in two stages by Rad3 ph osphorylation followed by autophosphorylation. The activated Cds1 phosphorylates downstream targets th at block cell cycle progression and prevent fork collapse. This novel two-step kin ase cascade provides sensitivity and noise immunity, properties required for the replication checkpoint. Eukaryotic cells irradiated with high doses of UV exhibit responses, referred to as the G1/S, intra-S or G2/M checkpoints, that function to delay particular cell cycle transitions. It is generally thought that the checkpoint-mediated delays provide time for repair of UV photoproducts. Using computer-aided time-lapse microscopy to follow individual S. pombe cells, we found unexpectedly that these checkpoi nt responses are not activated when cells are irradiated with moderate UV doses that approxi mate sunlight exposure and could reasonably be expected to occur in nature. Instea d, cells irradiated in all phases of the cell cycle progress without significant delay into the following S phase and then arrest for hours after the completion of bulk DNA synthesis. This post-replication replication checkpoint response is entirely dependent upon initiation of DNA replication and the checkpoint kinase, Chk1. Failure of the post-replication checkpoint response results in gr oss chromosomal damage and loss of viability only after UV lesions are propagated through S phase and the subseque nt mitosis. Thus, the DNA damage checkpoint appears to be triggered by structures (probably single-stranded gaps) in the chromosomal DNA that are generated when replication forks encounter UV le sions. It follows that the likely physiologic role of the checkpoint is to protect the genome by providing time for post-replication repair. Biography of Thomas J. Kelly, M.D., Ph.D Dr. Thomas J. Kelly, M.D., Ph.D., joined Memorial Sloan-Kettering Cancer Center in 2002 as director of Sloan-Kettering Institute afte r a 30-year career at The John s Hopkins University School of Medicine, where he served as director of the De partment of Molecular Biology and Genetics and director of the Institute for Basic Biomedical Sciences. Dr. Kellys research focuses on how the genome is duplicated during the eukaryotic cell cycle with particular emphasis on the ways DNA replication is initiated and controlled. Using animal viruses as models, Dr. Ke llys laboratory developed the first cell-free systems for studying the bioche mistry of DNA replication in human cells. More recently he has focused on the links between DNA re plication and the progression of the cell cycle in human cells and in the fission yeast, Schizosacchromyces pombe, which shares many properties with higher eukaryotes. He was a co-winner of the 2004 Alfred P. Sloan Jr. Prize for his seminal contributions to the understanding of the molecula r mechanisms of eukaryotic DNA replication. Dr. Kelly is a member of the National Academy of Scie nces, the American Academy of Arts and Sciences, and the American Philosophical Society.
5 Plant mitochondrial genomes: unexpected bounties of lateral gene transfer Palmer JD Department of Biology, Indiana University, Bloomington, IN Horizontal gene transfer (HGT) is now recogniz ed as a major evolutionary genetic force driving genomic and phenotypic change in prokaryotes and many unicellular eukaryotes. In contrast, there is little published evidence that HGT is common or important in the major groups of multicellular eukaryotes (animals, plants, and fungi). We have discovered that HGT of mitochondrial genes in plants is both widespread and re cent and have now expanded this work in several directions. Our recent work has provided insight into mechanisms of HGT (it frequently occurs by direct physical contract between parasitic plants and their host pl ants and seems to involve direct fusion between donor and recipient mitochondria), has identifie d a plant whose mitochondrial genome has been radically shaped by HGT (it contains numerous gene s acquired by HGT, and from a wide variety of donors, from other flowering plants to mosses and algae), and has shown that many horizontal transfers give rise to chimeric genes in which pieces of foreign genes are past ed into native copies. Biography of Jeffrey D. Palmer, Ph.D Jeffrey Palmer received his Ph.D. from Stanford Unive rsity. He joined the faculty of Indiana University in 1989, is a Distinguished Professor in the depa rtment of biology, and holds the Class of 1955 Endowed Professorship. He served as chairman of the department from 1996 to 2003. Under his leadership the department grew substantially in size and stature. His academic interests in biology focus on plant evolution, molecular evolution, and genomics. He was the recipient of a Presidential Young Investigator Award from the National Science Foundation (1985-1990). He received the David Starr Jordan Prize in 1990, the Wilhelmine E. Key Award from the American Genetics Association in 1998, a Merit Award from the Botanical Society of America in 2003, the Stebbins Medal in 2005, and a Guggenheim Fellowship for 2005-2006. He was el ected to the American Academy of Arts & Sciences in 1999, to the National Academy of Scie nces in 2000, and to the American Association for the Advancement of Science in 2005. With more than 200 influential scientific papers to his credit, Dr. Palmer was named in 2002 as one of the top 15 most highly cited researchers worldwide in the field of plant and animal sciences by the Institute for Scientific Information. He is widely considered to be a world leader in evolutionary genomics.
6 Isolation, induction and use of adult neural progenitor cells Goldman SA Departments of Neurology, Neurosurgery, and Pediatrics, University of Rochester Medical Center, Rochester, NY The major progenitor pools of the human brain, each of which has now been isolated, include ventricular zone neural stem cells, VZ neuronal pr ogenitors, hippocampal neuronal progenitors, and parenchymal glial progenitor cells (GPCs). The most abundant of these are the GPCs, which can generate astrocytes and oligodendrocytes in a context-dependent fashion. When isolated and transplanted into neonatal shiverer mice, whos e brains lack myelin, human GPCs produced oligodendrocytes that mediated substantial and ex tensive myelination, and substantially extended the survival of the implanted mice. Yet whereas adult gl ial progenitors generated only oligodendrocytes and astrocytes in vivo, upon removal from the tissue environmen t they produced neurons as well as glia, and expanded as neurospheres. Glial progenitor cells thus seem restricted to glial phenotype by their local environment, but nonetheless retain multilineage competence in isolation. By purifying these cells and assessing their patterns of differential gene expression, relative to their parental tissue environments, we identified a set of signaling pathwa ys that regulate their homeostatic self-renewal in vivo, and whose perturbation allows the directed indu ction of astrocytic and oligodendrocytic fate. Endogenous stem cells of the ventricular subependym a may also be targeted for induction. These cells may be induced to generate new neurons by over-exp ressing BDNF, and this pr ocess is potentiated by the BMP inhibitor noggin, which suppresses glial d ifferentiation. The new neurons mature as medium spiny neurons, and integrate into the existing stri atal neuronal network. Remarkably, the new neurons successfully project to the globus pallidus, extendin g processes over several mm of normal adult striatum. This strategy for inducing new medium sp iny neurons in the adult neostriatum has also been assessed in the R6/2 mouse, a transgenic model of Huntingtons disease. R6-2 mice treated with adenoviral BDNF and noggin exhibit both improved motor performance and longer survival than untreated controls, suggesting th e potential efficacy of this strategy for replacing medium spiny neurons lost to Huntingtons. These experiments argue that as our understanding of the biology of adult neural progenitor cells becomes more extens ive, our ability to target, induce and implant these cells for therapeutic purposes will become increasingly manifest. Biography of Steven A. Goldman, M.D., Ph.D. Dr. Steven A. Goldman is a professor of neurolog y, neurosurgery, and pediatrics, and chief of the division of cell and gene therapy at the Universit y of Rochester Medical Center. At Rochester, he holds the Dean Zutes Chair in Biology of the Agin g Brain, and serves as attending neurologist at Strong Memorial Hospital. He moved to Rocheste r in 2003 from the Weill Medical College of Cornell University, where he was the Nathan Cummings Professor of Neurology and Neuroscience, and attending neurologist at New York Presbyterian Hospital. A summa cum laude graduate of the University of Pennsylvania, he obtained his Ph.D. from Rockefeller University in 1983, and his M.D. from Cornell in 1984. His thesis work, with Fernan do Nottebohm at Rockefeller, included the first report of neuronal production in the adult vertebra te brain. Dr. Goldman then interned in medicine and completed his residency in neurology at New York Hospital-Cornell and at the Memorial SloanKettering Cancer Center. In 1988, after serving as chie f resident in neurology, he joined the faculty at Cornell and New York Hospital, where in 1997, at 39, he was promoted to professor of neurology. Dr. Goldmans laboratory is interested in neural re generation and cell genesis in the adult nervous system, with focus on neural stem and progenitor cells and their use in treating demyelinating and degenerative diseases of the brain and spinal cord He has published over a hundred papers in his field as first or senior author. He is a recipient of the Jacob Javits Neuroscience Investigator Award of the NIH, and has been elected to the American Neurological Association and American Society for Clinical Investigation. He has served as a cons ultant to both Aventis Pharmaceuticals and Merck Research Labs, and is a co-founder of Q Therapeutics in Salt Lake City.
7 Whole genome optical mapping Waterman MS Departments of Biological Sciences, Mathematics, and Computer Science, University of Southern California, Los Angeles, CA An innovative new technology, optical mapping, is used to infer the genome map of the location of short sequence patterns called restriction sites. The technology, developed by David Schwartz, allows the visualization of the maps of randomly lo cated single molecules around a million base pairs in length. The genome map is constructed from overlapping these shorter maps. The mathematical and computational challenges come from modeling the measurement errors and from the process of map assembly. Biography of Michael S. Waterman, Ph.D. Michael Waterman holds an endowed associates ch air at the University of Southern California. He came to USC in 1982 after positions at Los Alamos National Laboratory and Idaho State University. He has a bachelors degree in mathematics from Oregon State University, and a Ph.D. in statistics and probability is from Michigan State Universit y. He was named a Guggenheim Fellow (1995), was elected to the American Academy of Art and Sciences (1995), and was elected to the National Academy of Sciences (2001). He is also a Fellow of the American Association for the Advancement of Science and Fellow of the Institute of Mathematical Statistics. He has held visiting positions at the University of Hawaii (1979-80), the University of California at San Francisco (1982), Mt. Sinai Medical School (1988), Chalmers University (2000), and in 2000-2001 he held the Aisenstadt Chair at University of Montreal. He is professor-at-large at th e Keck Graduate Institute of Life Sciences and in fall 2000 he became the first Fellow of Celera Ge nomics. In 2002 he received a Gairdner Foundation International Award and in 2005 he was elected to the French Acadmie des Sciences. In fall 2003, he became faculty master of Parkside Internationa l Residence College, a residental college that is home to over 600 undergraduates and serves as a center for internationally oriented cultural, academic and social events. Professor Waterman works in the area of computational biology, concentrating on the creation and application of mathematics, statistics and computer science to molecular biology, particularly to DNA, RNA and prot ein sequence data. He is the co-developer of the Smith-Waterman algorithm for sequence compar ison and of the Lander-Waterman formula for physical mapping. He is a founding editor of Journ al of Computational Biology, is on the editorial board of seven journals, and is co-author of the texts Computational Genome Analysis: An Introduction and Introduction to Computational Biology: Maps, Sequences and Genomes.
8 Genetics in vitro: synthesizing the RNA world Joyce GF It is believed that an RNA-based genetic system, usually referred to as the RNA world, preceded the DNA and protein-based genetic system that has existed on Earth for the past 3.5 billion years. Questions concerning how the RNA world arose and the degree of complexity it attained can be addressed through laboratory experiments in preb iotic chemistry and RNA biochemistry. Central to the operation of the RNA world is the ability of RNA to catalyze the replication of RNA, thereby enabling RNA-based evolution. Through methods of in vitro evolution, we have developed RNA enzymes that catalyze the template-directed join ing of RNA substrates. One such molecule was converted to a format that allows it to produce additional copies of itself by joining two component subunits. It subsequently was converted to a crosscatalytic format whereby two RNA enzymes direct each others synthesis from a total of four component substrates. These molecules, however, operate with a highly restricted set of RNA substrates an d cannot undergo evolution on their own. Using a variety of approaches, we are employing in vitro evolution to develop other RNA enzymes that have enhanced potential to replicate and evolve in a self-sustained manner. Biography of Gerald F. Joyce, M.D., Ph.D. Gerald F. Joyce is a professor in the departme nts of chemistry and molecular biology and an investigator with the Skaggs Institute for Chemical Biology at The Scripps Research Institute in La Jolla, Calif. He received his B.A. from the Universit y of Chicago in 1978 and both an M.D. and Ph.D. from the University of California, San Diego, in 1984 He carried out postgraduate medical training at Mercy Hospital in San Diego and postdoctoral resear ch training at The Salk Institute before joining the faculty of The Scripps Research Institute in 1989. Dr. Joyces research involves the test-tube evolution of nucleic acids and the application of these methods to the development of novel RNA and DNA enzymes. He also has a longstanding interest in the origins of life and the role of RNA in the early history of life on Earth. He has published ov er 100 scientific papers and is the inventor or coinventor of 11 issued patents. He was elected to the U.S. National Academy of Sciences in 2001, and in 2005 received the H.C. Urey Award, presented ever y six years by the International Society for the Study of the Origin of Life. Dr. Joyce has lectur ed extensively around the world, including at the Pontifical Academy and the Royal Swedish Academy of Sciences.
9 Embryonic, adult and cancer stem cells in vitro and in vivo Steindler DA Department of Neuroscience, McKnight Brain In stitute, University of Florida, Gainesville, FL The adult rodent and human brain harbors a population of stem/progenitor cells (NSCs) that can both self-renew and generate progeny along the three lin eage pathways of the central nervous system (CNS), but their in vivo identification has proven elusive. Our recent studies have further characterized a multipotent astrocytic stem cell (MASC) that reside s within brain marrow (the adult subventricular zone) using an in vitro system that recapitulates neuropoiesis in vitro as it occurs in vivo. Live cell imaging, immunophenotypic analysis, and electrophysiol ogical studies have revealed a cell that exhibits astrotypic characteristics, but can give rise to large numbers of neuroblasts. These cells occupy a distinct neurogenic niche in the adult CNS, but inju ries and disease may induce what appear to be fully differentiated astrocytes to assume a neurogenic ro le whereby they upregulate their expressions of developmentally-regulated proteins and attempt to recapitulate neurogenic programs. Neurogenic astroctye-like cells change their biochemistry thro ughout the neuraxis at the end of the second postnatal week in mice and assume a more differentiated phenotype in vivo where their activities are related to standard operating procedur es associated with the normal CNS. In vitro conditions can be created, relying on information gained from studie s of embryonic and cancer stem cells, that restore neurogenic programs of apparently differentiated brai n cells, and the goal of new studies is to resolve the precise molecular cascades responsible for lifelo ng plasticity of CNS cells. This way, reactive neurogenesis could be exploited for neural repair in many different neurological disorders. In addition to the hope offered by stem-like cells in the mature human brain, these cells may also be involved in tumorigenesis that warrants further investigation. Genetic analysis of adaptive traits in forest trees Davis JM* School of Forest Resources and Conservati on, University of Florida, Gainesville, FL The health of our terrestrial ecos ystems exerts many direct and indirect influences on the human population on earth, with impacts ranging from th e global carbon balance to renewable energy. Since forests are such a prominent class of terrestrial ecos ystem, the application of genomics approaches in forest tree species is expected to have significant, positive impacts on the understanding of ecosystem function. Genomics approaches are now feasible in Populus and Pinus, two distinct genera of forest trees with complementary phenotypic traits, ecologic al niches and genomic toolkits available for their analysis. The extent of natural genetic diversity in both of these systems is remarkably high, which creates the opportunity to make causal connections between natural allelic variation (i.e. genotype) and adaptive traits (i.e., phenotype) in properly design ed experiments. The outcom es of this research are expected to enhance the efficiency of genetic improvement programs, and to improve understanding of forest ecosystem form and function. = U.F. Genetics Institute Faculty
10 Pharmacogenomics Johnson JA* Director, UF Center for Pharmacogenomics University of Florida, Gainesville, FL Pharmacogenomics is a field aimed at discovery of the genetic basis for variable drug response, with the potential to improve the safe and effective utilization of existing drug therapies, and identify new drug targets. This session will provide an overview of literat ure from the field, experimental approaches that are used, and future clinical potential of pharmacogenomi cs. There will be a particular focus on work at the University of Florida, which is focused on the pharmacogenomics of drugs used in the treatment of hypertension, heart failure and clotting disorders. Th ere will also be discussion of the UF Center for Pharmacogenomics, and its mission and activities. Darwins abominable mystery: developmental genetic/genomic insights into angiosperm evolution Soltis DE* The flower is the central identifying feature of the angios perms. Yet, little is known about the origin of the flower and the subsequent early and rapid evolutiona ry diversification of flowering plants (Darwins Abominable Mystery). Synergistic research involving paleobotany, phylogenetics, classical developmental studies, genomics, and developmental genetics has affo rded valuable new data regarding floral evolution in general, as well as the early flower in particular. Re cent progress in understan ding angiosperm phylogeny provides a solid framework for evaluating evolutionary innovation, and identifies the taxa that provide the best insights into key innovations. The interplay of genomics, developmental genetics, and focused studies of morphology, has provided rapid advances in the un derstanding of flower evolution. Phylogenetic studies of floral gene families suggest that most, if not all, of the fundamental genetic components of the classic Arabidopsis-based ABC model of floral organ identity were present in the ancestral angiosperm genome. Developmental genetic studies of diverse flowering plan ts reveal that there is no single model of floral organ identity that applies to all angiosperms; rather there are likely multiple models that apply depending on the phylogenetic position and floral structure of th e group in question. The classic ABC (or ABCE) model may work well for most highly derived angiosperms. However, modifications are needed for basal angiosperms. We offer the fading borders as a te stable hypothesis for the basalmost angiosperms and, by inference, perhaps some of the earliest (now extinct) angiosperms, as well. Development of tumor vasculature targeted nanopl exes for treatment of malignant brain cancer Sullivan, SM* Department of Pharmaceutics, Unive rsity of Florida, Gainesville, FL Plasmid DNA isolated from E. Coli is formulated with cationic amphiphiles to form toroids <200nm in diameter, termed nanoplexes. Peptide ligands that bi nd to receptors expressed on tumor cells and tumor endothelium are derivatized to the nanoplex surface yielding targeted gene delivery to these cells. Rat arterial organ cultures (arteriographs) are used to op timize the targeting paramete rs of the nanoplexes and also study the role of receptor density in achieving selective gene transfer. Expression of cytotoxic genes engineered to kill the tumor cells and tumor endothelia l cells is restricted to only these cells using a proliferating endothelial promoter. Th e cytotoxic genes are the product of molecular biological engineering with the end result being expression of gene products that are secreted from the transfected cells killing not only the transfected cells but all the nearby surro unding cells. Hence, transfection of a small number of cells results in a large impact on tumor growth.
11 Posters = U.F. Genetics Institute Faculty 1. Altering plant architecture of bahiagrass by constitutive expression of a gibberellin-catabolizing enzyme Agharkar M, Lomba P, Altpeter F*, Zhang H, Kenworthy K*, Sinclair T Department of Agronomy and Plant Molecular and Ce llular Biology Program, University of Florida, Gainesville, FL Bahiagrass is a low input, drought tolerant and di sease resistant warm seas on turfgrass used for residential lawns and along highways in the sout heastern US. Turf quality of bahiagrass is compromised by prolific seedhead production, op en growth habit and light green color. The objective of this study was to improve the turf quality of bahiagrass by over-expression of a gibberellin catabolizing enzyme, Gibberellin 2-oxi dase (GA2ox). GA2ox1 and GA2ox8 ORFs were isolated from Arabidopsis and sub-cloned under the control of the constitutive ubiquitin or 35S promoters. Co-transfer of constitutive nptII an d GA-2 oxidase expression cassettes into seed derived callus cultures from turf-type, apomictic bahiagrass (cv. Argentine) was carried out by biolistic gene transfer. Transgenic nature of the regenerated plants was confirmed by NPTII ELISA (Agdia), PCR, Southern blot analysis and RT-PCR. Initial phenotypic characterization was carried out with hydroponic or soil grown plants under controlled environment conditions. Five independent transgenic lines were established in a replicated small field plot trail at the PSREU near Citra, FL. Data collected from controlled and field environment on plant height, number of vegetative and generative tillers, seedhead le ngth, flowering time establishment rate and biomass of clippings will be presented. 2. Peripheral myelin protein 22 modulates the cytoskeletal organization of epithelia and Schwann cells Amici S, Bachow S, Notterpek L* Department of Neuroscience, Unive rsity of Florida, Gainesville, FL Peripheral myelin protein 22 (PMP22) is a tetraspan glycoprotein involved in the formation and maintenance of myelin in the peripheral nervou s system. If PMP22 is misexpressed, inherited neuropathies arise; however, the function of PMP22 at the plasma membrane remains unclear. We have recently shown that PMP22 and 6 4 integrin are in a complex in Schwann cells of the peripheral nervous system and in a human colon adenocarcinoma cell line (clone A cells). Integrins are transmembrane receptors that link th e extracellular matrix with the cytoskeleton. They control signaling events critical for ce ll survival, morphology and movement, which are often modulated by the Rho family of GTPases. To explore if PMP22 is involved in modifying cell morphology, adhesion and migration, we have employed in vitro models to evaluate the intrinsic properties of cells with altered levels of PMP22. Schwann cells derived from PMP22-deficient mice exhibit reduced adhesion and slowed migration. Overexpressed human wild type PMP22 traffics to the cell surface of clone A epithelia and is associated with reduced proliferation. By immunostaining, phosphorylated myosin light chai n, a downstream target of Rho kinase, is prominent at focal contacts when PMP22 is overex pressed, as compared to vector control cells. The increase in phosphorylated myosin light ch ain prompted us to examine cellular adhesive properties. Indeed, the overexpression of PMP 22 strengthens the adhesion of epithelia to laminin. These results indicate, through dire ct or indirect mechanisms, PMP22 modulates cytoskeletal organization, and provide novel insights into the biological function of this diseaselinked gene.
12 3. Transposon insertion needed for normal seed development? Braunschweig M, Settles AM* Department of Horticultural Sciences, University of Florida, Gainesville, FL Carotenoids are a widely-distributed group of pi gments having a long carbon chain and a red, yellow or orange color. In plants, carotenoids pl ay roles in energy transfer in photosynthesis, and are precursors to flavor/aroma molecules and hormones such as abcisic acid (ABA). The maize viviparous rough (vp-rgh) mutant is characterized by a shrunken seed with low starch accumulation, germinates early while on the mother plant, and the white seedling dies because it lacks pigmentation for photosynthesis. We cl oned the vp-rgh locus, and the sequence was found to be from the phytoene desaturase (pds ) locus. Pds is an enzyme in the catoenoid biosynthetic pathway. Vp-rgh individuals were found to be polymorphic at the pds locus with PCR, having either large(700bp) and/or small(500bp) bands. Normal individ uals posses at least one large band, but individuals with only the small band have the lethal mutant phenotype. Sequence alignment showed the large and small alle les to be identical, except the normal allele has one more exon. The normal allele for the pds locus can be amplified with a TIR primer, while the mutant allele can not, suggesting that the normal allele has a transposon insertion. A Southern blot may tell us if the transposon inse rtion is affecting copy number of the gene. To follow, we must learn how this transposon insertio n in a carotenoid biosynthetic gene is affecting seed development. 4. Genomic analysis of the human Hsp70 protein family Brocchieri L1,*, Conway de Macario E2, Macario AJL2 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2University of Maryland Biotechnolo gy Institute, Baltimore, MD Hsp70 proteins play crucial roles in cell physiolo gy and pathology. In a definitive survey made feasible by the availability of the complete human genome sequence, we identified 47 loci encoding hsp70 candidates. Sixteen of them had an N-terminal nucleotide-binding domain (NBD) typical of Hsp70 conserved in various degrees, but could be separated into sub-groups by distinctive C-terminal domains. Eight of the ge nes corresponded to typical hsp70s, encoding proteins of approximately 70kDa and possessing th e characteristic C-terminal substrate-binding domain (SBD). The products of these eight gene s were found to reside in the cytosol/nucleus (HSPA2, HSPA8, HSPA6, HSPA1A, HSPA1B, and HSPA1L), in the endoplasmic reticulum (HSPA5), and in the mitochondrion (HSPA9B). In two other genes the SBD domain was truncated (HSPA14) or missing (STCH). The C-terminal domains were unrelated to the typical Hsp70 SBD in the remaining six genes, of which four (HSP A4, HSPA4L, HSPH1, HYOU1) encoded heavier proteins (100-170 kDa) and two (HSPA12A and HS PA12B) showed regions of differentiation that extended also to large parts of the NBD. Among the remaining 31 sequences, one was characterized by one internal frame-shift and, although reportedly transcribed under certain conditions, its functionality remains to be estab lished. Thirty hsp70-related sequences presented multiple frame-shifts and in-frame stop codons ch aracteristic of pseudogenes. We also analyzed exon-intron structure, protein isoforms, alternative-transcription variants, patterns of expression, and evolutionary relationships of all 47 hsp70 candidates. Exon-intron structure analysis and evolutionary-tree reconstructions revealed a centra l role for HSPA8 in the evolution of the human hsp70 family. The results indicated that five of the six genes encoding 70-kDa cytosolic proteins and the vast majority of the 30 hsp70 pseudogenes originated from HSPA8 by retrotransposition. This finding unveils the importance of LINE retr o-transposon elements in the evolution of the human Hsp70 family.
13 5. The murine autosomal Ant4 gene is essential for survival of male meiotic germ cells Brower J1, Rodic N1, Seki T2, Fliess N2 McCarrey J3 Oh P2, Terada N1,* 1Department of Pathology, University of Florida, Gainesville, FL 2Department of Physiology and Functional Ge nomics, University of Florida, Gainesville, FL 3Department of Cellular and Structural Biology, Un iversity of Texas Health Sciences Center at San Antonio, TX The Adenine nucleotide translocases (Ant) are nucle ar encoded DNA gene products that facilitate the transport of ADP and ATP by an antiport mechanism across the inner mitochondrial membrane. We have recently identified a novel memb er of the Ant family, Ant4 in mouse, which is highly conserved among mammals. In contrast to the closest paralog Ant2, which is encoded by the X chromosome and ubiquitously expresse d in somatic cells, Ant4 is encoded by an autosome, chromosome 3. Ant4 expression begins at the transition that occurs between type B spermatogonia as they progress into primar y spermatocytes. The primary spermatocytes represent the cell type within the testis that enters meiosis. Concomitant with the entry of the spermatocyte into meiosis is th e inactivation of the X and Y ch romosomes which begins during prophase I. Indeed, real-time PCR analysis of the different spermatogenic cell types of spermatogenesis, demonstrate a decrease in Ant2 and an increase in Ant4 transcript upon entry into meiosis. Thus Ant4 appears to be evol utionary conserved in mammals in order to compensate the loss of Ant2 expression due to the meiotic sex chromosome inactivation (MSCI) that occurs during male germ cell development. Furthermore, Ant4 ablation by homologous recombination based gene silencing has led to an approximate 3 fold reduction in testis mass. Ant4 deficient male mice exhibit severe mo rphological aberrations in the process of spermatogenesis, with only spermatogonia presen t in the adult testis. The Ant4 deficient adult testis exhibit high levels of apoptosis in comparis on to wild type and Ant4 heterozygous testis, as shown by TUNEL analysis. These data clearly indica te that the autosomal gene Ant4 is essential for survival of male meiotic germ cells. 6. Network analysis reveals HIV-1 activa tion of macrophages is independent of tolllike receptors Brown J1, Kohler J1,2, Coberley CR1, Sleasman J4, Goodenow M1-3,* 1Department of Pathology, Immunology, and La boratory Medicine, University of Florida, Gainesville, FL 2All Children's Hospital, Gainesville, FL 3Pediatrics, Division of Immunology an d Infectious Diseases, Gainesville, FL 4University of South Florida, St. Petersburg, FL Macrophages offer important longlived reservoirs for HIV-1. Esta blishment of viral reservoirs within macrophages is not a benign process. We hypothesized that genetic networks involved in regulating the immune response and signal tran sduction cascades would be impacted by HIV-1 infection, and these complex interrelated proces ses would be better und erstood from a global genomic perspective. Monocyte-derived macrophage s from four individual healthy donors were treated with a macrophage-tropic HIV-1JR-FL virus or mock treated. At da ys 2, 4, and 7 post infection cells were lysed and RNA was collected and prepared for hybridization on Affymetrix HG-U95A microarray GeneChips. Resulting data was imported into a custom engineered relational database designed for functional genomic analysis of HIV-1 infection. Through database queries, a subset of genes that varied by more than 5-fold relative to mock-treated cultures in any given donor was selected and utilized in hierarchical a gglomerative clustering. Mutual exclusive analysis
14 was performed to further identify genes detected from either virusor mock-treated cultures alone. Functional analysis of gene expression during HIV-1 treatment identified complex genetic networks where crosstalk, synergy, and antagonism occur at multiple levels. Global analysis of intracellular proteins and secreted cytokines demons trated evidence of an early shift, by day 2, to a predominant Th2-type milieu in primary macr ophages induced by HIV-1. Network systems analysis identified altered expression of factor s across several networking signal transduction pathways. The network analysis approach highlig hted the uniqueness of the Toll-like receptor (TLR) pathway because few factors within the path way were impacted by HIV-1. Viral escape of TLR recognition was confirmed using a bimodal a pproach. Collectively, this study emphasizes the utility of network analysis in high-throughput ex periments that assimilates data across both the genome and the proteome. 7. Adeno-associated virus serotype 8 produces efficient gene delivery to widespread neuroendocrine and epithelial cell types Tenace L1, Cruz P2, Conlon T2, Clarke TJ1,*, Foust K2, Song S3,*, Zolotukhin S2,*, Flotte TR2,*, Campbell-Thompson M1,* 1Department of Pathology, Immunology, and La boratory Medicine, University of Florida, Gainesville, FL 2Department of Pediatrics, University of Florida, Gainesville, FL 3Department of Pharmaceutics, University of Florida, Gainesville, FL Adeno-associated virus (AAV) serotypes have th e potential to become important resources for clinical gene therapy by targeting specific ce ll populations. Among the novel AAV serotypes engineered for production of recombinant vect ors, AAV8 has shown tremendous potential for in vivo gene delivery in rodents with nearly complete transduction of many tissues such as liver and heart after intravascular infusion. In this study, AAV8 carrying the green fluorescent protein (GFP) reporter gene was administered by intravascular infusion in mice and neuroendocrine cell populations examined for GFP expression to further define AAV8 transduction potential and efficiency. The ability to transduce cell types was evaluated by GFP positive cell percentage. Transduction of hepatocytes and cardiomyocytes was used as an index for maximum potential. Widespread GFP expression was found in gastri c antrum, pancreas (acinar and islets), ovary (follicle, corpus luteum), and adrenal gland wi th the highest intensity found in the zona fasciculata in the adrenal gland. GFP positive cells were also found in epithelium in the gastrointestinal and reproductive tracts. These findings suggest that AAV8 could prove useful for neuroendocrine-directed systemic gene therapy and show potential for gene therapy of epithelial cells in the gastrointestinal an d reproductive tracts as well. 8. Microarray profiling of Persea americana flowers: in search of organ-specific gene expression and developmental pathways. Chanderbali A1, Altman N2, Leebens-Mack J3, Soltis PS4,*, Soltis DE1,* 1Department of Botany, Universit y of Florida, Gainesville, FL 2Department of Statistics, Pennsylvania State Un iversity, University Park, State College, PA 3Department of Biology, Pennsylvania State University, University Park, State College, PA 4Florida Museum of Natural History, University of Florida, Gainesville, FL As part of our goals to investigate the origin conservation, and diversification of the genetic architecture of the flower, a microarray approa ch to study floral gene expression has been developed for Persea americana (avocado). This species is one of a set of plant exemplars selected, based on recent phylogenetic studies, to represent the basal angiosperm groups, and to
15 bridge the gap between the plant models Arabidopsis and rice. The microarrays, printed by Agilent Technologies, contain in-situ synthesize d 60-mer oligonucleotide probes, representing approximately 6200 unique avocado floral transcripts collected and sequenced by the Floral Genome Project (http://fgp.bio.psu.edu/fgp/index.ht ml). We have examined gene expression in young and medium floral buds, tepals, stamens, ca rpels, initiating fruit, and leaves using a double loop design. Using a cut-off of two-fold difference, we identified 1,354 floral transcripts that were up-regulated in at least one floral tissue compared to leaves. Among the florally upregulated genes are homologues of AGAMOUS, APETALA3, PISTILLATA and SEPALLATA3, bonafide floral genes in Arabidopsis. Hierarchical clustering identified several tissue-specific gene clusters. One cluster of ca. 60 genes was primarily expressed in stamens, another group of ca. 60 genes was restricted to tepals, and one of ca. 80 genes was up-regulated in both carpels and fruit. A single cluster of ca. 20 genes was up-reg ulated in fruit alone, and one of ca. 50 genes was up-regulated only early in floral development. Clustering of tissues suggested that tepals and stamens have similar expression pr ofiles, while carpels and fruits formed a separate cluster. The ca. 20 Persea genes supporting the clustering of tepals with stamens includes several without Arabidopsis homologues or for which the function of Arabidopsis homologues is unknown. These genes are likely participants in a developmental pathway shared between stamens and tepals of Persea. 9. Regulation of -globin expression through the re cruitment of chromatin modifying enzymes by TFII-I and USF Crusselle-Davis VJ1-4, Anantharaman A1-4, Dodev T1-4, Bungert J1-4 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2Powell Gene Therapy Center, Univer sity of Florida, Gainesville, FL 3Center for Mammalian Genetics, University of Florida, Gainesville, FL 4UF Shands Cancer Center, Gainesville, FL The human -globin locus contains five functional genes which are arranged in the order of their developmental expression. Gene proximal cis-regu latory DNA elements and interacting proteins restrict expression of the genes to the embryonic, fetal, or adult stages of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region also contributes to the temporal regulation of the genes. To more fully understand adult -globin gene regulation, we examined the downstream promot er and found that transcription factors TFII-I and USF interact with elements within this region in erythroid cells. TFII-I was found to act as a repressor of -globin expression while USF proteins were found to act as activators of -globin expression. It is becoming increasingly clear that one role DNA binding proteins play is to recruit co-activators or co-repressors that modify histones or mobilize nucleosomes at regulatory sites. Therefore, to understand the mechanism behind the regulation of -globin expression by TFII-I and USF we investigated the recruitment of chromatin modifying enzymes to the -globin gene locus by these proteins in both an embryoni c and adult environment. TFII-I was found to interact with HDAC3 exclusively in embryonic environment. Suz12, a component of the Polycomb group complexes 2,3, and 4 which contains histone methylase activity, was also found to interact at the -globin promoter in an embryonic environment but not in an adult environment suggesting a role in repression. USF was found to interact with activators in an adult environment. The role of USF in -globin expression was also furt her investigated in transgenic mice which express a dominant-negative prot ein to USF exclusively in erythroid cells.
16 10. A developmental interaction between the embryo and endosperm in maize is revealed by the rough endosperm 3 (rgh3) seed mutant. Diego F1, Elisa G2, Joaquin R2, Tesfaye B1, Gregorio H2, Settles AM1,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Universidad Alcala de Henares, Spain. Higher plant seeds develop embryo and endosper m tissues from the two products of double fertilization. The embryo and endosperm interact during seed development. Maize seeds require a specific ratio of maternal to paternal chromo somes, and the embryo and endosperm are known to show tissue growth interactions. However, little is known about the molecular mechanisms of embryo-endosperm interactions. The rough endosper m3 (rgh3) mutation disrupts a locus that is involved in these interactions at a developmental le vel. We have shown that Rgh3 is required in the embryo for the normal development of endosperm tissues using marked genetic mosaic seeds. An analysis of cell type specific marker s in recessive mutant seeds suggest that the primary endosperm defect occurs after cell type specification. The rgh3 endosperm developmental defects are then reflected in tota l seed composition. Mutant rgh3 seeds also germinate at a low frequency and result in lethal seedlings suggesting an additional function of Rgh3 after seed development is completed. We mapped the Rgh3 locus to the long arm of chromosome 5 (5L). Complementation tests with other seed mutants mapped to 5L indicated that Rgh3 is a novel locus. We have cloned a transposon-tag that is closely-linked to the rgh3 mutant. This tag will be used to identify the Rg h3 locus with the goal of understanding the molecular mechanism by which the embr yo affects endosperm development. 11. Modeling adaptive point mutation me chanism of biofilm bacteria using phylogenetic models Feng H1, Joyce P2 1Department of Pharmacy Practice, University of Florida, Gainesville FL 2Department of Mathematics, University of Idaho, Moscow, ID Biofilms are implicated in a significant amount of human bacterial infections. Biologic biofilm experiments provided evidences that biofilm bact eria have wider diversities of genotype and phenotype. The wider variation in biofilm bacteria might be explained by adaptive mechanism, in which point mutation could play an important ro le. In this study, stochastic functions were developed to understand the point mutation dyna mics under the models of Jukes-Cantor and Kimuras 2-parameter. The simulation results reve led that 1) larger phenotypic variation results from higher proportion of non-synonymous mutation; 2) The ratio of non-synonymous differences (dN) to synonymous differences (dS) is positively related with the probability of fixation; and 3) the probability of at least one non-synonymous substitution at the end of a branch increases as the non-synonymous substitution rate per codon increases. Those developed models were applied to explain the fin dings in the artificial E.coli biofilm. 12. Stochastic simulation models of popula tion size estimates in genetic sampling Feng H1, Joyce P2 1Department of Pharmacy Practice, University of Florida, Gainesville FL 2Department of Mathematics, University of Idaho, Moscow, ID
17 Recent advances in genetics provide a way to st udy wildlife populations by extracting DNA from animal samples of hair and scat without handlin g the animals. However, despite the promise of the new molecular approach to the non-invasive genetic sampling, there are several sources of errors associated with the molecular identificat ion techniques during polymerase chain reaction (PCR) amplification. Those errors could significantly bias population estimates. Based on both of CAPWIRE estimator and Peter-Lincoln estimator, we use stochastic simulation algorithm to develop bias correction methods that correct for the effect of genotyping error and heterogeneous capturabilities. The methods grea tly improve the accuracy of the population abundance estimates, allowing for the true be nefits of non-invasive genetic sampling to be realized. 13. Transplastomic tobacco with expression of the L-aspartate-alpha-decarboxylase from Escherichia coli is more tolerant to high temperature stress Fouad W, Altpeter F* Department of Agronomy and Plant Molecular and Ce llular Biology Program, University of Florida, Gainesville, FL Elevated beta-alanine levels were recently repo rted to be associated with enhanced heat tolerance. The Escherichia coli L-aspartate-alpha-decarboxylase (AspDC), coded by the panD gene, catalyzes the decarboxylation of L-aspartate to generate beta-alanine and carbon dioxide. This is a pyruvoyl-dependent enzyme that under goes limited self-processing required for its activities and it is unique to prokaryotes. The ob jective of the current study is to integrate the E. coli panD into the tobacco chloroplast via homo logous recombination. The panD gene was isolated from E. coli by PCR using gene specific primers and subcloned under the control of plastid Prrn promoter and rbcL 3UTR to generate a chloroplast expression unit. This cassette was physically linked to the aadA selectable marker expression cassette and both cassettes were flanked by the tobacco rbcL / accD sequences to direct site specific homologous recombination into the tobacco chloroplast geno me using biolistic gene transfer. Molecular and physiological data correlating transgene expression and plant performance under heat stress conditions will be presented. 14. Genetic diversity of the rare endemic plants from the Lake Wales Ridge, Florida using microsatellites Germain-Aubrey C1, Gitzendanner M1,*, Weekley C2, Menges E2 1Department of Botany, Universit y of Florida, Gainesville, FL 2Archbold Biological Research Station, Sebring, FL The Lake Wales Ridge is one of the most threatened endemic-rich ecosystems of the nation and remains a conservation priority. Understanding the genetic diversity of some of its unique plants will help us protect this quickly disappearing ancient sand dune. 15. ADP-glucose pyrophosphorylase subun its exhibiting vastly different rates of sequence divergence are equally sensitive to amino acid changes altering enzyme activity Georgelis N, Shaw JR, Hannah LC* Department of Horticultural Sciences and Prog ram in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL
18 It is generally thought that the rate of evolutiona ry change in an amino acid sequence reflects, to a great extent, the importance of the monitored amino acids to the function of the protein. In the case of the enzyme ADP-glucose pyrophosphor ylase (AGPase) this premise has led to the hypothesis that the small subunit of the enzyme is more conserved than is the large subunit because it provides a function not shared wi th the large subunit. Accordingly, greater evolutionary constraints are pl aced on the amino acid sequence of the small subunit. Two independent approaches were used to test this hypothesis. First, the probability that a random, PCR-induced, missense mutation altered AGPase ac tivity was calculated for each subunit of the major maize endosperm isoform. Both subuni ts exhibited the same probability. Second, functional maize endosperm large and small subunit genes from heavily mutagenized populations were sequenced and the ratio of missense to sile nt mutations was calculated. This ratio was the same for both subunits. Our results indicate that the small and the large subunit genes of AGPase are equally predisposed to activity-altering misse nse mutations. The subunits are equally robust to random amino acid changes affecting AGPase ac tivity. Alternatively, we suggest that the small subunit is more evolutionarily conserved than is the large subunit because it has to form a functional enzyme complex with multiple large subu nit partners in multiple cellular environments. We base this hypothesis on the fact that, in planta, the small subunit generally interacts with multiple large subunits. To test specifically whethe r this places additional constraints on amino acid changes in the small subunit, we expressed a collection of maize endosperm small subunit mutants with each of two different large subunits and monitored resultant enzyme activity. Approximately 25 percent of the small subunit mutants interacted differently with the two large subunits. 16. Metabolic rate controls genotypic and phenotypic evolution McCoy M, Gillooly J* Department of Zoology, University of Florida, Gainesville, FL Determining the relative importance of neutral versus adaptive processes in shaping rates of phenotypic evolution is a central aim of evolutiona ry biology. Many evolutionary biologists, while agreeing that most changes to the genotype are neutral, would argue that most changes to the phenotype are adaptive. However, the relative importance of neutral mutation versus natural selection in driving phenotypic change has been difficult to quantify. Here we apply the body sizetemperature model of molecular evolution pr oposed by Gillooly et al. (2005) to address the importance of neutral processes in controlling rates of phenotypic evolution. Gillooly et al. (2005) showed that neutral rates of DNA evolution are pr oportional to mass-spec ific metabolic rate. In this study we show that for both mitochondria l and nuclear genes, non-neutral rates of DNA evolution, as well as rates of amino acid (i.e., phenotypic) evolution show the same size-and temperature dependence as neutral molecular evolut ion. This indicates that rates of phenotypic evolution, like neutral evolution, are directly pr oportional to mass-specific metabolic rate. More generally, these results suggest that the primary co ntrols on individual meta bolic rate (i.e., size and temperature) may also constrain the overall rate of evolution in organisms through their effects on mutation rate. Ultimately, our hope is that this work can be extended to better understand rates of speciation and patterns in biodiversity. 17. Analysis of LAT promoter deletion mutant 17DPst for altered histone modifications during latency and reac tivation in rabbit trigeminal ganglia Giordani NV1, Neumann DM2, Bhattacharjee PS2, McAnany PK1, Hill JM2, Bloom DC1,* 1Department of Molecular Genetics & Microbiology, University of Florida, Gainesville, FL
19 2Department of Ophthalmology, Louisiana State Un iversity Health Sciences Center, New Orleans, LA Only the latency-associated transcript (LAT) regi on of the Herpes Simplex Virus Type 1 (HSV-1) genome is abundantly transcribed during latency. While the LAT locus plays a significant role in reactivation, its precise function in the proces s is unknown. Since promoter deletion mutant 17dPst is highly restricted for reactivation in the rabbit, and to a much lesser degree in the mouse, we examined transcriptional permissivene ss of the LAT locus using the rabbit eye model to determine whether the greater restriction in reactivation in the rabbit (in vivo) compared to the mouse (in vitro) lies in the establishment of a specific chromatin profile. Trigeminal ganglia (TG) from rabbits latently-infected with either HSV-1 wild-type strain 17syn+ or 17dPst were assessed for histone H3 K9, K14 acetylation and H3 K4 dimethylation during latency. Similar to results obtained from the mouse model (13), an alyses in the rabbit reveal a transcriptional repression of lytic genes relative to LAT region ta rgets. In contrast to latency in the mouse, the viral genome in the rabbit appears to display a higher level of H3 K9, K14 acetylation and H3 K4 dimethylation at the region just upstream of the Pst-Pst deletion than at the 5exon/enhancer. Analyses of TG following epinephrine induct ion will determine whether 17dPsts defect in reactivation results from an inability of the LA T enhancer or ICP0 to be remodeled during the initial stages of reactivation. 18. Genetic analysis of BMP2 and BMP4 in the limb apical ectoderm ridge Choi KS, Maatouk DM, Harfe BD* Department of Molecular Genetics and Microb iology, University of Florida, Gainesville, FL During early limb development the apical ectodermal ridge (AER), a specialized epithelium on the distal tip of the limb bud, expres ses a number of signaling protei ns including bone morphogenetic proteins (BMPs) 2, 4 and 7. BMPs in the lim b are expressed in both the limb mesoderm and ectoderm and have been proposed to play a role in digit patterning. To date, it has not been possible to directly examine the role BMPs play in limb development since mice deficient in BMP2 or BMP4 die before digit condensation occurs. Pr evious studies have circumvented this problem by conditionally removing a BMP receptor (BMPR-IA) or expressing a BMP antagonist (noggin) in the AER. These studies concluded that BMPs are essential for normal limb development. Because each of the previous studies blocked all BMP signaling, the functions of each individual BMP remains unknown. Our studies take advantage of an Msx2-cre ectoderm-specific transgene to delete BMPs in the limb AER. We have shown that removable of BMP2 from the limb ectoderm has no affect on digit patterning. Lo ss of ectodermal BMP4 typically causes formation of a posterior nubbin. Double knockout of BMP2 and BMP4 in the ectoderm results in limb polydactyly and syndactyly. Using whole mount in situ hybridization we show that the expression domains of FGF4 and FGF8 in the AER were expa nded along the anterior and posterior axis in double mutants. There was also a decrease in cell death in interdigital tissue, which resulted in webbing between the digits. Further in situ ana lysis shows the expression domains of Hoxd11, Hoxa13, and Hoxd13 were extended anteriorly in the limb mesenchyme. These results demonstrate that ectodermal BMPs are partially functional redundant in the limb ectoderm and play an important role in cell death and ante roposterior patterning of the developing limb.
20 19. Phase I clinical trials of intram uscular injection of rAAV2 and rAAV1pseudotyped versions of an alpha-1 antitryp sin (AAT) vector in AAT-deficient adults. Brantly ML2,4,*, Spencer LT 1, Humphries M1,4, Conlon TJ1,4, Spencer CT1,4, Poirier A 1,4, Garlington W1, Baker D1, Song S 4,5,*, Berns KI3,4,*, Muzyczka N3,4,*, Snyder RO3,4,*, Washer S6, Byrne BJ1,3,4,*, Flotte TR1,3,4,* 1Department of Pediatrics, University of Florida, Gainesville, FL 2Department of Medicine, University of Florida, Gainesville, FL 3Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 4Powell Gene Therapy Center, Univer sity of Florida, Gainesville, FL 5Department of Pharmaceutics, University of Florida, Gainesville, FL 6Applied Genetic Technologies Corp., Alachua, FL A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) alpha-1 antitrypsin (AAT) vector (utiliz ing a CMV enhancer/beta actin promoter/hybrid intron [CBA] expression cassette ) was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were either homozygous fo r the most common AAT mutation (a missense designated PI*Z) or compound heterozygous fo r PI*Z and another mutation known to cause disease. There were four dosage cohorts, ranging from 2.1x1012 vector genomes (vg) to 6.9x1013 vg, with 3 subjects per cohort. Subjects we re injected sequentially in a dose escalating fashion with a minimum of 14 days between pati ents. Subjects who had been on AAT protein replacement discontinued that therapy 28 days pr ior to vector administration. There were no vector-related serious adverse events in any of the 12 participants. Vector DNA sequences were detected in the blood between 1 and 3 days afte r injection in nearly all patients receiving doses of 6.9x1012 vg or higher. Anti-AAV2 capsid an tibodies were present and rose after vector injection, but no other immune responses were detected. One subject who had not been on protein replacement exhibited low-level expressi on of wild-type M-AAT in the serum (82nM), which was detectable 30 days after receiving an in jection of 2.1x1013 vg. Unfortunately, residual but declining M-AAT levels from the wash-out of the protein replacement elevated background levels sufficiently to obscure any possible vector ex pression in that range in most of the other individuals in the higher dose cohorts. Based on a desire to increase the potency for M-AAT expression per vector genome, we further pursued pseudotyping of this same vector genome into AAV serotype 1 capsids (rAAV1-AAT). Preclinical data indicated that the potency per vector genome was approximately 500-fold higher in mice with this cassette. Toxicology and biodistribution studies in mice and rabbits indica ted that IM injection of rAAV1-AAT posed some risk of injection-site inflammation and biodistr ibution to distant sites, including gonads and semen, both of which were dose-related, but ne ither of which were significant within the dose range (per kg) planned for the rAAV1 clinical tria l. Based on this, a phase I trial has now been initiated with the pseudotyped vector. Three subjects have now been injected with 6.9x1012 vg of rAAV1-AAT. One of the three showed a level of total AAT peaking at 11microM 30 days after injection, but it is unclear whether this was du e to vector expression or to an intercurrent bacterial epididymitis, with it s associated fever and acute phase reaction, which could have induced endogenous Z-AAT to be transiently elevated. To date, there are no vector-related adverse events noted. Nine additional subjects are anticipated to receive doses ranging up to 1x1014 vg. Supported by grants from NHLBI, NCRR, and Applied Genetic Technologies Corporation.
21 20. The three endothelin receptors in the killifish, Fundulus heteroclitus: physiological and phylogenetic relationships Hyndman KA, Miyamoto MM*, Evans DH Department of Zoology, Universit y of Florida, Gainesville, FL The purpose of this study was to sequence and characterize the endothelin receptors (ETRs) from the gill of the killifish, Fundulus heteroclitus, where the ET signaling cascade is hypothesized to be involved in control of lo cal blood flow and ion transport. In mammals there are two ETRs termed ETA and ETB. Interestingly, in non-ma mmalian vertebrates there are three ETRs: ETA, ETB1, and ETB2 (ETC in frogs). Using standard cloning and sequencing, we have sequenced the three ETRs from killifish gill cDNA. Our phylogenetic an alysis supports previous findings that the three ETRs are produced by separate genes, and are not simply splice variants. To further characterize these receptors, tissue distributi on and quantitative PCR mRNA analyses were performed. Given that animals from fishes to birds have three ETRs while mammals have only two, we hypothesize that mammals have lost the ETB2 gene. 21. Fingerprinting cultivars: DNA polymorphisms of bermudagrass (Cynodon spp.) microsatellite loci Kamps TL1, Ortega VM1, Williams NR2, Chamusco KC1, Scully BT3, Chase CD1,* 1Department of Horticultural Sciences, University of Florida, Gainesville, FL 2Monsanto Corp., Soybean Trait Integr ation Group, Des Moines, Iowa 3UF/IFAS Indian River Research and Education Center, Fort Pierce, FL Bermudagrass is a complex of interbreeding Cynodon species that includes cultivars developed specifically for diverse utilizations ranging from close-cut golf greens to pastures. Breeding programs have produced high value seeding and strictly vegetatively propagated turf varieties designed for specific uses and responses to environmental conditions. Fast and reliable molecular marker fingerprinting is necessary to overcome the inherent difficulties of cultivar verification based on subtle morphological char acteristics that can also be confounded by environmental influences. By developing an SSR based marker system that is technically simple, highly reproducible, and amenable to high-thr oughput analysis, we have made a strong improvement above the previous, cumberso me AFLP and DAF methods for genetically fingerprinting bermudagrasses. Primer pairs fo r PCR were designed from sequences flanking SSRs of clones from a (CA)n enriched genomic library of Tifway 419. Utility of the resulting set of 20 markers is demonstrated by genetic characteri zation by gel electrophoresis and/or capillary analysis of the PCR products generated from th e DNA of a diploid (2N) and a tetraploid (4N) plant introduction (PI) lines used in turf breed ing, and 12 named turf type bermudagrasses. PCR amplification identified 2 -13 alleles for the geno types included in this study. Polymorphism information content (PIC) is high, ranging from 0.42 0.86 with an average of 0.75. Degree of revealed genetic diversity between accessions was consistent with expectations of the breeding method employed and this type of genetic marker system. Genetic relationships between accessions and purported progenitors were also analyzed and are reported.
22 22. Program for the isolation, identification, and characterization of pheromones from C. elegans Alborn H1, Bruschweiler R2, de Bono M3, Dossey A4, Edison AS4, Jorgensen E5, Kaplan F4, OBrian N4, Rocca JR4, Srinivasan J6, Stadler M4, Sternberg P6, Teal P1, Truong L5, White J5, Zhang F2 1USDA Laboratory, Gainesville, FL 2National High Magnetic Fiel d Laboratory, Tallahassee, FL 3MRC Laboratory of Molecular Biology, Cambridge, UK 4Department of Biochemistry & Molecular Biolog y, University of Florida, Gainesville, FL 5Department of Biology, University of Utah, Salt Lake City, UT 6Department of Biology, Cal Tech, Pasadena, CA The small soil nematode Caenorhabditis elegans is one of the best-s tudied and understood animals. Despite its wealth of genetic, cellular, and anatomical information, very little is known about the chemistry that C. elegans uses to communicate between individuals and its environment. Members of this research team an d other scientists have identified mating and aggregation behaviors that are also likely medi ated through chemical communication between animals. We hypothesize that C. elegans utilizes an extensive chemical language that allows individuals to communicate an d regulates basic behaviors such as feeding, mating, and population density control. Our goal is to identify several different molecules produced by the worm in order to begin to decipher its chemical language. This information will not only add to the extensive scientific knowledge of C. elegans but may provide important new clues for the biological control of other nematode parasites of plants or animals. We are currently pursuing the problem from two independent directions. First, we are using a traditional forward approach of growing large amounts of culture, separating compounds by various chromatographic methods, testing for biological ac tivity, further purification, and identification by mass spectrometry and nuclear magnetic resonanc e (NMR). Second, we are experimenting with a reverse approach by using NMR to compare mixtures of cultures produced both with and without worms. From the mixture of compounds, we ar e attempting to identify resonances that are unique to worms and not present in bacteria. We will use these unique resonances to guide HPLC purification, test the compounds for biological activity, and identify them with NMR and MS. Updates from both directions will be presented. 23. Silencing of Ant4 gene expression by the E2F6 repressor complex Kehoe S1, Oka M2, Gaubatz S3, Terada N1,* 1Department of Pathology, University of Flor ida College of Medicine, Gainesville, FL 32610, USA 2Department of Frontier Bioscience s, Osaka University, Osaka, Japan 3Department of Physiological Chemistry, Un iversity of Wuerzburg, Wuerzburg, Germany Adenine nucleotide (ADP/ATP) translocase 4 (Ant4), is a gene with testis-specific expression recently discovered by our lab. Its critical role in spermatogenesis and male fertility is currently being elucidated. Ant4 is initially expressed in embryonic stem (ES) cells but is downregulated upon ES cell differentiation. Here we focus on the mechanisms by which Ant4 repression occurs during ES cell differentiation. We have previo usly shown that DNA methylation of the Ant4 promoter by DNA methyltransferase 3 (Dnmt3) is required for Ant4 repression. We now aim to determine how Dnmt3 is recruited to the Ant4 pr omoter. More specifically, we show using gel mobility shift and luciferase assays that E2F6 a known repressor of male germ cell-specific genes, binds to the Ant4 promoter and represses its transcription. Similarly, using site-directed mutagenesis, we demonstrate that mutation of an E2F6 binding site at the Ant4 promoter prevents E2F6-induced repression of Ant4 transcription. Furthermore, we show that in E2F6
23 knockout mouse embryonic fibroblasts, Ant4 is no longer repressed. Our findings suggest the existence of an E2F6 repressor complex whereby E2F6 recruits Dnmt3 to the Ant4 promoter to repress its transcription. 24. Human and Florida manatee (Trichechus manatus latirostris) comparative genome maps generated by cross-species fluorescence in-situ hybridization (ZooFISH) Kellogg ME1, Dennis TR2, Burkett S3, Bonde RK4, Zori RT2, McGuire PM5, Stanyon R6 1Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 2Department of Pediatrics, Division of Geneti cs, University of Florida, Gainesville, FL 3Comparative Molecular Cytogenetics Core, National Cancer Institute Frederick, Frederick, MD 4U.S. Geological Survey, Florida Integrated Science Center, Sirenia Project, Gainesville, FL 5Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 6Laboratorio di Antropologia, Dipartimento di Biol ogia Animale e Genetica, Universit degli Studi di Firenze, Firenze, Italy Although anatomical and molecular sequencing studies have arranged the 18 extant placental (Eutherian) orders into four super-orders, Afrotheria, Xenarthra, Euarchontoglires and Laurasiatheria, unresolved questions remain. Th ere is still no consensus for the assembly and evolutionary position of the Afrotheria in whic h morphologically dissimilar African placentals were united by corresponding genetic sequences. In a ddition, nuclear DNA evidence places Afrotheria as the most ancient mammalian clade, whereas re cent comparative cytogenetic information does not rule out the possibility that Xenarthra is basal. Chromosome painting can provide an alternative source of data to test hypotheses of placental ma mmalian phylogeny and taxonomy. Sirenia (manatee and dugong) and Hyracoidea (h yrax) are the two Afrotherian orders not yet examined using comparative painting techniques. Presented here are the human and Florida manatee (Trichechus manatus latirostris) comparative genome maps. Cross-species fluorescence in-situ hybridization (Zoo-FISH) investigatio ns into the taxonomic assemblage will define chromosomal homologies and identify evolutio nary chromosome breakpoints. Human (HSA) chromosome painting probes were constructe d by degenerate oligonucleotide-primed PCR amplification of flow-sorted chromosomes. A co mparative genome map is presented based on the hybridization pattern. These maps identifie d 41 evolutionarily conserved segments on 21 (of 23) autosomal manatee chromosomes and the X. Nine human chromosomes demonstrated conserved synteny, retaining complete and exclusive hybridization to a single chromosome pair in the Florida manatee. Eight human chromosomes delineated whole chromosome homology in the manatee karyotype. The unique Afrotherian sy ntenic associations of HSA 1/19 and 5/21 were found in manatee and phylogenetically support th e Afrotherian super-order assemblage. These chromosome painting data o ffer non-DNA sequence support for the Afrotherian clade. Comparative human and manatee genomic maps provide new insights into the karyotype organization of the proto-afrotherian, and the an cestral Eutherian karyotype. These studies also facilitate high-resolution genetic mapping of th e manatee genome, as well as intraspecific and interspecific comparisons to other non-human species.
24 25. The evolution of the AGL6-like MADS-box genes Kim S1, Zahn LM2, Chanderbali A1, Zheng Z1, Oppenheimer DG1,*, Ma H2, Soltis DE1,*, Soltis PS3,* 1Department of Botany, Universit y of Florida, Gainesville, FL 2Department of Biology, Huck Institute of the L ife Sciences and Institute for Molecular Genetics and Evolution, The Pennsylvania State University 3Florida Museum of Natural History, University of Florida, Gainesville, FL AGL6-like genes form one of the major subgroups of the MADS-box gene family and are closely related to the SEP (E-class) and SQUA (A-class) subfamilies of MADS-box genes. In Arabidopsis, AGL6 and AGL13 have been reported from the AGL6 subfamily, but detailed expression studies of these genes have not yet been performed. We identified several new AGL6-like genes from pivotal taxa in angiosperm phylogeny and ana lyzed them together with previously reported AGL6like genes. Structural analyses showed 1) a one-aa gap in the I-domain in all AGL6-like genes relative to SEP-like and SQUA-like genes, 2) a seven-aa insertion in the C-domain of genes from asterids, and 3) a one-aa insertion in the C-do main of genes from gymnosperms. Phylogenetic analyses showed that AGL6-like genes are sister to AGL2-like genes, and SQUA-like genes are sister to these two groups. These relationships were strongly supported in both amino acid and DNA analyses. The phylogenetic tree of AGL6-like genes generally tracks organismal phylogeny as inferred from multigene data sets. Unlike other subgroups of MADS-box genes (e.g., DEF, SQUA, and SEP), no major duplication of AGL6-like genes occurred in the common ancestor of core eudicots. However, several recent gene duplications were detected (e.g., within Magnoliaceae). We investigated the expression of AGL6 genes from Arabidopsis, Eschscholzia, Magnolia, Liriodendron, Persea, Nuphar, and Amborella using relative-quantitative RT-PCR and in situ hybridization, and consider the expressi on patterns in a phylogenetic context. 26. Bayesian mapping QTL for dynamic quantitative traits. Li JH*, Yang RQ1 1School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, China Quantitative traits whose phenotypic values ch ange with time are called dynamic quantitative traits. Genetic analysis of dynamic traits is usua lly conducted in one of two ways. One is to treat phenotypic values collected at different time points as repeated measurements of the same traits, which are analyzed in the framework of multivariate theory. Alternatively, a growth curve may be fitted to those observations, and inferenc e can be made through the parameters of the growth trajectories. The latter has been used in QTL mapping for dynamic quantitative traits. We demonstrate that one can fit dynamic traits with the Legendre polynomials, which are sufficiently general for any shape of trajectories by selectin g different orders of the Legendre polynomials. Then we take a Bayesian approach implemen ted via the Markov Chain Monte Carlo (MCMC) algorithm to estimate the positions and effects of multiple QTL. The entire genome is divided into a finite number of regions, and QTL position s and effects of all regions are evaluated simultaneously. With this method, regions with no actual QTL will have negligible estimated QTL effects. We present this method using simulated da ta as well as data on the growth of leaf age collected from published experiments of DH population of rice.
25 27. Sequence-indexed mutations in maize using the UniformMu transposon-tagging population Li L1, Holding DR2, Tan BC1, Latshaw SP1, Liu J3, Suzuki M1, OBrien BA1, Fajardo DS1, Wroclawska E1, Tseung CW1, Lai J4, Hunter III CT1, Avigne WT1, Baier J1, Lonon D1, Messing J4, Hannah LC1,*, Koch KE1,*, Becraft PW3, Larkins BA2, McCarty DR1,*, Settles AM1,* 1Horticultural Sciences Department, Univ ersity of Florida, Gainesville, FL 2Department of Plant Sciences, Unive rsity of Arizona, Tucson, AZ 3Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 4Waksman Institute, Rutgers University, Piscataway, NJ Gene knockouts are a critical resource for f unctional genomics. In plant species such as Arabidopsis or rice, gene knockout collections have been generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic in sertions that fail to segregate in subsequent generations. Transpos on insertion sites from >100 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subs et of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. We conclude from these data that the FSTs from the UniformMu population iden tify stable, germinal insertion sites in maize. We also infer that the larger collection of 1, 882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse ge netics to generate hypotheses about gene function. 28. Expression of a synthetic Bacillus thuringiensis endotoxin to enhance resistance against fall armyworm in bahiagrass Luciani G1, Yactayo-Chang J1, Altpeter F1,*, Zhang H1, Gallo M1,*, Meagher R2, Wofford D1,* 1Department of Agronomy, Plant Molecular and Ce llular Biology Program, University of Florida, Gainesville, FL 2USDA, ARS, CMAVE, Gainesville, FL Bahiagrass is a low input, drought tolerant and di sease resistant warm seas on turfgrass used for forage and turf in the Southeastern US and other subtropical regions of the world. Fall armyworm, Spodoptera frugiperda (J. E. Smith) is a destructive migratory pests of many tropical and subtropical grasses including bahiagrass. Th e objective of this study was to improve the insect resistance of bahiagrass by constitu tive expression of an optimized, synthetic Bacillus thuringiensis endotoxin. A gene encoding an optimized, synthetic Bacillus thuringiensis endotoxin (Bt toxin) was sub-cloned under the control of th e constitutive ubiquitin promoter. Co-transfer of constitutive nptII and Bt-toxin expression casset tes into seed derived callus cultures from turftype, apomictic bahiagrass (cv. Argentine) was ca rried out by biolistic ge ne transfer. Transgenic nature of the regenerated plants was confirmed by PCR, Southern blot analysis, RT-PCR and immunoassays. Data on an initial screening for re sistance of transgenic and wildtype bahiagrass leaves against laboratory reared neonate larv ae of the fall armyworm will be presented.
26 29. Rapid and accurate pyrosequencing of angiosperm plastid genomes Moore MJ1, Dhingra A2, Soltis PS3,*, Shaw R4, Farmerie WG4,*, Folta KM2,*, Soltis DE1,* 1Department of Botany, Universit y of Florida, Gainesville, FL 2Horticultural Sciences Department, Un iversity of Florida, Gainesville, FL 3Florida Museum of Natural History, University of Florida, Gainesville, FL 4Interdisciplinary Center for Biotechnology Genome Sequencing Service Laboratory, University of Florida, Gainesville, FL Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid ge nomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing th rough the successful use of a newly available pyrosequencing platform, the Genome Sequen cer 20 (GS 20) System (454 Life Sciences Corporation), to sequence the whole plastid genomes of eight species of angiosperms, representing many of the basal lineages of eudi cots. More than 99.3% of each plastid genome was obtained during GS 20 sequence runs, to an average depth of coverage of ~20. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence was generated for two of the eight species, Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae) using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors as sociated with regions of extensive homopolymer runs. No substitution errors were present in ei ther genome. Error rates were generally higher in the single-copy and noncoding regions of both pl astid genomes relative to the inverted repeat and coding regions. The high accuracy of GS 20 plastid genome sequence is particularly important because it was generated for a significan t reduction in time and cost over traditional shotgun-based genome sequencing techniques. The GS 20 System should be broadly applicable to most plastid genome sequencing projects, and therefore promises to expand the scale of plant genetic and phylogenetic research dramatically. 30. SNP discovery of Voltage-gated calcium channel beta 2 subunit Niu Y, Moss J, Langaee TY, Gong Y, Johnson JA* Center for Pharmacogenomics/Department of Pharmacy Practice, University of Florida, Gainesville, FL The gene encoding the beta 2 subunit of the Vo ltage-gated calcium channels (CACNB2) affects multiple channel properties such as voltage-depe ndent activation, inactivation rates, G-protein modulation, drug sensitivity, and cell surface expression. We undertook a single nucleotide polymorphism (SNP) discovery effort to characte rize polymorphisms in CACNB2 using genomic DNA from 60 ethnically diversed individuals by dire ct DNA sequencing. We identified a total of 25 SNPs in CACNB2 of which 7 were population-spe cific SNPs. Fourteen of the polymorphisms (56%) are novel SNPs not previously reported. The codi ng block contained no non-synonymous SNP and only 3 synonymous SNPs, located in the last co ding exon. The other 22 SNPs were located in promoter, intronic, or untranslated regions. Allele frequencies ranged from less than 1.7% to 42.5% and 19 SNPs had a minor allele frequenc y greater than 5%. A lack of strong linkage disequilibrium among the 25 SNPs was observ ed in all three race/ethnicity groups.
27 31. Phylogenetic analysis to identify th e most informative regions of the human mitochondrial genome Non AL, Kitchen A, Mulligan CJ* Department of Anthropology, University of Florida, Gainesville, FL Analysis of complete mitochondrial genome sequences is becoming increasingly common in genetic studies. The availability of full genome da tasets enables an analysis of the information content distributed throughout the mitochondrial genome in order to optimize the research design of future evolutionary studies. The goal of our study was to identify informative regions of the human mitochondrial genome using two criteria: 1) accurate reconstruction of a phylogeny and 2) consistent estimates of time to most recent common ancestor (TMRCA). We created two series of datasets by deleting individual genes of varied length and by deleting 10 equal-size fragments throughout the coding region. Phyloge nies were statistically compared to the fullcoding-region tree, while coalescent methods were used to estimate the TMRCA and associated credible intervals of defined clades. In general, deletion of equal-size fragments had a greater effect on the phylogenetic analysis than deleti on of genes. Individual fragments important for maintaining a phylogeny similar to the fu ll-coding-region tree encompassed bp 577-2122 and 11399-16023, including all or part of 12S rRNA, 16S rRNA, ND4, ND5, ND6, and cytb. The control region-only tree was the most poorly resolved with the majority of the tree manifest as an unresolved polytomy. Coalescent estimates of TM RCA were less sensitive to removal of any particular fragment(s) than reconstruction of a co nsistent phylogeny. Overall, we discovered that half the genome, i.e. bp 3669-11398, could be re moved with no significant change in the phylogeny (pAU=0.077) while still maintaining ov erlap of TMRCA 95% credible intervals. 32. Intermittent fasting diet improves motor performance of hereditary neuropathic mice and is associated with altera tions in peripheral nerve proteins Madorsky I1, Waber A1, Dunn Jr. WA2, Notterpek L1,2,* 1Departments of Neuroscience, Unive rsity of Florida, Gainesville, FL 2Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL Intermittent fasting (IF) is known to increase longevity and protect neurons against degenerative processes in experimental models In the CNS, the underlying mechanisms appear to involve the stimulation of chaperones (heat shock proteins, HSPs) and neurotrophins. In this study we asked whether IF is an effective inducer of HSPs and au tophagy in the PNS, and if it could alleviate the neuropathic phenotype of Trembler J (TrJ) mice, carrying a Leu-16-Pro mutation in peripheral myelin protein 22 (PMP22). Nine-week old, male w ild type (wt) and heterozygous TrJ mice were maintained on the IF regimen for 4 months (food provided or removed at 12 noon each day). Littermates received food ad libitum (AL). The body weight of wt and TrJ animals was monitored at regular intervals, and decreased <10% on the IF regimen. After 4 months on the IF diet, TrJ mice exhibited improved motor performance on th e accelerated rota-rod, when compared to AL littermates. Biochemical analysis revealed altera tions in several protein groups, including HSPs, myelin proteins and markers of degradative pathways. The levels of HSP70, and of the autophagy-related proteins, beclin, Gsa7 and LC3, were elevated. Significantly, the expression of myelin proteins (PMP22, myelin basic protein) was increased in nerves of diet restricted mice. The differences between IF and AL mice were usua lly more pronounced in TrJ, as compared to wt. These results suggest that the chaperone and autophagic pathways are potential therapeutic targets for the neural disorders involving protei n misfolding. Supported by funds from the MDA and NIH-NINDS.
28 33. Development of animal model for pulmonary arterial hypertension (PAH) Hong K-H, Lee Y-J, Park SO, Jang D-S, Seki T, Oh SP* Department of Physiology and Functional Geno mics, University of Florida, Gainesville, FL Idiopathic Pulmonary Arterial Hypertension (IPAH) is a rare but life-threatening pulmonary vascular disorder characterized by the sustained elevation of mean pulmonary artery pressure. Genetic linkage analyses indicated that two TGFsuperfamily receptors activin receptor-like kinase-1 (ALK1) and bone morphogenetic protei n receptor type II (BMPR2) predispose PAH. Heterozygous mutation of BMPR2 is responsible for about 70% and 40%of cases of familial PAH and of sporadic PAH, respectively. However, lo w disease penetrance of heterozygous mutation suggests that the loss of one allele of BMPR2 gene is not sufficient to cause pathogenesis of PAH. Recent studies raise the possibility that addition al environmental and/or genetic factor(s) along with heterozygous mutation of BMPR2 (or ALK1 ) are involved in the development of PAH. We have previously demonstrated that Alk1 is pred ominantly expressed in arterial endothelium during embryonic and neonatal stages. In adult mice, Alk1 expression is greatly diminished in systemic vessels, yet persistent in pulmonary vasculature. We also showed that 9.2kb Alk1 promoter/enhancer is sufficient for spatiotemporal expression of Alk1 in transgenic mice. By using the 9.2kb Alk1 promoter/enhancer, we have generated a novel Cre mouse line showing a strong and consistent Cre activity in the pulm onary vascular endothelial cells (ECs) during development and postnatal life. Be cause heterozygous Bmpr2 mice display only mild elevation of pulmonary blood pressure, we investigated whet her the second genetic mutation in the Bmpr2 gene only within pulmonary ECs is one of the aforementioned additional factors for triggering the pathophysiology of PAH. We generated ho mozygous Bmpr2loxp/loxp mice containing the ALK1-Cre transgene. The Bmpr2loxp/loxp;Alk1-Cre mice develop severe pulmonary hypertension with right ventricular hypertrophy and significant increase in the thickness of smooth muscle layers of pulmonary vessels. This result indicates that Bm pr2loxp/loxp;Alk1-Cre mice are an unique animal model for PPH which will provide valuable resources to enhance our understanding of the etiology and pathogenesis of PPH. 34. In vivo assessment of the ALK1-ALK5 balance for mediating TGFsignal in endothelium during vascular development in mice Park SO, Seki T, Hong K-H, Lee Y, Fliess N, and Oh SP* Department of Physiology and Functional Ge nomics, University of Florida, Gainesville, FL TGFtransmits signals through a hetero-tetrameric cell-surface complex of type II (Tgfbr2) and type I (Alk5) serine/threonine kinase receptors. We have shown that another type I receptor, Alk1, can also mediate TGFsignals via BMP-activated Smads in vascular endothelial cells (ECs), and had proposed a hypothesis that two TGFsignaling pathways via Alk1 and Alk5 in vascular ECs may play a balancing role for controlling an giogenesis. Mounting evidence has clearly shown that such a balance exists and functions in cultured ECs, and that Alk5 is required for Alk1 signaling. In contrast to these in vitro data, our in vivo expression mapping studies using lacZknockin reporter strains showed that Alk1 is predominantly expressed in arterial endothelium, whereas Alk5 expression is localized in the medi al and adventitial layers of large blood vessels but undetectable in the intimal layer of blood vessels. These mutually exclusive expression patterns of Alk1 and Alk5 in blood vessels suggest that each type I receptor has its own unique function in vascular development. We hypothesize that Alk1 is the sole TGFtype I receptor in the vascular endothelium. Therefore, Tgfbr2, but not Alk5, is required for TGFsignaling in the endothelium during vascular development. To te st this hypothesis, we have generated Alk1conditional knockout mice and a transgenic mouse line which expresses Cre recombinase in a
29 subset of vascular endothelium. Using this Cre expressor, we have delete d Alk1, Alk5, or Tgfbr2 in the subset of endothelium. Blood vessels where Alk1 gene is deleted showed extensive dilation and arteriovenous malformation (AVM), recapitulating typical HHT vascular lesions, indicating that it will be served as an outsta nding animal model for studying pathogenesis of HHT. On the other hand, mice harboring the A lk5 deletion by the same Cre expressor appeared to be viable and show no apparent vascular abnormality. These results strongly support our hypothesis and demonstrate that Alk1, but not Alk5 is essential in endothelium for the vascular development and function. 35. Centromere mapping in sweet orange, Citrus sinensis O'Malley DM, Chaparro JX Department of Horticultural Sciences, University of Florida, Gainesville, FL As part of a collaborative effort in citrus ge nomics, we developed 32 EST-based microsatellite markers for a large interspecific Citrus sinens is x Poncirus trifoliata family, the mapping population. These markers and more microsatellites were located on a genetic map at University of California, Riverside. A fundamental goal of geno mics is alignment of genetic, cytological, and physical maps. Microsatellite markers develope d from ESTs can be readily integrated with physical maps. The major cytological feature of chromosomes is the centromere. Centromere DNA contains highly repetitive satellite DNA, wi th flanking middle repetitive DNA sequences. Gene density is lower and recomb ination is suppressed near cent romeres. Although centromeres are stably inherited, they evolve rapidly so are difficult to locate from DNA sequence alone. The mapping population included 40 50 spontaneous triploid progeny. To recognize triploids that received two identical sweet orange alleles, ge ne dosage was interpreted from peak height differences in microsatellite trace files from an automated fluorescent capillary DNA sequencing instrument (Megabase 1000). Our analysis of the triploid microsatellite marker genotypes provides strong evidence that the spontaneous tr iploids arose from 2N ovules in sweet orange through meiotic diplospory and second division rest itution (SDR). We used half tetrad analysis to estimate marker:centromere map distances for 18 microsatellite markers from sweet orange (C. sinensis). Knowledge of centromere locations pro vide useful information on the completeness of the genetic map and triploids has implications fo r seedless cultivar breeding. We have obtained preliminary gene:centromere distances from 58 additional UCR microsatellite markers and are working towards locating centromere regions on the consensus map. 36. A novel regulator of actin organization in plants Zhang X, Oppenheimer DG* Department of Botany and Plant Molecular and Ce llular Biology Program, University of Florida, Gainesville, FL We use Arabidopsis trichomes (epidermal hairs) as a model to probe the molecular and genetic mechanisms that control plant cell shape. By identifying mutants that show altered trichome shape and then cloning the genes identified by the mutations, we are dissecting the pathways that control plant cell shape. Arabidopsis trichomes are single cells that have three or four branches on the end of a stalk. We isolated a ne w class of mutants, the irregular trichome branch (itb) mutants, that show dramatic changes in trichome branch position and length. These mutations therefore identify genes that particip ate in the branch formation step of trichome morphogenesis. We cloned the ITB genes usin g a map-based approach. One of these genes, ITB3, encodes a novel protein of unknown function that is conserved in land plants, but is not found in algae, fungi, or animals. Because th e plant cytoskeleton plays a central role in
30 controlling plant cell expansion, and hence shap e, we used indirect immunofluorescence and confocal microscopy to examine the microtubule and actin cytoskeletons in developing itb3 mutant trichomes. We found that the micr otubule cytoskeleton was normal, but actin organization was profoundly disrupted in developing trichomes of itb3 mutants. In addition to a preponderance of thick, bundled actin cables in the mutant, many actin rings of various sizes were prominent. Fusions of ITB3 with GFP were constructed and used to transform itb3 mutant plants. Rescue of the mutant trichome phenot ype showed that the ITB3-GFP fusions were functional. The ITB3-GFP fusion proteins were found in the cytoplasm, consistent with the hypothesis that ITB3 functions as a regulator of the actin cytoskeleton. This work was supported by a grant from NSF (IOB 0352916) 37. A preliminary analysis of gene flow and population structure of the Florida manatee (Trichechus manatus latirostris) Pause KC1, Clark AM2, Bonde RK3, McGuire PM1 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 2Interdisciplinary Center for Biotechnology Re search, University of Florida, Gainesville, FL 3U.S. Geological Survey, Florida Integrated Science Center, Sirenia Project, Gainesville, FL Previous studies with allozymes and mitochondria l DNA have demonstrated low genetic diversity, and failed to resolve the population structure fo r Florida manatees. Micros atellite libraries have been developed and screened to identify loci for population studies, pedigree analysis, and individual identification using genetic fingerpri nts. Eight previously published microsatellite primers and 10 additional unpublished primers are being screened on approximately 400 Florida manatees. Six of the 8 previously published loci an d eight of the ten newly developed loci appear to be in Hardy-Weinberg equilibrium. Average he terozygosities for the es tablished primers range from 0.23 to 0.60, whereas the new primers rang e from 0.31 to 0.73. Primers from closely related species were also tested on the Florida an imals. Although products of the expected sizes were amplified, no polymorphism was detected. Additional loci from our manatee-specific libraries are being optimized and tested for po lymorphism to help increase the power of the estimates. The polymorphic loci will be instrument al in examining the genetic appropriateness of the current management units and estimating gene flow within the state of Florida. Preliminary analyses suggest no clear genetic subdivision in the Florida population. 38. Liver X receptor; (LXRA) genotype and response to intensive lipid-lowering therapy with statins Price ET1, Arant CB2, Wessel TR2, Schofield RS2, Zineh I1 1Department of Pharmacy Practice and Center for Pharmacogenomics, University of Florida, Gainesville, FL 2Division of Cardiovascular Medicine, Un iversity of Florida, Gainesville, FL Intensive lipid lowering with statins is a preferred treatment strategy in certain patient populations. The benefit of high-dose treatment is thought to be due to both greater low-density lipoprotein (LDL) and C-reactive protein (CRP) re duction. However, variability in LDL and CRP responses exist, and genetic factors may contribu te. We investigated whether a single nucleotide polymorphism (SNP) in the LXRA gene, a potential nucl ear site of statin action, is associated with either LDL or CRP responses to at orvastatin 80 mg. Subjects were eligible if they were at least 18 years old without CHD, CHD risk equivalents, or contraindications to statins. Subjects received atorvastatin 80 mg daily for 8 weeks. Baseline and 8-week lipids and CRP were obtained from the university hospital clinical laboratory. Genotype determination of the LXRA rs12221497 G/A
31 SNP was performed by pyrosequencing. Biom arker changes were tested by t-test and multivariate analysis. A total of 61 subjects (59% women; 79% white) were analyzed. Baseline age, total cholesterol, LDL, HDL, triglycerid es, and CRP were 3213 years, 17838 mg/dl, 9831 mg/dl, 6218 mg/dl, 9754 mg/dl, and 1.82.9 mg/L, respectively. The variant A allele frequency was 16%. There were no differences in lipid changes by genotype. However, wild-type homozygotes (G/G) had an 11% reduction in CR P compared with a 25% increase in variant carriers (p= 0.047). In multivariate analysis, ag e (p=0.01), baseline CRP (p=0.02), and LXRA genotype (p=0.04) were significant predictors of CRP response (model p=0.002; r2=0.23). LXRA genotype did not affect LDL response to 8 w eeks of high-dose atorvastatin. However, LXRA genotype was associated with atorvastatin-media ted CRP changes. This is the first study to demonstrate a genetic association with the CRP stat in response and should be further evaluated. 39. Rearranged MEF2D and DAZAP1 functional domains exhibit transforming properties in the variant t(1;19)(q23;p13) reciprocal translocation products Prima VI, Hunger SP Department of Pediatrics, University of Florida, Gainesville, FL We analyzed the TS-2 acute lymphoblastic leukemia (ALL) cell line that contains a t(1;19)(q23;p13.3) but lacks E2A-PBX1 fusion typically present in leukemias with this translocation. We found that the t(1;19) in TS-2 fuses the 19p13 gene DAZAP1 to the 1q23 gene MEF2D, leading to expression of reciprocal in-frame DAZAP1/MEF2D and MEF2D/DAZAP1 transcripts. MEF2D is a member of the MEF2 family of DNA binding proteins that activate transcription of genes involved in control of differentiation and survival of lymphoid, neural and muscle cells. DAZAP1 is a novel RNA binding pr otein involved in mRNA processing/trafficking and expressed most abundantly in the testis. We demonstrate that MEF2D/DAZAP1 binds avidly and specifically to DNA in a manner indistinguishable fr om that of native MEF2D and is a substantially more potent transcriptional activator than ME F2D. We also show that DAZAP1/MEF2D is a sequence-specific RNA-binding protein. MEF2D ha s been identified as a candidate oncogene in murine retroviral insertional mutagenesis studies. Our studies of cultured cells transduced with retroviruses expressing DAZAP1/MEF2D and MEF2D/ DAZAP1 show transforming properties of the fusion proteins. These data implicate MEF2D in human cancer and suggest that MEF2D/DAZAP1 and/or DAZAP1/MEF2D contribute to leukemogen esis by altering signaling pathways normally regulated by wild-type MEF2D and DAZAP1. 40. Asynchronous teams for probe selection problems Meneses CN1, Pardalos PN2,3,4,*, Ragle MA2 1Department of Transportation Engineering, University of Sao Paulo, Brazil 2Department of Industrial and Systems Engineering, University of Florida, Gainesville, FL 3Biomedical Engineering Department, University of Florida, Gainesville, FL 4Computer & Information Science & Engineering Depa rtment, University of Florida, Gainesville, FL We propose the use of the Asynchronous Team (A-Team) technique to try to select good oligonucleotide probe sets from large sets of candidate probes. An A-Team is comprised of several different heuristic algorithms that communi cate with each other via shared memories. We designed A-Teams for two probe selection problems called the Maximum Distinguishing Probe Set (MDPS) and the Minimum Cost Probe Set (MCPS) and developed a C++ program with graphical user interface (GUI) to run instances of the pr oblems. When compared to other methods, our approach produced better or comparable re sults in a small frac tion of the time.
32 41. Poxvirus-encoded tumor necrosis factor-binding proteins: variation in ligand binding specificities Rahman MM1, Barrett JW2, Brouckaert P3, McFadden G1,* 1Emerging Pathogens Initiative, Department of Mo lecular Genetics and Microbiology, University of Florida, Gainesville, FL 2BioTherapeutics Research Group, Roba rts Research Institute, London, ON 3Molecular Pathophysiology and Experimental Ther apy Unit, Department of Molecular Biomedical Research, Ghent University and VIB, Ghent, Belgium The Yatapoxviruses encode a distinct class of secreted TNF-binding protein (TNF-BP) that resembles an MHC class I heavy chain but dist inct from any other known TNF inhibitor. Characterization of these viral TNF inhibitors fr om Tanapox virus, Yaba monkey tumor virus and a closely related version from Swinepox virus re vealed dramatically differential TNF binding specificities for different mammalian species. Th e Tanapox virus 2L protein (TPV-2L) formed inhibitory complexes with human TNF, and interacted with monkey and canine TNF with high affinity but rabbit TNF with low affinity. On the other hand, YMTV-2L bound human and monkey TNF with high affinity but rabbit TNF with only low affinity. The TNF-BP from swinepox virus (SPV003/148) only interacted with porcine TNF with high affinity. The observed TNF binding analysis mirrored the biological activity of th ese TNFbinding protein to block TNF-induced cellular cytolysis. TPV-2L and YMTV-2L also inhi bited the human TNF-mediated signaling in cells but TPV-2L exhibited higher affinity for human TNF (KD=43pM) compared to monkey (KD=120pM) whereas for YMTV-2L, the affinit ies were reversed (human TNF KD=440 pM; monkey TNF KD=230 pM). The interaction domain of human TNF with TNF-binding proteins is significantly different from that of TNFRs, as de termined using human TNF mutants. We conclude that these poxvirus TNF-binding pr oteins represent a new class of TNF inhibitors and are distinct from the viral TNF-receptor homologues characterized to date. 42. Inhibitors of Hsp90 modulate the formation of peripheral myelin protein 22 aggregates Rangaraju S, Madorsky I, Go J, Notterpek L* Department of Neuroscience, Unive rsity of Florida, Gainesville, FL The correct expression of peripheral myelin protein 22 (PMP22) is required for normal Schwann cell function and myelination. Duplication (C22 mo use), deletion or point mutations (TrJ mouse) in PMP22 are associated with a range of demyelinating peripheral neuropathies. Neuropathic Schwann cells exhibit abnormal protein traffic king with decreased turnover rate of PMP22 resulting in intracellular accumulation and subs equent aggregate formation. To aid the folding and trafficking of the wild type PMP22 in neurop athic models, we modulated the levels of heat shock proteins (Hsps), Hsp90 and Hsp70, by activa ting the heat shock response. In this study, we screened 15 novel Hsp90 inhibitor compounds (analogs of geldanamycin, GA) provided by Conforma Therapeutics. These novel GA-analogs at 50-500 nM show no significant cellular toxicity, as determined by MTT assays. Sixteen ho ur exposure of cells to these concentrations induces Hsp70 levels. To explore the influence of elevated Hsps on the formation of PMP22 aggregates, Schwann cells were treated simultaneously with the proteasome inhibitor, lactacystin (10 M) and Hsp90 inhibitor compounds (50 nM) for 16 h. Biochemical an alyses of total cell lysates revealed a modest reduction in slow-migra ting polyubiquitinated proteasome substrates in response to combined treatment, as compared to Lc alone. The aggregate reducing effect of these compounds (50 nM) upon spontaneous aggr egate containing neuropathic cells was also investigated and was associated with a modest re duction in polyubiquitin. Significantly, improved
33 trafficking and processing of PMP22 in neurop athic mouse Schwann cells correlated with increased myelin production in dorsal root ga nglion explant cultures. These results demonstrate that Hsp90 inhibitors are potential ther apeutic agents for PMP22 neuropathies. 43. Episodic selection drives the in vivo evolution/amplification of HIV-1 X4 coreceptor switch mutants associated with disease progression Salemi M1, *, Burkhardt BR1, Pomeroy S1, Lowe A1, Ghaffari G2, *, Sleasman KW3, Goodenow MM1,2,* 1Department of Pathology, Immunology and La boratory Medicine, University of Florida, Gainesville, FL 2Department of Pediatrics, Division of Immuno logy, Rheumatology, and Infectious Diseases, University of Florida, Gainesville, FL 3Department of Pediatrics, Division of Allergy, Immunology, and Rheumatology, University of South Florida and All Children's Hospital, St. Petersburg, FL During the course of HIV-1 infection the phenom enon of coreceptor switch from viruses using CCR5 as coreceptor (R5) to virus es using CXCR4 as co receptor (X4), which are characterized by high charge amino acid residues in the V3 loop of the envelope gp120 protein, is associated with disease progression. Despite the importance of X4 viral strains emergence for pathogenesis, little is know on the in vivo evolution of coreceptor switch mutants. We used high-resolution phylodynamic analysis to investigate the emerge nce of X4 virus populations in longitudinal peripheral blood mononuclear (P BMC) samples, and lymphoid and non-lymphoid tissues collected post mortem from therapy nave patients. Reco nstruction of ancestral HIV-1 sequences showed that HIV-1 quasispecies undergo several popula tion bottlenecks, driven by episodic positive selection, and that the X4 strains in vivo naturally emerge, in the absence of anti-retroviral therapy, as a fitter population surving the bottlen eck. The evolution of X4 strains is, in fact, a multi-step temporally structured process that requ ires the initial fixation of positively selected mutations in V1, V2 and C2 domains of the gp120, without which high charge V3 mutants cannot survive. Moreover, migration analysis based on coalescent theory showed that the thymus constitutes a viral reservoir where the X4 quasispe cies is primarily amplified. The key-role of the thymus in the emergence of X4 variants during the course of HIV-1 infection has important consequences for the development of effective therapeutic strategies, based on both conventional antiretroviral regimens and receptor-blocking drugs, aiming at the eradication the virus. R5 coreceptor-blocking agents may be extr emely efficient, during the early stage of the disease, in avoiding the emergence of the X4 qu asispecies associated wi th disease progression, while the development of novel drugs targeting the export of X4 infected nave T-cells from the thymus could be critical to control HIV1 infection in advanced disease stages. 44. Understanding genetic variation in Rhabditid nematodes Salomon M1, Blanton D1, Bour W1, Brown J1, Custer A1, Keller T1, Levy L1, Ostrow DG1, Phillips N2, Rosenbloom J1, Baer C1,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Biology, Arcadia University, Glenside, PA Over the past several years our lab has been inve stigating the properties of new mutations in a model nematode system. Mutations have been allo wed to accumulate in the (relative) absence of natural selection, thus allowing us to estimate the genetic variance introduced by new mutation (VM) for two species of Rhabditid nematodes, Caenorhabditis elegans and C. briggsae. However this begs the question, what is the relationship of VM to the standing genetic variance (VG) and
34 what can this relationship tell us about the nature and magnitude of selection acting on these species? To complement our previous studie s and generate an estimate of VG within C. elegans and C. briggsae, estimates of VG in wild worms are needed. We assayed 40 wild strains of C. elegans and 8 wild strains of C. briggsae for fecundity and body size at 20C. The primary goal of this study is to characterize the extent of st anding life-history variation in these two species. Comparisons of VM to VG between our mutation ac cumulation lines and the natural isolates allow us to infer the magnitude and pattern of constrai nt on phenotypic evolution, as well as provide valuable information about the forces responsibl e for the genetic diversity within and between these two species. Our results suggest that the standing genetic variance in C. briggsae is much greater than in C. elegans, with C. briggsae displaying approximately twice the variance of C. elegans. The data also suggests evidence for Mutation Selection-Balance acting on the life history trait of fitness, with a VG to VM ratio much sma ller than 4Ne. Also, the persistence time of a new mutation was found to be longer in C. briggsae than in C. elegans. 45. Assessment of pollen mediated, intr aspecific gene transfer from apomictic, transgenic bahiagrass cultivar Argentine Sandhu S1,*, Altpeter F1,*, Blount A2 1Department of Agronomy, Plant Molecular and Ce llular Biology Program, University of Florida, Gainesville, FL 2Department of Agronomy, North Florida Research and Education Center, University of Florida, Marianna, FL Bahiagrass is the predominant fora ge grass in the southeastern United States. Sexual, diploid (2n=20) and tetraploid (2n=40) apomictic cyto types of bahiagrass (Paspalum notatum Flugge) are grown. The tetraploid cytotypes, includin g the commercially important cultivar Argentine are considered obligate apomicts, whereas, the sexual types are cross pollinating. Argentine bahiagrass is being genetically modified in our labora tory to increase its stress tolerance, turf and forage quality. Compared to sexually reproducin g grasses apomictic bahi agrass might reduce the risk of unintended transgene spread. Thus, it is important to evaluate pollen mediated intraspecific gene transfer from apomictic, transg enic bahiagrass. Glufosin ate resistant Argentine bahiagrass lines were generated by biolistic gene transfer and herbicide resistance was used as a marker to study the intraspecific gene flow from transgenic apomictic Argentine bahiagrass to wild type diploids under field and greenhouse cond itions. Data on herbicide resistance of seed progeny and intraspecific gene transfer frequencies under greenhouse and field conditions will be presented. 46. An essential role for Dicer and microRNAs in skeletal muscle development Seay HR1, O'Rourke JR1, Goldhamer DJ2, McManus MT3,*, Swanson MS1,*, Harfe BD1,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Department of Molecular and Cell Biology, University of Connecticut, Stors, CT 3Diabetes Center, Department of Microbiology and Immunology, University of California, San Francisco, CA MicroRNAs (miRNAs) are a class of evolutionary conserved, small (~22nt) and non-coding RNAs that regulate gene expression post-transcriptiona lly. miRNAs originate as long primary transcripts that are cleaved by the endonucleases Drosha and Dicer. Dicer is responsible for the production of the functional miRNA, and loss of Dicer results in the accumulation of the non-functional precursors and depletion of miRNAs. Dicer is ubiquitously expressed in mice and humans. However, miRNAs exhibit tissue-specific expressi on patterns, suggesting that these molecules
35 function in tissue differentiation. Three miRNAs miR-1, miR-133 and miR-206, have been shown to be specifically enriched in skeletal muscle. To gain insight into the roles of Dicer and miRNAs in skeletal muscle, we deleted Dicer and functional miRNAs in mouse skeletal muscles by crossing mice containing Dicer conditional alleles to thos e expressing a MyoDcre transgene. The resulting Dicer cond/cond; MyoDcre animals die perinatally Mutant embryos have an increased number of apoptotic cells and reduced skeletal muscle mass in the limb buds. Our results indicate that miRNA are required for normal myogenesis and proper fiber formation. 47. Cellular origin of the penile urethra an d temporal fate of the urethral epithelium Seifert AW1, Harfe B2,*, Cohn MJ1,* 1Department of Zoology, University of Florida, Gainesville, FL 2Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Hypospadias are a congenital malformation of the penis, affecting 1:125 live male births. As hypospadias affects both proper location of th e urethral opening and correct formation of the tubular urethra, a detailed understanding of th e embryonic origins and fate of the urethral epithelium (UE) is vital to a correct model of ge nital tubercle (GT) organogenesis. The prevailing view is that the distal (glandar) urethra arises fr om ectodermal cells invaginating from the distal tip of the glans. We tested the hypothesis that the glandar urethra originates from endoderm, by using Sonic Hedgehog (Shh), a marker of hindgu t endoderm, to genetically label and follow UE cells during mouse GT developmen t. We used a gfp-cre fusion cassette knocked into the Shh locus (Shhgfpcre) to activate the Rosa26 reporter in endodermal cells to permanently label all cells that express Shhgfpcre and their descendants. Our detailed fate-map revealed an endodermal origin for the glandar urethra, based on lacZ expression in newborn mice. In addition to fate-mapping the entire urethra, we investig ated the temporal development of the urethra to determine if different parts of the urethra are laid down at different stages. We utilized a tamoxifen-inducible allele of cre knocked into the Shh locus (ShhcreErt2) to label UE cells expressing Shh at daily intervals. Our results indica te that the entire urethra is determined prior to developmental stage E14.5, suggesting that development of the urethra does not follow a proximal to distal progression like that found in the limb. 48. A haplotype of 2-adrenoceptor is associated with the increased risk for transplant or death in heart failure patients Shin J1, Lobmeyer M1, Gong Y1, Zineh I1, Langaee TY1, *, Yarandi H1, Pauly DF2, Aranda Jr. JM2, Schofield RS2, Hill JA2, Johnson JA1,* 1Center for Pharmacogenomics/Department of Pharmacy Practice, University of Florida, Gainesville, FL 2Division of Cardiovascular Medicine/Department of Medicine, University of Florida, Gainesville, FL BACKGROUND: Heart failure (HF) is characterized by neurohormonal activation of sympathetic nervous and renin-angiotensin systems. We tested whether genetic polymorphisms in these systems are associated with death or hear t transplantation in patients with HF. METHODS: 227 HF patients were enrolled from a tertiary care HF clinic and followed for outcomes every 6 months for up to 4 years. We genotyped 8 polymorphisms in 6 genes including: 1 adrenergic receptor (ADRB1, S 49G and R389G), 2 adrenergic receptor (ADRB2, G16R and Q27E), 2c adrenergic receptor (ADRA2C, Insertion/Deletion (I/D) 322-325), angiotensinogen (AGT, M235T), angiotensin rece ptor type 1 (AGTR1, A1166C), and angiotensin converting enzyme (ACE, I/D in intron 16). HA P software was used for haplotype assignment for
36 ADRB1 and ADRB2. Cox proportional hazard re gression was performed to model the outcomes with genotypes/haplotypes, adjusting for non-genetic predictors of HF. RESULTS: During a median 2.5 year follow-up period, 78 patients had an adverse outcome. Ninety five percent and 81% of the patients received an ACE inhibitor/angiotensin receptor blocker and a -blocker at baseline, respectively. Significant predictors are shown in the Table. The ADRB2 RQ haplotype was the only genotype/h aplotype associated with adverse outcomes. Variable Hazard ratio 95% co nfidence interval P-value NYHA class 2.56 1.84-3.56 <0.0001 Creatinine clearance 0.99 0.98-0.99 0.0016 Sodium 0.91 0.86-0.97 0.0019 Male gender 2.33 1.33-4.10 0.0032 ADRB2 RQ haplotype 1.40 1.01-1.93 0.047 CONCLUSIONS: In addition to other traditio nal predictors, ADRB2 haplotype may have a significant effect on risk for heart transplant/death in HF patients who receive contemporary HF pharmacotherapy. 49. Accurate breast cancer prognosis through the combination of clinical and genetic markers Sun Y1, Goodison S2, Li J3, Li L1, Farmerie W1,* 1Interdisciplinary Center for Biotechnology Re search, University of Florida, Gainesville, FL 2Department of Surgery, University of Florida, Gainesville, FL 3Department of Electrical and Computer Engin eering, University of Florida, Gainesville, FL Motivation: Accurate prognosis of breast cancer can spare a significant number of breast cancer patients from receiving unnecessary adjuvant sy stemic treatment and its related expensive medical costs. Recent studies have demonstrat ed the potential value of gene expression signatures in assessing the risk of post-surgical disease recurrence. However, these studies all attempt to develop genetic marker-based prognost ic systems to replace the existing clinical criteria, while ignoring the rich information cont ained in established clinical markers. Given the complexity of breast cancer prognosis, a more prac tical strategy would be to utilize both clinical and genetic marker information that may be complementary. Methods: A computational study is performed on publicly available microarray data which has spawned a 70-gene prognostic signature. The rece ntly proposed I-RELIEF algorithm is used to identify a hybrid signature through the combination of both genetic and clinical markers. A rigorous experimental protocol is used to esti mate the prognostic performance of the hybrid signature and other prognostic approaches. Sur vival data analyses are performed to compare different prognostic approaches. Results: The hybrid signature performs significantly better than other methods, including the 70gene signature, clinical makers alone and th e St Gallen consensus criterion. At the 90% sensitivity level, the hybrid signature achieves 69% specificity, as compared to 47% for the 70gene signature and 48% for the clinical makers The odds ratio of the hybrid signature for developing distant metastases within five year s between the patients with a good prognosis signature and the patients with a bad prognosis is 23.0 (95% CI: 7.0-75.0), far higher than either genetic or clinical markers alone.
37 50. A role in arsenic resistance for a novel glutaredoxin from hyperaccumulator fern Pteris vittata L. Sundaram S, Rathinasabapathi B* Plant Molecular and Cellular Biology Program, Ho rticultural Science Department, University of Florida, Gainesville, FL Chinese brake fern Pteris vittata L. has an unusual ability to tolerate and hyperaccumulate arsenic and hence is valued for phytoremediation of arsenic-contaminated soil and water. In a functional genomics project to identify arsenic re sistance genes from this fern, PVGrx5-6, a cDNA for a glutaredoxin (GRX) was isolated based on its ability to confer arsenic resistance upon expression in Escherichia coli. Deduced amino acid sequence of PVGrx5-6 showed high homology with an Arabidopsis GRX and protein kinase C-interacting cousin of thioredoxin homology domain (PICOT-HD) proteins. PVGRX contained two domain s the N-terminus region (1-128 aa) that was unique, and the C-terminus region (128-214 aa ) similar to glutraredoxins known in many different organisms. Each domain had a CXXS catalytic motif. Pur ified recombinant PVGRX exhibited classical glutaredoxin activity which was activated 1.8-fold by the addition of 10 mM arsenate in the assay. To understand the nature of arsenic resistance conferred by PVGrx5-6, it was expressed in E. coli mutants lacking the arsenic resistance genes of the ars operon (AW3110), a glutathione-deficient strain (JTG10), an arsenate reductase-deficient strain (WC3110), and an aquaglyceroporin-deficient (OSBR1) strain. Except in OSBR1, expression of PVGrx5-6 increased cellular arsenic tolerance, su ggesting that PVGrx5-6 had a role in arsenic tolerance independent of the arsenic operon genes. Cells expressing PVGrx5-6 had significantly lower levels of arsenite compared to vector cont rols when cultured in medium containing 2.5 mM arsenate. Our results are consistent with PVGRX ha ving a role in regulating intracellular arsenite levels directly or indirectly through modulating an aquaglyceroporin previously known in arsenite transport. 51. Application of suppression subtra ctive hybridization (SSH) to cloning differentially expressed cDNA in nematode resistant and susceptible cultivars of peanut (Arachis hypogaea L.) under nematode attack Varma TSN1, Gallo M1,2,* 1Department of Agronomy and Unive rsity of Florida, Gainesville, FL 2Plant Molecular and Cellular Biology Progra m, University of Florida, Gainesville, FL Annual economic losses in cultivated peanut (Arachis hypogaea L.) due to nematodes are estimated at $102 million. Root-knot nematodes (Meloidogyne spp.) are the most important nematode pathogens of peanut, and the predominan t pathogenic species in the southern United States is M. arenaria. Resistance to root-knot nematodes from wild peanut species has been introgressed into cultivated peanut. Following a backcrossing program using Florunner, the nematode resistant, nearly isogenic cultivar, NemaTAM, was released. To identify possible candidate genes conferring resistance to this pe st, we have constructed a cDNA library enriched with differentially expressed sequences obtained by Mirror Orientation Selection PCR from root tissues of the nematode resistant cv. NemaTAM (Ne) and the nematode susceptible cv. Florunner (FL) through suppression subtractive hybridization. Randomly picked clones (480) from the tester Ne-specific library and the driver FL-specific library were used for differential screening. From this screen, 140 Ne -specific clones and 123 FL -s pecific clones were obtained. Virtual northern blot analysis was performed to confirm the differe ntial screening results, and verified clones were sequenced. Sequence analysis revealed that a majority of the identified NemaTAM-specific sequences had pathogenesis-related and stress-r elated functions, and the Florunner-specific
38 sequences were involved in signal transduction and cellular communication. This work provides the foundation for the identification and study of genes involved in the molecular defense mechanism of nematode resistance in peanut. 52. Analysis of 6-phosphogluconate dehydrogenase enzyme activity during seed development in maize Tseung CW, Nguyen R, Settles AM* Department of Horticultural Sciences, University of Florida, Gainesville, FL The 6-phosphogluconate dehydrogenase enzyme (6PGDH) has a central role in the oxidative pentose phosphate pathway and carbon metabo lism. 6PDGH carries out the oxidation and decarboxylation of glucose-6-ph osphate to produce ribulose-5-phosphate and NADPH. Ribulose5-phosphate is used for the synthesis of metabo lic intermediates such as nucleotides, and NADPH is used for reductive biosynthesis reactions within the cell. In plants, 6PGDH is found in both the cytosol and the plastid. There are three known loci that are predicted to encode 6PGDH enzymes in maize. Pgd1 and Pgd2 encode cytosolic enzy mes, and the PGD3 protein is predicted to be chloroplast-localized. Ho mozygous mutants in both pgd1 and pgd2 show normal plant and seed development. Here we show that a pgd3 mutant is linked to a rough endosperm (rgh) kernel phenotype. The rgh/pgd3 mutant shows reduce d grain-fill as well as embryo morphogenesis defects suggesting that PGD3 is an important en zyme for seed development. Native gel activity assays with pgd1, pgd2, and pgd3 mutants showed that PGD3 is an active 6PGDH enzyme and that PGD3 is induced during seed development. These results suggest that chloroplast-localized 6PGDH is necessary during seed development and that cytosolic 6PGDH enzymes cannot complement the pgd3 mutant. 53. Proteolytic mapping of AAV serotypes Van Vliet K1, Brument N1,5, Blouin V1,5, Mohiuddin Y1, Moullier P1,5, Agbandje-McKenna M2,* Snyder RO1,3,4,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 2Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 3Department of Pediatrics, University of Florida, Gainesville, FL 4Powell Gene Therapy Center, Universi ty of Florida, Gainesville, FL 5Laboratoire de Therapie Geniqu e, Nantes Cedex 44035, France Adeno-associated virus (AAV) is a very promising ge ne delivery system that is being tested in the clinic. In excess of 100 AAV serotypes have been id entified to date. Unique viral surface features are associated with different serotypes that pr ovide different efficiencies and tissue tropism. AAV2 is the most studied of the AAV serotypes; ho wever, clinical trials for other AAV serotypes with different tissue tropism have begun. AAV2 vect ors have been shown to elicit a neutralizing antibody response after the first administration that render subsequent treatments with AAV2 ineffective. AAV pseudotyping, a process in whic h the therapeutic transgene is flanked by the AAV2 ITRs and packaged into the capsid of anot her serotype is common. Pseudotyping allows comparison of different AAV serotypes expressing the same transgene cassette when evaluating gene transfer efficiency to specific organs. Pseudotyping may also be useful to provide an effective treatment for patients needing repeat dosing. AAV manufactured in compliance with current Good Manufacturing Practices (cGMP) re gulations must undergo product identity testing before being released for use in the clinic. Cu rrently, identity testing involves sequencing the packaged vector DNA and verifying the sequence against the GenBank database. Assays to verify the capsid serotype are not common and rely on immunological reagents that can cross react
39 with multiple serotypes. In the present study, we utilize proteolytic mapping as a tool for AAV capsid serotype identification. Treatment of AAV capsids with a variety of proteases results in unique fragmentation patterns for different AAV se rotypes, and we are using these reagents to develop standardized identity tests for AAV. By characterizing different serotypes with different proteases, we are developing a fingerprint data base that can be used to identify several AAV serotypes. These assays and databases provide be tter product characterization and protection of patients. 54. Characterization of photoreceptor gene s regulating apothecial morphogenesis of Sclerotinia sclerotiorum Veluchamy S, Rollins JA* Department of Plant Pathology, Un iversity of Florida, Gainesville, FL Apothecial development is a particularly important step in the developmental life cycle of Sclerotinia sclerotiorum, as ascospores are the primary source of initial inoculums and each apothecium is capable of producing millions of ascospores. This morphogenesis is light dependent requiring light in the UV-A waveleng ths. We have identified a number of putative photoreceptor encoding genes from our collaborative S. sclerotiorum Whole Genome Sequencing Project. In an attempt to identify a UV-A photoreceptor we been studying a gene homology to cryptochromes (scry1) in S. sclerotiorum. The predicted scry1 protein sequence shows a high degree of sequence identity (50%) to N. crassa cry1. scry1 transcript accumulates only after exposure to UV-A and not in other wavelengths of light including constant dark, white light, blue, red or green. Northern blot analyses revealed the accumulation of scry11 transcript in all stages of apothecia and vegetative mycelia but not in sc lerotia Different UV-A light fluence rates (from 1.2 to 14 moles/m2/s) were tested to study transcript accumulation kinetics in mycelia and apothecia. In apothecia, scry1 transcript was si gnificantly increased by two and six hours of UV-A light treatment at 3.5 moles/m2/s). We are currently working to functionally disrupt the scry1 gene to determine its involvement in light dependent apothecial development. 55. The regulation of peripheral myelin protein 22 expression by micoRNAs Verrier JD1, Lau P2, Hudson LD2, Notterpek L1,* 1Departments of Neuroscience and Anatomy and Cell Bi ology, University of Florida, Gainesville, FL 2Section of Developmental Genetics, National In stitute of Neurological Disorders and Stroke, National Institutes of Health MicroRNAs (miRNAs) are small ~22 nucleotide RNA molecules that have been shown to regulate gene expression at the post-transcriptional le vel primarily through interaction with the mRNAs 3UTR region. Recently miRNAs have been shown to be involved in many cellular processes, including development and apoptosis, as well as disease states. Using several miRNA prediction models (TargetScan, miRBase, and miRanda), we have identified two miRNAs, mir-9 and mir-29, that are likely to interact with Peripheral Myelin Protein 22 (PMP22). PMP22 is an essential myelin protein synthesized by Schwann cells in the peripheral nervous system. Underand overexpression of this protein are linked to hereditary peripheral neuropathies. To investigate the potential roles of mir-9 and mir-29 in the regula tion of pmp22 in rat Schwann cells, we first overexpressed the miRNAs through transfection of plasmids and analyzed PMP22 RNA levels by Northern blots. Using an emerald-green fluores cent protein (EmGFP) encoding plasmid, the transfection efficiency of the cells was ~30%. Tw enty-four hours post-transfection, we observed a notable reduction in the levels of PMP22 mRNA with both miRNAs, when compared to control cells. Steady-state levels of PMP22 protein were similarly reduced, as compared to EmGFP-alone
40 expressing cells. In agreement, fluorescence mi croscopy revealed a reduction of PMP22-like immunoreactivity in miRNA-positive cells. Steady-state protein levels are more affected by mir-9, as compared to mir-29. These experiments reve al a new mechanism in the regulation of the PMP22 gene and identify a potentially novel th erapeutic target for PMP22-linked diseases. 56. Functional characterization of myrosinases and interacting proteins in Arabidopsis Wang Y-YE*, Vijayvargiga K*, Godsmith J*, Chen S* Department of Botany, University of Florida, Gainesville, FL Plants are unique in their ability to produce a wide array of secondary compounds used for foods, flavors, medicines, and industrial materials. Gluc osinolates are a group of such compounds, the degradation products of which are not only beneficial to humans but also play an important role in plant growth, development, and interactions with insect herbivores. Hydrolysis of glucosinolates is known to be catalyzed by myro sinase, the only known S-glucosidases in nature. In model plant Arabidopsis, there are 6 myrosinase genes and many genes encoding myrosinase associated proteins and binding proteins. The functions of these genes and their interactions are not well understood. In our lab, we took multidis ciplinary approaches including molecular biology, biochemistry, proteomics, reverse genetics an d physiology to study gene functions in glucosinolate metabolism. We have cloned genes of myrosinase TGG1 and TGG2, myrosinase associated protein MyAP1 and myrosinase binding protein MBP1 in E. coli. The genes were transformed into yeast S. cerevisiae for protein expression and interaction studies. Western blot analysis has shown positive protein expression The heterologously expressed proteins are currently tested for enzyme activities, substrate prof iles and interactions with each other. In the meantime, vacuolar pr oteomics studies in Arabidopsis have revealed vacuolar localization of these proteins. Myrosinase complex formation w ill be analyzed using purified vacuoles from Arabidopsis. Reverse genetics tools were also implemented to characterize in vivo protein functions. T-DNA knockout mutants including sing le and double mutants of myrosinase TGG1 and TGG2 were obtained. Recent results will be presented and discussed. 57. SLCO1B1 gene variation and apolip oprotein response to atorvastatin Welder GJ1, Arant CB2, Wessel TR2, Schofield RS2,3, Langaee TY1,*, Zineh I1 1Center for Pharmacogenomics/Department of Pharmacy Practice, University of Florida, Gainesville, FL 2Division of Cardiovascular Medicine, University of Florida College of Medicine, Gainesville, FL 3Department of Veterans Affair s Medical Center, Gainesville, FL Studies suggest that apolipoprotein (apo) B, apoA1, and apoB/apoA1 ratio may be superior predictors of cardiovascular disease (CVD) risk over low-density lipoprotein (LDL) cholesterol. There are no pharmacogenetic studies evaluating variable statin effects on apos. The SLCO1B1 gene encodes a hepatic transporter responsible for statin uptake into their site of action. We investigated whether genetic variants in SLCO1B1 impact the apo response to atorvastatin. Fortyfive subjects (64% women) without CVD or risk equivalents were analyzed. Fasting apos were measured at baseline and after 8 weeks of ator vastatin 80 mg daily. SLCO1B1 genotypes and haplotypes were determined by PCR and pyro sequencing. T-tests were used to test the hypothesis that 174Ala variant carriers and/or su bjects with the *5 haplotype had diminished apo responses when compared to wildtype homozygotes (*1a/*1a), since it has been shown that these variants have diminished transporter activity. Baseline age, apoB, and apoA1 were: 3415 yrs, 8123 mg/dl, and 14431 mg/dl respectively. There were no differences in baseline
41 characteristics between genotype groups. 174Ala ca rriers (N=18) had an apoB reduction of 33% compared with 41% in *1a/*1a subjects (p=0.10) and a 12% greater increase in apoA1 (p=0.08). Those with the SLCO1B1*5 haplotype (N =6) had an apoB reduction of 34% compared to *1a/*1a subjects (p=0.07). An apoB/apoA1 reduction of 32% was seen in this haplotype group compared to 41% in wild-type homozy gotes (p=0.07). SLCO1B1 polymorphisms were associated with differences in the apo resp onse to atorvastatin 80 mg daily. Further investigations into SLCO1B1 variations are warra nted to confirm differences in the therapeutic effects of statin treatment. 58. The ankyrin repeat, host range protein of myxoma virus, M-T5, activates Akt and can be functionally replaced by PIKE-A Werden SJ 1,2, Barrett JW2, McFadden G1,* 1Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Fl. 2Department of Microbiology and Immunology University of Wester n Ontario and Robarts Research Institute, London, Ontario, Canada. The myxoma virus (MV) ankyrin-repeat host ra nge factor, M-T5, has the ability to bind and activate cellular Akt, leading to permissive MV replication in a variety of diverse human cancer cell lines (Wang et al. Proc. Natl. Acad. Sci. U.S.A. 103:46404645, 2006). The susceptibility of permissive human cancer cells to MV infection is directly correlated with the basal or induced levels of phosphorylated Akt. When M-T5 is de leted from MV, the knockout virus, vMyxT5KO, can no longer productively infect a subset of the human cancer cells (designated Type II) that exhibit little or no endogenous phosphor ylated Akt. In searching for a host counterpart of M-T5 we noted sequence similarity of M-T5 to a recently identified ankyrin-repeat cellular binding protein of Akt called PIKE-A. PIKE-A binds and activates the kinase activity of Akt in a GTP-dependent manner and promotes the invasiveness of human cancer cell lines. Here we demonstrate that transfected PIKE-A is able to rescue the ability of vMyxT5KO to productively infect Type II human cancer cells that were previously resistant to infe ction. As well, cancer cells that were completely non-permissive for both wild-type and vMyxT5KO infection (called Type III) were rendered fully permissive following ectopic expression of PIKE -A. We conclude that the MV M-T5 host range protein is functionally interchangeable with the ho st PIKE-A protein, and that the activation of host Akt by either M-T5 or PIKE-A is critical fo r the permissiveness of human cancer cells by MV. 59. Transgenic approaches to enhance abiotic stress tolerance of bahiagrass Xiong X1, James V1, Lomba P1, Halbig SM1, Altpeter F1,*, Blount A2 1Department of Agronomy, Plant Molecular an d Cellular Biology Program, Gainesville, FL 2Department of Agronomy, North Florida Rese arch and Education Center, Marianna, FL Bahiagrass (Paspalum notatum Flugge) is an important turf and forage grass in the southern United States and subtropical regions of th e world. The productivity and performance of bahiagrass is limited by abiotic stresses, such as drought, salt, and low temperature (chilling and freezing). Altering the transcriptional regulation of stress response genes is a successful strategy to enhance stress tolerance in plants. Stress in ducible transcription factors like HvWRKY38 and OsMYB4 have the potential to enhance the expressi on of stress protective genes. We generated transgenic bahiagrass plants constitutively ov er-expressing HvWRKY38 or OsMYB4 via biolistic gene transfer. We will present molecular and ph ysiological data allowing the correlation of transcription factor over-expression and plant performance under abiotic stress.
42 60. Characterization of a bipartite rAAV ve ctor for AAVS1 site specific integration ex vivo therapy Zhang C, Cortez N, Berns KI* Department of Molecular Genetics and Microb iology, University of Florida, Gainesville, FL Wild type adeno-associated virus 2 (AAV2) can establish a latent infection in cell culture by integrating at a specific site (AAVS1) on the q arm of human chromosome 19. Requirements for site specific integration include a permissive intr acellular milieu (e.g., absence of a helper virus co-infection), the AAV Rep 68/78 protein in trans, the AAV inverted terminal repeat (itr) in cis (this may not be essential if the P5 promoter, which contains a Rep binding site (RBS) is present), and AAVS1. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain the rep gene or the P5 promoter and consequently do not undergo site specific integration. Development of an AAV vector which could integrate into AAVS1 might offer significant advantages in the engineer ing of progenitor cells and stem cells ex vivo. Appropriately engineered cells could be expanded, cloned, characterized and used for human gene therapy. In this report we describe the development of a bipartite AAV vector which can preferentially integrate the transgene at AAVS1 while the rep gene remains extrachromosomal. Our results demonstrated that with the optimal MOI and ratio of rAAVSVAV2 and rAAVUF11, the transgene integrated at AAVS1 specifically with high efficiency (83.3%). Most importantly, the cloned cell lines with the AAVS1 site specific integrated GFP were healthy and continuously and stably expressing GFP. In discussion we prop ose a model of AAVS1 site specific integration ex vivo gene therapy of rAAV. 61. Lamprey type II collagen and Sox9 reveal an ancient origin of the vertebrate collagenous skeleton Zhang G, Miyamoto MM*, Cohn MJ* Department of Zoology, University of Florida, Gainesville, FL Type II collagen is the major cartilage matrix prot ein in the jawed vertebrate skeleton. Lampreys and hagfishes, by contrast, are thought to ha ve non-collagenous cartilage.This difference in skeletal structure has led to the hypothesis th at the vertebrate common ancestor had a noncollagenous skeleton, with type II collagen beco ming the predominant cartilage matrix protein after the divergence of jawless fish from the jawed vertebrates, approximately 500 million years ago. Here we report that lampreys have two ty pe II collagen (Col2a1) genes that are expressed during development of the cartilaginous skeleton. We also demonstrate that the lamprey skeleton is comprised of Col2a1 protein. Furthermore, we have isolated Sox9, a direct transcriptional regulator of Col2a1 in jawed vertebrates, and show that it is co-expressed with both Col2a1 genes during development of the lamprey skelet on. These results reveal a highly conserved genetic pathway for chondrogenesis in lamp reys and gnathostomes, and suggest that a collagenous skeleton evolved surprisingly close to the origin of vertebrates.
43 62. Altering plant architecture of bahiagrass by constitutive expression of the Arabidopsis ATHB16 gene, a repressor of cell expansion Zhang H, Lomba P, Altpeter F* Department of Agronomy, Plant Molecular an d Cellular Biology Program, Gainesville, FL Bahiagrass (Paspalum notatum Flugge) is a popular low-input turfgrass in subtropical regions. However its turf quality and widespread use as re sidential turf is limited by up to 60 cm tall seedheads and its open, sparse looking growth habit. Due to rising mowing costs and the absence of a dwarf bahiagrass cultivar, plant growth retardants (PGRs) are being used increasingly to suppress seedheads and leaf growth in bahiagrass. However, PGRs have to be applied frequently and may result in phytotoxici ty. Improvement of turf quality by genetic engineering represents a promising alternative. In this study, the ATH16 transcription factor gene was isolated from Arabidopsis, cloned in an ex pression cassette between the CaMV 35S promoter and nos terminator and genetically transferred to bahiagrass via particle bombardment. The integration of ATHB16 in the bahiagrass genome an d its constitutive expression were confirmed by Southern and RT-PCR analysis respectively. Mo rphological characteristics of bahiagrass overexpressing ATHB16 under controlled environment co nditions will be presented in comparison to non-transgenic bahiagrass. 63. Analysis of -globin locus activation during eryt hroid differentiation of embryonic stem cells Zhou Z, Crusselle-Davis V, I-Chun L, Levings P, Bungert J* Department of Agronomy, Plant Molecular and Ce llular Biology Program, University of Florida, Gainesville The -globin gene locus consists of five developm entally regulated genes that are under control of a locus control region (LCR) located far upst ream of the genes. The LCR is composed of several subregions exhibiting high sensitivity to DNaseI in erythroid cells, called HS sites, which function together to mediate high level globin expression. Recent studies have shown that individual HS sites recruit transcription complexe s. We found that RNA polymerase II (RNA Pol II) is recruited to LCR HS elements prior to erythr oid differentiation. In order to understand the temporal order of transcription factor recruitm ent and modification of chromatin structure, we continued our analysis of the globin locus during in vitro differentiation using chromatin immunoprecipitation. Several transcription fact ors, including the ubiquitously expressed helixloop-helix protein USF are detectable at LCR elem ents prior to erythroid differentiation. We have recently developed an in vitro system to study the transfer of activities from the LCR to the globin gene. We found that RNA Pol II can be tr ansferred from immobilized LCR constructs to a -globin;-globin gene, that the transfer is sensit ive to mutations in the basal promoter of the globin;-globin gene, and that the hematopoietic tr anscription factor NF-E2 facilitates the transfer from the LCR to the globin gene promoter. We continued this analysis and found that other proteins are transferred from the LCR to the gl obin gene promoter, including USF, Fog-1, and GATA-1. Our data suggest that NF-E2 decreases the affinity of RNA Pol II to the LCR and at the same time increases the binding affinity to the -globin gene promoter. Taken together our data support the proposition that transcription co mplexes are first recruited to the LCR and subsequently transferred to the globin genes.